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WO2025194124A1 - Modified st1cas9 guide nucleic acids - Google Patents

Modified st1cas9 guide nucleic acids

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Publication number
WO2025194124A1
WO2025194124A1 PCT/US2025/020065 US2025020065W WO2025194124A1 WO 2025194124 A1 WO2025194124 A1 WO 2025194124A1 US 2025020065 W US2025020065 W US 2025020065W WO 2025194124 A1 WO2025194124 A1 WO 2025194124A1
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WIPO (PCT)
Prior art keywords
domain
sequence
nucleic acid
stlcas9
chemically modified
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
PCT/US2025/020065
Other languages
French (fr)
Other versions
WO2025194124A8 (en
Inventor
Xiaolong DONG
Anne Helen Bothmer
Aamir MIR
Luciano Henrique APPONI
Alan MENDOZA
Zsanett JANCSO
Cecilia Giovanna Silvia COTTA-RAMUSINO
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Tessera Therapeutics Inc
Original Assignee
Tessera Therapeutics Inc
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Filing date
Publication date
Application filed by Tessera Therapeutics Inc filed Critical Tessera Therapeutics Inc
Publication of WO2025194124A1 publication Critical patent/WO2025194124A1/en
Publication of WO2025194124A8 publication Critical patent/WO2025194124A8/en
Pending legal-status Critical Current
Anticipated expiration legal-status Critical

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • C—CHEMISTRY; METALLURGY
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C12N15/102—Mutagenizing nucleic acids
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
    • C12N15/88—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation using microencapsulation, e.g. using amphiphile liposome vesicle
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
    • C12N15/90—Stable introduction of foreign DNA into chromosome
    • C12N15/902—Stable introduction of foreign DNA into chromosome using homologous recombination
    • C12N15/907—Stable introduction of foreign DNA into chromosome using homologous recombination in mammalian cells
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/20—Type of nucleic acid involving clustered regularly interspaced short palindromic repeats [CRISPR]
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/30—Chemical structure
    • C12N2310/34—Spatial arrangement of the modifications
    • C12N2310/343—Spatial arrangement of the modifications having patterns, e.g. ==--==--==--
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
    • C12N9/22—Ribonucleases [RNase]; Deoxyribonucleases [DNase]
    • C12N9/222—Clustered regularly interspaced short palindromic repeats [CRISPR]-associated [CAS] enzymes
    • C12N9/226—Class 2 CAS enzyme complex, e.g. single CAS protein

Definitions

  • Alpha- 1 antitrypsin deficiency is characterized by low circulating levels of alpha- 1 antitrypsin (AAT).
  • AAT is produced primarily in liver cells and secreted into the blood, but it is also made by other cell types including lung epithelial cells and certain white blood cells.
  • AAT inhibits several serine proteases secreted by inflammatory cells (most notably neutrophil elastase [NE], proteinase 3, and cathepsin G) and thus protects organs, such as the lung, from protease-induced damage, especially during periods of inflammation.
  • the two most common clinical variants of AAT are E264V (PiS) and E342K (PiZ).
  • the clinical E342K (PiZ) mutation (also referred to as the Z mutation) is caused by a single base-pair substitution in the SERPINA gene (referred to as the Z allele) and results in a glutamic acid to lysine mutation at position 342 of AAT. Inheritance of the Z allele is autosomal codominant and more than half of AATD patients harbor at least one copy of the Z allele.
  • the E342K mutation leads to structurally unstable and/or inactive AAT-Z protein that causes toxicity in the liver and is inactive in the lungs.
  • the E342K mutation is located at the hinge between the beta sheet and the Reactive Center Loop (RCL) of the AAT protein and causes a loop-sheet dimer that can extend to form long chains of loop-sheet polymers. These polymers form aggregates that accumulate inside the rough endoplasmic reticulum of hepatocytes during translation and are therefore not secreted into the bloodstream. Consequently, circulating AAT levels in individuals homozygous for the Z allele (PiZZ) are markedly reduced; only approximately 15% of mutant AAT-Z protein folds correctly and is secreted by the cell.
  • RCL Reactive Center Loop
  • a gain-of - function phenotype presents as the accumulation of polymerized AAT-Z protein in hepatocytes results in a gain-of-function cytotoxicity that can result in cellular stress, inflammation, fibrosis, cirrhosis, hepatocellular carcinoma (HCC), and neonatal liver disease in 12% of patients. This accumulation may spontaneously remit but can be fatal in a small number of children.
  • a loss-of- function phenotype results from the reduced systemic levels of AAT that lead to increased protease digestion of connective tissue in the lower airway.
  • COPD Chronic Obstructive Pulmonary Disease
  • PiZZ individuals account for the majority of patients with clinically relevant AATD lung disease.
  • a milder form of AATD is associated with the SZ genotype in which a patient has a Z allele and an S allele.
  • the S allele is associated with somewhat reduced levels of circulating AAT but the AAT S-protein is not hepatotoxic. Accordingly, the SZ genotype is associated with clinically significant lung disease but not liver disease. Frcgoncsc and Stolk, Orphanct J Rare Dis. 2008; 33:16.
  • the deficiency of circulating AAT in subjects with the SZ genotype results in dysregulated protease activity that degrades lung tissue over time and can result in emphysema, particularly in smokers.
  • Augmentation therapy involves administration (weekly infuction) of a human AAT protein concentrate purified from pooled from healthy donor plasma. Although infusions of plasma protein have been shown to improve survival or slow the rate of emphysema progression, augmentation therapy is often insufficient under challenging conditions (e.g., active lung infection). Augmentation therapy also fails to restore the normal physiological regulation of AAT in patients and efficacy has been difficult to demonstrate. In addition, augmentation therapy does not remedy liver disease driven by the toxic gain-of-function of the Z allele. Accordingly, there is a need for new and more effective treatments for AATD.
  • This disclosure relates to novel compositions, systems, and methods for altering a genome at one or more locations in a host cell, tissue, or subject, in vivo or in vitro.
  • the disclosure provides, for instance, gene modifying systems that comprise a gene modifying polypeptide comprising a reverse transcriptase (RT) domain and a StlCas9 domain, and a template RNA comprising a variant gRNA scaffold that has been engineered for improved performance, e.g., when used in concert with the StlCas9 domain.
  • RT reverse transcriptase
  • the disclosure also provides gene modifying systems that are capable of modulating (e.g., inserting, altering, or deleting sequences of interest) alpha- 1 antitrypsin (AAT) activity and methods of treating alpha- 1 antitrypsin deficiency (AATD) by administering one or more such systems to alter a genomic sequence at a single nucleotide to correct the SERPINA1 PiZ mutation that causes AATD.
  • AAT alpha- 1 antitrypsin
  • AATD alpha- 1 antitrypsin deficiency
  • the present disclosure provides a system for modifying DNA to correct a human SERPINA1 gene mutation that causes AATD comprising (a) a nucleic acid encoding a gene modifying polypeptide capable of target primed reverse transcription, the polypeptide comprising (i) a reverse transcriptase domain and (ii) a St1Cas9 nickase that binds DNA and has endonuclease activity, and (b) a template RNA comprising (i) a gRNA spacer that is complementary to a first portion of the human SERPINA1 gene, (ii) a gRNA scaffold that binds the polypeptide, (iii) a heterologous object sequence comprising a mutation region to correct the SERPINA1 gene mutation, and (iv) a primer binding site (PBS) sequence comprising at least 3, 4, 5, 6, 7, or 8 bases of 100% homology to a target DNA strand at the 3' end of the template RNA.
  • the SERPINA1 gene may comprise
  • a gRNA spacer may comprise at least 15 bases of 100% homology to a target DNA at the 5' end of the template RNA.
  • a template RNA may further comprise a PBS sequence comprising at least 5 bases of at least 80% homology to a target DNA strand.
  • a template RNA may comprise one or more chemical modifications.
  • Domains of a gene modifying polypeptide may be joined by a peptide linker.
  • a polypeptide may comprise one or more peptide linkers.
  • a gene modifying polypeptide may further comprise a nuclear localization signal.
  • a polypeptide may comprise more than one nuclear localization signal, e.g., multiple adjacent nuclear localization signals or one or more nuclear localization signals in different regions of the polypeptide, e.g., one or more nuclear localization signals in the N-terminus of the polypeptide and one or more nuclear localization signals in the C-terminus of the polypeptide.
  • a nucleic acid encoding a gene modifying polypeptide may encode one or more intein domains.
  • Introduction of a system of the present disclosure into a target cell may result in insertion of at least 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 500, or 1000 base pairs of exogenous DNA.
  • Introduction of a system of the present disclosure into a target cell may result in a deletion, wherein the deletion is less than 2, 3, 4, 5, 10, 50, or 100 base pairs of genomic DNA upstream or downstream of an insertion.
  • Introduction of a system of the present disclosure into a target cell may result in substitution, e.g., substitution of 1, 2, or 3 nucleotides, e.g., consecutive nucleotides.
  • a heterologous object sequence may be at least 5, 10, 25, 50, 100, 150, 200, 250, 300, 400, 500, 600, or 700 base pairs.
  • the present disclosure provides a pharmaceutical composition comprising a system described herein and a pharmaceutically acceptable excipient or carrier, wherein the pharmaceutically acceptable excipient or carrier is selected from the group consisting of a plasmid vector, a viral vector, a vesicle, and a lipid nanoparticle.
  • the present disclosure provides a pharmaceutical composition
  • a pharmaceutical composition comprising a system described herein and multiple pharmaceutically acceptable excipients or carriers, wherein the pharmaceutically acceptable excipients or carriers are selected from the group consisting of a plasmid vector, a viral vector, a vesicle, and a lipid nanoparticle, e.g., where the system described above is delivered by two distinct excipients or carriers, e.g., two lipid nanoparticles, two viral vectors, or one lipid nanoparticle and one viral vector.
  • a viral vector may be an adeno- associated virus (AAV).
  • the disclosure relates to a host cell (e.g., a mammalian cell, e.g., a human cell) comprising the system described above.
  • a host cell e.g., a mammalian cell, e.g., a human cell
  • the present disclosure provides a method of correcting a mutation in the human SERPINA1 gene in a cell, tissue or subject, the method comprising administering a system described herein to the cell, tissue or subject, wherein optionally the correction of the mutant SERPINA1 gene comprises an amino acid substitution of K342E (i.e., reversing the pathogenic E342K mutation).
  • a system described herein may be introduced in vivo, in vitro, ex vivo, or in situ.
  • a nucleic acid of a system described herein may be integrated into the genome of a host cell. In some embodiments, a nucleic acid of a system described herein is not integrated into the genome of a host cell.
  • a heterologous object sequence is inserted at only one target site in a host cell genome.
  • a heterologous object sequence may be inserted at two or more target sites in a host cell genome, e.g., at the same corresponding site in two homologous chromosomes or at two different sites on the same or different chromosomes.
  • a heterologous object sequence may encode a mammalian polypeptide, or a fragment or a variant thereof.
  • Components of a system described herein may be delivered on 1, 2, 3, 4, or more distinct nucleic acid molecules.
  • a system of the present disclosure may be introduced into a host cell by electroporation or by using at least one vehicle selected from a plasmid vector, a viral vector, a vesicle, and a lipid nanoparticle.
  • compositions or methods can include one or more of the following enumerated embodiments. Enumerated Embodiments
  • a nucleic acid comprising a Repeat: anti-repeat (RAR) region which comprises a chemically modified nucleotide.
  • RAR anti-repeat
  • nucleic acid of embodiment 1 which further comprises a stem-loop 1 (SL1) region (e.g., wherein the SL1 region is 3’ of the RAR region), wherein optionally the SL1 region comprises a chemically modified nucleotide.
  • SL1 region e.g., wherein the SL1 region is 3’ of the RAR region
  • optionally the SL1 region comprises a chemically modified nucleotide.
  • nucleic acid of embodiment 1 or 2 which further comprises a stem loop 2 (SL2) region (e.g., wherein the SL2 region is 3’ of the SL1 region), wherein optionally the SL2 region comprises a chemically modified nucleotide.
  • SL2 region e.g., wherein the SL2 region is 3’ of the SL1 region
  • optionally the SL2 region comprises a chemically modified nucleotide.
  • a nucleic acid comprising a stem-loop 1 (SL1) region which comprises a chemically modified nucleotide.
  • nucleic acid of embodiment 4 which further comprises an RAR region (e.g., wherein the RAR region is 5’ of the SL1 region), wherein optionally the RAR region comprises a chemically modified nucleotide.
  • a nucleic acid molecule comprising: an StlCas9 scaffold; wherein the StlCas9 scaffold comprises a chemically modified nucleotide.
  • a nucleic acid molecule comprising: an StlCas9 scaffold; wherein the StlCas9 scaffold comprises a chemically modified nucleotide at one or more of (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38 or all of) positions 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, and 42 relative to SEQ ID NO: 25999.
  • the StlCas9 scaffold comprises a chemically modified nucleotide at one or more of (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, and 42 relative to SEQ ID NO: 25999.
  • a nucleic acid molecule comprising: an StlCas9 scaffold comprising: a) a Repeat: anti-repeat (RAR) region, wherein optionally the RAR region comprises a RAR lower stem, a RAR upper stem, and an RAR loop (c.g., a tctraloop); b) a stem- loop 1 (SL1) region that is optionally 3’ of the RAR region, and c) optionally, a stem loop 2 (SL2) region that is optionally 3’ of the SL1 region; wherein the StlCas9 scaffold comprises a chemically modified nucleotide in one or both of the RAR region or the SL1 region.
  • RAR anti-repeat
  • SL1 stem- loop 1
  • SL2 stem loop 2
  • the StlCas9 scaffold comprises: a) a Repeat: anti-repeat (RAR) region, wherein optionally the RAR region comprises a RAR lower stem, a RAR upper stem, and an RAR loop (e.g., a tetraloop); b) a stem-loop 1 (SL1) region that is 3’ of the RAR region, and c) optionally, a stem loop 2 (SL2) region that is 3’ of the SL1 region; wherein the StlCas9 scaffold comprises a chemically modified nucleotide in one or both of the RAR region or the SL1 region.
  • RAR repeat: anti-repeat
  • SL1 stem-loop 1
  • SL2 stem loop 2
  • the StlCas9 scaffold comprises a chemically modified nucleotide at one or more of (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or all of) positions 4, 5, 6, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 34, 35, 36, 37, 38, 39, 40, 41, and 42.
  • a chemically modified nucleotide at one or more of (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or all of) positions 4, 5, 6, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 34, 35, 36, 37, 38, 39, 40, 41, and 42.
  • the StlCas9 scaffold comprises a RAR region, wherein the RAR region comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,
  • the StlCas9 scaffold comprises a SL1 region, wherein the SL1 region comprises 1, 2, 3, 4, 5, 6, 7, or 8 chemically modified nucleotides (e.g., wherein the chemically modified nucleotides have the same chemical modification).
  • the St 1 Cas9 scaffold comprises a chemically modified nucleotide in the RAR region and a chemically modified nucleotide in the SL1 region.
  • the StlCas9 scaffold comprises between 10-21 (e.g., 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or 21) chemically modified nucleotides in the RAR region.
  • the StlCas9 scaffold comprises between 0-8 (e.g., 0, 1, 2, 3, 4, 5, 6, 7, or 8) chemically modified nucleotides in the SL1 region.
  • nucleic acid of any of the preceding embodiments wherein positions 1, 2, and 3 (if present) do not comprise a 2’-O-methyl chemically modified nucleotide.
  • the StlCas9 scaffold comprises a first insertion (e.g., of 4 nucleotides) between positions
  • the StlCas9 scaffold comprises an insertion (e.g., of 10 nucleotides) between positions 15 and 18, and a deletion of positions 16 and 17, wherein optionally the insertion has a sequence according to GACUUCGGUC.
  • the StlCas9 scaffold comprises a chemically modified nucleotide (e.g., 2’-O-methyl) at one or more of (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or all of) positions 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, and 25.
  • a chemically modified nucleotide e.g., 2’-O-methyl
  • the StlCas9 scaffold comprises a chemically modified nucleotide (e.g., 2’-O-methyl) at one or more of (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or all of) positions 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 50, 51, 52, 53, and 54.
  • a chemically modified nucleotide e.g., 2’-O-methyl
  • the StlCas9 scaffold comprises a chemically modified nucleotide (e.g., 2’-O-methyl) at one or more of (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or all of) positions 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 51, 52, 53, and 54. 40.
  • a chemically modified nucleotide e.g., 2’-O-methyl
  • the StlCas9 scaffold comprises a chemically modified nucleotide (e.g., 2’-O-methyl) at one or more of (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or all of) positions 4, 5, 6, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 34, 35, 36, 37, 38, 39, 40, 41, and 42.
  • a chemically modified nucleotide e.g., 2’-O-methyl
  • the StlCas9 scaffold comprises a chemically modified nucleotide (e.g., 2’-O-methyl) at one or more of (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, or all of) positions 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, and 54.
  • a chemically modified nucleotide e.g., 2’-O-methyl
  • the StlCas9 scaffold comprises a chemically modified nucleotide (e.g., 2’-O-methyl) at one or more of (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, or all of) positions 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, and 53.
  • a chemically modified nucleotide e.g., 2’-O-methyl
  • the StlCas9 scaffold comprises a chemically modified nucleotide (e.g., 2’-O-methyl) at one or more of (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, or all of) positions 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, and 52.
  • a chemically modified nucleotide e.g., 2’-O-methyl
  • a chemically modified nucleotide e.g., 2’-O-methyl
  • the StlCas9 scaffold comprises a chemically modified nucleotide (e.g., 2’-O-methyl) at one or more of (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or all of) positions 4,
  • a chemically modified nucleotide e.g., 2’-O-methyl
  • chemically modified nucleotide e.g., 2’-O-methyl
  • the St 1 Cas9 scaffold comprises a chemically modified nucleotide (c.g., 2’-O-mcthyl) at one or more of (c.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or all of) positions 4, 5, 6, 12, 13, 14, 15, 16, 17, 18, 19, 20, and 21.
  • a chemically modified nucleotide c.g., 2’-O-mcthyl
  • the StlCas9 scaffold comprises a chemically modified nucleotide (e.g., 2’-O-methyl) at one or more of (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, or all of) positions 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, and 24.
  • a chemically modified nucleotide e.g., 2’-O-methyl
  • a chemically modified nucleotide e.g., 2’-O-methyl
  • the StlCas9 scaffold comprises a chemically modified nucleotide (e.g., 2’-O-methyl) at one or more of (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, or all of) positions 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 34, 35, 36, 37, 38, 39, 40, 41, and 42.
  • a chemically modified nucleotide e.g., 2’-O-methyl
  • the StlCas9 scaffold comprises a chemically modified nucleotide (e.g., 2’-O-methyl) at one or more of (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, or all of) positions 4, 5, 6, 12,
  • a chemically modified nucleotide e.g., 2’-O-methyl
  • the StlCas9 scaffold comprises a chemically modified nucleotide (e.g., 2’-O-methyl) at one or more of (e.g., 2, 3,
  • the StlCas9 scaffold comprises a chemically modified nucleotide (e.g., 2’-O-methyl) at one or more of (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or all of) positions 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 43, and 44.
  • a chemically modified nucleotide e.g., 2’-O-methyl
  • the StlCas9 scaffold comprises a chemically modified nucleotide (e.g., 2’-O-methyl) at one or more of (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, or all of) positions 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, and 43.
  • the St 1 Cas9 scaffold comprises a chemically modified nucleotide (c.g., 2’-O-mcthyl) at one or more of (c.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, or all of) positions 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, and 44.
  • the StlCas9 scaffold comprises a chemically modified nucleotide (e.g., 2’-O-methyl) at one or more of (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, or all of) positions 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, and 45.
  • a chemically modified nucleotide e.g., 2’-O-methyl
  • the StlCas9 scaffold comprises a chemically modified nucleotide (e.g., 2’-O-methyl) at one or more of (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, or all of) positions 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, and 46.
  • a chemically modified nucleotide e.g., 2’-O-methyl
  • the StlCas9 scaffold comprises a chemically modified nucleotide (e.g., 2’-O-methyl) at one or more of (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, or all of) positions 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, and 47.
  • a chemically modified nucleotide e.g., 2’-O-methyl
  • the StlCas9 scaffold comprises a chemically modified nucleotide (e.g., 2’-O-methyl) at one or more of (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, or all of) positions 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, and 49.
  • a chemically modified nucleotide e.g., 2’-O-methyl
  • the StlCas9 scaffold comprises a chemically modified nucleotide (e.g., 2’-O-methyl) at one or more of (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, or all of) positions 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, and 50, or the StlCas9 scaffold comprises a chemically modified nucleotide (e.g., 2’-O-methyl) at one or more of (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, or all of) positions 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, and 51.
  • the StlCas9 scaffold comprises a chemically modified nucleotide (e.g., 2’-O-methyl) at one or more of (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, or all of) positions 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, and 51.
  • the StlCas9 scaffold comprises a chemically modified nucleotide (e.g., 2’-O-methyl) at each of positions 12 through 21; b) the StlCas9 scaffold comprises a chemically modified nucleotide (e.g., 2’-O-methyl) at each of positions 34 through 36; c) the StlCas9 scaffold comprises a chemically modified nucleotide (e.g., 2’-Fluoro) at one or more of positions 37 through 42; d) the StlCas9 scaffold comprises a chemically modified nucleotide (e.g., 2’-Fluoro) at each of positions 37 through 42; e) the StlCas9 scaffold comprises a chemically modified nucleotide (e.g., 2’-Fluoro) at one or more of positions 45 through 47; f) the StlCas9
  • nucleic acid of any of the preceding embodiments which comprises a plurality of chemically modified nucleotides in the StlCas9 scaffold, wherein the plurality of chemically modified nucleotides in the StlCas9 scaffold have the same chemical modification.
  • nucleic acid of any of embodiments 1-63 which comprises a plurality of chemically modified nucleotides in the StlCas9 scaffold, wherein the plurality of chemically modified nucleotides in the StlCas9 scaffold have two or more different chemical modifications.
  • the St 1 Cas9 scaffold comprises a sequence having the chemically modified nucleotides set out in Table 22, 21, 20, E3, E7, E8, E9, E10, E12, or E13.
  • LNA locked nucleic acid
  • nucleic acid of any of the preceding embodiments which further comprises a second chemically modified nucleotide.
  • nucleic acid of any of the preceding embodiments which comprises a sequence according to SEQ ID NO: 26000, or a sequence having at least 70%, 80%, 90%, 95%, 97%, 98%, or 99% identity thereto.
  • nucleic acid of any of the preceding embodiments which comprises a sequence of Tables 42, 43, or 44, or a sequence having at least 70%, 80%, 90%, 95%, 97%, 98%, or 99% identity thereto.
  • nucleic acid of any of the preceding embodiments which comprises a sequence according to SEQ ID NO: 25999, or a sequence having at least 70%, 80%, 90%, 95%, 97%, 98%, or 99% identity thereto.
  • nucleic acid of any of the preceding embodiments which comprises one or more (e.g., at least 1, 2, or 3, and optionally no more than 10, 20, or 30) sequence differences (e.g., insertions, deletions, or substitutions) relative to SEQ ID NO: 25999.
  • nucleic acid of any of the preceding embodiments wherein the SL2 region has a length of 20-30 nucleotides (e.g., 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides).
  • SS nucleic acid of any of embodiments 1-69, which comprises an SL2 region.
  • nucleic acid of any of the preceding embodiments which further comprises a gRNA spacer situated 5’ of the StlCas9 scaffold region.
  • nucleic acid of any of the preceding embodiments which further comprises a heterologous object sequence situated 3’ of the StlCas9 scaffold region.
  • nucleic acid of any of the preceding embodiments which further comprises a primer binding site (PBS) sequence situated 3’ of the StlCas9 scaffold region, e.g., wherein the PBS sequence is situated 3’ of the heterologous object sequence.
  • PBS primer binding site
  • PBS primer binding site
  • the template RNA of embodiment 85 which further comprises a chemical modification outside of the chemically modified StlCas9 scaffold.
  • a gRNA scaffold c.g., a StlCas9 scaffold, c.g., a chemically modified StlCas9 scaffold of any of claims 1-80;
  • PBS primer binding site
  • heterologous object sequence further comprises one or more (e.g., 2) additional chemically modified nucleotides, e.g., 2’- Fluoro nucleotides.
  • position +4 of the heterologous object sequence is a chemically modified nucleotide (e.g., a 2’-Fluoro nucleotide);
  • position +5 of the heterologous object sequence is a chemically modified nucleotide (e.g., a 2’-Fluoro nucleotide);
  • position +6 of the heterologous object sequence is a chemically modified nucleotide (e.g., a 2’-Fluoro nucleotide);
  • positions +4, +5, and +6 of the heterologous object sequence are each a chemically modified nucleotide (e.g., each is a 2’-Fluoro nucleotide);
  • the 3’ most nucleotide in the template RNA is a chemically modified nucleotide (e.g., a 2’-O-methyl nucleotide); the second nucleotide from the 3’ end of the template RNA is a chemically modified nucleotide (e.g., a 2’-O-methyl nucleotide); the third nucleotide from the 3’ end of the template RNA is a chemically modified nucleotide (e.g., a 2’-O-methyl nucleotide); the 3’ most nucleotide and the second and third nucleotides from the 3’ end of the template RNA are each a chemically modified nucleotide (e.g., each is a 2’-O-methyl nucleotide); the twelfth nucleotide from the 3’ end of the template RNA is a chemically modified nucleotide
  • RNA or nucleic acid of embodiment 95 wherein the deletion is between 1-32 (e.g., 2-29, 2-20, 2-10, or 10-20) nucleotides in length.
  • RNA or nucleic acid of embodiment 95 wherein the StlCas9 scaffold comprises a deletion of part of the second single stranded region (e.g., 1, 2, 3, or 4 nucleotides at the 3’ end of the single stranded region).
  • the chemically modified StlCas9 scaffold comprises a variant StlCas9 scaffold having one or both of a lengthened RAR upper stem or a substitution resulting in a G-C base pair in the RAR upper stem.
  • the template RNA or nucleic acid of embodiment 101 or 102, wherein the lengthened RAR upper stem comprises one or more base pairs that are new relative to SEQ ID NO: 25999, wherein the one or more new base pairs comprise one or more chemically modified nucleotides.
  • RNA or nucleic acid of any of the preceding embodiments wherein the tetraloop comprises a sequence chosen from: AACA, AAUA, ACCA, ACUA, AGUA, AGCA, AUCA, AUUA, CAAC, CUCG, CUUG, GAAA, GAGA, GCAA, GCGA, GGAA, GGAG, GGGA, GUAA, GUGA, UAAC, UACG, UCAC, UCCG, UGAA, UGAC, UGCG, UUAC, or UUCG.
  • RNA or nucleic acid of any of the preceding embodiments which comprises a sequence according to any of Tables 39, 24, 40, 57, 41, 42, 45, 56, or 12-62 or a sequence having at least 80%, 85%, 90%, 95%, 98%, or 99% identity thereto.
  • RNA or nucleic acid of any of the preceding embodiments which comprises a sequence according to SEQ ID NO: 27131, or a sequence having at least 80%, 85%, 90%, 95%, 98%, or 99% identity thereto.
  • RNA or nucleic acid of any of the preceding embodiments which comprises a sequence according to SEQ ID NO: 27133, or a sequence having at least 80%, 85%, 90%, 95%, 98%, or 99% identity thereto.
  • RNA or nucleic acid of any of the preceding embodiments which comprises a sequence according to SEQ ID NO: 27134, or a sequence having at least 80%, 85%, 90%, 95%, 98%, or 99% identity thereto.
  • the chemically modified StlCas9 scaffold comprises a variant gRNA scaffold comprising a sequence according to Table 23, or a sequence having no more than 1, 2, or 3 sequence alterations (e.g., substitutions) relative thereto.
  • a system comprising: a nucleic acid or template RNA of any of the preceding embodiments; and a polypeptide comprising a StlCas9 domain, or a nucleic acid encoding the polypeptide.
  • a Cas9 domain e.g., a StlCas9 domain
  • the gene modifying system of embodiment 120 or 121, wherein the gene modifying polypeptide comprises, in an N-terminal to C-terminal direction, a first NLS, the StlCas9 nickase domain, a linker, an RT domain, and a second NLS.
  • the first NLS comprises a sequence of SEQ ID NO: 11,095
  • the second NLS comprises a sequence of SEQ ID NO: 11,099.
  • linker has a sequence according to SEQ ID NO: 5006, or a sequence having at least 70%, 80%, 90%, 95%, 97%, 98%, or 99% identity thereto.
  • RT domain has a sequence according to SEQ ID NO: 26006, or a sequence having at least 70%, 80%, 90%, 95%, 97%, 98%, or 99% identity thereto.
  • the gene modifying system of embodiment 120 wherein the gene modifying polypeptide has a sequence according to SEQ ID NO: 26002, or a sequence having at least 70%, 80%, 90%, 95%, 97%, 98%, or 99% identity thereto.
  • a pharmaceutical composition comprising the template RNA of nucleic acid of any one of embodiments 1-117 or the system of any one of embodiments 118-131, and a pharmaceutically acceptable excipient or carrier.
  • composition of embodiment 132 wherein the pharmaceutically acceptable excipient or carrier is selected from the group consisting of a plasmid vector, a viral vector, a vesicle, and a lipid nanoparticle (LNP).
  • the pharmaceutically acceptable excipient or carrier is selected from the group consisting of a plasmid vector, a viral vector, a vesicle, and a lipid nanoparticle (LNP).
  • a host cell e.g., a mammalian cell, e.g., a human cell
  • a host cell comprising the gene modifying system, template RNA, or nucleic acid of any one of the preceding claims.
  • a method for modifying a target site e.g., a target site in the human SERPINA1 gene
  • the method comprising contacting the cell with the gene modifying system of any one of embodiments 118-131, or DNA encoding the same, or the pharmaceutical composition of embodiment 132 or 133, thereby modifying the target site.
  • a method for treating a subject having a disease or condition associated with a mutation in a gene comprising administering to the subject the gene modifying system of any one of embodiments 118-131, or DNA encoding the same, or the pharmaceutical composition of embodiment 132 or 133, thereby treating the subject having a disease or condition.
  • AATD alpha-1 antitrypsin deficiency
  • a method for treating a subject having AATD comprising administering to the subject the gene modifying system of any one of embodiments 118-131, or DNA encoding the same, or the pharmaceutical composition of embodiment 132 or 133, thereby treating the subject having AATD.
  • FIG. 1 is a diagram depicting components of a gene modifying system as described herein.
  • FIG. 1A is a diagram showing a gene modifying polypeptide comprising a Cas nickase domain (e.g., spCas9 N863A) and a reverse transcriptase domain (RT domain) which are linked by a linker.
  • FIG. IB is a diagram showing a template RNA comprising, from 5’ to 3’, a gRNA spacer, a gRNA scaffold, a heterologous object sequence, and a primer binding site sequence (PBS sequence).
  • a heterologous object sequence can comprise a mutation region that comprises one or more sequence differences relative to a target site.
  • a heterologous object sequence can also comprise a pre-edit homology region and a post-edit homology region, which flank a mutation region.
  • a gRNA spacer of a template RNA binds to a second strand of a target site in the genome
  • a gRNA scaffold of the template RNA binds to a gene modifying polypeptide, e.g., localizing the gene modifying polypeptide to a target site in the genome.
  • a Cas domain of a gene modifying polypeptide nicks a target site (e.g., a first strand of the target site), e.g., allowing a PBS sequence to bind to a sequence adjacent to the target site to be altered on the first strand of the target site.
  • an RT domain of a gene modifying polypeptide uses a first strand of a target site that is bound to a complementary sequence comprising a PBS sequence of a template RNA as a primer and a heterologous object sequence of the template RNA as a template to, e.g., polymerize a sequence complementary to the heterologous object sequence.
  • reverse transcription can then proceed through a pre-edit homology region, then through a mutation region, and then through a post-edit homology region, thereby producing a DNA strand comprising a mutation specified by a heterologous object sequence.
  • FIG. 2 is a diagram illustrating the hypothesized secondary structure of a wild-type StlCas9 gRNA scaffold and is overlaid with description of valiants described herein.
  • FIG. 3A is a bar graph showing the rewriting performance of exemplary StlCas9-based gene modifying systems comprising exemplary template RNAs comprising various scaffolds truncated in the stem loop 2 region as depicted in FIG. 2.
  • FIG. 4A is a bar graph showing the rewriting efficiency of exemplary gene modifying systems comprising different StlCas9-compatible template RNAs comprising modified scaffold sequences.
  • FIG. 4C is a bar graph showing the rewriting efficiency of additional exemplary gene modifying systems comprising different StlCas9-compatible template RNAs comprising modified scaffold sequences.
  • FIG. 5 is a bar graph showin the rewriting efficiency of exemplary gene modifying systems comprising StlCas9-based gene modifying polypeptide.
  • FIG. 6A is a bar graph showing the rewriting efficiency of exemplary gene modifying systems comprising StlCas9-based gene modifying polypeptide, with and without ngRNA.
  • FIG. 8B is a bar graph showing percent rewriting achieved using an exemplary gene modifying system comprising different StlCas9-compatible template RNAs comprising variant scaffolds containing various exemplary variant tetraloop structures in HEK293T cells treated with a high dose of exemplary gene modifying system.
  • FIG. 8D is a diagram illustrating the hypothesized secondary structure of a dSL2 truncated StlCas9 gRNA scaffold and is overlaid with description of valiants described herein.
  • FIG. 9A is a bar graph showing the rewriting activity of exemplary StlCas9-based gene modifying systems comprising variant template RNAs having the nucleotide sequence of exemplary template RNA RNACS6681 with various 2-O'-methyl chemical modifications in the gRNA scaffold region.
  • FIGs. 9B-9K are schematic diagrams of tested chemical modification pattern designs of exemplary StlCas9-based gene modifying systems comprising variant template RNAs having the nucleotide sequence of exemplary template RNA RNACS6681, with 2’-O-methyl chemical modifications shown in bold.
  • FIG. 10A is a bar graph showing the rewriting activity of exemplary StlCas9-based gene modifying systems comprising variant template RNAs having the nucleotide sequence of exemplary template RNA RNACS9201 (containing a dSL2 variant gRNA scaffold) with various 2-O'-methyl chemical modifications in the gRNA scaffold region.
  • FIG. 10B is a bar graph showing the rewriting activity of exemplary StlCas9-based gene modifying systems comprising variant template RNAs having a scaffold comprising three modified nucleotides at a time with 2’-O-methyl chemical modifications.
  • FIG. IOC is a diagram illustrating the positions of poorly tolerated (lowercase,), somewhat tolerated positions (capital), and tolerated positions (italic) to 2’-O-methyl chemical modification in the dSL2 StlCas9 scaffold sequence.
  • FIGs. 10D-10L are diagrams illustrating exemplary design patterns of 2’-O-methyl chemical modified nucleotides in the dSL2 StlCas9 scaffold sequence in FIG. 10A.
  • Bold bases represent2'-O-methyl modified nucleotide positions.
  • FIG. 11 is a bar graph showing the rewriting efficiency of exemplary gene modifying systems comprising different StlCas9-compatible template RNAs comprising different patterns of 2’-O-methyl chemical modifications in the dSL2 StlCas9 scaffold in primary hepatocytes.
  • FIG. 12A is a bar graph showing the rewriting activity in the livers of mice administered with exemplary gene modifying systems comprising different StlCas9-compatible template RNAscontaining a dSL2 variant gRNA scaffold and formulated in lipid nanoparticles (LNP).
  • LNP lipid nanoparticles
  • FIG. 12B is a bar graph showing the % indel levels in the livers of mice administered with exemplary gene modifying systems comprising different StlCas9-compatible template RNAscontaining a dSL2 variant gRNA scaffold and formulated in LNP.
  • FIG. 12C is a bar graph showing the concentration of hAlAT in the serum of mice administered with exemplary gene modifying systems comprising different StlCas9-compatible template RNAscontaining a dSL2 variant gRNA scaffold and formulated in LNP.
  • FIG. 13A is a bar graph showing of the percent of perfect rewriting in the liver of mice administered with exemplary gene modifying systems that comprises different StlCas9- compatible template RNAs comprising different patterns of 2’-O-methyl chemical modifications in the dSL2 StlCas9 scaffold and are formulated in LNP.
  • FIG. 13B is a bar graph showing of the % indel levels in the liver of mice administered with exemplary gene modifying systems that comprises different StlCas9-compatible template RNAs comprising different patterns of 2’-O-methyl chemical modifications in the dSL2 StlCas9 scaffold and are formulated in LNP.
  • FIG. 13C is a bar graph showing the concentration of hAlAT in the serum of mice administered with exemplary gene modifying systems that comprises different StlCas9- compatible template RNAs comprising different patterns of 2’-O-methyl chemical modifications in the dSL2 StlCas9 scaffold and arc formulated in LNP.
  • FIG. 14A is a diagram illustrating the positions of the reference dSL2 StlCas9 scaffold sequence.
  • FIG. 14B is a diagram illustrating the positions of the reference wild-type StlCas9 scaffold sequence.
  • FIG. 14C is a diagram illustrating the hypothesized structure of RNACS 13597, having RAR+4_UUCG mutations relative to dSL2.
  • FIG. 14D is a diagram illustrating the hypothesized structure of RNACS17210, having RAR+4_AGCA mutations relative to dSL2.
  • FIG. 15 are schematics of three exemplary template RNAs comprising chemical modifications. Closed circles are 2’F modifications, open circles are 2’0Me modifications, diamonds are phosphorothioate modifications.
  • FIG. 16A is a bar graph showing the rewriting efficiency in the livers of mice administered with exemplary gene modifying systems comprising different template RNAs comprising scaffold chemical modifications.
  • FIG. 16B is a bar graph showing the % indel levels in the livers of mice administered with the exemplary gene modifying systems evaluated in FIG. 16A.
  • FIG. 17A is a bar graph showing the rewriting efficiency in the livers of mice administered with exemplary gene modifying systems comprising different template RNAs comprising scaffold chemical modifications in combination with fluoro modifications at the heterologous object sequence.
  • FIG. 17B is a bar graph showing the % indel levels in the livers of mice administered with the exemplary gene modifying systems evaluated in FIG. 17A.
  • FIG. 18A is a bar graph showing the rewriting efficiency in the livers of mice administered with exemplary gene modifying systems comprising different gene modifying polypeptides and different template RNAs comprising scaffold chemical modifications in combination with fluoro modifications at the heterologous object sequence.
  • FIG. 18B is a bar graph showing the % indel levels in the livers of mice administered with the exemplary gene modifying systems evaluated in FIG. 18A.
  • FIG. 19A is a bar graph showing the % corrected genomic DNA in the livers of hSERPINAl E342K mice by administered with exemplary gene modifying systems comprising RNAIVT6241 or RNAIVT6898 polypeptides over evaluated dosages.
  • FIG. 19B is a bar graph showing the % corrected genomic DNA in the livers of NSG-PiZ mice by exemplary RNAIVT6898 systems over evaluated dosages at 7 days post-administration.
  • FIG. 19C is a bar graph showing the % corrected genomic DNA in the livers of NSG-PiZ mice by exemplary RNAIVT6898 systems over evaluated dosages at 21 days postadministration.
  • FIG. 19D is a bar graph showing the % edited mRNA in the livers of hSERPINAl E342K and NSG-PiZ mice by exemplary RNAIVT6898 systems over evaluated dosages at 21 days postadministration.
  • FIG. 19E is a bar graph showing the % indels introduced into the livers of hSERPINAl E342K by exemplary gene modifying polypeptides RNAVT6241 and RNAIVT6838 over evaluated dosages.
  • FIG. 19F is a bar graph showing the % indels introduced into the livers of NSG-PiZ mice by exemplary RNAIVT6898 systems over evaluated dosages at 7 days post-administration.
  • FIG. 19G is a bar graph showing the % indels introduced into the livers of NSG-PiZ mice by exemplary RNAIVT6898 systems over evaluated dosages at 21 days post-administration.
  • FIG. 20A is a bar graph showing the serum concentration of human Al AT in hSERPINAl E342K mice administered with exemplary gene modifying systems comprising RNAIVT6241 or RNAIVT6898 polypeptides over evaluated dosages.
  • FIG. 20B is a bar graph showing the serum concentration of human Al AT in NSG-PiZ mice administered with exemplary RNAIVT6898 systems over evaluated dosages at 7 days postadministration.
  • FIG. 20C is a bar graph showing the serum concentration of human Al AT in NSG-PiZ mice administered with exemplary RNAIVT6898 systems over evaluated dosages at 21 days postadministration.
  • FIG. 21A is a bar graph showing the % liver area occupied by globules in NSG-PiZ mice administered with exemplary RNAIVT6898 systems over evaluated dosages at 7 days postadministration.
  • FIG. 21B is a bar graph showing the % liver area occupied by globules in NSG-PiZ mice administered with exemplary RNAIVT6898 systems over evaluated dosages at 21 days postadministration.
  • FIG. 22A is a bar graph showing the % corrected genomic DNA in the livers of NSG-PiZ mice by exemplary gene modifying systems comprising RNAIVT9315 gene modifying polypeptide and RNACS22230 template RNA over evaluated dosages.
  • FIG. 22B is a bar graph showing the % edited mRNA in the livers of NSG-PiZ mice by exemplary gene modifying systems comprising RNAIVT9315 gene modifying polypeptide and RNACS22230 template RNA over evaluated dosages.
  • FIG. 22C is a bar graph showing the % indels introduced into the livers of NSG-PiZ mice by exemplary gene modifying systems comprising RNAIVT9315 gene modifying polypeptide and RNACS22230 template RNA over evaluated dosages.
  • FIG. 23A is a bar graph showing the serum concentration of human Al AT in NSG-PiZ mice administered with exemplary gene modifying systems comprising RNAIVT9315 gene modifying polypeptide and RNACS22230 template RNA over evaluated dosages.
  • FIG. 23B is a line graph showing the serum concentration of human Al AT as a function of % genomic DNA rewriting in NSG-PiZ mice administered with exemplary gene modifying systems comprising RNAIVT9315 gene modifying polypeptide and RNACS22230 template RNA over evaluated dosages.
  • FIG. 24A is a bar graph showing the % corrected genomic DNA in the livers of hSERPINAl E342K mice by exemplary gene modifying systems comprising RNAIVT9315 or RNAIVT9318 gene modifying polypeptides and RNACS22230 template RNA over evaluated dosages.
  • FIG. 24B is a bar graph showing the % indels introduced in the livers of hSERPINAl E342K mice by exemplary gene modifying systems comprising RNAIVT9315 or RNAIVT9318 gene modifying polypeptides and RNACS22230 template RNA over evaluated dosages.
  • a “gRNA scaffold,” as used herein, refers to a portion of a nucleic acid that can bind a Cas protein and can, together with a gRNA spacer, target the Cas protein to the target nucleic acid.
  • the gRNA scaffold comprises a crRNA sequence, tetraloop, and tracrRNA sequence.
  • StlCas9 scaffold refers to a gRNA scaffold that can bind an StlCas9 protein and can, together with a gRNA spacer, target the StlCas9 protein to the target nucleic acid.
  • an StlCas9 scaffold comprises a crRNA sequence, tetraloop, and tracerRNA sequence.
  • An exemplary position of StlCas9 scaffold within an exemplary template RNA is illustrated in FIG. 1.
  • a gene modifying polypeptide is capable of integrating a sequence substantially without relying on host machinery. In some embodiments, a gene modifying polypeptide integrates a sequence into a random position in a genome. In some embodiments, a gene modifying polypeptide integrates a sequence into a specific target site. In some embodiments, a gene modifying polypeptide includes one or more domains that, collectively, facilitate 1) binding a template nucleic acid, 2) binding a target DNA molecule, and 3) integration of at least a portion of the template nucleic acid into the target DNA.
  • Exemplary gene modifying polypeptides, and systems comprising the same can be used in methods provided herein and described, e.g., in PCT/US2021/020948, which is incorporated herein by reference with respect to gene modifying polypeptides that comprise a retroviral reverse transcriptase domain.
  • a gene modifying polypeptide integrates a sequence into a gene.
  • a gene modifying polypeptide integrates a sequence into a sequence outside of a gene.
  • domain refers to a structure of a biomolecule that contributes to a specified function of the biomolecule.
  • a domain may comprise a contiguous region (e.g., a contiguous sequence) or distinct, non-contiguous regions (e.g., non-contiguous sequences) of a biomolcculc.
  • protein domains include, but arc not limited to, an endonuclease domain, a DNA binding domain, a reverse transcription domain; an example of a domain of a nucleic acid is a regulatory domain, such as a transcription factor binding domain.
  • a domain e.g., a Cas domain
  • exogenous when used with reference to a biomolecule (such as a nucleic acid sequence or polypeptide) means that the biomolecule was introduced into a host genome, cell or organism by the hand of man.
  • a nucleic acid that is as added into an existing genome, cell, tissue or subject using recombinant DNA techniques or other methods is exogenous to the existing nucleic acid sequence, cell, tissue or subject.
  • first strand and second strand as used to describe the individual DNA strands of target DNA, distinguish the two DNA strands based upon which strand a reverse transcriptase domain initiates polymerization, e.g., based upon where target primed synthesis initiates.
  • a “first strand” refers to the strand of a target DNA upon which a reverse transcriptase domain initiates polymerization, e.g., where target primed synthesis initiates.
  • a “second strand” refers to the other strand of the target DNA.
  • First and second strand designations do not describe a target site DNA strands in other respects; for example, in some embodiments the first and second strands are nicked by a polypeptide described herein, but the designations ‘first’ and ‘second’ strand have no bearing on the order in which such nicks occur.
  • heterologous polypeptide, nucleic acid molecule, construct or sequence refers to (a) a polypeptide, nucleic acid molecule or portion of a polypeptide or nucleic acid molecule sequence that is not native to a cell in which it is expressed, (b) a polypeptide or nucleic acid molecule or portion of a polypeptide or nucleic acid molecule that has been altered or mutated relative to its native state, or (c) a polypeptide or nucleic acid molecule with an altered expression as compared to the native expression levels under similar conditions.
  • a heterologous regulatory sequence e.g., promoter, enhancer
  • a heterologous domain of a polypeptide or nucleic acid sequence e.g., a DNA binding domain of a polypeptide or nucleic acid encoding a DNA binding domain of a polypeptide
  • a heterologous nucleic acid molecule may exist in a native host cell genome but may have an altered expression level or have a different sequence or both.
  • heterologous nucleic acid molecules may not be endogenous to a host cell or host genome but instead may have been introduced into a host cell by transformation (e.g., transfection, electroporation), wherein the added molecule may integrate into the host genome or can exist as extra-chromosomal genetic material either transiently (e.g., mRNA) or semi-stably for more than one generation (e.g., episomal viral vector, plasmid or other self-replicating vector).
  • insertion of a sequence into a target site refers to the net addition of DNA sequence at a target site, e.g., where there are new nucleotides in a heterologous object sequence with no cognate positions in the unedited target site.
  • a nucleotide alignment of a PBS sequence and heterologous object sequence to a target nucleic acid sequence would result in an alignment gap in the target nucleic acid sequence.
  • a “deletion” generated by a heterologous object sequence in a target site refers to the net deletion of DNA sequence at the target site, e.g., where there are nucleotides in the unedited target site with no cognate positions in the heterologous object sequence.
  • a nucleotide alignment of the PBS sequence and heterologous object sequence to the target nucleic acid sequence would result in an alignment gap in the molecule comprising the PBS sequence and heterologous object sequence.
  • ITRs inverted terminal repeats
  • AAV viral ciselements named so because of their symmetry.
  • These elements promote efficient multiplication of an AAV genome. It is hypothesized that the minimal elements for ITR function are a Repbinding site (RBS; 5'-GCGCGCTCGCTCGCTC-3' for AAV2; SEQ ID NO: 4601) and a terminal resolution site (TRS; 5'-AGTTGG-3' for AAV2; SEQ ID NO: 4602) plus a variable palindromic sequence allowing for hairpin formation.
  • an ITR comprises at least these three elements (RBS, TRS, and sequences allowing the formation of a hairpin).
  • ITR refers to ITRs of known natural AAV serotypes (e.g., ITR of a serotype 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or 11 AAV), to chimeric ITRs formed by the fusion of ITR elements derived from different serotypes, and to functional variants thereof.
  • “Functional variant” refers to a sequence presenting a sequence identity of at least 80%, 85%, 90%, preferably of at least 95% with a known ITR and allowing multiplication of the sequence that includes said ITR in the presence of Rep proteins.
  • mutant region refers to a region in a template RNA having one or more sequence difference relative to the corresponding sequence in a target nucleic acid.
  • the one or more sequence difference may comprise, for example, a substitution, insertion, frameshift, or deletion.
  • mutated when applied to nucleic acid sequences means that nucleotides in a nucleic acid sequence are inserted, deleted, or changed compared to a reference (e.g., native) nucleic acid sequence.
  • a single alteration may be made at a locus (a point mutation), or multiple nucleotides may be inserted, deleted, or changed at a single locus.
  • one or more alterations may be made at any number of loci within a nucleic acid sequence.
  • a nucleic acid sequence may be mutated by any method known in the ail.
  • Nucleic acid molecule refers to both RNA and DNA molecules including, without limitation, complementary DNA (“cDNA”), genomic DNA (“gDNA”), and messenger RNA (“mRNA”), and also includes synthetic nucleic acid molecules, such as those that are chemically synthesized or recombinantly produced, such as RNA templates, as described herein.
  • a nucleic acid molecule can be double- stranded or single-stranded, circular’, or linear. If single- stranded, a nucleic acid molecule can be a sense strand or an antisense strand.
  • nucleic acid comprising SEQ ID NO: 1 refers to a nucleic acid, at least a portion which has either (i) the sequence of SEQ ID NO: 1, or (ii) a sequence complimentary to SEQ ID NO: 1.
  • the choice between the two is dictated by the context in which SEQ ID NO: 1 is used. For instance, if a nucleic acid is used as a probe, the choice between the two is dictated by the requirement that the probe be complementary to a desired target.
  • Nucleic acid sequences of the present disclosure may be modified chemically or biochemically or may contain non-natural or derivatized nucleotide bases, as will be readily appreciated by those of skill in the art. Such modifications include, for example, labels, methylation, substitution of one or more naturally occurring nucleotides with an analog, inter-nucleotide modifications such as uncharged linkages (for example, methyl phosphonates, phosphotriesters, phosphoramidates, carbamates, etc.), charged linkages (for example, phosphorothioates, phosphorodithioates, etc.), pendant moieties, (for example, polypeptides), intcrcalators (for example, acridine, psoralen, etc.), chelators, alkylators, and modified linkages (for example, alpha anomeric nucleic acids, etc.).
  • uncharged linkages for example, methyl phosphonates, phosphotriesters, phosphoramidates, carbamates, etc
  • ⁇ are chemically modified bases (see, for example, Table 32), backbones (see, for example, Table 33), and modified caps (see, for example, Table 34).
  • synthetic molecules that mimic polynucleotides in their ability to bind to a designated sequence via hydrogen bonding and other chemical interactions. Such molecules are known in the art and include, for example, those in which peptide linkages substitute for phosphate linkages in the backbone of a molecule, e.g., peptide nucleic acids (PNAs).
  • PNAs peptide nucleic acids
  • Other modifications can include, for example, analogs in which the ribose ring contains a bridging moiety or other structure such as modifications found in “locked” nucleic acids (LNAs).
  • nucleic acids are in operative association with additional genetic elements, such as tissue-specific expressioncontrol sequence(s) (e.g., tissue-specific promoters and tissue-specific microRNA recognition sequences), as well as additional elements, such as inverted repeats (e.g., inverted terminal repeats, such as elements from or derived from viruses, e.g., AAV ITRs) and tandem repeats, inverted repeats/direct repeats, homology regions (segments with various degrees of homology to a target DNA), untranslated regions (UTRs) (5k 3k or both 5' and 3" UTRs), and various combinations of the foregoing.
  • tissue-specific expressioncontrol sequence(s) e.g., tissue-specific promoters and tissue-specific microRNA recognition sequences
  • additional elements such as inverted repeats (e.g., inverted terminal repeats, such as elements from or derived from viruses, e.g., AAV ITRs) and tandem repeats, inverted repeats/direct repeats, homology regions (
  • Nucleic acid elements of systems disclosed in the present application may be provided in a variety of topologies, including single-stranded, doublestranded, circular, linear, linear with open ends, linear with closed ends, and particular versions of these, such as doggybone DNA (dbDNA), and closed-ended DNA (ceDNA).
  • dbDNA doggybone DNA
  • ceDNA closed-ended DNA
  • chemically modified nucleotide refers to a nucleotide comprising one or more structural differences relative to the canonical ribonucleotides (i.e., G, U, C, and .A).
  • a chemically modified nucleotide may have (relative to a canonical nucleotide) a chemically modified nudeobase, a chemically modified sugar, a chemically modified phosphodiester linkage, or a combination thereof.
  • a chemically modified nucleotide is a 2'-O-methyl nucleotide, e.g., 2'-O-methyl- Adenosine, 2'-O-methyl-Cytidine, 2'-O- methyl-Guanosine, or 2'-O-methyl-Uridine.
  • a chemically modified nucleotide can be produced directly by chemical synthesis, or by covalently modifying a canonical nucleotide.
  • the term “chemical modification,” as used herein, refers to a structural difference of a chemical modified nucleotide relative to die canonical ribonucleotides (i.c., G, U, C. and A).
  • a chemical modification may comprise a modification resulting in a chemically modified nucleobase, a chemically modified sugar, a chemically modified phosphodiester linkage, or a combination thereof.
  • a chemical modification is 2'-O-methylation or 2’- fluoro modification. No particular process of making is implied; for instance, a chemical modification can be produced directly by chemical synthesis, or by covalently modifying a canonical nucleotide.
  • the term “position” with respect to an StlCas9 scaffold refers to anucleotide of the StlCas9 scaffold that aligns with a corresponding nucleotide of a reference sequence of SEQ ID NO: 25999. The positions of the reference sequence are illustrated in FIG, 14. Alignments of nucleic acid or polypeptide sequences can be performed by using a routine sequence analysis tool such as Basic Local Alignment Search Tool (BLAST), for instance NIH megablast using default parameters.
  • BLAST Basic Local Alignment Search Tool
  • a position of an StlCas9 scaffold can be identified by providing an alignment of an StlCas9 scaffold (query sequence) to a reference sequence of SEQ ID NO: 25999 (a full length wild-type sequence, see e.g., FIG 14B) or SEQ ID NO: 26000 (a truncation mutant, see e.g., FIG. 14A), and identifying the position in the query sequence that corresponds to the position in the reference sequence.
  • the substituted position is position 1.
  • an StlCas9 scaffold consisting of the sequence of SEQ ID NO: 25999 except that a single new nucleotide is inserted just 5’ of the 5’ most G, the G is still position 1.
  • nucleotides 3’ of the insert maintain their original position number.
  • the U of position 2 is still position 2 rather than position n+2.
  • a nucleotide that is inserted relative to the reference sequence need not be assigned a position number.
  • a range of nucleotides includes all nucleotides in that range regardless of whether they are assigned a number; for example, if a scaffold comprises a chemically modified nucleotide at each of positions 12 through 21 , and the scaffold comprises inserted nucleotides anywhere between positions 12 and 21, then the scaffold comprises chemically modified nucleotides at each of the inserted nucleotides situated anywhere between positions 12 and 21 (which inserted nucleotides do not have a position number in this example), as well as chemically modified nucleotides at positions 12, 13, 14, 15, 16, 17, 18, 19, 20, and 21.
  • a “gene expression unit” is a nucleic acid sequence comprising at least one regulatory nucleic acid sequence operably linked to at least one effector sequence.
  • a first nucleic acid sequence is operably linked with a second nucleic acid sequence when the first nucleic acid sequence is placed in a functional relationship with the second nucleic acid sequence.
  • a promoter or enhancer is operably linked to a coding sequence if the promoter or enhancer affects the transcription or expression of the coding sequence.
  • Operably linked DNA sequences may be contiguous or non-contiguous. Where necessary to join two protein-coding regions, operably linked sequences may be in the same reading frame.
  • host genome refers to a cell and/or its genome into which protein and/or genetic material has been introduced. It should be understood that such terms are intended to refer not only to the particular subject cell and/or genome, but to the progeny of such a cell and/or the genome of the progeny of such a cell. Because certain modifications may occur in succeeding generations due to either mutation or environmental influences, such progeny may not, in fact, be identical to the parent cell, but are still included within the scope of the term “host cell” as used herein.
  • a host genome or host cell may be an isolated cell or cell line grown in culture, or genomic material isolated from such a cell or cell line, or may be a host cell or host genome which composing living tissue or an organism.
  • a host cell may be an animal cell or a plant cell, e.g., as described herein.
  • a host cell may be a mammalian cell, a human cell, avian cell, reptilian cell, bovine cell, horse cell, pig cell, goat cell, sheep cell, chicken cell, or turkey cell.
  • a host cell may be a corn cell, soy cell, wheat cell, or rice cell.
  • operative association describes a functional relationship between two nucleic acid sequences, such as a 1) promoter and 2) a heterologous object sequence, and means, in such example, the promoter and heterologous object sequence (e.g., a gene of interest) are oriented such that, under suitable conditions, the promoter drives expression of the heterologous object sequence.
  • a template nucleic acid carrying a promoter and a heterologous object sequence may be single-stranded, e.g., either the (+) or (-) orientation.
  • an “operative association” between the promoter and the heterologous object sequence in this template means that, regardless of whether the template nucleic acid will be transcribed in a particular state, when it is in the suitable state (e.g., is in the (+) orientation, in the presence of required catalytic factors, and NTPs, etc.), it is accurately transcribed. Operative association applies analogously to other pairs of nucleic acids, including other tissue-specific expression control sequences (such as enhancers, repressors and microRNA recognition sequences), IR/DR, ITRs, UTRs, or homology regions and heterologous object sequences or sequences encoding a retroviral RT domain.
  • PBS sequence refers to a portion of a template RNA capable of binding to a region in a target nucleic acid sequence
  • a PBS sequence is a nucleic acid sequence comprising at least 3, 4, 5, 6, 7, or 8 bases with 100% identity to a region in a target nucleic acid sequence.
  • a primer region comprises at least 5, 6, 7, 8 bases with 100% identity to a region in a target nucleic acid sequence.
  • a template RNA comprises a PBS sequence and a heterologous object sequence
  • the PBS sequence binds to a region in a target nucleic acid sequence, allowing a reverse transcriptase domain to use that region as a primer for reverse transcription, and to use the heterologous object sequence as a template for reverse transcription.
  • a “stem-loop sequence” refers to a nucleic acid sequence (e.g., RNA sequence) with sufficient self-complementarity to form a stem-loop, e.g., having a stem comprising at least two (e.g., 3, 4, 5, 6, 7, 8, 9, or 10) base pairs, and a loop with at least three (e.g., four) base pairs.
  • the stem may comprise mismatches or bulges.
  • tissue-specific expression-control sequence means nucleic acid elements that increase or decrease the level of a transcript comprising the heterologous object sequence in a target tissue in a tissue-specific manner, e.g., preferentially in on-target tissue(s), relative to off-target tissue(s).
  • a tissue- specific expression-control sequence preferentially drives or represses transcription, activity, or the half-life of a transcript comprising the heterologous object sequence in the target tissue in a tissue- specific manner, e.g., preferentially in an on-target tissue(s), relative to an off-target tissue(s).
  • tissuespecific expression-control sequences include tissue-specific promoters, repressors, enhancers, or combinations thereof, as well as tissue-specific microRNA recognition sequences.
  • Tissue specificity refers to on-target (tissuc(s) where expression or activity of the template nucleic acid is desired or tolerable) and off-target (tissue(s) where expression or activity of the template nucleic acid is not desired or is not tolerable).
  • a tissue-specific promoter drives expression preferentially in on-target tissues, relative to off-target tissues.
  • a microRNA that binds the tissue-specific microRNA recognition sequences is preferentially expressed in off-target tissues, relative to on-target tissues, thereby reducing expression of a template nucleic acid in off-target tissues.
  • a promoter and a microRNA recognition sequence that are specific for the same tissue, such as the target tissue have contrasting functions (promote and repress, respectively, with concordant expression levels, i.e., high levels of the microRNA in off-target tissues and low levels in on-target tissues, while promoters drive high expression in on-target tissues and low expression in off-target tissues) with regard to the transcription, activity, or half-life of an associated sequence in that tissue.
  • a genetic modification is a point mutation.
  • point mutation refers to a substitution that replaces one of the nucleotides.
  • This disclosure provides methods for treating alpha- 1 antitrypsin deficiency (AATD) and compositions for targeting, editing, modifying or manipulating a DNA sequence (e.g., inserting a heterologous object sequence into a target site of a mammalian genome) at one or more locations in a DNA sequence in a cell, tissue or subject, e.g., in vivo or in vitro.
  • a heterologous object DNA sequence may include, e.g., a substitution.
  • the disclosure provides methods for treating AATD using reverse transcriptase-based systems for altering a genomic DNA sequence of interest, e.g., by inserting, deleting, or substituting one or more nucleotides into/from a sequence of interest.
  • the disclosure provides methods for treating AATD using a gene modifying system comprising a gene modifying polypeptide component and a template nucleic acid (e.g., template RNA) component.
  • a gene modifying system can be used to introduce an alteration into a target site in a genome.
  • a gene modifying polypeptide component comprises a writing domain (e.g., a reverse transcriptase domain), a DNA-binding domain, and an endonuclease domain (e.g., a nickase domain).
  • a template nucleic acid (e.g., template RNA) comprises a sequence (e.g., a gRNA spacer) that binds a target site in the genome (e.g., that binds to a second strand of a target site), a sequence (e.g., a gRNA scaffold) that binds a gene modifying polypeptide component, a heterologous object sequence, and a PBS sequence.
  • a sequence e.g., a gRNA spacer
  • a target site in the genome e.g., that binds to a second strand of a target site
  • a sequence e.g., a gRNA scaffold
  • a template nucleic acid binds to the second strand of a target site in the genome and binds to a gene modifying polypeptide component (e.g., localizing the gene modifying polypeptide component to the target site in the genome).
  • a gene modifying polypeptide component e.g., localizing the gene modifying polypeptide component to the target site in the genome.
  • the endonuclease e.g., nickase
  • cuts a target site e.g., the first strand of the target site
  • a PBS sequence to bind to a sequence adjacent to a site to be altered on the first strand of the target site.
  • a writing domain e.g., reverse transcriptase domain
  • a gene modifying polypeptide component uses the first strand of a target site that is bound to a complementary sequence comprising a PBS sequence of a template nucleic acid as a primer and a heterologous object sequence of the template nucleic acid as a template to, e.g., polymerize a sequence complementary to the heterologous object sequence.
  • selection of an appropriate heterologous object sequence can result in substitution, deletion, and/or insertion of one or more nucleotides at the target site.
  • a gene modifying system described herein comprises: (A) a gene modifying polypeptide or a nucleic acid encoding the gene modifying polypeptide, wherein the gene modifying polypeptide comprises (i) a reverse transcriptase domain, and either (x) an endonuclease domain that contains DNA binding functionality or (y) an endonuclease domain and separate DNA binding domain; and (B) a template RNA.
  • a gene modifying polypeptide acts as a substantially autonomous protein machine capable of integrating a template nucleic acid sequence into a target DNA molecule (e.g., in a mammalian host cell, such as a genomic DNA molecule in the host cell), substantially without relying on host machinery.
  • a gene modifying polypeptide may comprise a DNA-binding domain, a reverse transcriptase domain, and an endonuclease domain.
  • a DNA-binding function may involve an RNA component that directs a gene modifying polypeptide to a DNA sequence, e.g., a gRNA spacer.
  • a gene modifying polypeptide may comprise a reverse transcriptase domain and an endonuclease domain.
  • An RNA template element of a gene modifying system may be heterologous to a gene modifying polypeptide element and provides an object sequence to be inserted (reverse transcribed) into a host genome.
  • a gene modifying polypeptide is capable of target primed reverse transcription.
  • a gene modifying polypeptide is capable of second-strand synthesis.
  • a gene modifying system is combined with a second polypeptide.
  • a second polypeptide may comprise an endonuclease domain.
  • a second polypeptide may comprise a polymerase domain, e.g., a reverse transcriptase domain.
  • a second polypeptide may comprise a DNA-dependent DNA polymerase domain.
  • a second polypeptide aids in completion of a genome edit, e.g., by contributing to second-strand synthesis or DNA repair resolution.
  • a functional gene modifying polypeptide can be made up of unrelated DNA binding, reverse transcription, and endonuclease domains.
  • This modular structure allows combining of functional domains, e.g., dCas9 (DNA binding), MMLV reverse transcriptase (reverse transcription), FokI (endonuclease).
  • functional domains e.g., dCas9 (DNA binding), MMLV reverse transcriptase (reverse transcription), FokI (endonuclease).
  • multiple functional domains may arise from a single protein, e.g., Cas9 or Cas9 nickase (DNA binding, endonuclease).
  • a gene modifying polypeptide includes one or more domains that, collectively, facilitate 1) binding a template nucleic acid, 2) binding a target DNA molecule, and 3) integration of at least a portion of the template nucleic acid into the target DNA.
  • a gene modifying polypeptide is an engineered polypeptide that comprises one or more amino acid substitutions to a corresponding naturally occurring sequence.
  • a gene modifying polypeptide comprises two or more domains that are heterologous relative to each other, e.g., through a heterologous fusion (or other conjugate) of otherwise wild-type domains, or well as fusions of modified domains, e.g., by way of replacement or fusion of a heterologous sub-domain or other substituted domain.
  • an RT domain is heterologous to a DNA-binding domain (DBD); a DBD is heterologous to an endonuclease domain; or an RT domain is heterologous to an endonuclease domain.
  • a template RNA molecule for use in a system of the present disclosure comprises, from 5' to 3' (1) a gRNA spacer; (2) a gRNA scaffold; (3) a heterologous object sequence; and (4) a primer binding site (PBS) sequence.
  • a gRNA spacer is about!8 to -22 nucleotides in length (e.g., about 20 nucleotides in length).
  • a gRNA scaffold comprises one or more hairpin loops, e.g., 1, 2, or 3 loops for associating a template RNA with a Cas domain, e.g., a nickase Cas9 domain.
  • a gRNA scaffold comprises the sequence, from 5' to 3', GTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAA AGTGGGACCGAGTCGGTCC (SEQ ID NO: 5008).
  • a heterologous object sequence is, e.g., 7-74, e.g., 10-20, 20-30, 30-40, 40-50, 50-60, 60-70, or 70-80 nucleotides or, 80-90 nucleotdies in length.
  • a first (i.e., 5 '-most) base of a heterologous object sequence is not C.
  • a PBS sequence that binds a target priming sequence after nicking occurs is e.g., 3-20 nucleotides, e.g., 7-15 nucleotides, e.g., 12-14 nucleotides in length. In some embodiments, a PBS sequence has 40-60% GC content.
  • a second gRNA associated with a system of the present disclosure may help drive complete integration.
  • a second gRNA may target a location that is 0-200 nucleotides away from a first-strand nick, e.g., 0-50, 50-100, 100-200 nucleotides away from the first-strand nick.
  • a second gRNA can only bind its target sequence after an edit is made, e.g., the gRNA binds a sequence present in a heterologous object sequence, but not in the initial target sequence.
  • a gene modifying system described herein is used to make an edit in HEK293, K562, U2OS, or HeLa cells.
  • a gene modifying system is used to make an edit in primary cells, e.g., primary liver cells or primary lung cells.
  • a gene modifying polypeptide as described herein comprises a reverse transcriptase or RT domain (e.g., as described herein) that comprises a MoMLV RT sequence or variant thereof.
  • a MoMLV RT sequence comprises one or more mutations selected from D200N, L603W, T330P, T306K, W313F, D524G, E562Q, D583N, P51L, S67R, E67K, T197A, H204R, E302K, F309N, L435G, N454K, H594Q, D653N, R110S, and K103L.
  • a MoMLV RT sequence comprises a combination of mutations, such as D200N, L603W, and T33OP, optionally further including T306K and/or W313F.
  • an endonuclease domain (e.g., as described herein) is Cas9. In some embodiments, an endonuclease domain is nCas9. In some embodiments, an endonuclease domain comprises an N863A mutation (e.g., in spCas9). In some embodiments, an endonuclease domain comprises a H840A mutation.
  • a heterologous object sequence (e.g., of a system as described herein) is about 1-50, 50-100, 100-200, 200-300, 300-400, 400-500, 500-600, 600-700, 700-800, 800-900, 900-1000, or more, nucleotides in length.
  • RT and endonuclease domains are joined by a flexible linker.
  • a linker comprises the amino acid sequence SGGSSGGSSGSETPGTSESATPESSGGSSGGSS (SEQ ID NO: 5006).
  • an endonuclease domain is N-terminal relative to an RT domain. In some embodiments, an endonuclease domain is C-terminal relative to an RT domain.
  • a system of the present disclosure incorporates a heterologous object sequence into a target site by target primed reverse transcription (TPRT), e.g., as described herein.
  • TPRT target primed reverse transcription
  • a gene modifying polypeptide comprises a DNA binding domain (DBD). In some embodiments, a gene modifying polypeptide comprises an RNA binding domain. In some embodiments, an RNA binding domain comprises an RNA binding domain of B-box protein, MS2 coat protein, dCas, or an element of a sequence of a Table herein. In some embodiments, an RNA binding domain is capable of binding to a template RNA with greater affinity than a reference RNA binding domain.
  • a gene modifying system is capable of producing an insertion of at least 45, at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, or at least 100 nucleotides (and optionally no more than 500, no more than 400, no more than 300, no more than 200, or no more than 100 nucleotides) in a target site.
  • a gene modifying system is capable of producing an insertion of at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, or at least 100 nucleotides (and optionally no more than 500, no more than 400, no more than 300, no more than 200, or no more than 100 nucleotides) in a target site.
  • a gene modifying system is capable of producing an insertion of at least 0.2, at least 0.3, at least 0.4, at least 0.5, at least 0.6, at least 0.7, at least 0.8, at least 0.9, at least 1, at least 1.5, at least 2, at least 2.5, at least 3, at least 3.5, at least 4, at least 4.5, at least 5, at least 5.5, at least 6, at least 6.5, at least 7, at least 7.5, at least 8, at least 8.5, at least 9, at least 9.5 or at least 10 kilobases (and optionally no more than 1, 5, 10, or 20 kilobases) into a target site.
  • a gene modifying system is capable of producing a deletion of at least 81, at least 85, at least 90, at least 95, at least 100, at least 110, at least 120, at least 130, at least 140, at least 150, at least 160, at least 170, at least 180, at least 190, or at least 200 nucleotides (and optionally no more than 500, no more than 400, no more than 300, or no more than 200 nucleotides).
  • a gene modifying system is capable of producing a deletion of at least 81, at least 85, at least 90, at least 95, at least 100, at least 110, at least 120, at least 130, at least 140, at least 150, at least 160, at least 170, at least 180, at least 190, or at least 200 nucleotides (and optionally no more than 500, 400, 300, or 200 nucleotides).
  • a gene modifying system is capable of producing a deletion of at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, or at least 100 nucleotides (and optionally no more than 500, no more than 400, no more than 300, no more than 200, or no more than 100 nucleotides).
  • a gene modifying system is capable of producing a deletion of at least 0.2, at least 0.3, at least 0.4, at least 0.5, at least 0.6, at least 0.7, at least 0.8, at least 0.9, at least 1, at least 1.5, at least 2, at least 2.5, at least 3, at least 3.5, at least 4, at least 4.5, at least 5, at least 5.5, at least 6, at least 6.5, at least 7, at least 7.5, at least 8, at least 8.5, at least 9, at least 9.5 or at least 10 kilobases (and optionally no more than 1, 5, 10, or 20 kilobases).
  • a gene modifying system is capable of producing a substitution of at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, or at least 100, or more nucleotides in a target site.
  • a gene modifying system is capable of producing a substitution of 1 -2, 2-3, 3-4, 4-5, 5-10, 10-15, 15-20, 20-30, 30-40, 40- 50, 50-60, 60-70, 70-80, 80-90, or 90-100 nucleotides in a target site.
  • a substitution is a transition mutation. In some embodiments, a substitution is a transversion mutation. In some embodiments, a substitution converts an adenine to a thymine, an adenine to a guanine, an adenine to a cytosine, a guanine to a thymine, a guanine to a cytosine, a guanine to an adenine, a thymine to a cytosine, a thymine to an adenine, a thymine to a guanine, a cytosine to an adenine, a cytosine to a guanine, or a cytosine to a thymine.
  • an insertion, deletion, substitution, or combination thereof increases or decreases expression (e.g., transcription or translation) of a gene.
  • an insertion, deletion, substitution, or combination thereof increases or decreases expression (e.g,. transcription or translation) of a gene by altering, adding, or deleting sequences in a promoter or enhancer, e.g., sequences that bind transcription factors.
  • an insertion, deletion, substitution, or combination thereof alters translation of a gene (e.g., alters an amino acid sequence), inserts or deletes a start or stop codon, or alters or fixes the translation frame of a gene.
  • an insertion, deletion, substitution, or combination thereof alters splicing of a gene, e.g., by inserting, deleting, or altering a splice acceptor or donor site. In some embodiments, an insertion, deletion, substitution, or combination thereof alters transcript or protein half-life. In some embodiments, an insertion, deletion, substitution, or combination thereof alters protein localization in the cell (e.g., from the cytoplasm to a mitochondria, from the cytoplasm into the extracellular space (e.g., adds a secretion tag)). In some embodiments, an insertion, deletion, substitution, or combination thereof alters (e.g., improves) protein folding (e.g., to prevent accumulation of misfolded proteins). In some embodiments, an insertion, deletion, substitution, or combination thereof, alters, increases, decreases the activity of a gene, e.g., a protein encoded by the gene.
  • Exemplary gene modifying polypeptides and retroviral RT domain sequences are also described, e.g., in International Application No. PCT/US21/20948, filed March 4, 2021, e.g., at Table 30, Table 31 , and Table 44 therein; the entire application is incorporated by reference herein with respect to retroviral RTs, e.g., in said sequences and Tables.
  • a gene modifying polypeptide described herein may comprise an amino acid sequence according to any of the Tables mentioned in this paragraph, or a domain thereof (e.g., a retroviral RT domain), or a functional fragment or variant of any of the foregoing, or an amino acid sequence having at least 70%, 80%, 85%, 90%, 95%, or 99% identity thereto.
  • a polypeptide for use in any of the systems described herein can be a molecular reconstruction or ancestral reconstruction based upon the aligned polypeptide sequence of multiple homologous proteins.
  • a reverse transcriptase domain for use in any of the systems described herein can be a molecular reconstruction or an ancestral reconstruction, or can be modified at particular residues, based upon alignments of reverse transcriptase domains from the same or different sources.
  • a skilled artisan can, based on the Accession numbers provided herein, align polypeptides or nucleic acid sequences, e.g., by using routine sequence analysis tools as Basic Local Alignment Search Tool (BLAST) or CD- Search for conserved domain analysis.
  • BLAST Basic Local Alignment Search Tool
  • Molecular reconstructions can be created based upon sequence consensus, e.g., using approaches described in Ivies et al., Cell 1997, 501 - 510; Wagstaff et al., Molecular Biology and Evolution 2013, 88-99.
  • a gene modifying polypeptide possesses the functions of DNA target site binding, template nucleic acid (e.g., template RNA) binding, DNA target site cleavage, and template nucleic acid (e.g., template RNA) writing (e.g., reverse transcription).
  • each function is contained within a distinct domain.
  • a function may be attributed to two or more domains (e.g., two or more domains, together, exhibit the functionality).
  • two or more domains may have the same or similar function (e.g., two or more domains each independently have DNA-binding functionality, e.g., for two different DNA sequences).
  • one or more domains may be capable of enabling one or more functions, e.g., a Cas9 domain enabling both DNA binding and target site cleavage.
  • domains are all located within a single polypeptide.
  • a first domain is in one polypeptide and a second domain is in a second polypeptide.
  • sequences may be split between a first polypeptide and a second polypeptide, e.g., wherein the first polypeptide comprises a reverse transcriptase (RT) domain and wherein the second polypeptide comprises a DNA-binding domain and an endonuclease domain, c.g., a nickase domain.
  • RT reverse transcriptase
  • a first polypeptide and a second polypeptide each comprise a DNA binding domain (e.g., a first DNA binding domain and a second DNA binding domain).
  • a first polypeptides and a second polypeptide may be brought together post- translationally via a split-intein to form a single gene modifying polypeptide.
  • a gene modifying polypeptide described herein comprises an StlCas9 domain.
  • An StlCas9 domain can comprise a naturally occurring StlCas9 amino acid sequence, or a variant thereof.
  • an StlCas9 domain is a nickase.
  • an StlCas9 domain comprises a sequence according to SEQ ID NO: 23818, or a sequence having at least 80%, 85%, 90%, 95%, 98%, or 99% identity thereto.
  • a gene modifying polypeptide comprising an StlCas9 domain is used together with a compatible template RNA comprising a variant gRNA scaffold described herein.
  • a gene modifying polypeptide described herein comprises (e.g., a system described herein comprises a gene modifying polypeptide that comprises): 1) a Cas domain (e.g., a Cas nickase domain, e.g., a Cas9 nickase domain); 2) a reverse transcriptase (RT) domain of Table 7 or Table 8, or a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% identity thereto, wherein the RT domain is C-terminal of the Cas domain; and a linker is disposed between the RT domain and the Cas domain, wherein the linker has a sequence from the same row of Table 7 or Table 8 as the RT domain, or a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% identity thereto.
  • a Cas domain e.g., a Cas nickase domain,
  • an RT domain has a sequence with 100% identity to an RT domain of Table 7 or Table 8and a linker has a sequence with 100% identity to the linker sequence from the same row of Table 7 or Table 8as the RT domain.
  • a Cas domain comprises a sequence of Table 4, or a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% identity thereto.
  • a gene modifying polypeptide comprises an amino acid sequence according to any one of SEQ ID NOs: 1-3332 in the sequence listing, or a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% identity thereto.
  • a gene modifying polypeptide comprises a GG amino acid sequence between a Cas domain and a linker, an AG amino acid sequence between an RT domain and a second nuclear localization seqeuence (NLS), and/or a GG amino acid sequence between the linker and the RT domain.
  • a gene modifying polypeptide comprises a sequence of SEQ ID NO: 4000 which comprises a first NLS and a Cas domain, or a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% identity thereto.
  • a gene modifying polypeptide comprises a sequence of SEQ ID NO: 4001 which comprises a second NLS, or a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% identity thereto.
  • a writing domain of a gene modifying system of the present disclosure possesses reverse transcriptase activity and is also referred to as a reverse transcriptase domain (an RT domain).
  • an RT domain comprises an RT catalytic portion and an RNA-binding region (c.g., a region that binds a template RNA).
  • a nucleic acid encoding a reverse transcriptase is altered from its natural sequence to have altered codon usage, e.g., improved for human cells.
  • a reverse transcriptase domain is a heterologous reverse transcriptase from a retrovirus.
  • an RT domain has been mutated from its original amino acid sequence, e.g., has at least about 1, at least about 2, at least about 3, at least about 4, at least about 5, at least about 6, at least about 7, at least about 8, at least about 9, at least about 10, at least about 20, at least about 30, at least about 40, at least about 50, at least about 60, at least about 70, at least about 80, at least about 90, or at least about 100 substitutions.
  • an RT domain is derived from an RT of a retrovirus, e.g., HIV-1 RT, Moloney Murine Leukemia Virus (MMLV) RT, avian myeloblastosis virus (AMV) RT, or Rous Sarcoma Virus (RSV) RT.
  • a retrovirus e.g., HIV-1 RT, Moloney Murine Leukemia Virus (MMLV) RT, avian myeloblastosis virus (AMV) RT, or Rous Sarcoma Virus (RSV) RT.
  • a retroviral reverse transcriptase (RT) domain exhibits enhanced stringency of target-primed reverse transcription (TPRT) initiation, e.g., relative to an endogenous RT domain.
  • TPRT target-primed reverse transcription
  • an RT domain initiates TPRT when the 3 nucleotides in a target site immediately upstream of a first strand nick, e.g., genomic DNA priming of an RNA template, have at least 66% or 100% complementarity to 3 nucleotides of homology in the RNA template.
  • an RT domain initiates TPRT when there are less than 5 nucleotides mismatched (e.g., less than 1, less than 2, less than 3, less than 4, or less than 5 nt mismatched) between an RNA template and a target DNA priming reverse transcription.
  • an RT domain is modified such that the stringency for mismatches in priming a TPRT reaction is increased, e.g., wherein the RT domain does not tolerate any mismatches or tolerates fewer mismatches in a priming region relative to a wild-type (e.g., unmodified) RT domain.
  • an RT domain comprises a HIV-1 RT domain.
  • an HIV-1 RT domain initiates lower levels of synthesis even with three nucleotide mismatches relative to an alternative RT domain (e.g., as described by Jamburuthugoda and Eickbush J Mol Biol 407(5):661-672 (2011); incorporated herein by reference in its entirety).
  • an RT domain forms a dimer (e.g., a heterodimer or homodimer).
  • an RT domain is monomeric.
  • an RT domain naturally functions as a monomer or as a dimer (e.g., heterodimer or homodimer).
  • an RT domain naturally functions as a monomer, e.g., is derived from a virus wherein it functions as a monomer.
  • an RT domain is selected from an RT domain from murine leukemia virus (MLV ; sometimes referred to as MoMLV) (e.g., P03355), porcine endogenous retrovirus (PERV) (e.g., UniProt Q4VFZ2), mouse mammary tumor virus (MMTV) (e.g., UniProt P03365), Avian reticuloendotheliosis vims (AVIRE) (e.g., UniProtKB accession: P03360); Feline leukemia virus (FLV or FeLV) (e.g., e.g., UniProtKB accession: P10273); Mason-Pfizer monkey vims (MPMV) (e.g., UniProt P07572), bovine leukemia virus (BLV) (e.g., UniProt
  • MMV murine
  • an RT domain is dimeric in its natural functioning.
  • an RT domain is derived from a virus wherein it functions as a dimer.
  • an RT domain is selected from an RT domain from avian sarcoma/leukemia vims (ASLV) (e.g., UniProt A0A142BKH1), Rous sarcoma virus (RSV) (e.g., UniProt P03354), avian myeloblastosis vims (AMV) (e.g., UniProt Q83133), human immunodeficiency vims type I (HIV-1) (e.g., UniProt P03369), human immunodeficiency virus type II (HIV-2) (e.g., UniProt P15833), simian immunodeficiency virus (SIV) (e.g., UniProt P05896), bovine immunodeficiency vims (BIV) (e.g.,
  • Naturally heterodimeric RT domains may, in some embodiments, also be functional as homodimers.
  • dimeric RT domains are expressed as fusion proteins, e.g., as homodimeric fusion proteins or heterodimeric fusion proteins.
  • an RT function ofa system of the present disclosure is fulfilled by multiple RT domains (e.g., as described herein).
  • multiple RT domains are fused or separate, e.g., may be on the same polypeptide or on different polypeptides.
  • a gene modifying system described herein comprises an integrase domain, e.g., wherein the integrase domain may be part of an RT domain.
  • an RT domain e.g., as described herein
  • an RT domain c.g., as described herein
  • a gene modifying system described herein comprises an RNase H domain, e.g., wherein the RNase H domain may be part of an RT domain.
  • an RNase H domain is not part of an RT domain and is covalently linked via a flexible linker.
  • an RT domain e.g., as described herein
  • comprises an RNase H domain e.g., an endogenous RNAse H domain or a heterologous RNase H domain.
  • an RT domain e.g., as described herein
  • an RT domain e.g., as described herein
  • a gene modifying polypeptide comprises an inactivated endogenous RNase H domain.
  • an endogenous RNase H domain of a polypeptide is genetically removed such that it is not included in the polypeptide, e.g., the endogenous RNase H domain is partially or completely truncated from the polypeptide.
  • one or more mutations of an RNase H domain yields a polypeptide exhibiting lower RNase activity, e.g., as determined by the methods described in Kotewicz et al.
  • an RT domain is mutated to increase fidelity compared to an otherwise similar domain without the mutation.
  • a YADD or YMDD motif in an RT domain e.g., in a reverse transcriptase
  • YVDD a YADD or YMDD motif in an RT domain
  • replacement of a YADD, YMDD, or YVDD motif results in higher fidelity in retroviral reverse transcriptase activity (e.g., as described in Jamburuthugoda and Eickbush J Mol Biol 2011; incorporated herein by reference in its entirety).
  • Table 1 Exemplary reverse transcriptase domains from retroviruses
  • an RT domain may be engineered to have improved tolerance to inhibitors as compared to a reference RT domain. In some embodiments, an RT domain may be engineered to be faster as compared to a reference RT domain. In some embodiments, an RT domain may be engineered to better tolerate modified nucleotides in an RNA template as compared to a reference RT domain. In some embodiments, an RT domain may be engineered to be capable of inserting modified DNA nucleotides. In some embodiments, an RT domain is engineered to bind a template RNA.
  • a gene modifying polypeptide further comprises one additional amino acid at the C-terminus of the sequence of amino acids 659-1329 of NP_057933.
  • a gene modifying polypeptide comprises an RNaseHl domain (e.g., amino acids 1178-1318 of NP_057933).
  • an M-MLV RT domain comprises, relative to the M-MLV (WT) sequence above, one or more mutations, e.g., selected from D200N, L603W, T330P, T306K, W313F, D524G, E562Q, D583N, P51L, S67R, E67K, T197A, H204R, E302K, F309N, L435G, N454K, H594Q, D653N, R110S, K103L, or a combination thereof.
  • WT M-MLV
  • an M-MLV RT domain comprises, relative to the M-MLV (WT) sequence above, a combination of mutations including D200N, L603W, and T330P, and optionally further including T306K and W313F.
  • an M-MLV RT used herein comprises D200N, L603W, T33OP, T306K and W313F mutations.
  • a mutant M-MLV RT comprises the following amino acid sequence:
  • a writing domain (e.g., an RT domain) comprises an RNA-binding domain, e.g., that specifically binds to an RNA sequence.
  • a template e.g., a sequence of RNA sequences.
  • RNA comprises an RNA sequence that is specifically bound by an RNA-binding domain of a writing domain (e.g., an RT domain).
  • a writing domain e.g., an RT domain
  • an RT domain only recognizes and reverse transcribes a specific template, e.g., a template RNA of a system of the preset disclosure.
  • a template RNA comprises a sequence or structure that enables recognition and reverse transcription by a reverse transcription domain.
  • a template RNA comprises a sequence or structure that enables association with an RNA-binding domain of a gene modifying polypeptide component of a system described herein.
  • a system of the present disclosure preferably reverse transcribes a template comprising an association sequence over a template lacking an association sequence.
  • a writing domain may also comprise DNA-dependent DNA polymerase activity, e.g., comprise enzymatic activity capable of writing DNA into the genome from a template DNA sequence.
  • DNA-dependent DNA polymerization is employed to complete second-strand synthesis of a target site edit.
  • DNA-dependent DNA polymerase activity is provided by a DNA polymerase domain in a gene modifying polypeptide.
  • DNA-dependent DNA polymerase activity is provided by an RT domain that is also capable of DNA-dependent DNA polymerization, e.g., second-strand synthesis.
  • DNA-dependent DNA polymerase activity is provided by a second polypeptide of a system of the present disclosure. In some embodiments, DNA-dependent DNA polymerase activity is provided by an endogenous host cell polymerase that is optionally recruited to a target site by a component of a system of the present disclosure.
  • an RT domain has a lower probability of premature termination rate (P off ) in vitro relative to a reference RT domain.
  • a reference RT domain is a viral RT domain, e.g., the RT domain from M-MLV.
  • an RT domain has a lower probability of premature termination rate (P off ) in vitro of less than about 5 x 10 -3 /nucleotides, less than about 5 x 10 -4 / nucleotides, or less than about 5 x 10 -6 / nucleotides, e.g., as measured on a 1094 nucleotide RNA.
  • P off probability of premature termination rate
  • an in vitro premature termination rate is determined as described in Bibillo and Eickbush (2002) J Biol Chem 277(38):34836-34845 (incorporated by reference herein its entirety).
  • quantifying integrations in cells comprises counting the fraction of integrations that contain at least about 75%, about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99%, or 100% of the DNA sequence corresponding to a template RNA (e.g., a template RNA having a length of at least 0.05, at least 0.1, at least 0.5, at least 0.6, at least 0.7, at least 0.8, at least 0.9, at least 1, at least 1.5, at least 2, at least 3, at least 4, or at least 5 kb, e.g., a length between 0.5-0.6, 0.6-0.7, 0.7-0.8, 0.8-0.9, 1.0-1.2, 1.2-1.4, 1.4- 1.6, 1.6-1.8, 1.8-2.0, 2-3, 3-4, or 4-5 kb).
  • a template RNA e.g., a template RNA having a length of at least 0.05, at least 0.1, at least 0.5, at least 0.6, at least
  • an RT domain performs reverse transcription at least 5 or 10 times more efficiently (e.g., by cDNA production), e.g., when converting its RNA template to cDNA, for example, as compared to an RNA template lacking a protein binding motif (e.g., a 3' UTR).
  • efficiency of reverse transcription is measured as described in Yasukawa et al. (2017) Biochem Biophys Res Commun 492(2): 147- 153 (incorporated by reference herein in its entirety).
  • an RT domain specifically binds a specific RNA template with higher frequency (e.g., about 5 or 10-fold higher frequency) than any endogenous cellular RNA, e.g., when expressed in cells (e.g., HEK293T cells, primary liver cells, or primary lung cells).
  • frequency of specific binding between an RT domain and a template RNA is measured by CLIP-seq, e.g., as described in Lin and Miles (2019) Nucleic Acids Res 47(l l):5490-5501 (incorporated herein by reference in its entirety).
  • a gene modifying polypeptide contains regions capable of associating with a template nucleic acid (e.g., template RNA).
  • a template nucleic acid binding domain is an RNA binding domain.
  • an RNA binding domain is a modular domain that can associate with RNA molecules containing specific signatures, e.g., structural motifs.
  • a template nucleic acid binding domain (e.g., RNA binding domain) is contained within an RT domain, e.g., the reverse transcriptase- derived component has a known signature for RNA preference.
  • a template nucleic acid binding domain (e.g., RNA binding domain) is contained within a target DNA binding domain.
  • a DNA binding domain is a CRIS PR-associated protein that recognizes the structure of a template nucleic acid (e.g., a template RNA) comprising a gRNA.
  • a gene modifying polypeptide comprises a DNA-binding domain comprising a CRISPR-associated protein that associates with a gRNA scaffold that allows the DNA-binding domain to bind a target genomic DNA sequence.
  • a gRNA scaffold and a gRNA spacer is comprised within a template nucleic acid (e.g., template RNA), thus, in some embodiments, a DNA-binding domain is also a template nucleic acid binding domain.
  • a gene modifying polypeptide possesses RNA binding function in multiple domains, e.g., can bind a gRNA structure in a CRISPR-associated DNA binding domain and an additional sequence or structure in an RT domain.
  • an RNA binding domain is capable of binding to a template RNA with greater affinity than a reference RNA binding domain.
  • a reference RNA binding domain is an RNA binding domain from Cas9 of S. pyogenes.
  • an RNA binding domain is capable of binding to a template RNA with an affinity between 100 pM - 10 nM (e.g., between 100 pM-I nM or I nM - 10 nM).
  • the affinity of an RNA binding domain for a template RNA is measured in vitro, e.g., by thermophoresis, e.g., as described in Asmari et al. Methods 146:107-119 (2016) (incorporated by reference herein in its entirety).
  • the affinity of an RNA binding domain for a template RNA is measured in cells (e.g., by FRET or CLIP-Seq).
  • an RNA binding domain is associated with a template RNA in vitro at a frequency at least about 5-fold higher or at least about 10-fold higher than with a scrambled RNA.
  • the frequency of association between an RNA binding domain and a template RNA or scrambled RNA is measured by CLIP-scq, c.g., as described in Lin and Miles (2019) Nucleic Acids Res 47(11 ) :5490-5501 (incorporated by reference herein in its entirety).
  • an RNA binding domain is associated with a template RNA in cells (e.g., in HEK293T cells, primary liver cells, or primary lung cells) at a frequency at least about 5-fold or at least about 10-fold higher than with a scrambled RNA.
  • the frequency of association between an RNA binding domain and a template RNA or scrambled RNA is measured by CLIP-seq, e.g., as described in Lin and Miles (2019), supra.
  • an RT domain (e.g., as listed in Table 1) comprises one or more mutations as listed in Table 2A below. In some embodiment, an RT domain as listed in Table 1 comprises one, two, three, four, five, or six of the mutations listed in the corresponding row of Table 2 below.
  • Table 2 Exemplary RT domain mutations (relative to corresponding wild-type sequences as listed in the corresponding row of Table 1)
  • a domain has two or more functions.
  • the endonuclease domain is also a DNA-binding domain.
  • an endonuclease domain is also a template nucleic acid (e.g., a template RNA) binding domain.
  • a gene modifying polypeptide comprises a CRISPR- associated endonuclease domain that binds a template RNA comprising a gRNA, binds a target DNA sequence (e.g., with complementarity to a portion of the gRNA), and cuts the target DNA sequence.
  • an endonuclease domain or endonuclease/DNA-binding domain from a heterologous source can be used or can be modified (e.g., by insertion, deletion, or substitution of one or more residues) in a gene modifying system described herein.
  • a nucleic acid encoding an endonuclease domain or endonuclease/DNA binding domain is altered from its natural sequence to have altered codon usage, e.g. improved for human cells.
  • the endonuclease element is a heterologous endonuclease element, such as a Cas endonuclease (e.g., Cas9), a type-II restriction endonuclease (e.g., Fokl), a meganuclease (e.g., I-Scel), or other endonuclease domain.
  • a DNA-binding domain of a gene modifying polypeptide described herein is selected, designed, or constructed for binding to a desired host DNA target sequence.
  • a DNA-binding domain of a gene modifying polypeptide is a heterologous DNA-binding element.
  • a heterologous DNA binding element is a zinc-finger element or a TAL effector element, e.g., a zinc-finger or TAL polypeptide or functional fragment thereof.
  • a heterologous DNA binding element is a sequence-guided DNA binding element, such as Cas9, Cpfl, or other CRISPR- related protein that has been altered to have no endonuclease activity.
  • a heterologous DNA binding element retains endonuclease activity. In some embodiments, a heterologous DNA binding element retains partial endonuclease activity to cleave ssDNA, e.g., possesses nickase activity. In some embodiments, a heterologous DNA-binding domain comprises a Cas9 domain, a TAL domain, a ZF domain, a Myb domain, a combination thereof, or multiples thereof.
  • a DNA-binding domain is modified, for example by site-specific mutation, increasing or decreasing DNA-binding elements (for example, number and/or specificity of zinc fingers), etc., to alter DNA-binding specificity and affinity.
  • a nucleic acid sequence encoding a DNA binding domain is altered from its natural sequence to have altered codon usage, e.g., improved for human cell expression.
  • a DNA binding domain comprises one or more modifications relative to a wildtype DNA binding domain, e.g., a modification via directed evolution, e.g., phage-assisted continuous evolution (PACE).
  • PACE phage-assisted continuous evolution
  • a DNA binding domain comprises a meganuclease domain (e.g., as described herein), or a functional fragment thereof.
  • a meganuclease domain possesses endonuclease activity, e.g., double-strand cleavage and/or nickase activity.
  • a meganuclease domain has reduced activity, e.g., lacks endonuclease activity, e.g., the meganuclease is catalytically inactive.
  • a catalytically inactive meganuclease is used as a DNA binding domain, e.g., as described in Fonfara et al. Nucleic Acids Res 40(2):847-860 (2012), incorporated herein by reference in its entirety.
  • a gene modifying polypeptide comprises a modification to a DNA-binding domain, e.g., relative to the wild-type polypeptide.
  • a DNA-binding domain comprises an addition, deletion, replacement, or modification to the amino acid sequence of the original DNA-binding domain.
  • a DNA-binding domain is modified to include a heterologous functional domain that binds specifically to a target nucleic acid (e.g., DNA) sequence of interest.
  • a functional domain replaces at least a portion (e.g., the entirety of) the prior DNA-binding domain of the gene modifying polypeptide.
  • a functional domain comprises a zinc finger (e.g., a zinc finger that specifically binds to the target nucleic acid (e.g., DNA) sequence of interest.
  • a functional domain comprises a Cas domain (e.g., a Cas domain that specifically binds to a target nucleic acid (e.g., DNA) sequence of interest.
  • a Cas domain comprises a Cas9 or a mutant or a variant thereof (e.g., as described herein).
  • a Cas domain is associated with a guide RNA (gRNA), e.g., as described herein.
  • gRNA guide RNA
  • a Cas domain is directed to a target nucleic acid (e.g., DNA) sequence of interest by a gRNA.
  • a Cas domain is encoded by the same nucleic acid (e.g., RNA) molecule as a gRNA.
  • a Cas domain is encoded by a different nucleic acid (e.g., RNA) molecule from the gRNA.
  • a DNA binding domain is capable of binding to a target sequence (e.g., a dsDNA target sequence) with greater affinity than a reference DNA binding domain.
  • a reference DNA binding domain is a DNA binding domain from Cas9 of S. pyogenes.
  • a DNA binding domain is capable of binding to a target sequence (e.g., a dsDNA target sequence) with an affinity between 100 pM - 10 nM (e.g., between 100 pM-1 nM or 1 nM - 10 nM).
  • the affinity of a DNA binding domain for a target sequence is measured in vitro, e.g., by thermophoresis, e.g., as described in Asmari et al. Methods 146:107-119 (2016) (incorporated by reference herein in its entirety).
  • a DNA binding domain is capable of binding to a target sequence (e.g., dsDNA target sequence), e.g, with an affinity between 100 pM - 10 nM (e.g., between 100 pM-1 nM or 1 nM - 10 nM) in the presence of a molar excess of scrambled sequence competitor dsDNA, e.g., of about 100-fold molar excess.
  • a target sequence e.g., dsDNA target sequence
  • a DNA binding domain is found associated with a target sequence (e.g., a dsDNA target sequence) more frequently than any other sequence in the genome of a target cell, e.g., human target cell, e.g., as measured by ChlP-seq (e.g., in HEK293T cells), e.g., as described in He and Pu (2010) Curr. Protoc Mol Biol Chapter 21 (incorporated herein by reference in its entirety).
  • a target sequence e.g., a dsDNA target sequence
  • a DNA binding domain is found associated with a target sequence (e.g., a dsDNA target sequence) at least about 5-fold or at least about 10-fold more frequently than any other sequence in the genome of a target cell, e.g., as measured by ChlP-seq (e.g., in HEK293T cells), e.g., as described in He and Pu (2010), supra.
  • a target sequence e.g., a dsDNA target sequence
  • ChlP-seq e.g., in HEK293T cells
  • an endonuclease domain has nickase activity and cleaves one strand of a target DNA. In some embodiments, nickase activity reduces the formation of doublestranded breaks at a target site. In some embodiments, an endonuclease domain creates a staggered nick structure in the first and second strands of a target DNA. In some embodiments, a staggered nick structure generates free 3’ overhangs at a target site. In some embodiments, free 3’ overhangs at a target site improve editing efficiency, e.g., by enhancing access and annealing of a 3’ homology region of a template nucleic acid. In some embodiments, a staggered nick structure reduces the formation of double-stranded breaks at a target site.
  • an endonuclease domain cleaves both strands of a target DNA, e.g., results in blunt-end cleavage of a target with no ssDNA overhangs on either side of the cutsite.
  • the amino acid sequence of an endonuclease domain of a gene modifying system described herein may be at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% identical to the amino acid sequence of an endonuclease domain described herein, e.g., an endonuclease domain described in Table 4.
  • a heterologous endonuclease is Fokl or a functional fragment thereof.
  • a heterologous endonuclease is a Holliday junction resolvase or homolog thereof, such as the Holliday junction resolving enzyme from Sulfolobus solfataricus — Ssol Hje (Govindaraju et al., Nucleic Acids Research 44:7, 2016).
  • a heterologous endonuclease is an endonuclease of a large fragment of a spliceosomal protein, such as Prp8 (Mahbub et al., Mobile DNA 8:16, 2017).
  • a heterologous endonuclease is derived from a CRIS PR-associated protein, e.g., Cas9.
  • a heterologous endonuclease is engineered to have only ssDNA cleavage activity, e.g., only nickase activity, e.g., be a Cas9 nickase, e.g., SpCas9 with D10A, H840A, or N863A mutations.
  • Table 4 provides exemplary Cas proteins and mutations associated with nickase activity.
  • an endonuclease domain is modified, for example by site-specific mutation, to alter DNA endonuclease activity.
  • an endonuclease domain is modified to reduce DNA-sequence specificity, e.g., by truncation to remove domains that confer DNA- sequence specificity or mutation to inactivate regions conferring DNA-sequence specificity.
  • an endonuclease domain has nickase activity and does not form double-stranded breaks.
  • an endonuclease domain forms single-stranded breaks at a higher frequency than double-stranded breaks, e.g., at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% of the breaks are single-stranded breaks, or less than 10%, less than 5%, less than 4%, less than 3%, less than 2%, or less than 1% of the breaks are double-stranded breaks.
  • an endonuclease domain forms substantially no double- stranded breaks.
  • an endonuclease domain does not form detectable levels of double- stranded breaks.
  • an endonuclease domain has nickase activity that nicks the first strand of a target site DNA.
  • an endonuclease domain cuts the genomic DNA of a target site near to the site of alteration on the strand that will be extended by a writing domain (e.g., an RT domain).
  • a writing domain e.g., an RT domain
  • an endonuclease domain has nickase activity that nicks the first strand of a target site DNA and does not nick the second strand of the target site DNA.
  • a gene modifying polypeptide comprises a CRISPR- associated endonuclease domain having nickase activity
  • said CRISPR- associated endonuclease domain nicks a target site DNA strand containing a PAM site (e.g., and does not nick the target site DNA strand that does not contain the PAM site).
  • a gene modifying polypeptide comprises a CRISPR-associated endonuclease domain having nickase activity
  • said CRISPR-associated endonuclease domain nicks a target site DNA strand that does not containa PAM site (e.g., and does not nick the target site DNA strand that contains the PAM site).
  • an endonuclease domain has nickase activity that nicks the first strand and the second strand of a target site DNA.
  • a writing domain e.g., an RT domain
  • a gene modifying polypeptide described herein polymerizes (e.g., reverse transcribes) from a heterologous object sequence of a template nucleic acid (e.g., a template RNA)
  • the cellular DNA repair machinery must repair the nick on the first DNA strand.
  • the target site DNA now contains two different sequences for the first DNA strand: one corresponding to the original genomic DNA (e.g., having a free 5' end) and a second corresponding to that polymerized from the heterologous object sequence (e.g., having a free 3' end). It is thought that the two different sequences equilibrate with one another, first one hybridizing the second strand, then the other, and which sequence the cellular DNA repair apparatus incorporates into its repaired target site may be a stochastic process. Without wishing to be bound by any particular theory, it is thought that introducing an additional nick to the second-strand may bias the cellular DNA repair machinery to adopt the heterologous object sequence-based sequence more frequently than the original genomic sequence (Anzalone et al.
  • an additional nick is positioned at least 10, at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 100, at least 105, at least 110, at least 115, at least 120, at least 125, at least 130, at least 135, at least 140, at least 145, or at least 150 nucleotides 5' or 3' of a target site modification (e.g., an insertion, deletion, or substitution) or to a nick on the first strand.
  • a target site modification e.g., an insertion, deletion, or substitution
  • an additional nick to the second strand may promote second- strand synthesis.
  • synthesis of a new sequence corresponding to the insertion/substitution in the second strand is necessary.
  • a gene modifying polypeptide comprises a single domain having endonuclease activity (e.g., a single endonuclease domain) and said domain nicks both the first strand and the second strand.
  • an endonuclease domain may be a CRISPR-associated endonuclease domain
  • a template nucleic acid e.g., template RNA
  • a gRNA spacer that directs nicking of the first strand and an additional gRNA spacer that directs nicking of the second strand.
  • a gene modifying polypeptide comprises a plurality of domains having endonuclease activity, and a first endonuclease domain nicks the first strand and a second endonuclease domain nicks the second strand (optionally, the first endonuclease domain does not (e.g., cannot) nick the second strand and the second endonuclease domain does not (e.g., cannot) nick the first strand).
  • an endonuclease domain is capable of nicking a first strand and a second strand.
  • first and second strand nicks occur at the same position in a target site but on opposite strands.
  • a second strand nick occurs in a staggered location, e.g., upstream or downstream, from a first nick.
  • an endonuclease domain generates a target site deletion if a second strand nick is upstream of a first strand nick.
  • an endonuclease domain generates a target site duplication if a second strand nick is downstream of a first strand nick.
  • an endonuclease domain generates no duplication and/or deletion if a first and second strand nicks occur in the same position of a target site.
  • an endonuclease domain has altered activity depending on protein conformation or RNA-binding status, e.g., which promotes the nicking of the first or second strand (e.g., as described in Christensen et al. PNAS 2006; incorporated by reference herein in its entirety).
  • an endonuclease domain comprises a meganuclease, or a functional fragment thereof. In some embodiments, an endonuclease domain comprises a homing endonuclease, or a functional fragment thereof. In some embodiments, an endonuclease domain comprises a mcganuclcasc from the LAGLID ADG, GIY-YIG, HNH, His-Cys Box, or PD-(D/E) XK families, or a functional fragment or variant thereof, e.g., which possess conserved amino acid motifs, e.g., as indicated in the family names.
  • an endonuclease domain comprises a meganuclease, or fragment thereof, chosen from, e.g., I- SmaMI (Uniprot F7WD42), I-Scel (Uniprot P03882), I- Anil (Uniprot PO388O), I-Dmol (Uniprot P21505), I-Crel (Uniprot P05725), I-TevI (Uniprot P13299), LOnuI (Uniprot Q4VWW5), or I- Bmol (Uniprot Q9ANR6).
  • I- SmaMI Uniprot F7WD42
  • I-Scel Uniprot P03882
  • I- Anil Uniprot PO388O
  • I-Dmol Uniprot P21505
  • I-Crel Uniprot P05725)
  • I-TevI Uniprot P13299
  • LOnuI Uniprot Q
  • a meganuclease is naturally monomeric, e.g., I-Scel, I-TevI, or dimeric, e.g., I-Crel, in its functional form.
  • LAGLID ADG meganucleases with a single copy of the LAGLID ADG motif generally form homodimers, whereas members with two copies of the LAGLID ADG motif arc generally found as monomers.
  • a meganuclease that normally forms as a dimer is expressed as a fusion, e.g., two subunits are expressed as a single open reading frame (ORF) and, optionally, connected by a linker, e.g., an I-Crel dimer fusion (Rodriguez-Fomes et al. Gene Therapy 2020; incorporated by reference herein in its entirety).
  • a meganuclease, or a functional fragment thereof is altered to favor nickase activity for one strand of a doublestranded DNA molecule, e.g., I-Scel (K122I and/or K223I) (Niu et al.
  • a meganuclease or functional fragment thereof possessing this preference for single-strand cleavage is used as an endonuclease domain, e.g., with nickase activity.
  • an endonuclease domain comprises a meganuclease, or a functional fragment thereof, which naturally targets or is engineered to target a safe harbor site, e.g., an I-Crel targeting SH6 site (Rodriguez-Fomes et al., supra).
  • an endonuclease domain comprises a meganuclease, or a functional fragment thereof, with a sequence-tolerant catalytic domain, e.g., I-TevI recognizing the minimal motif CNNNG (Kleinstiver et al. PNAS 2012).
  • a target sequence-tolerant catalytic domain is fused to a DNA binding domain, e.g., to direct activity, e.g., by fusing I-TevI to: (i) zinc fingers to create Tev-ZFEs (Kleinstiver et al. PNAS 2012), (ii) other meganucleases to create MegaTevs (Wolfs et al. Nucleic Acids Res 2014), and/or (iii) Cas9 to create TevCas9 (Wolfs et al. PNAS 2016).
  • an endonuclease domain comprises a restriction enzyme, e.g., a Type IIS or Type IIP restriction enzyme.
  • a gene modifying polypeptide comprises a modification to an endonuclease domain, e.g., relative to a wild-type Cas protein.
  • an endonuclease domain comprises an addition, deletion, replacement, or modification to the amino acid sequence of a wild-type Cas protein.
  • an endonuclease domain is modified to include a heterologous functional domain that binds specifically to and/or induces endonuclease cleavage of a target nucleic acid (e.g., DNA) sequence of interest.
  • an endonuclease domain comprises a zinc finger.
  • an endonuclease domain comprising a Cas domain is associated with a guide RNA (gRNA), e.g., as described herein.
  • gRNA guide RNA
  • an endonuclease domain is modified to include a functional domain that does not target a specific target nucleic acid (e.g., DNA) sequence.
  • an endonuclease domain comprises a FokI domain.
  • an endonuclease domain is associated with a target dsDNA in vitro at a frequency at least about 5-fold or at least about 10-fold higher than with a scrambled dsDNA. In some embodiments, an endonuclease domain is associated with a target dsDNA in vitro at a frequency at least about 5-fold or at least about 10-fold higher than with a scrambled dsDNA, e.g., in a cell (e.g., a HEK293T cell, a primary liver cell, or a primary lung cell).
  • a cell e.g., a HEK293T cell, a primary liver cell, or a primary lung cell.
  • the frequency of association between an endonuclease domain and a target DNA or scrambled DNA is measured by ChlP-seq, e.g., as described in He and Pu (2010) Curr. Protoc Mol Biol Chapter 21 (incorporated by reference herein in its entirety).
  • an endonuclease domain can catalyze the formation of a nick at a target sequence, e.g., to an increase of at least about 5-fold or at least about 10-fold relative to a non-target sequence (e.g., relative to any other genomic sequence in the genome of the target cell).
  • the level of nick formation is determined using NickScq, e.g., as described in Elacqua et al. (2019) bioRxiv doi.org/10.1101/867937 (incorporated herein by reference in its entirety).
  • an endonuclease domain releases a target after cleavage.
  • release of a target is indicated indirectly by assessing for multiple turnovers by an enzyme, e.g., as described in Yourik at al. RNA 25( 1 ):35-44 (2019) (incorporated herein by reference in its entirety) and shown in FIG. 2 therein.
  • the k eX p of an endonuclease domain is 1 x 10’ 3 - 1 x 10’ 5 min’ 1 as measured by such methods.
  • an endonuclease domain has a catalytic efficiency (fc C at/ m) greater than about 1 x 10 8 s’ 1 M’ 1 in vitro. In some embodiments, an endonuclease domain has a catalytic efficiency greater than about 1 x 10 5 , greater than about 1 x 10 6 , greater than about 1 x 10 7 , or greater than about 1 x 10 8 , s’ 1 M’ 1 in vitro. In some embodiments, catalytic efficiency is determined as described in Chen et al. (2016) Science 360(6387):436-439 (incorporated herein by reference in its entirety).
  • an endonuclease domain has a catalytic efficiency (fcat/ ⁇ m) greater than about 1 x 10 8 s" 1 M' 1 in cells. In some embodiments, an endonuclease domain has a catalytic efficiency greater than about 1 x 10 5 , greater than about 1 x 10 6 , greater than about 1 x 10 7 , or greater than about 1 x 10 8 s 1 M 1 in cells.
  • a gene modifying polypeptide described herein comprises a Cas domain.
  • a Cas domain can direct a gene modifying polypeptide to a target site specified by a gRNA spacer, thereby modifying a target nucleic acid sequence in “cis”.
  • a gene modifying polypeptide is fused to a Cas domain.
  • a gene modifying polypeptide comprises a CRISPR/Cas domain (also referred to herein as a CRISPR-associated protein).
  • CRISPR systems are adaptive defense systems originally discovered in bacteria and archaea.
  • CRISPR systems use RNA-guided nucleases termed CRISPR-associated or “Cas” endonucleases (e. g., Cas9 or Cpfl) to cleave foreign DNA.
  • CRISPR-associated or “Cas” endonucleases e. g., Cas9 or Cpfl
  • an endonuclease is directed to a target nucleotide sequence (e. g., a site in the genome that is to be sequence-edited) by sequence-specific, non-coding “guide RNAs” that target single- or double-stranded DNA sequences.
  • target nucleotide sequence e. g., a site in the genome that is to be sequence-edited
  • guide RNAs target single- or double-stranded DNA sequences.
  • Three classes (I-III) of CRISPR systems have been identified.
  • the class II CRISPR systems use a single Cas endonuclease (rather than multiple Cas proteins).
  • One class II CRISPR system includes a type II Cas endonuclease such as Cas9, a CRISPR RNA (“crRNA”), and a trans-activating crRNA (“tracrRNA”).
  • the crRNA contains a “spacer” sequence, a typically about 20-nucleotide RNA sequence that corresponds to a target DNA sequence (“protospacer”).
  • crRNA also contains a region that binds to the tracrRNA to form a partially doublestranded structure that is cleaved by RNase III, resulting in a crRNA/tracrRNA hybrid molecule.
  • a crRNA/tracrRNA hybrid then directs the Cas endonuclease to recognize and cleave a target DNA sequence.
  • a target DNA sequence is generally adjacent to a “protospacer adjacent motif’ (“PAM”) that is specific for a given Cas endonuclease and required for cleavage activity at a target site matching the spacer of the crRNA.
  • PAM protospacer adjacent motif
  • CRISPR endonucleases identified from various prokaryotic species have unique PAM sequence requirements, e.g., as listed for exemplary Cas enzymes in Table 3; examples of PAM sequences include 5'-NGG (Streptococcus pyogenes; SEQ ID NO: 11,019), 5'-NNAGAA (Streptococcus thermophilus CRISPR1; SEQ ID NO: 11,020), 5 '-NGGNG (Streptococcus thermophilus CRISPR3; SEQ ID NO: 11,021), and 5'- NNNGATT (Neisseria meningiditis; SEQ ID NO: 11,022).
  • 5'-NGG Streptococcus pyogenes
  • 5'-NNAGAA Streptococcus thermophilus CRISPR1; SEQ ID NO: 11,020
  • 5 '-NGGNG Streptococcus thermophilus CRISPR3; SEQ ID NO: 11,021
  • Some endonucleases e.g., Cas9 endonucleases, are associated with G-rich PAM sites, e. g., 5'-NGG (SEQ ID NO: 11,023), and perform blunt-end cleaving of the target DNA at a location 3 nucleotides upstream from (5' from) the PAM site.
  • Another class II CRISPR system includes the type V endonuclease Cpfl, which is smaller than Cas9; examples include AsCpfl (from Acidaminococcus sp.) and LbCpfl (from Lachnospiraceae sp.).
  • Cpfl -associated CRISPR arrays are processed into mature crRNAs without the requirement of a tracrRNA; in other words, a Cpfl system, in some embodiments, comprises only Cpfl nuclease and a crRNA to cleave a target DNA sequence.
  • Cpfl endonucleases are typically associated with T-rich PAM sites, e. g., 5'-TTN. Cpfl can also recognize a 5'-CTA PAM motif.
  • Cpfl typically cleaves a target DNA by introducing an offset or staggered double-strand break with a 4- or 5-nucleotide 5' overhang, for example, cleaving a target DNA with a 5-nucleotide offset or staggered cut located 18 nucleotides downstream from (3' from) a PAM site on the coding strand and 23 nucleotides downstream from the PAM site on the complimentary strand; the 5-nucleotide overhang that results from such offset cleavage allows more precise genome editing by DNA insertion by homologous recombination than by insertion at blunt-end cleaved DNA. See, e.g., Zetsche et al. (2015) Cell, 163:759 - 771.
  • Cas proteins A variety of CRISPR associated (Cas) genes or proteins can be used in the technologies provided by the present disclosure and the choice of Cas protein will depend upon the particular conditions of the method. Specific examples of Cas proteins include class II systems including Casl, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9, CaslO, Cpfl, C2C1, or C2C3.
  • a Cas protein e.g., a Cas9 protein
  • a Cas protein may be from any of a variety of prokaryotic species.
  • a Cas protein, e.g., a Cas9 protein is selected to recognize a particular protospacer-adjacent motif (PAM) sequence.
  • PAM protospacer-adjacent motif
  • a DNA-binding domain or endonuclease domain includes a sequence targeting polypeptide, such as a Cas protein, e.g., Cas9.
  • a Cas protein e.g., a Cas9 protein
  • a Cas protein may be obtained from a bacteria or archaea or synthesized using known methods.
  • a Cas protein may be from a gram-positive bacteria or a gram-negative bacteria.
  • a Cas protein may be from a Streptococcus (e.g., a S. pyogenes, or a S. thermophilus), a Francisella (e.g., an F.
  • novicida a Staphylococcus (e.g., an S. aureus), an Acidaminococcus (e.g., an Acidaminococcus sp. BV3L6), a Neisseria (e.g., an N. meningitidis), a Cryptococcus, a Corynebacterium, a Haemophilus, a Eubacterium, a Pasteurella, a Prevotella, a Veillonella, or a Marinobacter.
  • Staphylococcus e.g., an S. aureus
  • an Acidaminococcus e.g., an Acidaminococcus sp. BV3L6
  • Neisseria e.g., an N. meningitidis
  • Cryptococcus e.g., a Corynebacterium, a Haemophilus, a Eubacterium, a Pasteurella, a Prevotella, a Veillon
  • a gene modifying polypeptide may comprise the amino acid sequence of SEQ ID NO: 4000, or a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity thereto.
  • the amino acid sequence of SEQ ID NO: 4000, or a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity thereto is positioned at the N- terminal end of a gene modifying polypeptide.
  • the amino acid sequence of SEQ ID NO: 4000 is positioned within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, or 30 amino acids of the N-terminal end of a gene modifying polypeptide.
  • a gene modifying polypeptide may comprise the amino acid sequence of SEQ ID NO: 4001, or a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity thereto.
  • the amino acid sequence of SEQ ID NO: 4001, or a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity thereto is positioned at the C- terminal end of a gene modifying polypeptide.
  • an amino acid sequence of SEQ ID NO: 4001 below is positioned within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, or 30 amino acids of the C -terminal end of a gene modifying polypeptide.
  • a gene modifying polypeptide may comprise a Cas domain as listed in Table 3 or Table 4, or a functional fragment thereof, or a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity thereto.
  • a Cas protein requires a protospacer adjacent motif (PAM) to be present in or adjacent to a target DNA sequence for the Cas protein to bind and/or function.
  • a PAM is or comprises, from 5' to 3', NGG (SEQ ID NO: 11,024), YG (SEQ ID NO: 11,025), NNGRRT (SEQ ID NO: 11,026), NNNRRT (SEQ ID NO: 11,027), NGA (SEQ ID NO: 11,029), TYCV (SEQ ID NO: 11,030), TATV (SEQ ID NO: 11,031), NTTN (SEQ ID NO: 11,032), or NNNGATT (SEQ ID NO: 11,033), where N stands for any nucleotide, Y stands for C or T, R stands for A or G, and V stands for A or C or G.
  • a Cas protein is a protein listed in Table 3 or Table 4.
  • a Cas protein comprises one or more mutations altering its PAM.
  • a Cas protein comprises E1369R, E1449H, and R1556A mutations or analogous substitutions to the amino acids corresponding to said positions.
  • a Cas protein comprises E782K, N968K, and R1015H mutations or analogous substitutions to the amino acids corresponding to said positions.
  • a Cas protein comprises DI 135V, R1335Q, and T1337R mutations or analogous substitutions to the amino acids corresponding to said positions.
  • a Cas protein comprises S542R and K607R mutations or analogous substitutions to the amino acids corresponding to said positions. In some embodiments, a Cas protein comprises S542R, K548V, and N552R mutations or analogous substitutions to the amino acids corresponding to said positions. Exemplary advances in the engineering of Cas enzymes to recognize altered PAM sequences are reviewed in Collias et al Nature Communications 12:555 (2021), incorporated herein by reference in its entirety.
  • a Cas protein is catalytically active and cuts one or both strands of a target DNA site. In some embodiments, cutting a target DNA site is followed by formation of an alteration, e.g., an insertion or deletion, e.g., by the cellular repair machinery.
  • a Cas protein is modified to deactivate or partially deactivate its nuclease, e.g., a nuclease-deficient Cas9.
  • a Cas protein is modified to deactivate or partially deactivate its nuclease, e.g., a nuclease-deficient Cas9.
  • wild-type Cas9 generates double-strand breaks (DSBs) at specific DNA sequences targeted by a gRNA
  • a number of CRISPR endonucleases having modified functionalities are available, for example: a “nickase” version of Cas9 that has been partially deactivated generates only a single-strand break; a catalytically inactive Cas9 (“dCas9”) does not cut target DNA.
  • dCas9 binding to a DNA sequence may interfere with transcription at that site by steric hindrance.
  • dCas9 binding to an anchor sequence may interfere with (e.g., decrease or prevent) genomic complex (e.g., ASMC) formation and/or maintenance.
  • a DNA-binding domain comprises a catalytically inactive Cas9, e.g., dCas9.
  • Many catalytically inactive Cas9 proteins arc known in the art.
  • dCas9 comprises mutations in each endonuclease domain of the Cas protein, e.g., D10A and H840A or N863A mutations.
  • a catalytically inactive or partially inactive CRISPR/Cas domain comprises a Cas protein comprising one or more mutations, e.g., one or more of the mutations listed in Table 3.
  • a Cas protein described on a given row of Table 3 comprises one, two, three, or all of the mutations listed in the same row of Table 3.
  • a Cas protein, e.g., not described in Table 3 comprises one, two, three, or all of the mutations listed in a row of Table 3 or a corresponding mutation at a corresponding site in that Cas protein.
  • a catalytically inactive, e.g., dCas9, or partially deactivated Cas9 protein comprises a Dl l mutation (e.g., Dl l A mutation) or an analogous substitution to the amino acid corresponding to said position.
  • a catalytically inactive Cas9 protein, c.g., dCas9, or partially deactivated Cas9 protein comprises a H969 mutation (c.g., H969A mutation) or an analogous substitution to the amino acid corresponding to said position.
  • a catalytically inactive Cas9 protein e.g., dCas9, or partially deactivated Cas9 protein comprises a N995 mutation (e.g., N995A mutation) or an analogous substitution to the amino acid corresponding to said position.
  • a catalytically inactive Cas9 protein e.g., dCas9, comprises mutations at one, two, or three of positions Dl l, H969, and N995 (e.g., D11A, H969A, and N995A mutations) or analogous substitutions to the amino acids corresponding to said positions.
  • a catalytically inactive Cas9 protein e.g., dCas9, or partially deactivated Cas9 protein comprises a DIO mutation (e.g., a D10A mutation) or an analogous substitution to the amino acid corresponding to said position.
  • a catalytically inactive Cas9 protein, e.g., dCas9, or partially deactivated Cas9 protein comprises a H557 mutation (e.g., a H557A mutation) or an analogous substitution to the amino acid corresponding to said position.
  • a catalytically inactive Cas9 protein e.g., dCas9
  • dCas9 comprises a DIO mutation (e.g., a D10A mutation) and a H557 mutation (e.g., a H557A mutation) or analogous substitutions to the amino acids corresponding to said positions.
  • a catalytically inactive Cas9 protein e.g., dCas9, or partially deactivated Cas9 protein comprises a D839 mutation (e.g., a D839A mutation) or an analogous substitution to the amino acid corresponding to said position.
  • a catalytically inactive Cas9 protein, e.g., dCas9, or partially deactivated Cas9 protein comprises a H840 mutation (e.g., a H840A mutation) or an analogous substitution to the amino acid corresponding to said position.
  • a catalytically inactive Cas9 protein e.g., dCas9, or partially deactivated Cas9 protein comprises a N863 mutation (e.g., a N863A mutation) or an analogous substitution to the amino acid corresponding to said position.
  • a catalytically inactive Cas9 protein e.g., dCas9, comprises a DIO mutation (e.g., D10A), a D839 mutation (e.g., D839A), a H840 mutation (e.g., H840A), and a N863 mutation (e.g., N863A) or analogous substitutions to the amino acids corresponding to said positions.
  • a catalytically inactive Cas9 protein e.g., dCas9, or partially deactivated Cas9 protein comprises a E993 mutation (e.g., a E993A mutation) or an analogous substitution to the amino acid corresponding to said position.
  • a catalytically inactive Cas9 protein, e.g., dCas9, or partially deactivated Cas9 protein comprises a D917 mutation (e.g., a D917A mutation) or an analogous substitution to the amino acid corresponding to said position.
  • a catalytically inactive Cas9 protein e.g., dCas9, or partially deactivated Cas9 protein comprises a a E1006 mutation (e.g., a E1006A mutation) or an analogous substitution to the amino acid corresponding to said position.
  • a catalytically inactive Cas9 protein, e.g., dCas9, or partially deactivated Cas9 protein comprises a D1255 mutation (e.g., a D1255A mutation) or an analogous substitution to the amino acid corresponding to said position.
  • a catalytically inactive Cas9 protein comprises a D917 mutation (e.g., D917A), a E1006 mutation (e.g., E1006A), and a D1255 mutation (e.g., D1255A) or analogous substitutions to the amino acids corresponding to said positions.
  • a catalytically inactive Cas9 protein e.g., dCas9, or partially deactivated Cas9 protein comprises a D16 mutation (e.g., a D16A mutation) or an analogous substitution to the amino acid corresponding to said position.
  • a catalytically inactive Cas9 protein, e.g., dCas9, or partially deactivated Cas9 protein comprises a D587 mutation (e.g., a D587A mutation) or an analogous substitution to the amino acid corresponding to said position.
  • a partially deactivated Cas domain has nickase activity.
  • a partially deactivated Cas9 domain is a Cas9 nickase domain.
  • the catalytically inactive Cas domain or dead Cas domain produces no detectable double strand break formation.
  • a catalytically inactive Cas9 protein, e.g., dCas9, or partially deactivated Cas9 protein comprises a H588 mutation (e.g., a H588A mutation) or an analogous substitution to the amino acid corresponding to said position.
  • a catalytically inactive Cas9 protein e.g., dCas9, or partially deactivated Cas9 protein comprises a N611 mutation (e.g., a N611A mutation) or an analogous substitution to the amino acid corresponding to said position.
  • a catalytically inactive Cas9 protein e.g., dCas9, comprises a D16 mutation (e.g., D16A), a D587 mutation (e.g., D587A), a H588 mutation (e.g., H588A), and a N611 mutation (e.g., N611A) or analogous substitutions to the amino acids corresponding to said positions.
  • a DNA-binding domain or endonuclease domain may comprise a Cas molecule comprising or linked (e.g., covalently) to a gRNA (e.g., a template nucleic acid, e.g., template RNA, comprising a gRNA).
  • a gRNA e.g., a template nucleic acid, e.g., template RNA, comprising a gRNA.
  • an endonuclease domain or DNA binding domain comprises a Streptococcus pyogenes Cas9 (SpCas9) or a functional fragment or variant thereof.
  • SpCas9 Streptococcus pyogenes Cas9
  • an endonuclease domain or DNA binding domain comprises a modified SpCas9.
  • a modified SpCas9 comprises a modification that alters protospacer- adjacent motif (PAM) specificity.
  • PAM has specificity for the nucleic acid sequence 5'-NGT-3'.
  • a modified SpCas9 comprises one or more amino acid substitutions, e.g., at one or more of positions LI 111, DI 135, G1218, E1219, A1322, or R1335, e.g., selected from Li l HR, DI 135V, G1218R, E1219F, A1322R, or R1335V.
  • a modified SpCas9 comprises the amino acid substitution T1337R and one or more additional amino acid substitutions, e.g., selected from Li l HR, D1135L, S1136R, G1218S, E1219V, D1332A, D1332S, D1332T, D1332V, D1332L, D1332K, D1332R, R1335Q, T1337, T1337L, T1337Q, T1337I, T1337V, T1337F, T1337S, T1337N, T1337K, T1337H, T1337Q, and T1337M, or corresponding amino acid substitutions thereto.
  • additional amino acid substitutions e.g., selected from Li l HR, D1135L, S1136R, G1218S, E1219V, D1332A, D1332S, D1332T, D1332V, D1332L, D1332K, D1332R, R1335Q, T1337,
  • a modified SpCas9 comprises: (i) one or more amino acid substitutions selected from D1135L, S1136R, G1218S, E1219V, A1322R, R1335Q, and T1337; and (ii) one or more amino acid substitutions selected from Li l HR, G1218R, E1219F, D1332A, D1332S, D1332T, D1332V, D1332L, D1332K, D1332R, T1337L, T1337I, T1337V, T1337F, T1337S, T1337N, T1337K, T1337R, T1337H, T1337Q, and T1337M, or corresponding amino acid substitutions thereto.
  • an endonuclease domain or DNA binding domain comprises a Cas domain, e.g., a Cas9 domain.
  • an endonuclease domain or DNA binding domain comprises a nuclease-active Cas domain, a Cas nickase (nCas) domain, or a nucleaseinactive Cas (dCas) domain.
  • an endonuclease domain or DNA binding domain comprises a nuclease-active Cas9 domain, a Cas9 nickase (nCas9) domain, or a nuclease-inactive Cas9 (dCas9) domain.
  • an endonuclease domain or DNA binding domain comprises a Cas9 domain of Cas9 (e.g., dCas9 and nCas9), Casl2a/Cpfl, Casl2b/C2cl, Casl2c/C2c3, Casl2d/CasY, Casl2e/CasX, Casl2g, Casl2h, or Casl2i.
  • Cas9 domain of Cas9 e.g., dCas9 and nCas9
  • an endonuclease domain or DNA binding domain comprises a Cas9 (e.g., dCas9 and nCas9), Casl2a/Cpfl, Casl2b/C2cl, Casl2c/C2c3, Casl2d/CasY, Casl2e/CasX, Casl2g, Casl2h, or Casl2i.
  • an endonuclease domain or DNA binding domain comprises an S. pyogenes or an S. thermophilus Cas9, or a functional fragment thereof.
  • an endonuclease domain or DNA binding domain comprises a Cas9 sequence, e.g., as described in Chylinski, Rhun, and Charpentier (2013) RNA Biology 10:5, 726-737; incorporated herein by reference.
  • an endonuclease domain or DNA binding domain comprises the HNH nuclease subdomain and/or the RuvCl subdomain of a Cas, e.g., Cas9, e.g., as described herein, or a variant thereof.
  • an endonuclease domain or DNA binding domain comprises Casl2a/Cpfl, Casl2b/C2cl, Casl2c/C2c3, Casl2d/CasY, Casl2e/CasX, Casl2g, Casl2h, or Casl2i.
  • an endonuclease domain or DNA binding domain comprises a Cas polypeptide (e.g., enzyme), or a functional fragment thereof.
  • a Cas polypeptide (e.g., enzyme) is selected from Cast, CaslB, Cas2, Cas3, Cas4, Cas5, Cas5d, Cas5t, Cas5h, Cas5a, Cas6, Cas7, Cas8, Cas8a, Cas8b, Cas8c, Cas9 (e.g., Csnl or Csxl2), CaslO, CaslOd, Casl2a/Cpfl, Casl2b/C2cl, Casl2c/C2c3, Casl2d/CasY, Casl2e/CasX, Casl2g, Casl2h, Casl2i, Csyl , Csy2, Csy3, Csy4, Csel, Cse2, Cse3, Cse4, Cse5e, Cscl, Csc2, Csa5, Csnl, Csn2, Csml
  • a Cas9 comprises one or more substitutions, e.g., selected from H840A, D10A, P475A, W476A, N477A, DI 125 A, W1126A, and D1127A.
  • a Cas9 comprises one or more mutations at positions selected from: DIO, G12, G17, E762, H840, N854, N863, H982, H983, A984, D986, and/or A987, e.g., one or more substitutions selected from D10A, G12A, G17A, E762A, H840A, N854A, N863A, H982A, H983A, A984A, and/or D986A.
  • an endonuclease domain or DNA binding domain comprises a Cas (e.g., Cas9) sequence from Corynebacterium ulcerans, Corynebacterium diphtheria, Spiroplasma syrphidicola, Prevotella intermedia, Spiroplasma taiwanense, Streptococcus iniae, Belliella baltica, Psychroflexus torquis, Streptococcus thermophilus, Listeria innocua, Campylobacter jejuni, Neisseria meningitidis, Streptococcus pyogenes, or Staphylococcus aureus, or a fragment or variant thereof.
  • Cas e.g., Cas9 sequence from Corynebacterium ulcerans, Corynebacterium diphtheria, Spiroplasma syrphidicola, Prevotella intermedia, Spiroplasma taiwanense, Streptococcus in
  • an endonuclease domain or DNA binding domain comprises a Cpfl domain, e.g., comprising one or more substitutions, e.g., at position D917, E1006A, D1255 or any combination thereof, e.g., selected from D917A, E1006A, D1255A, D917A/E1006A,
  • D917A/D1255A, E1006A/D1255A, and D917A/E1006A/D1255A are D917A/E1006A/D1255A.
  • an endonuclease domain or DNA binding domain comprises spCas9, spCas9-VRQR (SEQ ID NO: 5019), spCas9- VRER (SEQ ID NO: 5020), xCas9 (sp), saCas9, saCas9-KKH, spCas9-MQKSER (SEQ ID NO: 5021), spCas9-LRKIQK (SEQ ID NO: 5022), or spCas9- LRVSQL (SEQ ID NO: 5023).
  • a gene modifying polypeptide has an endonuclease domain comprising a Cas9 nickase, e.g., Cas9 H840A.
  • the Cas9 H840A has the following amino acid sequence:
  • a gene modifying polypeptide comprises a dCas9 sequence comprising a D10A and/or H840A mutation, e.g., the following sequence:
  • an endonuclease domain or DNA-binding domain comprises a TAL effector molecule.
  • a TAL effector molecule e.g., a TAL effector molecule that specifically binds a DNA sequence, typically comprises a plurality of TAL effector domains or fragments thereof, and optionally one or more additional portions of naturally occurring TAL effectors (e.g., N- and/or C-terminal of the plurality of TAL effector domains).
  • Many TAL effectors are known to those of skill in the art and are commercially available, e.g., from Thermo Fisher Scientific.
  • Naturally occurring TALEs are natural effector proteins secreted by numerous species of bacterial pathogens including the plant pathogen Xanlhomonas which modulates gene expression in host plants and facilitates bacterial colonization and survival.
  • the specific binding of TAL effectors is based on a central repeat domain of tandemly arranged nearly identical repeats of typically 33 or 34 amino acids (the repeat-variable di-residues, RVD domain).
  • Members of the TAL effectors family differ mainly in the number and order of their repeats.
  • the number of repeats typically ranges from 1.5 to 33.5 repeats and the C-tcrminal repeat is usually shorter in length (e.g., about 20 amino acids) and is generally referred to as a “half-repeat.”
  • Each repeat of the TAL effector generally features a one-repeat-to-one-base-pair correlation with different repeat types exhibiting different base-pair specificity (one repeat recognizes one base-pair on the target gene sequence).
  • the smaller the number of repeats the weaker the protein-DNA interactions.
  • a number of 6.5 repeats has been shown to be sufficient to activate transcription of a reporter gene (Scholze et al., 2010).
  • TAL effectors it is possible to modify the repeats of a TAL effector to target specific DNA sequences. Further studies have shown that the RVD NK can target G. Target sites of TAL effectors also tend to include a T flanking the 5' base targeted by the first repeat, but the exact mechanism of this recognition is not known. More than 113 TAL effector sequences are known to date. Non-limiting examples of TAL effectors from Xanthomonas include, Hax2, Hax3, Hax4, AvrXa7, AvrXalO and AvrBs3.
  • a TAL effector domain of a TAL effector molecule described herein may be derived from a TAL effector from any bacterial species (e.g., Xanthomonas species such as the African strain of Xanthomonas oryzae pv. Oryzae (Yu et al. 2011), Xanthomonas campestris pv. raphani strain 756C and Xanthomonas oryzae pv. Oryzicola strain BLS256 (Bogdanove et al. 2011).
  • Xanthomonas species such as the African strain of Xanthomonas oryzae pv. Oryzae (Yu et al. 2011), Xanthomonas campestris pv. raphani strain 756C and Xanthomonas oryzae pv. Oryzicola strain BLS256 (Bogdanove et al. 2011).
  • a TAL effector domain comprises an RVD domain as well as flanking sequence(s) (sequences on the N-terminal and/or C-terminal side of the RVD domain) also from the naturally occurring TAL effector. It may comprise more or fewer repeats than the RVD of the naturally occurring TAL effector.
  • a TAL effector molecule can be designed to target a given DNA sequence based on the above code and others known in the art. The number of TAL effector domains (e.g., repeats (monomers or modules)) and their specific sequence can be selected based on the desired DNA target sequence. For example, TAL effector domains, e.g., repeats, may be removed or added in order to suit a specific target sequence.
  • a TAL effector molecule comprises between 6.5 and 33.5 TAL effector domains, e.g., repeats.
  • a TAL effector molecule of the present invention comprises between 8 and 33.5 TAL effector domains, e.g., repeats, e.g., between 10 and 25 TAL effector domains, e.g., repeats, e.g., between 10 and 14 TAL effector domains, e.g., repeats.
  • a TAL effector molecule comprises TAL effector domains that correspond to a perfect match to a DNA target sequence.
  • a mismatch between a repeat and a target base-pair on a DNA target sequence is permitted as along as it allows for the function of the polypeptide comprising the TAL effector molecule. Wihtout wishing to be bound by any particular theory, TALE binding is inversely correlated with the number of mismatches.
  • a TAL effector molecule of a gene modfiying polypeptide comprises no more than 7 mismatches, no more than 6 mismatches, no more than 5 mismatches, no more than 4 mismatches, no more than 3 mismatches, no more than 2 mismatches, or no more than 1 mismatch, and optionally no mismatch, with a target DNA sequence.
  • the smaller the number of TAL effector domains in a TAL effector molecule the smaller the number of mismatches will be tolerated and still allow for the function of a gene modifying polypeptide comprising the TAL effector molecule. Binding affinity is thought to depend on the sum of matching repeat-DNA combinations. For example, TAL effector molecules having 25 TAL effector domains or more may be able to tolerate up to 7 mismatches.
  • a TAL effector molecule may comprise additional sequences derived from a naturally occurring TAL effector.
  • the length of C-terminal and/or N- terminal sequence(s) included on each side of a TAL effector domain portion of a TAL effector molecule can vary and be selected by one skilled in the art, for example based on the studies of Zhang et al. (2011). Zhang et al., have characterized a number of C-terminal and N-terminal truncation mutants in Hax3 derived TAL-effector based proteins and have identified key elements, which contribute to optimal binding to the target sequence and thus activation of transcription.
  • transcriptional activity is inversely correlated with the length of N-terminus.
  • C-terminus an important element for DNA binding residues within the first 68 amino acids of the Hax 3 sequence was identified. Accordingly, in some embodiments, the first 68 amino acids on the C-terminal side of a TAL effector domains of the naturally occurring TAL effector is included in a TAL effector molecule.
  • a TAL effector molecule comprises 1) one or more TAL effector domains derived from a naturally occurring TAL effector; 2) at least 70, at least 80, at least 90, at least 100, at least 110, at least 110, at least 120, at least 130, at least 140, at least 150, at least 160, at least 170, at least 180, at least 190, at least 200, at least 210, at least 220, at least 230, at least 240, at least 250, at least 260, at least 270, at least 280 or more amino acids from the naturally occurring TAL effector on the N-terminal side of the TAL effector domains; and/or 3) at least 68, at least 80, at least 90, at least 100, at least 110, at least 120, at least 130, at least 140, at least 150, at least 160, at least 170, at least 180, at least 190, at least 200, at least 210, at least 220, at least 230, at least 240, at least 250
  • an endonuclease domain or DNA-binding domain is or comprises a Zn finger molecule.
  • a Zn finger molecule comprises a Zn finger protein, e.g., a naturally occurring Zn finger protein or engineered Zn finger protein, or fragment thereof.
  • Many Zn finger proteins are known to those of skill in the art and are commercially available, e.g., from Sigma-Aldrich.
  • a Zn finger molecule comprises a non-naturally occurring Zn finger protein that is engineered to bind to a target DNA sequence of choice. See, for example, Beerli, et al. (2002) Nature Biotechnol. 20:135-141; Pabo, et al. (2001) Ann. Rev. Biochem. 70:313-340; Isalan, et al. (2001) Nature Biotechnol. 19:656-660; Segal, et al. (2001) Curr. Opin. Biotechnol. 12:632-637; Choo, et al. (2000) Curr. Opin. Struct. Biol. 10:411-416; U.S. Pat. Nos.
  • An engineered Zn finger protein may have a novel binding specificity, compared to a naturally-occurring Zn finger protein.
  • Engineering methods include, but are not limited to, rational design and various types of selection. Rational design includes, for example, using databases comprising triplet (or quadruplet) nucleotide sequences and individual Zn finger amino acid sequences, in which each triplet or quadruplet nucleotide sequence is associated with one or more amino acid sequences of zinc fingers which bind the particular triplet or quadruplet sequence. See, for example, U.S. Pat. Nos. 6,453,242 and 6,534,261, incorporated by reference herein in their entireties.
  • Exemplary selection methods including phage display and two-hybrid systems, are disclosed in U.S. Pat. Nos. 5,789,538; 5,925,523; 6,007,988; 6,013,453; 6,410,248; 6,140,466; 6,200,759; and 6,242,568; as well as International Patent Publication Nos. WO 98/37186; WO 98/53057; WO 00/27878; WO 01/88197; and GB 2,338,237.
  • enhancement of binding specificity for zinc finger proteins has been described, for example, in International Patent Publication No. WO 02/077227.
  • zinc finger domains and/or multifingered zinc finger proteins may be linked together using any suitable linker sequences, including for example, linkers of 5 or more amino acids in length. See, also, U.S. Pat. Nos. 6,479,626; 6,903,185; and 7,153,949 for exemplary linker sequences 6 or more amino acids in length.
  • the proteins described herein may include any combination of suitable linkers between the individual zinc fingers of the protein.
  • enhancement of binding specificity for zinc finger binding domains has been described, for example, in co-owned International Patent Publication No. WO 02/077227.
  • Zn finger proteins and methods for design and construction of fusion proteins are known to those of skill in the art and described in detail in U.S. Pat. Nos. 6,140,0815; 789,538; 6,453,242; 6,534,261; 5,925,523; 6,007,988; 6,013,453; and 6,200,759; International Patent Publication Nos.
  • Zn finger proteins and/or multifingered Zn finger proteins may be linked together, e.g., as a fusion protein, using any suitable linker sequences, including for example, linkers of 5 or more amino acids in length. See, also, U.S. Pat. Nos. 6,479,626; 6,903,185; and 7,153,949 for exemplary linker sequences 6 or more amino acids in length.
  • the Zn finger molecules described herein may include any combination of suitable linkers between the individual zinc finger proteins and/or multi-fingered Zn finger proteins of the Zn finger molecule.
  • a DNA-binding domain or endonuclease domain comprises a Zn finger molecule comprising an engineered zinc finger protein that binds (in a sequence-specific manner) to a target DNA sequence.
  • a Zn finger molecule comprises one Zn finger protein or fragment thereof.
  • a Zn finger molecule comprises a plurality of Zn finger proteins (or fragments thereof), e.g., 2, 3, 4, 5, 6 or more Zn finger proteins (and optionally no more than 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, or 2 Zn finger proteins).
  • a Zn finger molecule comprises at least three Zn finger proteins.
  • a Zn finger molecule comprises four, five or six Zn finger proteins.
  • a Zn finger molecule comprises 8, 9, 10, 11 or 12 finger proteins. In some embodiments, a Zn finger molecule comprising three Zn finger proteins recognizes a target DNA sequence comprising 9 or 10 nucleotides. In some embodiments, a Zn finger molecule comprising four Zn finger proteins recognizes a target DNA sequence comprising 12 to 14 nucleotides. In some embodiments, a Zn finger molecule comprising six Zn finger proteins recognizes a target DNA sequence comprising 18 to 21 nucleotides.
  • a Zn finger molecule comprises a two-handed Zn finger protein.
  • Two handed zinc finger proteins are those proteins in which two clusters of zinc finger proteins are separated by intervening amino acids so that the two zinc finger domains bind to two discontinuous target DNA sequences.
  • An example of a two-handed type of zinc finger binding protein is SIP1, where a cluster of four zinc finger proteins is located at the amino terminus of the protein and a cluster of three Zn finger proteins is located at the carboxyl terminus (see Remade, et al. (1999) EMBO Journal 18(18):5073-5084).
  • Each cluster of zinc fingers in these proteins is able to bind to a unique target sequence and the spacing between the two target sequences can comprise many nucleotides.
  • a gene modifying polypeptide may comprise a linker, e.g., a peptide linker, e.g., a linker as described in Table 6.
  • a gene modifying polypeptide comprises, in an N-terminal to C-terminal direction, a Cas domain (e.g., a Cas domain of Table 4), a linker of Table 6 (or a sequence having at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity thereto), and an RT domain (e.g., an RT domain of Table 1).
  • a gene modifying polypeptide comprises a flexible linker between an endonuclease and ab RT domain, c.g., a linker comprising the amino acid sequence SGGSSGGSSGSETPGTSESATPESSGGSSGGSS (SEQ ID NO: 11,002).
  • an RT domain of a gene modifying polypeptide may be located C-terminal to an endonuclease domain.
  • an RT domain of a gene modifying polypeptide may be located N-terminal to the endonuclease domain.
  • a linker of a gene modifying polypeptide comprises a motif chosen from: (SGGS) n (SEQ ID NO: 5025), (GGGS) compassion (SEQ ID NO: 5026), (GGGGS)n (SEQ ID NO: 5027), (G) n , (EAAAK) U (SEQ ID NO: 5028), (GGS) U , or (XP) U .
  • Candidate gene modifying polypeptides may be screened to evaluate a candidate’s gene editing ability.
  • an RNA gene modifying system designed for the targeted editing of a coding sequence in the human genome may be used.
  • such a gene modifying system may be used in conjunction with a pooled screening approach.
  • a library of gene modifying polypeptide candidates and a template guide may be used in conjunction with a pooled screening approach.
  • RNA may be introduced into mammalian cells to test the candidates’ gene editing abilities by a pooled screening approach.
  • a library of gene modifying polypeptide candidates is introduced into mammalian cells followed by introduction of tgRNA into the cells.
  • mammalian cells that may be used in screening include HEK293T cells, U2OS cells, HeLa cells, HepG2 cells, Huh7 cells, K562 cells, or iPS cells.
  • a gene modifying polypeptide candidate may comprise 1 ) a Cas-nuclease, for example a wild-type Cas nuclease, c.g., a wild-type Cas9 nuclease, a mutant Cas nuclease, c.g., a Cas nickase, for example, a Cas9 nickase such as a Cas9 N863A nickase, or a Cas nuclease selected from Table 3 or Table 4, 2) a peptide linker, e.g., a sequence from Table 6, Table 7, or Table 8, that may exhibit varying degrees of length, flexibility, hydrophobicity, and/or secondary structure; and 3) a reverse transcriptase (RT), e.g.
  • a Cas-nuclease for example a wild-type Cas nuclease, c.g., a wild-type Cas9 nuclease, a mutant Cas nu
  • a gene modifying polypeptide candidate library comprises: a plurality of different gene modifying polypeptide candidates that differ from each other with respect to one, two or all three of a Cas nuclease, a peptide linker or an RT domain component, or a plurality of nucleic acid expression vectors that encode such gene modifying polypeptide candidates.
  • a gene modifying component may comprise, for example, an expression vector, e.g., an expression plasmid or lentiviral vector, that encodes a gene modifying polypeptide candidate, for example, comprises a human codon-optimized nucleic acid that encodes a gene modifying polypeptide candidate, e.g., a Cas-linker-RT fusion as described above.
  • a lentiviral cassette comprises: (i) a promoter for expression in mammalian cells, e.g., a CMV promoter; (ii) a gene modifying library candidate, e.g. a Cas-linker-RT fusion comprising a Cas nuclease of Table 3 or Table 4, a peptide linker of Table 6, and an RT of Table 1, for example a Cas-linker-RT fusion as in Table 7 or Table 8; (iii) a self-cleaving polypeptide, e.g., a T2A peptide; (iv) a marker enabling selection in mammalian cells, e.g., a puromycin resistance gene; and (v) a termination signal, e.g., a poly A tail.
  • a promoter for expression in mammalian cells e.g., a CMV promoter
  • a gene modifying library candidate e.g. a Cas-linker-RT
  • a tgRNA component may comprise a tgRNA or expression vector, e.g., an expression plasmid, that produces the tgRNA, for example, utilizes a U6 promoter to drive expression of the tgRNA, wherein the tgRNA is a non-coding RNA sequence that is recognized by Cas and localizes it to the genomic locus of interest, and that also templates reverse transcription of a desired edit into the genome by an RT domain.
  • mammalian cells e.g., HEK293T or U2OS cells
  • pooled gene modifying polypeptide candidate expression vector preparations e.g., lentiviral preparations, of the gene modifying candidate polypeptide library.
  • lentiviral plasmids are utilized, and HEK293 Lenti-X cells are seeded in 15 cm plates ( ⁇ 12xl0 6 cells) prior to lentiviral plasmid transfection.
  • lentiviral plasmid transfection may be performed using the Lentiviral Packaging Mix (Biosettia) and transfection of the plasmid DNA for the gene modifying candidate library is performed the following day using Lipofectamine 2000 and Opti-MEM media according to the manufacturer’s protocol.
  • extracellular DNA may be removed by a full media change the next day and virus-containing media may be harvested 48 hours after.
  • Lentiviral media may be concentrated using Lenti-X Concentrator (TaKaRa Biosciences) and 5 mL lentiviral aliquots may be made and stored at -80°C. Lentiviral titering is performed by enumerating colony forming units post-selection, e.g., post Puromycin selection.
  • mammalian cells e.g., HEK293T or U2OS cells
  • carrying a target DNA may be utilized.
  • mammalian cells e.g., HEK293T or U2OS cells
  • carrying a target DNA genomic landing pad may be utilized.
  • a target DNA genomic landing pad may comprise a gene to be edited for treatment of a disease or disorder of interest.
  • a target DNA is a gene sequence that expresses a protein that exhibits detectable characteristics that may be monitored to determine whether gene editing has occurred.
  • a blue fluorescence protein (BFP)- or green fluorescence protein (GFP)-expressing genomic landing pad is utilized.
  • mammalian cells e.g., HEK293T or U2OS cells, comprising a target DNA, e.g., a target DNA genomic landing pad, are seeded in culture plates at 500x - 3000x cells per gene modifying library candidate and transduced at a 0.2 - 0.3 multiplicity of infection (MOI) to minimize multiple infections per cell.
  • Puromycin (2.5 ug/mL) may be added 48 hours post infection to allow for selection of infected cells. In such an embodiment, cells may be kept under puromycin selection for at least 7 days and then scaled up for tgRNA introduction, e.g., tgRNA electroporation.
  • mammalian cells containing a target DNA to be edited may be infected with gene modifying polypeptide library candidates then transfected with tgRNA designed for use in editing of the target DNA. Subsequently, the cells may be analyzed to determine whether editing of the target locus has occurred according to the designed outcome, or whether no editing or imperfect editing has occurred, e.g., by using cell sorting and sequence analysis.
  • BFP- or GFP- cxprcssing mammalian cells may be infected with gene modifying library candidates and then transfected or electroporated with tgRNA plasmid or RNA, e.g., by electroporation of 250,000 cells/well with 200 ng of a tgRNA plasmid designed to convert BFP- to-GFP or GFP-to-BFP, at a cell count ensuring >250x - lOOOx coverage per library candidate.
  • the genome-editing capacity of the various constructs in this assay may be assessed by sorting the cells by Fluorescence-Activated Cell Sorting (FACS) for expression of the color-converted fluorescent protein (FP) at 4-10 days post-electroporation.
  • FACS Fluorescence-Activated Cell Sorting
  • FP color-converted fluorescent protein
  • Cells are sorted and harvested as distinct populations of unedited cells (exhibiting original florescence protein signal), edited cells (exhibiting converted fluorescence protein signal), and imperfect edit (exhibiting no florescence protein signal) cells.
  • a sample of unsorted cells may also be harvested as the input population to determine candidate enrichment during analysis.
  • genomic DNA is harvested from the sorted cell populations, and analyzed by sequencing the gene modifying library candidates in each population.
  • gene modifying candidates may be amplified from the genome using primers specific to the gene modifying polypeptide expression vector, e.g., the lentiviral cassette, amplified in a second round of PCR to dilute genomic DNA, and then sequenced, for example, sequenced by a next-generation sequencing platform.
  • reads of at least about 1500 nucleotides and generally no more than about 3200 nucleotides are mapped to the gene modifying polypeptide library sequences and those containing a minimum of about an 80% match to a library sequence are considered to be successfully aligned to a given candidate for purposes of this pooled screen.
  • candidates capable of performing gene editing in the assay e.g., the BFP-to-GFP or GFP-to-BFP edit
  • the read count of each library candidate in the edited population is compared to its read count in the initial, unsorted population.
  • gene modifying candidates with genome-editing capacity are identified based on enrichment in the edited (converted FP) population relative to unsorted (input) cells.
  • an enrichment of at least 1.0, at least 1.5, at least 2.0, at least 2.5, at least 3.0, at least 4.0, at least 5.0, at least 6.0, at least 7.0, at least 8.0, at least 9.0, at least 10, at least 15, at least 20, at least 25, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, or at least 100-fold over the input indicates potentially useful gene editing activity, e.g., at least 2-fold enrichment.
  • the enrichment is converted to a log-value by taking the log base 2 of the enrichment ratio.
  • a log2 enrichment score of at least 0, at least 1, at least 2, at least 3, at least 4, at least 5, at least 5.5, at least 6.0, at least 6.1, at least 6.2, at least 6.3, at least 6.4, at least 6.5, or at least 6.6 indicates potentially useful gene editing activity, e.g., a log2 enrichment score of at least 1.0.
  • enrichment values observed for gene modifying candidates may be compared to enrichment values observed under similar conditions utilizing a reference, e.g., Element ID No: 17380.
  • multiple tgRNAs may be used to screen a gene modifying candidate library.
  • a plurality of tgRNAs may be utilized to optimize template/Cas- linker-RT fusion pairs, e.g., for gene editing of particular target genes, for example, gene targets for the treatment of disease.
  • a pooled approach to screening gene modifying candidates may be performed using a multiplicity of different tgRNAs in an arrayed format.
  • multiple types of edits e.g., insertions, substitutions, and/or deletions of different lengths, may be used to screen the gene modifying candidate library.
  • multiple target sequences may be used to screen the gene modifying candidate library.
  • multiple target sequences e.g., different fluorescent proteins
  • multiple cell types e.g., HEK293T or U2OS, may be used to screen a gene modifying candidate library.
  • gene modifying library candidates are screened across multiple parameters, e.g., with at least two distinct tgRNAs in at least two cell types, and gene editing activity is identified by enrichment in any single condition.
  • a candidate with more robust activity across different tgRNA and cell types is identified by enrichment in at least two conditions, e.g., in all conditions screened. For clarity, candidates found to exhibit little to no enrichment under any given condition are not assumed to be inactive across all conditions and may be screened with different parameters or reconfigured at the polypeptide level, e.g., by swapping, shuffling, or evolving domains (e.g., RT domain), linkers, or other signals (e.g., NLS).
  • a gene modifying polypeptide comprises a linker sequence and an RT sequence.
  • a gene modifying polypeptide comprises a linker sequence as listed in Table 7 or Table 8, or an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity thereto.
  • a gene modifying polypeptide comprises the amino acid sequence of an RT domain as listed in Table 7 or Table 8, or an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity thereto.
  • a gene modifying polypeptide comprises a linker sequence as listed in Table 7 or Table 8, or an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity thereto; and the amino acid sequence of an RT domain as listed in Table 7 or Table 8, or an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity thereto.
  • a gene modifying polypeptide comprises: (i) a linker sequence as listed in a row of Table 7 or Table 8, or an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity thereto; and (ii) the amino acid sequence of an RT domain as listed in the same row of Table 7 or Table 8, or an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity thereto.
  • a gene modifying polypeptide (e.g., a gene modifying polypeptide that is part of a system described herein) comprises an amino acid sequence of any one of SEQ ID NOs: 1-7743 of the sequence listing, or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity thereto.
  • a gene modifying polypeptide comprises an amino acid sequence of any one of SEQ ID NOs: 1 -7743, or an amino acid sequence having at least 80% identity thereto.
  • a gene modifying polypeptide comprises an amino acid sequence of any one of SEQ ID NOs: 1 -7743, or an amino acid sequence having at least 90% identity thereto. In some embodiments, a gene modifying polypeptide comprises an amino acid sequence of any one of SEQ ID NOs: 1-7743, or an amino acid sequence having at least 95% identity thereto. In some embodiments, a gene modifying polypeptide comprises an amino acid sequence of any one of SEQ ID NOs: 1-7743, or an amino acid sequence having at least 99% identity thereto. In some embodiments, a gene modifying polypeptide comprises an amino acid sequence of any one of SEQ ID NOs: 1-7743.
  • a gene modifying polypeptide comprises an amino acid sequence of any one of SEQ ID NOs: 6001-7743, or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity thereto. In some embodiments, a gene modifying polypeptide comprises an amino acid sequence of any one of SEQ ID NOs: 4501-4541, or an amino acid sequence having at least at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity thereto.
  • a gene modifying polypeptide described herein comprises an RT and linker sequence from any of SEQ ID NOs: 1-7743, or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity thereto and a StlCas9 domain described herein.
  • a gene modifying polypeptide described herein comprises an RT and linker sequence from any of SEQ ID NOs: 1-7743, and a StlCas9 domain described herein.
  • a gene modifying polypeptide comprises an amino acid sequence as listed in Table 12, or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity thereto.
  • a gene modifying polypeptide comprises an amino acid sequence as listed in Table 7, or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity thereto.
  • a gene modifying polypeptide comprises a linker comprising a linker sequence as listed in Table 7, or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity thereto.
  • a gene modifying polypeptide comprises an RT domain comprising an RT domain sequence as listed in Table 7, or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity thereto.
  • a gene modifying polypeptide comprises: (i) a linker comprising a linker sequence as listed in a row of Table 7, or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity thereto; and (ii) an RT domain comprising an RT domain sequence as listed in the same row of Table 7, or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity thereto.
  • a gene modifying polypeptide comprises an amino acid sequence as listed in Table 8, or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity thereto.
  • a gene modifying polypeptide comprises a linker comprising a linker sequence as listed in Table 8, or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity thereto.
  • a gene modifying polypeptide comprises an RT domain comprising an RT domain sequence as listed in Table 8, or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity thereto.
  • a gene modifying polypeptide comprises: (i) a linker comprising a linker sequence as listed in a row of Table 8, or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity thereto; and (ii) an RT domain comprising an RT domain sequence as listed in the same row of Table 8, or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity thereto.
  • a gene modifying polypeptide comprises, in N-terminal to C- terminal order, one or more (e.g., 1, 2, 3, 4, 5, or all 6) of an N-terminal methionine residue, a first nuclear localization signal (NLS), a DNA binding domain, a linker, an RT domain, and/or a second NLS.
  • NLS nuclear localization signal
  • a gene modifying polypeptide comprises, in N-terminal to C-terminal order, a NLS (e.g., a first NLS), a DNA binding domain, a linker, and an RT domain, wherein the linker and RT domain are the linker and RT domain of a gene modifying polypeptide of any one of SEQ ID NOs: 1-7743, or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity to said linker and RT domain.
  • a NLS e.g., a first NLS
  • the linker and RT domain are the linker and RT domain of a gene modifying polypeptide of any one of SEQ ID NOs: 1-7743, or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity to said linker and RT domain.
  • a gene modifying polypeptide comprises, in N-terminal to C-terminal order, a DNA binding domain, a linker, an RT domain, and an NLS (e.g., a second NLS) wherein the linker and RT domain are the linker and RT domain of a gene modifying polypeptide of any one of SEQ ID NOs: 1-7743, or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity to said linker and RT domain.
  • a gene modifying polypeptide comprises, in N-terminal to C-terminal order, a first NLS, a DNA binding domain, a linker, an RT domain, and a second NLS, wherein the linker and RT domain are the linker and RT domain of a gene modifying polypeptide of any one of SEQ ID NOs: 1-7743, or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity to said linker and RT domain.
  • the gene modifying polypeptide further comprises an N-terminal methionine residue.
  • a gene modifying polypeptide comprises, in N-terminal to C- terminal order, one or more (e.g., 1, 2, 3, 4, 5, or all 6) of an N-terminal methionine residue, a first nuclear localization signal (NLS) (e.g., of a gene modifying polypeptide of any one of SEQ ID NOs: 1-7743 and/or as listed in any of Table 12, Table 7, or Table 8, or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity thereto), a DNA binding domain (e.g., a Cas domain, e.g., a SpyCas9 domain, e.g., as listed in Table 4, or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity thereto; or a DNA binding domain of a gene modifying polypeptide
  • NLS
  • the gene modifying polypeptide further comprises (e.g., C-terminal to the second NLS) a T2A sequence and/or a puromycin sequence (e.g., of a gene modifying polypeptide of any one of SEQ ID NOs: 1-7743 and/or as listed in any of Table 12, Table 7, or Table 8, or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity thereto).
  • a T2A sequence e.g., C-terminal to the second NLS
  • a puromycin sequence e.g., of a gene modifying polypeptide of any one of SEQ ID NOs: 1-7743 and/or as listed in any of Table 12, Table 7, or Table 8, or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity thereto.
  • a nucleic acid encoding a gene modifying polypeptide encodes a T2A sequence, e.g., wherein the T2A sequence is situated between a region encoding the gene modifying polypeptide and a second region, wherein the second region optionally encodes a selectable marker, e.g., puromycin.
  • the first NLS comprises a first NLS sequence of a gene modifying polypeptide having an amino acid sequence of any one of SEQ ID NOs: 1-7743, or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity thereto.
  • the first NLS comprises a first NLS sequence of a gene modifying polypeptide as listed in any of Table 12, Table 7, or Table 8, or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99%idcntity thereto.
  • a gene modifying polypeptide further comprises a spacer sequence between a first NLS and a DNA binding domain.
  • a spacer sequence between a first NLS and a DNA binding domain comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids.
  • a spacer sequence between a first NLS and a DNA binding domain comprises the amino acid sequence GG.
  • a DNA binding domain comprises a DNA binding domain of a gene modifying polypeptide of any one of SEQ ID NOs: 1-7743, or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity thereto.
  • a DNA binding domain comprises a DNA binding domain of a gene modifying polypeptide as listed in any of Table 12, Table 7, or Table 8, or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity thereto.
  • a DNA binding domain comprises a Cas domain (e.g., as listed in Table 4).
  • a DNA binding domain comprises the amino acid sequence of a SpyCas9 polypeptide (e.g., as listed in Table 4, e.g., a Cas9 N863A polypeptide), or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity thereto.
  • a DNA binding domain comprises the amino acid sequence:
  • a gene modifying polypeptide further comprises a spacer sequence between a DNA binding domain and a linker.
  • a spacer sequence between a DNA binding domain and a linker comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids.
  • a spacer sequence between a DNA binding domain and a linker comprises the amino acid sequence GG.
  • a linker comprises a linker sequence of a gene modifying polypeptide of any one of SEQ ID NOs: 1-7743, or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity thereto.
  • the linker comprises a linker sequence of a gene modifying polypeptide as listed in any of Table 12, Table 7, or Table 8, or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity thereto.
  • the linker comprises an amino acid sequence as listed in Table 6, Table 7 or Table 8 , or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity thereto.
  • a gene modifying polypeptide further comprises a spacer sequence between a linker and an RT domain.
  • a spacer sequence between a linker and an RT domain comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids.
  • a spacer sequence between a linker and an RT domain comprises the amino acid sequence GG.
  • an RT domain comprises an RT domain sequence of a gene modifying polypeptide of any one of SEQ ID NOs: 1-7743, or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity thereto.
  • an RT domain comprises a RT domain sequence of a gene modifying polypeptide as listed in any of Table 12, Table 7, or Table 8, or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity thereto.
  • an RT domain comprises an amino acid sequence as listed in Table 7, Table 8, or Table 1, or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity thereto.
  • an RT domain has a length of about 400-500, about 500-600, about 600-700, about 700-800, about 800-900, or about 900-1000 amino acids.
  • a gene modifying polypeptide further comprises a spacer sequence between an RT domain and a second NLS.
  • a spacer sequence between an RT domain and a second NLS comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids.
  • a spacer sequence between an RT domain and a second NLS comprises the amino acid sequence AG.
  • a second NLS comprises a second NLS sequence of a gene modifying polypeptide of any one of SEQ ID NOs: 1-7743. In some embodiments, a second NLS comprises a second NLS sequence of a gene modifying polypeptide as listed in any of Table 12, Table 7, or Table 8. In some embodiments, a second NLS sequence comprises a plurality of partial NLS sequences.
  • an NLS sequence e.g., a second NLS sequence
  • a first partial NLS sequence e.g., comprising the amino acid sequence KRTADGSEFE (SEQ ID NO: 11,097), or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity thereto.
  • an NLS sequence e.g., a second NLS sequence, comprises a second partial NLS sequence.
  • an NLS sequence e.g., a second NLS sequence, comprises an SV40A5 NLS, e.g., a bipartite SV40A5 NLS, e.g., comprising the amino acid sequence KRTADGSEFESPKKKAKVE (SEQ ID NO: 11,098), or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity thereto.
  • SV40A5 NLS e.g., a bipartite SV40A5 NLS, e.g., comprising the amino acid sequence KRTADGSEFESPKKKAKVE (SEQ ID NO: 11,098), or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity thereto.
  • an NLS sequence e.g., a second NLS sequence, comprises the amino acid sequence KRTADGSEFEKRTADGSEFESPKKKAKVE (SEQ ID NO: 11,099), or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity thereto.
  • a gene modifying polypeptide further comprises a spacer sequence between a second NLS and a T2A sequence and/or puromycin sequence.
  • a spacer sequence between a second NLS and a T2A sequence and/or puromycin sequence comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids.
  • a spacer sequence between a second NLS and a T2A sequence and/or puromycin sequence comprises the amino acid sequence GSG.
  • a gene modifying polypeptide comprises a linker (e.g., as described herein) and an RT domain (e.g., as described herein). In some embodiments, a gene modifying polypeptide comprises, in N-tcrminal to C-tcrminal order, a linker (e.g., as described herein) and an RT domain (e.g., as described herein).
  • a linker comprises a linker sequence as listed in Table 6, or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity thereto.
  • the linker comprises a linker sequence of any one of SEQ ID NOs: 1-7743, or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity thereto.
  • a linker comprises a linker sequence of any one of SEQ ID NOs: 6001-7743, or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity thereto. In some embodiments, a linker comprises a linker sequence present in any one of SEQ ID NOs: 4501-4541, or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity thereto.
  • a linker comprises a linker sequence of an exemplary gene modifying polypeptide listed in any of Table 12, Table 7, or Table 8, or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity thereto.
  • an RT domain comprises an RT domain sequence as listed in Table 1, or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity thereto.
  • an RT domain comprises an RT domain sequence of an exemplary gene modifying polypeptide listed in any of Table 12, Table 7, or Table 8, or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity thereto.
  • a gene modifying polypeptide comprises a portion of a gene modifying polypeptide of any one of SEQ ID NOs: 1-7743, wherein the portion comprises a linker and RT domain, or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity to said portion.
  • a gene modifying polypeptide comprises a linker of a gene modifying polypeptide of any one of SEQ ID NOs: 1-7743, or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity to said linker.
  • a gene modifying polypeptide comprises a linker of a gene modifying polypeptide of any one of SEQ ID NOs: 6001-7743, or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity to said linker.
  • a gene modifying polypeptide comprises a linker of a gene modifying polypeptide of any one of SEQ ID NOs: 4501-4541, or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity to said linker.
  • a gene modifying polypeptide comprises a linker of a gene modifying polypeptide as listed in any of Table 12, Table 7, or Table 8, or a linker comprising an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity thereto.
  • a gene modifying polypeptide comprises an RT domain of a gene modifying polypeptide of any one of SEQ ID NOs: 1-7743, or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity to said RT domain.
  • a gene modifying polypeptide comprises an RT domain of a gene modifying polypeptide of any one of SEQ ID NOs: 6001-7743, or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity said RT domain.
  • a gene modifying polypeptide comprises an RT domain of a gene modifying polypeptide of any one of SEQ ID NOs: 4501-4541, or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity said RT domain.
  • a gene modifying polypeptide comprises an RT domain of a gene modifying polypeptide as listed in any of Table 12, Table 7, or Table 8, or an RT domain comprising an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity thereto.
  • a linker and an RT domain of a gene modifying polypeptide comprise the amino acid sequences of a linker and RT domain (or amino acid sequences having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity thereto) of a gene modifying polypeptide having the amino acid sequence of any one of SEQ ID NOs: 1-7743.
  • a linker and an RT domain of a gene modifying polypeptide comprise amino acid sequences of a linker and RT domain having at least 80% identity to the linker and RT domains of any one of SEQ ID NOs: 1-7743.
  • a linker and an RT domain of a gene modifying polypeptide comprise amino acid sequences of a linker and RT domain having at least 90% identity to the linker and RT domains of any one of SEQ ID NOs: 1-7743. In some embodiments, a linker and an RT domain of a gene modifying polypeptide comprise amino acid sequences of a linker and RT domain having at least 95% identity to the linker and RT domains of any one of SEQ ID NOs: 1-7743.
  • a linker and an RT domain of a gene modifying polypeptide comprise amino acid sequences of a linker and RT domain having at least 99% identity to the linker and RT domains of any one of SEQ ID NOs: 1-7743.
  • a linker and an RT domain of a gene modifying polypeptide comprise the amino acid sequences of a linker and RT domain (or amino acid sequences having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity thereto) of a gene modifying polypeptide having the amino acid sequence of any one of SEQ ID NOs: 6001-7743.
  • a linker and an RT domain of a gene modifying polypeptide comprise the amino acid sequences of a linker and RT domain (or amino acid sequences having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity thereto) of a gene modifying polypeptide having the amino acid sequence of any one of SEQ ID NOs: 4501-4541.
  • a linker and am RT domain of a gene modifying polypeptide comprise the amino acid sequences of a linker and RT domain (or amino acid sequences having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity thereto) from a single row of any of Table 12, Table 7, or Table 8 (e.g., from a single exemplary gene modifying polypeptide as listed in any of Table 12, Table 7, or Table 8).
  • a linker and an RT domain of a gene modifying polypeptide comprise the amino acid sequences of a linker and RT domain (or amino acid sequences having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity thereto) from two different amino acid sequences selected from SEQ ID NOs: 1-7743.
  • a linker and an RT domain of a gene modifying polypeptide comprise the amino acid sequences of a linker and RT domain (or amino acid sequences having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity thereto) from different rows of any of Table 12, Table 7, or Table 8.
  • a gene modifying polypeptide further comprises a first NLS (e.g., a 5’ NLS), e.g., as described herein. In some embodiments, a gene modifying polypeptide further comprises a second NLS (e.g., a 3’ NLS), e.g., as described herein. In some embodiments, a gene modifying polypeptide further comprises an N-terminal methionine residue.
  • a first NLS e.g., a 5’ NLS
  • a gene modifying polypeptide further comprises a second NLS (e.g., a 3’ NLS), e.g., as described herein.
  • a gene modifying polypeptide further comprises an N-terminal methionine residue.
  • a gene modifying polypeptide comprises the amino acid sequence of an RT domain sequence from a family selected from: AVIRE, BAEVM, FFV, FLV, FOAMV, GALV, KORV, MLVAV, MLVBM, MLVCB, MLVFF, ML VMS, PERV, SFV1, SFV3L, WMSV, XMRV6, BLVAU, BLVJ, HTL1A, HTL1C, HTL1L, HTL32, HTL3P, HTLV2, JSRV, MLVF5, MLVRD, MMTVB, MPMV, SFVCP, SMRVH, SRV1, SRV2, and WDSV.
  • a family selected from: AVIRE, BAEVM, FFV, FLV, FOAMV, GALV, KORV, MLVAV, MLVBM, MLVCB, MLVFF, ML VMS, PERV, SFV1, SFV3L, WMSV, XMRV6,
  • a gene modifying polypeptide comprises the amino acid sequence of an RT domain sequence from a family selected from: AVIRE, BAEVM, FFV, FLV, FOAMV, GALV, KORV, MLVAV, MLVBM, MLVCB, MLVFF, MLVMS, PERV, SFV1, SFV3L, WMSV, and XMRV6.
  • a gene modifying polypeptide comprises the amino acid sequence of an RT domain sequence from an MLVMS RT domain.
  • the amino acid sequence of an RT domain sequence comprises one or more point mutations as listed in column 1 of Table 9, or a point mutation corresponding thereto.
  • the amino acid sequence of an RT domain sequence comprises one or more point mutations as listed in column 3 of Table 9 (Genl MLVMS), or a point mutation corresponding thereto.
  • the amino acid sequence of an RT domain sequence comprises one or more point mutations at an amino acid position of the RT domain as listed in columns 1 and 2 of Table 10, or an amino acid position corresponding thereto.
  • a gene modifying polypeptide comprises the amino acid sequence of an RT domain sequence from an AVIRE RT domain.
  • the amino acid sequence of an RT domain sequence comprises one or more point mutations as listed in column 2 of Table 9, or a point mutation corresponding thereto.
  • the amino acid sequence of an RT domain sequence comprises one or more point mutations as listed in column 4 of Table 9 (Gen2 AVIRE), or a point mutation corresponding thereto.
  • the amino acid sequence of an RT domain sequence comprises one or more point mutations at an amino acid position of the RT domain as listed in columns 3 and 4 of Table 10, or an amino acid position corresponding thereto.
  • an RT domain comprises an IENSSP (e.g., at the C-terminus).
  • Table 9 Exemplary point mutations in ML VMS and AVIRE RT domains
  • a gene modifying polypeptide comprises a gamma retrovirus derived RT domain.
  • a gamma retrovirus-derived RT domain of a gene modifying polypeptide comprises the amino acid sequence of an RT domain sequence from a family selected from: AVIRE, BAEVM, FFV, FLV, FOAMV, GALV, KORV, MLVAV, MLVBM, MLVCB, MLVFF, ML VMS, PERV, SFV1 , SFV3L, WMSV, and XMRV6.
  • a gamma retrovirus-derived RT domain of a gene modifying polypeptide is not derived from PERV.
  • an RT domain includes one, two, three, four, five, six or more mutations shown in Table 2 and corresponding to mutations D200N, L603W, T330P, D524G, E562Q, D583N, P51L, S67R, E67K, T197A, H204R, E302K, F309N, W313F, L435G, N454K, H594Q, L67 IP, E69K, or D653N in the RT domain of murine leukemia virus reverse transcriptase.
  • a gene modifying polypeptide further comprises a linker having at least 99% identity to a linker domains of any one of SEQ ID NOs: 1-7743.
  • the gene modifying polypeptide further comprises a linker having at least 99% or 100% identity to SEQ ID NO: 5217 or SEQ ID NO: 11,041.
  • an RT domain comprises the amino acid sequence of an RT domain of an AVIRE RT (e.g., an AVIRE_P03360 sequence, e.g., SEQ ID NO: 8001), or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity thereto.
  • an RT domain comprises the amino acid sequence of an AVIRE RT further comprising one, two, three, four, or five mutations selected from the group consisting of D200N, G33OP, L605W, T306K, and W313F, or a corresponding position in a homologous RT domain.
  • an RT domain comprises the amino acid sequence of an AVIRE RT further comprising one, two, or three mutations selected from the group consisting of D200N, G33OP, and L605W, or a corresponding position in a homologous RT domain.
  • an RT domain comprises the amino acid sequence of an RT domain of a BAEVM RT (e.g., an BAEVM_P10272 sequence, e.g., SEQ ID NO: 8004), or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity thereto.
  • an RT domain comprises the amino acid sequence of a BAEVM RT further comprising one, two, three, four, or five mutations selected from the group consisting of D198N, E328P, L602W, T304K, and W311F, or a corresponding position in a homologous RT domain.
  • an RT domain comprises the amino acid sequence of a BAEVM RT further comprising one, two, or three mutations selected from the group consisting of D198N, E328P, and L602W, or a corresponding position in a homologous RT domain.
  • an RT domain comprises the amino acid sequence of an RT domain of an FFV RT (c.g., an FFV_O93209 sequence, c.g., SEQ ID NO: 8012), or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity thereto.
  • an RT domain comprises the amino acid sequence of an FFV RT further comprising one, two, three, or four mutations selected from the group consisting of D21N, T293N, T419P, and L393K, or a corresponding position in a homologous RT domain.
  • an RT domain comprises the amino acid sequence of an FFV RT further comprising one, two, or three mutations selected from the group consisting of D21N, T293N, and T419P, or a corresponding position in a homologous RT domain.
  • an RT domain comprises the amino acid sequence of an FFV RT further comprising the mutation D21N.
  • an RT domain comprises the amino acid sequence of an FFV RT further comprising one, two, or three mutations selected from the group consisting of T207N, T333P, and L307K, or a corresponding position in a homologous RT domain. In some embodiments, an RT domain comprises the amino acid sequence of an FFV RT further comprising one or two mutations selected from the group consisting of T207N and T333P, or a corresponding position in a homologous RT domain.
  • an RT domain comprises the amino acid sequence of an RT domain of an FLV RT (e.g., an FLV_P10273 sequence, e.g., SEQ ID NO: 8019), or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity thereto.
  • an RT domain comprises the amino acid sequence of an FLV RT further comprising one, two, three, or four mutations selected from the group consisting of D199N, L602W, T305K, and W312F, or a corresponding position in a homologous RT domain.
  • an RT domain comprises the amino acid sequence of an FLV RT further comprising one or two mutations selected from the group consisting of D199N and L602W, or a corresponding position in a homologous RT domain.
  • an RT domain comprises the amino acid sequence of an RT domain of a FOAMV RT (e.g., a FOAMV_P14350 sequence, e.g., SEQ ID NO: 8021), or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity thereto.
  • an RT domain comprises the amino acid sequence of a FOAMV RT further comprising one, two, three, or four mutations selected from the group consisting of D24N, T296N, S420P, and L396K, or a corresponding position in a homologous RT domain.
  • an RT domain comprises the amino acid sequence of a FOAMV RT further comprising one, two, or three mutations selected from the group consisting of D24N, T296N, and S420P, or a corresponding position in a homologous RT domain.
  • an RT domain comprises the amino acid sequence of a FOAMV RT further comprising the mutation D24N, or a corresponding position in a homologous RT domain.
  • an RT domain comprises the amino acid sequence of a FOAMV RT further comprising one, two, or three mutations selected from the group consisting of T207N, S331P, and L307K, or a corresponding position in a homologous RT domain.
  • an RT domain comprises the amino acid sequence of a FOAMV RT further comprising one or two mutations selected from the group consisting of T207N and S331P, or a corresponding position in a homologous RT domain.
  • an RT domain comprises the amino acid sequence of an RT domain of a GALV RT (e.g., an GALV_P21414 sequence, e.g., SEQ ID NO: 8027), or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity thereto.
  • an RT domain comprises the amino acid sequence of a GALV RT further comprising one, two, three, four, or five mutations selected from the group consisting of D198N, E328P, L600W, T304K, and W31 IF, or a corresponding position in a homologous RT domain.
  • an RT domain comprises the amino acid sequence of a GALV RT further comprising one, two, or three mutations selected from the group consisting of D198N, E328P, and L600W, or a corresponding position in a homologous RT domain.
  • an RT domain comprises the amino acid sequence of an RT domain of a KORV RT (e.g., an KORV_Q9TTC1 sequence, e.g., SEQ ID NO: 8047), or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity thereto.
  • an RT domain comprises the amino acid sequence of a GALV RT further comprising one, two, three, four, five, or six mutations selected from the group consisting of D32N, D322N, E452P, L274W, T428K, and W435F, or a corresponding position in a homologous RT domain.
  • an RT domain comprises the amino acid sequence of a GALV RT further comprising one, two, three, or four mutations selected from the group consisting of D32N, D322N, E452P, and L274W, or a corresponding position in a homologous RT domain. In some embodiments, an RT domain comprises the amino acid sequence of a GALV RT further comprising the mutation D32N.
  • an RT domain comprises the amino acid sequence of a KORV RT further comprising one, two, three, four, or five mutations selected from the group consisting of D231N, E361P, L633W, T337K, and W344F, or a corresponding position in a homologous RT domain.
  • an RT domain comprises the amino acid sequence of a KORV RT further comprising one, two, or three mutations selected from the group consisting of D231N, E361P, and L633W, or a corresponding position in a homologous RT domain.
  • an RT domain comprises the amino acid sequence of an RT domain of a MLVAV RT (e.g., an MLVAV_P03356 sequence, e.g., SEQ ID NO: 8053), or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity thereto.
  • an RT domain comprises the amino acid sequence of a MLVAV RT further comprising one, two, three, four, or five mutations selected from the group consisting of D200N, T33OP, L603W, T306K, and W313F, or a corresponding position in a homologous RT domain.
  • an RT domain comprises the amino acid sequence of a MLVAV RT further comprising one, two, or three mutations selected from the group consisting of D200N, T33OP, and L603W, or a corresponding position in a homologous RT domain.
  • an RT domain comprises the amino acid sequence of an RT domain of a MLVBM RT (e.g., an MLVBM_Q7SVK7 sequence, e.g., SEQ ID NO: 8056), or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity thereto.
  • an RT domain comprises the amino acid sequence of a MLVBM RT further comprising one, two, three, four, or five mutations selected from the group consisting of D199N, T329P, L602W, T305K, and W312F, or a corresponding position in a homologous RT domain.
  • an RT domain comprises the amino acid sequence of a MLVBM RT further comprising one, two, and three mutations selected from the group consisting of D200N, T33OP, and L603W, or a corresponding position in a homologous RT domain.
  • an RT domain comprises the amino acid sequence of an RT domain of a MLVCB RT (e.g., an MLVCB_P08361 sequence, e.g., SEQ ID NO: 8062), or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity thereto.
  • an RT domain comprises the amino acid sequence of a MLVCB RT further comprising one, two, three, four, or five mutations selected from the group consisting of D200N, T33OP, L603W, T306K, and W313F, or a corresponding position in a homologous RT domain.
  • an RT domain comprises the amino acid sequence of a MLVCB RT further comprising one, two, and three mutations selected from the group consisting of D200N, T33OP, and L603W, or a corresponding position in a homologous RT domain.
  • an RT domain comprises the amino acid sequence of an RT domain of a MLVFF RT, or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity thereto.
  • an RT domain comprises the amino acid sequence of a MLVFF RT further comprising one, two, three, four, or five mutations selected from the group consisting of D200N, T330P, L603W, T306K, and W313F, or a corresponding position in a homologous RT domain.
  • an RT domain comprises the amino acid sequence of a MLVFF RT further comprising one, two, and three mutations selected from the group consisting of D200N, T330P, and L603W, or a corresponding position in a homologous RT domain.
  • an RT domain comprises the amino acid sequence of an RT domain of a MLVMS RT (e.g., an MLVMS_reference sequence, e.g., SEQ ID NO: 8137; or an MLVMS_P03355 sequence, e.g., SEQ ID NO: 8070), or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity thereto.
  • MLVMS_reference sequence e.g., SEQ ID NO: 8137
  • MLVMS_P03355 sequence e.g., SEQ ID NO: 8070
  • an RT domain comprises the amino acid sequence of a MLVMS RT further comprising one, two, three, four, five, or six mutations selected from the group consisting of D200N, T330P, L603W, T306K, W313F, and H8Y, or a corresponding position in a homologous RT domain.
  • an RT domain comprises the amino acid sequence of a MLVMS RT further comprising one, two, three, four, or five mutations selected from the group consisting of D200N, T330P, L603W, T306K, and W313F, or a corresponding position in a homologous RT domain.
  • an RT domain comprises the amino acid sequence of a MLVMS RT further comprising one, two, or three mutations selected from the group consisting of D200N, T330P, and L603W, or a corresponding position in a homologous RT domain.
  • an RT domain comprises the amino acid sequence of an RT domain of a PERV RT (e.g., an PERV_Q4VFZ2 sequence, e.g., SEQ ID NO: 8099), or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity thereto.
  • an RT domain comprises the amino acid sequence of a PERV RT further comprising one, two, three, four, or five mutations selected from the group consisting of D196N, E326P, L599W, T302K, and W309F, or a corresponding position in a homologous RT domain.
  • an RT domain comprises the amino acid sequence of a PERV RT further comprising one, two, or three mutations selected from the group consisting of D196N, E326P, and L599W, or a corresponding position in a homologous RT domain.
  • an RT domain comprises the amino acid sequence of an RT domain of a SFV1 RT (e.g., an SFV1_P23O74 sequence, e.g., SEQ ID NO: 8105), or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity thereto.
  • an RT domain comprises the amino acid sequence of a SFV 1 RT further comprising one, two, three, or four mutations selected from the group consisting of D24N, T296N, N420P, and L396K, or a corresponding position in a homologous RT domain.
  • an RT domain comprises the amino acid sequence of a SFV 1 RT further comprising one, two, or three mutations selected from the group consisting of D24N, T296N, and N420P, or a corresponding position in a homologous RT domain.
  • an RT domain comprises the amino acid sequence of a SFV1 RT further comprising the D24N, or a corresponding position in a homologous RT domain.
  • an RT domain comprises the amino acid sequence of an RT domain of a SFV3L RT (e.g., an SFV3L_P27401 sequence, e.g., SEQ ID NO: 8111), or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity thereto.
  • an RT domain comprises the amino acid sequence of a SFV3L RT further comprising one, two, three, or four mutations selected from the group consisting of D24N, T296N, N422P, and L396K, or a corresponding position in a homologous RT domain.
  • an RT domain comprises the amino acid sequence of a SFV3L RT further comprising one, two, or three mutations selected from the group consisting of D24N, T296N, and N422P, or a corresponding position in a homologous RT domain. In some embodiments, an RT domain comprises the amino acid sequence of a SFV3L RT further comprising the mutation D24N, or a corresponding position in a homologous RT domain.
  • an RT domain comprises the amino acid sequence of a SFV3L RT further comprising one, two, or three mutations selected from the group consisting of T307N, N333P, and L307K, or a corresponding position in a homologous RT domain. In some embodiments, an RT domain comprises the amino acid sequence of a SFV3L RT further comprising one or two mutations selected from the group consisting of T307N and N333P, or a corresponding position in a homologous RT domain.
  • an RT domain comprises the amino acid sequence of an RT domain of a WMSV RT (e.g., an WMSV_P03359 sequence, e.g., SEQ ID NO: 8131), or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity thereto.
  • an RT domain comprises the amino acid sequence of a WMSV RT further comprising one, two, three, four, or five mutations selected from the group consisting of D198N, E328P, L600W, T304K, and W31 IF, or a corresponding position in a homologous RT domain.
  • an RT domain comprises the amino acid sequence of a WMSV RT further comprising one, two, or three mutations selected from the group consisting of D198N, E328P, and L600W, or a corresponding position in a homologous RT domain.
  • an RT domain comprises the amino acid sequence of an RT domain of a XMRV6 RT (e.g., an XMRV6_A1Z651 sequence, e.g., SEQ ID NO: 8134), or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity thereto.
  • an RT domain comprises the amino acid sequence of a XMRV6 RT further comprising one, two, three, four, or five mutations selected from the group consisting of D200N, T33OP, L603W, T306K, and W313F, or a corresponding position in a homologous RT domain.
  • an RT domain comprises the amino acid sequence of a XMRV6 RT further comprising one, two, or three mutations selected from the group consisting of D200N, T33OP, and L603W, or a corresponding position in a homologous RT domain.
  • an RT domain of a gene modifying polypeptide comprises the amino acid sequence of an RT domain of an AVIRE RT, or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity thereto.
  • an RT domain comprises the amino acid sequence of an RT domain comprised in a sequence listed in column 1 of Table 11, or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity thereto.
  • a gene modifying polypeptide further comprises a linker having at least 99% or 100% identity to SEQ ID NO: 5217 or SEQ ID NO:11,041.
  • an RT domain of a gene modifying polypeptide comprises the amino acid sequence of an RT domain of an MLVMS RT, or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity thereto.
  • an RT domain comprises the amino acid sequence of an RT domain comprised in a sequence listed in any of columns 2-6 of Table 11, or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity thereto.
  • a gene modifying polypeptide further comprises a linker having at least 99% or 100% identity to SEQ ID NO: 5217 or SEQ ID NO:11,041.
  • Table 11 Exemplary gene modifying polypeptides comprising an AVIRE RT domain or an MLVMS RT domain.
  • the present disclosure provides a system comprising a nucleic acid molecule encoding a gene modifying polypeptide (e.g., as described herein) and a template nucleic acid (e.g., a template RNA, e.g., as described herein).
  • a nucleic acid molecule encoding a gene modifying polypeptide comprises one or more silent mutations in the coding region (e.g., in the sequence encoding an RT domain) relative to a nucleic acid molecule as described herein.
  • a system further comprises a gRNA (e.g., a gRNA that binds to a polypeptide that induces a nick, e.g., in the opposite strand of a target DNA bound by a gene modifying polypeptide).
  • a gRNA e.g., a gRNA that binds to a polypeptide that induces a nick, e.g., in the opposite strand of a target DNA bound by a gene modifying polypeptide.
  • a nucleic acid molecule encoding a gene modifying polypeptide encodes a polypeptide having an amino acid sequence selected from SEQ ID NOs: 1-7743, or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity thereto.
  • a nucleic acid molecule encoding a gene modifying polypeptide encodes a polypeptide having an amino acid sequence selected from SEQ ID NOs: 6001-7743, or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity thereto.
  • a nucleic acid molecule encoding a gene modifying polypeptide encodes a polypeptide having an amino acid sequence selected from SEQ ID NOs: 4501-4541, or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity thereto.
  • a nucleic acid molecule encoding a gene modifying polypeptide encodes a polypeptide as listed in any of Tables 12, 7, or 8, or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity thereto.
  • the nucleic acid molecule encoding the gene modifying polypeptide comprises a sequence encoding a portion of an amino acid sequence selected from SEQ ID NOs: 1-7743, wherein the portion comprises a linker and RT domain, or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity to said portion.
  • the nucleic acid molecule encoding the gene modifying polypeptide comprises a sequence encoding a portion of an amino acid sequence selected from SEQ ID NOs: 6001-7743, wherein the portion comprises a linker and RT domain, or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity to said portion.
  • the nucleic acid molecule encoding the gene modifying polypeptide comprises a sequence encoding a portion of an amino acid sequence selected from SEQ ID NOs: 4501-4541, wherein the portion comprises a linker and RT domain, or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity to said portion.
  • the nucleic acid molecule encoding the gene modifying polypeptide comprises a sequence encoding a portion of a polypeptide listed in any of Table 12, Table 7, or Table 8, wherein the portion comprises a linker and RT domain, or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity to said portion.
  • a nucleic acid molecule encoding a gene modifying polypeptide comprises a sequence encoding a linker of an amino acid sequence selected from SEQ ID NOs: 1-7743, or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity thereto.
  • a nucleic acid molecule encoding a gene modifying polypeptide comprises a sequence encoding a linker of a polypeptide having an amino acid sequence selected from SEQ ID NOs: 6001-7743, or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity thereto.
  • a nucleic acid molecule encoding a gene modifying polypeptide comprises a sequence encoding a linker of a polypeptide having an amino acid sequence selected from SEQ ID NOs: 4501-4541, or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity thereto.
  • a nucleic acid molecule encoding a gene modifying polypeptide comprises a sequence encoding a linker of a polypeptide as listed in any of Table 12, Table 7, or Table 8, or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity thereto.
  • a nucleic acid molecule encoding a gene modifying polypeptide comprises a sequence encoding an RT domain of an amino acid sequence selected from SEQ ID NOs: 1-7743, or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity thereto.
  • a nucleic acid molecule encoding a gene modifying polypeptide comprises a sequence encoding an RT domain of a polypeptide having an amino acid sequence selected from SEQ ID NOs: 6001- 7743, or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity thereto.
  • a nucleic acid molecule encoding a gene modifying polypeptide comprises a sequence encoding an RT domain of a polypeptide having an amino acid sequence selected from SEQ ID NOs: 4501-4541 , or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity thereto.
  • a nucleic acid molecule encoding a gene modifying polypeptide comprises a sequence encoding an RT domain of a polypeptide as listed in any of Table 12, Table 7, or Table 8, or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity thereto.
  • the present disclosure provides a system comprising a gene modifying polypeptide (e.g., as described herein) and a template nucleic acid (e.g., a template RNA, e.g., as described herein).
  • a gene modifying polypeptide e.g., as described herein
  • a template nucleic acid e.g., a template RNA, e.g., as described herein.
  • a gene modifying polypeptide comprises a polypeptide having an amino acid sequence selected from any one of SEQ ID NOs: 1-7743, or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity thereto.
  • a gene modifying polypeptide comprises a polypeptide having an amino acid sequence selected from any one of SEQ ID NOs: 6001-7743, or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity thereto.
  • a gene modifying polypeptide comprises a polypeptide having an amino acid sequence selected from any one of SEQ ID NOs: 4501-4541, or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity thereto.
  • a gene modifying polypeptide comprises a polypeptide as listed in any of Table 12, Table 7, or Table 8, or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity thereto.
  • a gene modifying polypeptide comprises a portion of an amino acid sequence selected from SEQ ID NOs: 1-7743, wherein the portion comprises a linker and RT domain, or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity to said portion.
  • a gene modifying polypeptide comprises a portion of an amino acid sequence selected from SEQ ID NOs: 6001-7743, wherein the portion comprises a linker and RT domain, or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity to said portion.
  • a gene modifying polypeptide comprises a portion of an amino acid sequence selected from SEQ ID NOs: 4501-4541 , wherein the portion comprises a linker and RT domain, or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity to said portion.
  • a gene modifying polypeptide comprises a portion of a polypeptide listed in any of Table 12, Table 7, or Table 8, wherein the portion comprises a linker and RT domain, or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity to said portion.
  • a gene modifying polypeptide comprises a linker of an amino acid sequence selected from SEQ ID NOs: 1-7743, or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity thereto.
  • a gene modifying polypeptide comprises a sequence encoding the linker of a polypeptide having an amino acid sequence selected from SEQ ID NOs: 6001-7743, or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity thereto.
  • a gene modifying polypeptide comprises a sequence encoding the linker of a polypeptide having an amino acid sequence selected from SEQ ID NOs: 4501-4541, or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity thereto.
  • a gene modifying polypeptide comprises the linker of a polypeptide as listed in any of Table 12, Table 7, or Table 8, or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity thereto.
  • a gene modifying polypeptide comprises an RT domain of an amino acid sequence selected from SEQ ID NOs: 1-7743, or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity thereto.
  • a gene modifying polypeptide comprises a sequence encoding an RT domain of a polypeptide having an amino acid sequence selected from SEQ ID NOs: 6001-7743, or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity thereto.
  • a gene modifying polypeptide comprises a sequence encodingan RT domain of a polypeptide having an amino acid sequence selected from SEQ ID NOs: 4501-4541, or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity thereto.
  • a gene modifying polypeptide comprises an RT domain of a polypeptide as listed in any of Table 12, Table 7, or Table 8, or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity thereto.
  • Table 12 Exemplary amino acid sequences for gene modifying polypeptides
  • a gene modifying polypeptide comprises an amino acid sequence selected from SEQ ID NOs: 26002, 26004, 10001, 10011, 10118, 10119, 10120, 31453, 31454, 31455, 31458, or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity thereto. Localization sequences for gene modifying systems
  • a gene modifying system RNA further comprises an intracellular localization sequence, c.g., a nuclear localization sequence (NLS).
  • a gene modifying polypeptide comprises an NLS as comprised in SEQ ID NO: 4000 and/or SEQ ID NO: 4001, or an NLS having an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity thereto.
  • a nuclear localization sequence may be an RNA sequence that promotes the import of an RNA into the nucleus.
  • a nuclear localization signal is located on a template RNA.
  • a gene modifying polypeptide is encoded on a first RNA, and a template RNA is a second, separate, RNA, and a nuclear localization signal is located on the template RNA and not on an RNA encoding the gene modifying polypeptide.
  • an RNA encoding a gene modifying polypeptide is targeted primarily to the cytoplasm to promote its translation, while a template RNA is targeted primarily to the nucleus to promote insertion into the genome.
  • a nuclear localization signal is at the 3' end, 5' end, or in an internal region of a template RNA.
  • a nuclear localization signal is 3' of a heterologous sequence (e.g., is directly 3' of the heterologous sequence) or is 5' of the heterologous sequence (e.g., is directly 5' of the heterologous sequence).
  • a nuclear localization signal is placed outside of a 5' UTR or outside of a 3' UTR of a template RNA.
  • a nuclear localization signal is placed between a 5' UTR and a 3' UTR, wherein optionally the nuclear localization signal is not transcribed with a transgene (e.g., the nuclear localization signal is in an anti-sense orientation or is downstream of a transcriptional termination signal or polyadenylation signal).
  • a nuclear localization sequence is situated inside of an intron.
  • a plurality of the same or different nuclear localization signals are in an RNA, e.g., in a template RNA.
  • a nuclear localization signal is less than 5, less than 10, less than 25, less than 50, less than 75, less than 100, less than 150, less than 200, less than 250, less than 300, less than 350, less than 400, less than 450, less than 500, less than 600, less than 700, less than 800, less than 900 or less than 1000 bp in length.
  • RNA nuclear localization sequences can be used. For example, Lubelsky and Ulitsky, Nature 555 (107-111), 2018 describe RNA sequences which drive RNA localization into the nucleus.
  • a nuclear localization signal is a SINE-derived nuclear RNA localization (SIRLOIN) signal.
  • a nuclear localization signal binds a nuclear-enriched protein. In some embodiments, a nuclear localization signal binds the HNRNPK protein. In some embodiments, a nuclear localization signal is rich in pyrimidines, e.g., is a C/T rich, C/U rich, C rich, T rich, or U rich region. In some embodiments, a nuclear localization signal is derived from a long non-coding RNA. In some embodiments, a nuclear localization signal is derived from MALAT1 long non-coding RNA or is the 600 nucleotide M region of MALAT 1 (described in Miyagawa et al., RNA 18, (738-751), 2012).
  • a nuclear localization signal is derived from BORG long non-coding RNA or is a AGCCC motif (described in Zhang et al., Molecular and Cellular Biology 34, 2318-2329 (2014).
  • a nuclear localization sequence is described in Shukla et al., The EMBO Journal c98452 (2016).
  • a nuclear localization signal is derived from a retrovirus.
  • a gene modifying polypeptide described herein comprises one or more (e.g., 2, 3, 4, 5) nuclear targeting sequences, for example a nuclear localization sequence (NLS).
  • an NLS is a bipartite NLS.
  • an NLS facilitates the import of a protein comprising an NLS into the cell nucleus.
  • an NLS is fused to the N-terminus of a gene modifying polypeptide as described herein.
  • an NLS is fused to the C-terminus of a gene modifying polypeptide.
  • an NLS is fused to the N-terminus or the C-terminus of a Cas domain.
  • a linker sequence is disposed between an NLS and a neighboring domain of a gene modifying polypeptide.
  • an NLS comprises the amino acid sequence MDSLLMNRRKFLYQFKNVRWAKGRRETYLC (SEQ ID NO: 5009), PKKRKVEGADKRTADGSEFESPKKKRKV(SEQ ID NO: 5010), RKSGKIAAIWKRPRKPKKKRKV (SEQ ID NO: 5011) KRTADGSEFESPKKKRKV(SEQ ID NO: 5012), KKTELQTTNAENKTKKL (SEQ ID NO: 5013), or KRGINDRNFWRGENGRKTR (SEQ ID NO: 5014), KRPAATKKAGQAKKKK (SEQ ID NO: 5015), PAAKRVKLD (SEQ ID NO:4644), KRTADGSEFEKRTADGSEFESPKKKAKVE (SEQ ID NO: 4649), KRTADGSEFE (SEQ ID NO: 4650), KRTADGSEFESPKKKAKVE (SEQ ID NO: 4651), AGKRTADGSEFEKRTADGS
  • an NLS comprises an amino acid sequence as disclosed in Table 13.
  • An NLS of Table 13 may be utilized with one or more copies in a polypeptide in one or more locations in a polypeptide, e.g., 1, 2, 3 or more copies of an NLS in an N-terminal domain, between peptide domains, in a C- terminal domain, or in a combination of locations, in order to improve subcellular localization to the nucleus. Multiple unique sequences may be used within a single polypeptide.
  • Sequences may be naturally monopartite or bipartite, e.g., having one or two stretches of basic amino acids, or may be used as chimeric bipartite sequences. Sequence references correspond to UniProt accession numbers, except where indicated as SeqNLS for sequences mined using a subcellular localization prediction algorithm (Lin et al BMC Bioinformat 13:157 (2012), incorporated herein by reference in its entirety).
  • Table 13 Exemplary nuclear localization signals for use in gene modifying systems
  • an NLS is a bipartite NLS.
  • a bipartite NLS typically comprises two basic amino acid clusters separated by a spacer sequence (which may be, e.g., about 10 amino acids in length).
  • a monopartite NLS typically lacks a spacer.
  • An example of a bipartite NLS is the nucleoplasmin NLS, having the sequence KR[PAATKKAGQA]KKKK (SEQ ID NO: 5015), wherein the spacer is indicated in square brackets.
  • Another exemplary bipartite NLS has the sequence PKKKRKVEGADKRTADGSEFESPKKKRKV (SEQ ID NO: 5016).
  • Exemplary NLSs are described in International Application W02020051561, which is herein incorporated by reference in its entirety, including for its disclosures regarding nuclear localization sequences.
  • a gene editor system polypeptide (e.g., a gene modifying polypeptide as described herein) further comprises an intracellular localization sequence, e.g., a nuclear localization sequence and/or a nucleolar localization sequence.
  • a nuclear localization sequence and/or nucleolar localization sequence may be an amino acid sequences that promotes the import of the protein into the nucleus and/or nucleolus, where it can promote integration of heterologous sequence into the genome.
  • a gene editor system polypeptide (e.g., (e.g., a gene modifying polypeptide as described herein) further comprises a nucleolar localization sequence.
  • a gene modifying polypeptide is encoded on a first RNA
  • a template RNA is a second, separate, RNA
  • a nucleolar localization signal is encoded on the RNA encoding the gene modifying polypeptide and not on the template RNA.
  • a nucleolar localization signal is located at the N-terminus, C- terminus, or in an internal region of a gene modifying polypeptide.
  • a plurality of the same or different nucleolar localization signals are used.
  • a nuclear localization signal is less than 5, less than 10, less than 25, less than 50, less than 75, or less than 100 amino acids in length.
  • Various polypeptide nucleolar localization signals can be used.
  • a nucleolar localization signal may also be a nuclear localization signal.
  • a nucleolar localization signal may overlap with a nuclear localization signal.
  • a nucleolar localization signal may comprise a stretch of basic residues.
  • a nucleolar localization signal may be rich in arginine and lysine residues.
  • a nucleolar localization signal may be derived from a protein that is enriched in the nucleolus.
  • a nucleolar localization signal may be derived from a protein enriched at ribosomal RNA loci. In some embodiments, a nucleolar localization signal may be derived from a protein that binds rRNA. In some embodiments, a nucleolar localization signal may be derived from MSP58. In some embodiments, a nucleolar localization signal may be a monopartite motif. In some embodiments, a nucleolar localization signal may be a bipartite motif. In some embodiments, a nucleolar localization signal may comprise multiple monopartite or bipartite motifs. In some embodiments, a nucleolar localization signal may comprise a mix of monopartite and bipartite motifs.
  • a nucleolar localization signal may be a dual bipartite motif.
  • a nucleolar localization motif may be a KRASSQALGTIPKRRSSSRFIKRKK (SEQ ID NO: 5017).
  • a nucleolar localization signal may be derived from nuclear factor-KB-inducing kinase.
  • a nucleolar localization signal may be an RKKRKKK motif (SEQ ID NO: 5018) (described in Birbach et al., Journal of Cell Science, 117 (3615-3624), 2004).
  • the present disclosure provides evolved variants of gene modifying polypeptides as described herein.
  • Evolved variants can, in some embodiments, be produced by mutagenizing a reference gene modifying polypeptide, or one of the fragments or domains comprised therein.
  • one or more domains e.g., a reverse transcriptase domain
  • One or more of such evolved variant domains can, in some embodiments, be evolved alone or together with other domains.
  • An evolved variant domain or domains may, in some embodiments, be combined with unevolved cognate component(s) or evolved variants of the cognate component(s), e.g., which may have been evolved in either a parallel or serial manner.
  • a process of mutagenizing a reference gene modifying polypeptide, or fragment or domain thereof comprises mutagenizing a reference gene modifying polypeptide or fragment or domain thereof.
  • mutagenesis comprises a continuous evolution method (e.g., PACE) or non-continuous evolution method (e.g., PANCE), e.g., as described herein.
  • an evolved gene modifying polypeptide, or a fragment or domain thereof comprises one or more amino acid variations introduced into its amino acid sequence relative to the amino acid sequence of a reference gene modifying polypeptide, or fragment or domain thereof.
  • amino acid sequence variations may include one or more mutated residues (e.g., conservative substitutions, nonconservative substitutions, or a combination thereof) within the amino acid sequence of a reference gene modifying polypeptide, e.g., as a result of a change in the nucleotide sequence encoding the gene modifying polypeptide that results in, e.g., a change in the codon at any particular position in the coding sequence, the deletion of one or more amino acids (e.g., a truncated protein), the insertion of one or more amino acids, or any combination of the foregoing.
  • An evolved variant gene modifying polypeptide may include variants in one or more components or domains of the gene modifying polypeptide (e.g., variants introduced into a reverse transcriptase domain).
  • the present disclosure provides gene modifying polypeptides, systems, kits, and methods using or comprising an evolved variant of a gene modifying polypeptide, e.g., employs an evolved variant of a gene modifying polypeptide or a gene modifying polypeptide produced or producible by PACE or PANCE.
  • an unevolved reference gene modifying polypeptide is a gene modifying polypeptide as disclosed herein.
  • phage-assisted continuous evolution generally refers to continuous evolution that employs phage as viral vectors.
  • PACE phage-assisted continuous evolution
  • Examples of PACE technology have been described, for example, in International PCT Application No. PCT/US 2009/056194, filed September 8, 2009, published as WO 2010/028347 on March 11, 2010; International PCT Application, PCT/US2011/066747, filed December 22, 2011, published as WO 2012/088381 on June 28, 2012; U.S. Patent No. 9,023,594, issued May 5, 2015; U.S. Patent No. 9,771,574, issued September 26, 2017; U.S. Patent No.
  • PANCE phage-assisted non-continuous evolution
  • SP evolving selection phage
  • Genes inside the host cell may be held constant while genes contained in the SP continuously evolve. Following phage growth, an aliquot of infected cells may be used to transfect a subsequent flask containing host E. coli. This process can be repeated and/or continued until the desired phenotype is evolved, e.g., for as many transfers as desired.
  • a method of evolution of an evolved variant gene modifying polypeptide, of a fragment or domain thereof comprises: (a) contacting a population of host cells with a population of viral vectors comprising the gene of interest (the starting gene modifying polypeptide or fragment or domain thereof), wherein: (1) the host cell is amenable to infection by the viral vector; (2) the host cell expresses viral genes required for the generation of viral particles; (3) the expression of at least one viral gene required for the production of an infectious viral particle is dependent on a function of the gene of interest; and/or (4) the viral vector allows for expression of the protein in the host cell, and can be replicated and packaged into a viral particle by the host cell.
  • the method comprises (b) contacting the host cells with a mutagen, using host cells with mutations that elevate mutation rate (e.g., either by carrying a mutation plasmid or some genome modification — e.g., proofing- impaired DNA polymerase, SOS genes, such as UmuC, UmuD’, and/or RecA, which mutations, if plasmid-bound, may be under control of an inducible promoter), or a combination thereof.
  • mutation rate e.g., either by carrying a mutation plasmid or some genome modification — e.g., proofing- impaired DNA polymerase, SOS genes, such as UmuC, UmuD’, and/or RecA, which mutations, if plasmid-bound, may be under control of an inducible promoter
  • the method additionally comprises (c) incubating the population of host cells under conditions allowing for viral replication and the production of viral particles, wherein host cells are removed from the host cell population, and fresh, uninfected host cells are introduced into the population of host cells, thus replenishing the population of host cells and creating a flow of host cells.
  • the cells are incubated under conditions allowing for the gene of interest to acquire a mutation.
  • the method further comprises (d) isolating a mutated version of the viral vector, encoding an evolved gene product (e.g., an evolved variant gene modifying polypeptide, or fragment or domain thereof), from the population of host cells.
  • an evolved gene product e.g., an evolved variant gene modifying polypeptide, or fragment or domain thereof
  • a viral vector or a phage is a filamentous phage, for example, an M13 phage, e.g., an M13 selection phage.
  • a gene required for the production of infectious viral particles is the M13 gene III (gill).
  • a phage may lack a functional gill, but otherwise comprise gl, gll, gIV, gV, gVI, gVII, gVIII, glX, and a gX.
  • generation of infectious VSV particles involves the envelope protein VSV-G.
  • retroviral vectors for example, Murine Leukemia Virus vectors, or Lentiviral vectors.
  • retroviral vectors can efficiently be packaged with VSV-G envelope protein, e.g., as a substitute for the native envelope protein of the virus.
  • host cells are incubated according to a suitable number of viral life cycles, e.g., at least 10, at least 20, at least 30, at least 40, at least 50, at least 100, at least 200, at least 300, at least 400, at least, 500, at least 600, at least 700, at least 800, at least 900, at least 1000, at least 1250, at least 1500, at least 1750, at least 2000, at least 2500, at least 3000, at least 4000, at least 5000, at least 7500, at least 10000, or more consecutive viral life cycles, which in on illustrative and non-limiting examples of M13 phage is 10-20 minutes per virus life cycle.
  • a suitable number of viral life cycles e.g., at least 10, at least 20, at least 30, at least 40, at least 50, at least 100, at least 200, at least 300, at least 400, at least, 500, at least 600, at least 700, at least 800, at least 900, at least 1000, at least 1250, at least 1500, at least 1750,
  • conditions can be modulated to adjust the time a host cell remains in a population of host cells, e.g., about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, about 25, about 30, about 35, about 40, about 45, about 50, about 55, about 60, about 70, about 80, about 90, about 100, about 120, about 150, or about 180 minutes.
  • Host cell populations can be controlled in part by density of the host cells, or, in some embodiments, the host cell density in an inflow, e.g., 10 3 cells/ml, about 10 4 cells/ml, about IO 3 cells/ml, about 5 - 10 5 cells/ml, about 10 6 cells/ml, about 5 - 10 6 cells/ml, about 10 7 cells/ml, about 5 - 10 7 cells/ml, about 10 8 cells/ml, about 5 - 10 8 cells/ml, about 10 9 cells/ml, about 5-- 10 9 cells/ml, about 10 10 cells/ml, or about 5-- 10 10 cells/ml.
  • 10 3 cells/ml about 10 4 cells/ml, about IO 3 cells/ml, about 5 - 10 5 cells/ml, about 10 6 cells/ml, about 5 - 10 6 cells/ml, about 10 7 cells/ml, about 5 - 10 7 cells/ml, about 10 8 cells/ml, about 5 -
  • an intein-N (intN) domain may be fused to the N-terminal portion of a first domain of a gene modifying polypeptide described herein
  • an intein-C (intC) domain may be fused to the C-terminal portion of a second domain of a gene modifying polypeptide described herein for the joining of the N-terminal portion to the C-terminal portion, thereby joining the first and second domains.
  • the first and second domains are each independently chosen from a DNA binding domain, an RNA binding domain, an RT domain, and an endonuclease domain.
  • Inteins can occur as self-splicing protein intron (e.g., peptide), e.g., which ligates flanking N-terminal and C-terminal exteins (e.g., fragments to be joined).
  • An intein may, in some embodiments, comprise a fragment of a protein that is able to excise itself and join the remaining fragments (the exteins) with a peptide bond in a process known as protein splicing. Inteins are also referred to as “protein introns.” The process of an intein excising itself and joining the remaining portions of the protein is herein termed “protein splicing” or “intcin-mcdiatcd protein splicing.”
  • an intein of a precursor protein comes from two genes.
  • Such intein is referred to herein as a split intein (e.g., split intein-N and split intein-C).
  • an intein-based approach may be used to join a first polypeptide sequence and a second polypeptide sequence together.
  • DnaE the catalytic subunit a of DNA polymerase III, is encoded by two separate genes, dnaE-n and dnaE-c.
  • An intein-N domain such as that encoded by the dnaE- n gene, when situated as part of a first polypeptide sequence, may join the first polypeptide sequence with a second polypeptide sequence, wherein the second polypeptide sequence comprises an intein-C domain, such as that encoded by the dnaE-c gene.
  • a protein can be made by providing nucleic acid encoding the first and second polypeptide sequences (e.g., wherein a first nucleic acid molecule encodes the first polypeptide sequence and a second nucleic acid molecule encodes the second polypeptide sequence), and the nucleic acid is introduced into the cell under conditions that allow for production of the first and second polypeptide sequences, and for joining of the first to the second polypeptide sequence via an intein-based mechanism.
  • inteins for joining heterologous protein fragments is described, for example, in Wood et al., J. Biol. Chem.289(21); 14512-9 (2014) (incorporated herein by reference in its entirety).
  • the inteins IntN and IntC may recognize each other, splice themselves out, and/or simultaneously ligate the flanking N- and C- terminal exteins of the protein fragments to which they were fused, thereby reconstituting a full- length protein from the two protein fragments.
  • a synthetic intein based on the dnaE intein, the Cfa-N (e.g., split intein-N) and Cfa-C (e.g., split intein-C) intein pair is used.
  • inteins have been described, e.g., in Stevens et al., J Am Chem Soc. 2016 Feb. 24; 138(7):2162-5 (incorporated herein by reference in its entirety).
  • Non-limiting examples of intein pairs that may be used in accordance with the present disclosure include: Cfa DnaE intein, Ssp GyrB intein, Ssp DnaX intein, Ter DnaE3 intein, Ter ThyX intein, Rma DnaB intein and Cne Prp8 intein (e.g., as described in U.S. Pat. No. 8,394,604, incorporated herein by reference.
  • an intein-N domain and an intein-C domain may be fused to the N-tcrminal portion of the split Cas9 and the C-tcrminal portion of a split Cas9, respectively, for the joining of the N-terminal portion of the split Cas9 and the C-terminal portion of the split Cas9.
  • an intein-N is fused to the C- terminus of the N-terminal portion of the split Cas9, i.e., to form a structure of N — [N-terminal portion of the split Cas9] -[intein-N] ⁇ C.
  • an intein-C is fused to the N- terminus of the C-terminal portion of the split Cas9, i.e., to form a structure of N- [intein-C] ⁇ [C- tenninal portion of the split Cas9]-C.
  • the mechanism of intein-mediated protein splicing for joining the proteins the inteins are fused to is described in Shah et al., Chem Sci. 2014; 5(1):446-461, incorporated herein by reference.
  • a split refers to a division into two or more fragments.
  • a split Cas9 protein or split Cas9 comprises a Cas9 protein that is provided as an N-terminal fragment and a C-terminal fragment encoded by two separate nucleotide sequences.
  • the polypeptides corresponding to the N-terminal portion and the C-terminal portion of the Cas9 protein may be spliced to form a reconstituted Cas9 protein.
  • the Cas9 protein is divided into two fragments within a disordered region of the protein, e.g., as described in Nishimasu et al., Cell, Volume 156, Issue 5, pp.
  • a disordered region may be determined by one or more protein structure determination techniques known in the art, including, without limitation, X-ray crystallography, NMR spectroscopy, electron microscopy (e.g., cryoEM), and/or in silica protein modeling.
  • the protein is divided into two fragments at any C, T, A, or S, e.g., within a region of SpCas9 between amino acids A292- G364, F445-K483, or E565-T637, or at corresponding positions in any other Cas9, Cas9 variant (e.g., nCas9, dCas9), or other napDNAbp.
  • protein is divided into two fragments at SpCas9 T310, T313, A456, S469, or C574.
  • a process of dividing the protein into two fragments is referred to as splitting the protein.
  • a protein fragment ranges from about 2-1000 amino acids (e.g., between 2-10, 10-50, 50-100, 100-200, 200-300, 300-400, 400-500, 500-600, 600-700, 700-800, 800-900, or 900-1000 amino acids) in length. In some embodiments, a protein fragment ranges from about 5-500 amino acids (e.g., between 5-10, 10-50, 50-100, 100-200, 200-300, 300-400, or 400-500 amino acids) in length. In some embodiments, a protein fragment ranges from about 20- 200 amino acids (e.g., between 20-30, 30-40, 40-50, 50-100, or 100-200 amino acids) in length.
  • a portion or fragment of a gene modifying polypeptide is fused to an intein.
  • a nuclease can be fused to the N-terminus or the C-terminus of the intein.
  • a portion or fragment of a fusion protein is fused to an intein and fused to an AAV capsid protein.
  • An intein, nuclease and capsid protein can be fused together in any arrangement (e.g., nuclease-intein-capsid, intein-nuclease-capsid, capsid-intein-nuclease, etc.).
  • the N-terminus of an intein is fused to the C-terminus of a fusion protein and the C-terminus of the intein is fused to the N-terminus of an AAV capsid protein.
  • an endonuclease domain (e.g., a nickase Cas9 domain) is fused to intein-N and a polypeptide comprising an RT domain is fused to an intein-C.
  • nucleotide and amino acid sequences of intein-N domains and compatible intein-C domains are provided below:
  • a gene modifying polypeptide can bind a target DNA sequence and template nucleic acid (e.g., template RNA), nick a target site, and write (e.g., reverse transcribe) the template into DNA, resulting in a modification of the target site.
  • additional domains may be added to a gene modifying polypeptide to enhance the efficiency of the process.
  • a gene modifying polypeptide may contain an additional DNA ligation domain to join reverse transcribed DNA to the DNA of a target site.
  • a gene modifying polypeptide may comprise a heterologous RNA-binding domain.
  • a gene 195ubstitut polypeptide may comprise a domain having 5' to 3' exonuclease activity (e.g., wherein the 5' to 3' exonuclease activity increases repair of the alteration of the target site, e.g., in favor of alteration over the original genomic sequence).
  • a gene modifying polypeptide may comprise a domain having 3' to 5' exonuclease activity, e.g., proof-reading activity.
  • a writing domain e.g., an RT domain, has 3' to 5' exonuclease activity, e.g., proof-reading activity.
  • Gene modifying systems described herein can modify a host target DNA site using a template nucleic acid sequence.
  • gene modifying systems described herein transcribe an RNA sequence template into host target DNA sites by target-primed reverse transcription (TPRT).
  • TPRT target-primed reverse transcription
  • a gene modifying system can insert an object sequence into a target genome without the need for exogenous DNA sequences to be introduced into the host cell (unlike, for example, CRISPR systems), as well as eliminate an exogenous DNA insertion step.
  • a gene modifying system can also delete a sequence from a target genome or introduce a substitution using an object sequence. Therefore, a gene modifying system provides a platform for the use of customized RNA sequence templates containing object sequences, e.g., sequences comprising heterologous gene coding and/or function information.
  • a template nucleic acid comprises one or more sequence (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, or more sequences) that binds a gene modifying polypeptide.
  • a system or method described herein comprises a single template nucleic acid (e.g., template RNA). In some embodiments, a system or method described herein comprises a plurality of template nucleic acids (e.g., template RNAs).
  • a system described herein comprises a first RNA comprising (e.g., from 5' to 3") a sequence that binds a gene modifying polypeptide (e.g., a DNA-binding domain and/or an endonuclease domain, e.g., a gRNA) and a sequence that binds a target site (e.g., a second strand of a site in a target genome), and a second RNA (e.g., a template RNA) comprising (e.g., from 5' to 3") optionally a sequence that binds the gene modifying polypeptide (e.g., that specifically binds an RT domain), a heterologous object sequence, and a PBS sequence.
  • a gene modifying polypeptide e.g., a DNA-binding domain and/or an endonuclease domain, e.g., a gRNA
  • a target site e.g., a second
  • each nucleic acid comprises a conjugating domain.
  • a conjugating domain enables association of nucleic acid molecules, e.g., by hybridization of complementary sequences.
  • a first RNA comprises a first conjugating domain and a second RNA comprises a second conjugating domain, and the first and second conjugating domains are capable of hybridizing to one another, e.g., under stringent conditions.
  • stringent conditions for hybridization include hybridization in 4x sodium chloride/sodium citrate (SSC), at about 65 C, followed by a wash in IxSSC, at about 65 C.
  • a template nucleic acid comprises RNA. In some embodiments, a template nucleic acid comprises DNA (e.g., single stranded or double stranded DNA).
  • a template nucleic acid comprises one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) homology domains that have homology to the target sequence.
  • a homology domain is about 10-20, 20-50, or 50-100 nucleotides in length.
  • a template RNA can comprise a gRNA sequence, e.g., to direct a gene modifying polypeptide to a target site of interest.
  • a template RNA comprises (e.g., from 5' to 3') (i) optionally a gRNA spacer that binds a target site (e.g., a second strand of a site in a target genome), (ii) optionally a gRNA scaffold that binds a polypeptide described herein (e.g., a gene modifying polypeptide or a Cas polypeptide), (iii) a heterologous object sequence comprising a mutation region (optionally the heterologous object sequence comprises, from 5’ to 3’, a first homology region, a mutation region, and a second homology region), and (iv) a primer binding site (PBS) sequence comprising a 3' target homology domain.
  • PBS primer binding site
  • a template nucleic acid (e.g., template RNA) component of a genome editing system described herein typically is able to bind a gene modifying polypeptide of a system described herein.
  • a template nucleic acid e.g., template RNA
  • a binding region e.g., 3' region, may be a structured RNA region, e.g., having at least 1, 2 or 3 hairpin loops, capable of binding the gene modifying polypeptide of the system.
  • a binding region may associate with a template nucleic acid (e.g., template RNA) with any of the polypeptide modules.
  • a binding region of a template nucleic acid may associate with an RNA-binding domain in a gene modifying polypeptide.
  • a binding region of a template nucleic acid may associate with a reverse transcription domain of a gene modifying polypeptide (e.g., specifically bind to the RT domain).
  • a template nucleic acid e.g., a template RNA
  • a binding region may also provide DNA target recognition, e.g., a gRNA hybridizing to a target DNA sequence and binding a gene modifying polypeptide, e.g., a Cas9 domain.
  • a template nucleic acid e.g., template RNA
  • a template RNA has a poly-A tail at its 3' end. In some embodiments, a template RNA does not have a poly-A tail at its 3' end.
  • a template nucleic acid is a template RNA.
  • a template RNA comprises one or more modified nucleotides.
  • a template RNA comprises one or more deoxyribonucleotides.
  • regions of a template RNA are replaced by DNA nucleotides, e.g., to enhance stability of the molecule.
  • the 3' end of a template may comprise DNA nucleotides, while the rest of the template comprises RNA nucleotides that can be reverse transcribed.
  • a heterologous object sequence is primarily or wholly made up of RNA nucleotides (e.g., at least 90%, at least 95%, at least 98%, at least 99%, or 100% RNA nucleotides).
  • a PBS sequence is primarily or wholly made up of DNA nucleotides (e.g., at least 90%, at least 95%, at least 98%, at least 99%, or 100% DNA nucleotides).
  • a heterologous object sequence for writing into the genome may comprise DNA nucleotides.
  • DNA nucleotides in a template nucleic acid are copied into the genome by a domain capable of DNA-dependent DNA polymerase activity.
  • DNA-dependent DNA polymerase activity is provided by a DNA polymerase domain in a gene modifying polypeptide.
  • DNA-dependent DNA polymerase activity is provided by a reverse transcriptase domain that is also capable of DNA-dependent DNA polymerization, e.g., second strand synthesis.
  • a template nucleic acid molecule is composed of only DNA nucleotides.
  • a system described herein comprises two nucleic acids which together comprise the sequences of a template RNA described herein.
  • two nucleic acids are associated with each other non-covalently, e.g., directly associated with each other (e.g., via base pairing), or indirectly associated as part of a complex comprising one or more additional molecule.
  • a template RNA described herein may comprise, from 5’ to 3’: (1) a gRNA spacer; (2) a gRNA scaffold; (3) heterologous object sequence (4) a primer binding site (PBS) sequence.
  • PBS primer binding site
  • a template RNA described herein may comprise a gRNA spacer that directs a gene modifying system to a target nucleic acid, and a gRNA scaffold that promotes association of a template RNA with a Cas domain of a gene modifying polypeptide.
  • a gRNA scaffold has been engineered for improved performance with StlCas9.
  • Systems described herein can also comprise a gRNA that is not part of a template nucleic acid.
  • a gRNA that comprises a gRNA spacer and gRNA scaffold, but not a heterologous object sequence or a PBS sequence can be used, e.g., to induce second strand nicking, e.g., as described in the section herein entitled “Second Strand Nicking”.
  • variant gRNA scaffolds that are compatible with StlCas9.
  • variant gRNA scaffolds are used in a system comprising a gene modifying polypeptide that comprises an StlCas9 domain.
  • the wild-type StlCas9 gRNA scaffold has a hypothesized secondary structure, shown in FIG. 2.
  • the gRNA scaffold comprises: a region comprising a lower stem, an upper stem, and tetraloop (also collectively referred to as Repeat: anti-repeat duplex or RAR); a first single stranded region; a Stem loop 1, a second single stranded region; a Stem loop 2; and a third single stranded region.
  • the upper stem comprises three paired bases (nt 12-14 pair with nt 19-21) and the 4-nucleotide tetraloop is nt 15-18.
  • the next region is the first single stranded region which contains nt 35 and 36.
  • Stem loop 1 which comprises nucleotides 37-47.
  • the second single stranded region comprising nucleotides 48-53.
  • Stem loop 2 which comprises nucleotides 54-82.
  • 3’ of Stem loop 2 is a third single stranded region which comprises nucleotides 83-84.
  • the hypothesized structure represents the likely secondary structure of the StlCas9 gRNA scaffold under physiogically relevant conditions.
  • the named regions such as Stem loop 1, Stem loop 2, RAR upper stem, RAR lower stem, and tetraloop
  • the spacer is typically situated at the 5’ end of the gRNA scaffold.
  • a variant gRNA scaffold herein can comprise mutations in different regions of the gRNA scaffold.
  • a variant gRNA scaffold may comprise a mutation in the upper stem that results in the thermodynamic strengthening of RAR. More specifically, the upper stem may be lengthened, e.g., by 1-8 base pairs (e.g., 1, 2, 3, 4, 5, 6, 7, or 8 base pairs).
  • a variant gRNA scaffold may comprise a mutation in the tetraloop of RAR, which may optimize performance by improving the thermodynamic stability of RAR. More specifically, one or more nucleotides in the loop of the tetraloop may be substituted. In some embodiments, the loop region of the tetraloop may be lengthened, e.g., by 1 nucleotide, resulting in a loop 5 nucleotides in length.
  • a variant gRNA scaffold may comprise a truncation in the stem of Stem loop 2 and/or in one or both single stranded regions at its base (i.e., the second and third single stranded regions).
  • the stem of Stem loop 2 comprises truncations in 3’-5’ direction end ranging from 1- 32 nt.
  • a variant gRNA scaffold may comprise one or more mutations that destabilize the upper RAR stem relative to the wild-type sequence.
  • a variant gRNA scaffold has a deletion of one or more nucleotides of the upper RAR stem.
  • a variant gRNA scaffold has a deletion of one or more nucleotides in the region with bulges that is situated between the upper RAR stem and lower RAR stem.
  • a variant gRNA scaffold has a substitution wherein a G-C base pair in the upper RAR stem is replaced with a base pair other than G-C (e.g., an A-U base pair).
  • a variant gRNA scaffold comprises a mutation in the upper stem of the RAR and a mutation in the tetraloop of the RAR. More specifically, in some embodiments, the upper stem is lengthened, e.g., by 1-8 base pairs (e.g., 1, 2, 3, 4, 5, 6, 7, or 8 base pairs), and the tetraloop comprises a substitution or an insertion (e.g., a 1 nucleotide insertion).
  • a variant gRNA scaffold comprises a mutation in the upper stem of the RAR and a truncation in the stem of Stem loop 2. More specifically, in some embodiments, the upper stem is lengthened, e.g., by 1-8 base pairs (e.g., 1, 2, 3, 4, 5, 6, 7, or 8 base pairs), and the stem of Stem loop 2 comprises a truncation of from 1- 32 nt.
  • a variant gRNA scaffold comprises a mutation in the tetraloop of the RAR and a truncation in the stem of Stem loop 2. More specifically, in some embodiments the tetraloop comprises a substitution or an insertion (e.g., a 1 nucleotide insertion) and the stem of Stem loop 2 comprises a truncation of from 1- 32 nt.
  • a variant gRNA scaffold comprises: (1) a mutation in the upper stem of the RAR, (2) a mutation in the tetraloop of the RAR, and (3) a truncation in the stem of Stem loop 2.
  • the upper stem is lengthened, e.g., by 1-8 base pairs (e.g., 1, 2, 3, 4, 5, 6, 7, or 8 base pairs)
  • the tetraloop comprises a substitution or an insertion (e.g., a 1 nucleotide insertion)
  • the stem of Stem loop 2 comprises a truncation of from 1- 32 nt.
  • Exemplary variant gRNA scaffolds containing the alterations described in this section are provided in Table 26.
  • an StlCas9 scaffold comprises an insertion (e.g., of 10 nucleotides) between positions 15 and 18, and a deletion of positions 16 and 17.
  • an insertion has a sequence according to GACUUCGGUC (SEQ ID N0:30006).
  • an StlCas9 scaffold comprises an insertion (e.g., of 10 nucleotides) between positions 15 and 18, and a deletion of positions 16 and 17.
  • an insertion has a sequence according to CUAGAAAUAG (SEQ ID N0:30007).
  • an StlCas9 scaffold comprises an insertion (e.g., of 12 nucleotides) between positions 14 and 19, and a deletion of positions 15-18.
  • an insertion has a sequence according to CGCGGUAACGCG (SEQ ID N0:30008).
  • a variant StlCas9 scaffold has a substitution resulting in a G-C base pair in the RAR lower stem.
  • a substitution comprises a substitution of position 4 with a G and a template further comprises a substitution of position 31 with a C.
  • a template RNA comprises a substitution in the second single stranded region.
  • a substitution is a substitution of position 51 with U or a substitution of position 54 with C.
  • a gRNA is a short synthetic RNA composed of a scaffold sequence that participates in CRIS PR-associated protein binding and a user-defined ⁇ 20 nucleotide targeting sequence for a genomic target.
  • the structure of a complete gRNA was described by Nishimasu et al. Cell 156, P935-949 (2014).
  • a gRNA (also referred to as sgRNA for single-guide RNA) comprises crRNA- and tracrRNA-dcrivcd sequences connected by an artificial tetraloop.
  • the crRNA sequence can be divided into guide (20 nt) and repeat (12 nt) regions, whereas the tracrRNA sequence can be divided into anti-repeat (14 nt) and three tracrRNA stem loops (Nishimasu et al. Cell 156, P935-949 (2014)).
  • guide RNA sequences are generally designed to have a length of between 17 - 24 nucleotides (e.g., 19, 20, or 21 nucleotides) and be complementary to a targeted nucleic acid sequence. Custom gRNA generators and algorithms are available commercially for use in the design of effective guide RNAs.
  • a gRNA comprises two RNA components from the native CRISPR system, e.g., crRNA and tracrRNA.
  • a gRNA may also comprise a chimeric, single guide RNA (sgRNA) containing sequence from both a tracrRNA (for binding the nuclease) and at least one crRNA (to guide the nuclease to the sequence targeted for editing/binding).
  • sgRNA single guide RNA
  • a gRNA spacer comprises a nucleic acid sequence that is complementary to a DNA sequence associated with a target gene.
  • the region of a template nucleic acid, e.g., template RNA, comprising a gRNA adopts an underwound ribbon-like structure of gRNA bound to target DNA (e.g., as described in Mulepati et al. Science 19 Sep 2014:Vol. 345, Issue 6203, pp. 1479-1484).
  • this non-canonical structure is thought to be facilitated by rotation of every sixth nucleotide out of the RNA-DNA hybrid.
  • the region of a template nucleic acid, e.g., template RNA, comprising a gRNA may tolerate increased mismatching with a target site at some interval, e.g., every sixth base.
  • the region of a template nucleic acid, e.g., template RNA, comprising a gRNA comprising homology to a target site may possess wobble positions at a regular interval, e.g., every sixth base, that do not need to base pair with the target site.
  • a Cas9 derivative with enhanced activity may be used in a gene modification polypeptide.
  • a Cas9 derivative may comprise mutations that improve activity of the HNH endonuclease domain, e.g., SpyCas9 R221K, N394K, or mutations that improve R-loop formation, e.g., SpyCas9 L1245V, or comprise a combination of such mutations, e.g., SpyCas9 R221K/N394K, SpyCas9 N394K/L1245V, SpyCas9 R221K/L1245V, or SpyCas9 R221K/N394K/L 1245V (see, e.g., Spencer and Zhang Sci Rep 7:16836 (2017), the Cas9 derivatives and mutations thereof which arc incorporated herein by reference).
  • a Cas9 derivative may comprise one or more types of mutations described herein, e.g., PAM-modifying mutations, protein stabilizing mutations, activity enhancing mutations, and/or mutations partially or fully inactivating one or two endonuclease domains relative to the parental enzyme (e.g., one or more mutations to abolish endonuclease activity towards one or both strands of a target DNA, e.g., a nickase or catalytically dead enzyme).
  • PAM-modifying mutations e.g., protein stabilizing mutations, activity enhancing mutations, and/or mutations partially or fully inactivating one or two endonuclease domains relative to the parental enzyme (e.g., one or more mutations to abolish endonuclease activity towards one or both strands of a target DNA, e.g., a nickase or catalytically dead enzyme).
  • a Cas9 enzyme used in a system described herein may comprise mutations that confer nickase activity toward the enzyme (e.g., SpyCas9 N863A or H840A) in addition to mutations improving catalytic efficiency (e.g., SpyCas9 R221K, N394K, and/or L1245V).
  • a Cas9 enzyme used in a system described herein is a SpyCas9 enzyme or derivative that further comprises an N863A mutation to confer nickase activity in addition to R221K and N394K mutations to improve catalytic efficiency.
  • a template nucleic acid (e.g., template RNA) has at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, or at least 24 bases of at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% homology to a target site, e.g., at the 5’ end, e.g., comprising a gRNA spacer sequence of length appropriate to the Cas9 domain of a gene modifying polypeptide (Table 4).
  • Table 14 provides parameters to define components for designing gRNA and/or template RNAs to apply Cas variants listed in Table 4 for gene modifying.
  • the cut site indicates the validated or predicted protospacer adjacent motif (PAM) requirements, validated or predicted location of cut site (relative to the most upstream base of the PAM site).
  • a gRNA for a given enzyme can be assembled by concatenating the crRNA, tetraloop, and tracrRNA sequences, and further adding a 5 ' spacer of a length within Spacer (min) and Spacer (max) that matches a protospacer at a target site.
  • a gRNA scaffold described herein comprises a nucleic acid sequence comprising, in the 5’ to 3’ direction, a crRNA of Table 14, a tetraloop from the same row of Table 14, and a tracrRNA from the same row of Table 14, or a sequence having at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity thereto.
  • a gRNA or template RNA comprising a scaffold further comprises a gRNA spacer having a length within the Spacer (min) and Spacer (max) indicated in the same row of Table 14.
  • a gRNA or template RNA having a sequence according to Table 14 is incorporated in a system that further comprises a gene modifying polypeptide, wherein the gene modifying polypeptide comprises a Cas domain described in the same row of Table 14.
  • Table 14 Parameters to define components for designing gRNA and/or Template RNAs to apply Cas variants listed in Table 4 in gene modifying systems.
  • RNA sequence e.g., a template RNA sequence
  • a particular sequence e.g., a sequence of Table 14 or a portion thereof
  • RNA sequence may (and frequently does) comprise uracil
  • the RNA sequence may comprise U at every position shown as T in the sequence in Table 14. More specifically, the present disclosure provides an RNA sequence according to every gRNA scaffold sequence of Table 14, wherein the RNA sequence has a U in place of each T in the sequence in Table 14. Additionally, it is understood that terminal Us and Ts may optionally be added or removed from tracrRNA sequences and may be modified or unmodified when provided as RNA.
  • versions of gRNA scaffold sequences alternative to those exemplified in Table 14 may also function with the different Cas9 enzymes or derivatives thereof exemplified in Table 4, e.g., alternate gRNA scaffold sequences with nucleotide additions, substitutions, or deletions, e.g., sequences with stem-loop structures added or removed. It is contemplated herein that gRNA scaffold sequences represent a component of gene modifying systems that can be similarly optimized for a given system, Cas-RT fusion polypeptide, indication, target mutation, template RNA, or delivery vehicle.
  • a template RNA described herein may comprise a heterologous object sequence that a gene modifying polypeptide can use as a template for reverse transcription, to write a desired sequence into a target nucleic acid.
  • a heterologous object sequence comprises, from 5’ to 3’, a post-edit homology region, a mutation region, and a pre-edit homology region.
  • an RT performing reverse transcription on a template RNA first reverse transcribes a pre-edit homology region, then a mutation region, and then a post-edit homology region, thereby creating a DNA strand comprising a desired mutation with a homology region on either side.
  • a heterologous object sequence is at least 32, at least 33, at least 34, at least 35, at least 36, at least 37, at least 38, at least 39, at least 40, at least 41, at least 42, at least 43, at least 44, at least 45, at least 46, at least 47, at least 48, at least 49, at least 50, at least 51 , at least 52, at least 53, at least 54, at least 55, at least 56, at least 57, at least 58, at least 59, at least 60, at least 61, at least 62, at least 63, at least 64, at least 65, at least 66, at least 67, at least 68, at least 69, at least 70, at least 71, at least 72, at least 73, at least 74, at least 75, at least 76, at least 77, at least 78, at least 79, at least 80, at least 81 , at least 82, at least 83, at least 84, at least 85, at least 84, at least
  • a heterologous object sequence is no more than 33, no more than 34, no more than 35, no more than 36, no more than 37, no more than 38, no more than 39, no more than 40, no more than 41, no more than 42, no more than 43, no more than 44, no more than 45, no more than 46, no more than 47, no more than 48, no more than 49, no more than 50, no more than 51, no more than 52, no more than 53, no more than 54, no more than 55, no more than 56, no more than 57, no more than 58, no more than 59, no more than 60, no more than 61, no more than 62, no more than 63, no more than 64, no more than 65, no more than 66, no more than 67, no more than 68, no more than 69, no more than 70, no more than 71, no more than 72, no more than 73, no more than 74, no more than 75, no more than 76, no more than 77, no more
  • a heterologous object sequence is 30-1000, 40-1000, 50-1000, 60- 1000, 70-1000, 74-1000, 75-1000, 76-1000, 77-1000, 78-1000, 79-1000, 80-1000, 85-1000, 90- 1000, 100-1000, 120-1000, 140-1000, 160-1000, 180-1000, 200-1000, 500-1000, 30-500, 40- 500, 50-500, 60-500, 70-500, 74-500, 75-500, 76-500, 77-500, 78-500, 79-500, 80-500, 85-500, 90-500, 100-500, 120-500, 140-500, 160-500, 180-500, 200-500, 30-200, 40-200, 50-200, 60- 200, 70-200, 74-200, 75-200, 76-200, 77-200, 78-200, 79-200, 80-200, 85-200, 90-200, 100-200, 120-200, 140-500, 160-
  • a heterologous object sequence is 10-100, 10-90, 10-80, 10-70, 10-60, 10-50, 10-40, 10-30, or 10- 20 nt in length, e.g., 10-80, 10-50, or 10-20 nt in length, e.g., aboutl0-20 nt in length.
  • a heterologous object sequence is 8-30, 9-25, 10-20, 11-16, or 12-15 nucleotides in length, e.g., is 11-16 nt in length.
  • a larger insertion size, larger region of editing e.g., the distance between a first edit/substitution and a second edit/substitution in the target region
  • greater number of desired edits e.g., mismatches of the heterologous object sequence to the target genome
  • a template nucleic acid comprises a customized RNA sequence template which can be identified, designed, engineered and constructed to contain sequences altering or specifying host genome function, for example by introducing a heterologous coding region into a genome; affecting or causing exon structure/altemative splicing, e.g., leading to exon skipping of one or more exons; causing disruption of an endogenous gene, e.g., creating a genetic knockout; causing transcriptional activation of an endogenous gene; causing epigenetic regulation of an endogenous DNA; causing up-regulation of one or more operably linked genes, e.g., leading to gene activation or overexpression; causing down-regulation of one or more operably linked genes, e.g., creating a genetic knock-down; etc.
  • a customized RNA sequence template can be engineered to contain sequences coding for exons and/or transgenes, provide binding sites for transcription factor activators, repressors, enhancers, etc., and combinations thereof.
  • a customized template can be engineered to encode a nucleic acid or peptide tag to be expressed in an endogenous RNA transcript or endogenous protein operably linked to the target site.
  • the coding sequence can be further customized with splice donor sites, splice acceptor sites, or poly-A tails.
  • a template nucleic acid (e.g., a template RNA) of a system provided herin may comprise an object sequence (e.g., a heterologous object sequence) for writing a desired sequence into a target DNA.
  • An object sequence e.g., a heterologous object sequence
  • a template nucleic acid e.g., template RNA
  • a template nucleic acid e.g., template RNA
  • a template nucleic acid may contain a heterologous sequence, wherein reverse transcription will result in insertion of the heterologous sequence into a target DNA.
  • an RNA template may be designed to introduce a deletion into a target DNA.
  • a template nucleic acid e.g., template RNA
  • a template nucleic acid may be designed to introduce an edit into a target DNA.
  • a template RNA may match a target DNA sequence with the exception of one or more nucleotides, wherein reverse transcription will result in the copying of these edits into the target DNA, e.g., resulting in mutations, e.g., transition or trans vers ion mutations.
  • writing (e.g., reverse transcription) of an object sequence (e.g., a heterologous object sequence) into a target site results in the substitution of nucleotides, e.g., where the full length of the object sequence corresponds to a matching length of the target site with one or more mismatched bases.
  • a heterologous object sequence may be designed such that a combination of sequence alterations may occur, e.g., a simultaneous addition and deletion, addition and substitution, or deletion and substitution.
  • a heterologous object sequence may contain an open reading frame or a fragment of an open reading frame. In some embodiments, a heterologous object sequence has a Kozak sequence. In some embodiments, a heterologous object sequence has an internal ribosome entry site. In some embodiments, a heterologous object sequence has a selfcleaving peptide such as a T2A or P2A site. In some embodiments, a heterologous object sequence has a start codon. In some embodiments, a template RNA has a splice acceptor site. In some embodiments, a template RNA has a splice donor site.
  • a template RNA has a microRNA binding site downstream of a stop codon.
  • a template RNA has a polyA tail downstream of a stop codon of an open reading frame.
  • a template RNA comprises one or more exons.
  • a template RNA comprises one or more introns.
  • a template RNA comprises a eukaryotic transcriptional terminator.
  • a template RNA comprises an enhanced translation element or a translation enhancing element.
  • a termplate RNA comprises a human T-cell leukemia virus (HTLV-1) R region.
  • a template RNA comprises a posttranscriptional regulatory element that enhances nuclear export, such as that of Hepatitis B Virus (HPRE) or Woodchuck Hepatitis Virus (WPRE).
  • HPRE Hepatitis B Virus
  • WPRE Woodchuck Hepatitis Virus
  • a heterologous object sequence may contain a non-coding sequence.
  • a template nucleic acid e.g., a template RNA
  • a regulatory element e.g., a promoter or enhancer sequence or miRNA binding site.
  • integration of an object sequence (e.g., a heterologous object seqeucen) at a target site will result in upregulation of an endogenous gene.
  • integration of an object sequence (e.g., a heterologous object sequence)_at a target site will result in downregulation of an endogenous gene.
  • a template nucleic acid comprises a tissue specific promoter or enhancer, each of which may be unidirectional or bidirectional.
  • a promoter is an RNA polymerase I promoter, RNA polymerase II promoter, or RNA polymerase III promoter.
  • a promoter comprises a TATA element.
  • a promoter comprises a B recognition element.
  • a promoter has one or more binding sites for transcription factors.
  • a template nucleic acid (e.g., a template RNA) comprises a site that coordinates epigenetic modification.
  • a template nucleic acid (e.g., a template RNA) comprises a chromatin insulator.
  • a template nucleic acid (e.g., a template RNA) may comprise a CTCF site or a site targeted for DNA methylation.
  • a template nucleic acid (e.g., a template RNA) comprises a gene expression unit composed of at least one regulatory region operably linked to an effector sequence.
  • An effector sequence may be a sequence that is transcribed into RNA (e.g., a coding sequence or a non-coding sequence such as a sequence encoding a micro RNA).
  • a heterologous object sequence of a template nucleic acid is inserted into a target genome in an endogenous intron.
  • a heterologous object sequence of a template nucleic acid e.g., a template RNA
  • a heterologous object sequence of a template nucleic acid is inserted into a target genome and thereby acts as a new exon.
  • insertion of a heterologous object sequence into a target genome results in replacement of a natural exon or the skipping of a natural exon.
  • a template nucleic acid (e.g., a template RNA) can be designed to result in insertions, mutations, or deletions at a target DNA locus.
  • a template nucleic acid (e.g., a template RNA) may be designed to cause an insertion in a target DNA.
  • a template nucleic acid e.g., a template RNA
  • an RNA template may be designed to write a deletion into a target DNA.
  • a template nucleic acid may match a target DNA upstream and downstream of a desired deletion, wherein reverse transcription will result in copying of the upstream and downstream sequences from the template nucleic acid (e.g., the template RNA) without the intervening sequence, e.g., causing deletion of the intervening sequence.
  • a template nucleic acid e.g., a template RNA
  • a template RNA may match a target DNA sequence with the exception of one or more nucleotides, wherein reverse transcription will result in copying of these edits into the target DNA, e.g., resulting in mutations, e.g., transition or transversion mutations.
  • a pre-edit homology domain comprises a nucleic acid sequence having 100% sequence identity with a nucleic acid sequence comprised in a target nucleic acid molecule.
  • a post-edit homology domain comprises a nucleic acid sequence having 100% sequence identity with a nucleic acid sequence comprised in a target nucleic acid molecule.
  • a template nucleic acid (e.g., a template RNA) comprises a primer binding site (PBS) sequence.
  • PBS primer binding site
  • a PBS sequence is disposed 3' of a heterologous object sequence and is complementary to a sequence adjacent to a site to be modified by a system described herein, or comprises no more than 1, no more than 2, no more than 3, no more than 4, or no more than 5 mismatches to a sequence complementary to a sequence adjacent to a site to be modified by a system/gene modifying polypeptide.
  • a PBS sequence binds within 1 , 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides of a nick site in a target nucleic acid molecule.
  • binding of a PBS sequence to a target nucleic acid molecule permits initiation of target-primed reverse transcription (TPRT), e.g., with the 3' homology domain acting as a primer for TPRT.
  • a PBS sequence is 3-5, 5-10, 10-30, 10-25, 10-20, 10-19, 10-18, 10-17, 10-16, 10-15, 10-14, 10-13, 10- 12, 10-11, 11-30, 11-25, 11-20, 11-19, 11-18, 11-17, 11-16, 11-15, 11-14, 11-13, 11-12, 12-30, 12-25, 12-20, 12-19, 12-18, 12-17, 12-16, 12-15, 12-14, 12-13, 13-30, 13-25, 13-20, 13-19, 13- 18, 13-17, 13-16, 13-15, 13-14, 14-30, 14-25, 14-20, 14-19, 14-18, 14-17, 14-16, 14-15, 15-30, 15-25, 15-20, 15-19, 15-18, 15-17, 15-16, 16-30, 16-25, 16-20, 16-19, 16-19
  • a template nucleic acid may have some homology to a target DNA.
  • a template nucleic acid e.g., a template RNA
  • PBS sequence domain may serve as an annealing region to a target DNA, such that the target DNA is positioned to prime the reverse transcription of the template nucleic acid (e.g., the template RNA).
  • a template nucleic acid (e.g., a template RNA) has at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 110, at least 120, at least 130, at least 140, at least 150, at least 175, at least 200 or more bases of exact homology to a target DNA at the 3' end of the RNA.
  • a template nucleic acid (e.g., a template RNA) has at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 110, at least 120, at least 130, at least 140, at least 150, at least 175, at least 200 or more bases of at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% homology to a target DNA, e.g., at the 5' end of the template nucleic acid (e.g., the template RNA).
  • Exemplary template sequences e.g., at the 5' end of the template nucleic acid (e.g., the
  • a template RNA comprises a gRNA spacer comprising the core nucleotides of a gRNA spacer sequence of Table 15.
  • a gRNA spacer additionally comprises one or more (e.g., 2, 3, or all) consecutive nucleotides starting with the 3’ end of the flanking nucleotides of the gRNA spacer.
  • a template RNA comprising a sequence of Table 15 is comprised by a system that further comprises a gene modifying polypeptide having an RT domain listed in the same line of Table 15.
  • RT domain amino acid sequences can be found, e.g., in Table 15 herein.
  • Table 15 provides exemplary gRNAs for correcting the pathogenic E342K mutation in SERPINA1. Also provided is a listing of spacers, PAMs, and Cas variants for generating a nick at an appropriate position to enable installation of a desired genomic edit with a gene modifying system of the present disclsourc. Spacers in Table 15 arc designed to be used with a gene modifying polypeptide comprising a nickase variant of the Cas species indicated in Table 15.
  • Table 17, Table 16, and Table 18 detail other components of exemplary systems of the present disclosure and are organized such that the ID number shown in Column 1 (“ID”) of Table 15 is meant to correspond to the same ID number in Table 17, Table 16, and Table 18.
  • RNA sequence e.g., a template RNA sequence
  • a particular sequence e.g., a sequence of Table 15 or a portion thereof
  • T thymine
  • U uracil
  • the RNA sequence may comprise U at every position shown as T in the sequence in Table 15.
  • the present disclosure provides an RNA sequence according to every gRNA spacer sequence shown in Table 15, wherein the RNA sequence has a U in place of each T in the sequence in Table 15.
  • a heterologous object sequence comprises the core nucleotides of an RT template sequence from Table 16.
  • a heterologous object sequence additionally comprises one or more (e.g., 2, 3, 4, 5, 10, 20, 30, 40, or all) consecutive nucleotides starting with the 3’ end of the flanking nucleotides of an RT template sequence.
  • a heterologous object sequence comprises the core nucleotides of an RT template sequence of Table 16 that corresponds to a gRNA spacer sequence.
  • a first component “corresponds to” a second component when both components have the same ID number in the referenced table.
  • a heterologous object sequence additionally comprises one or more consecutive nucleotides starting with the 3’ end of the flanking nucleotides of the RT template sequence.
  • a primer binding site (PBS) sequence has a sequence comprising the core nucleotides of a PBS sequence from the same row of Table 16 as an RT template sequence.
  • a PBS sequence additionally comprises one or more (e.g., 1, 2, 3, 4, 5, 6, 7, or all) consecutive nucleotides starting with the 5’ end of the flanking nucleotides of a primer region.
  • Table 16 provides exemplary PBS sequences and heterologous object sequences (reverse transcription template regions) of a template RNA for correcting the pathogenic E342K mutation in SERPINA1.
  • gRNA spacers from Table 15 were filtered, e.g., filtered by occurrence within 15 nt of the desired editing location and use of a Tier 1 Cas enzyme.
  • PBS sequences and heterologous object sequences (reverse transcription template regions) were designed relative to a nick site directed by a cognate gRNA from Table 15. Tn some embodiments, these regions were designed to be 8-17 nt (priming) and 1-50 nt extended beyond the location of an edit (e.g., RT).
  • sequences are provided that use the maximum length parameters and comprise all templates of shorter length within the given parameters. Sequences are shown with uppercase letters indicating core sequence and lowercase letters indicating flanking sequence that may be truncated within the described length parameters.
  • RNA sequence e.g., a template RNA sequence
  • a particular sequence e.g., a sequence of Table 16 or a portion thereof
  • RNA sequence may (and frequently does) comprise uracil
  • an RNA sequence may comprise U at every position shown as T in the sequence in Table 16. More specifically, the present disclosure provides an RNA sequence according to every heterologous object sequence and PBS sequence shown in Table 16, wherein the RNA sequence has a U in place of each T in the sequence of Table 16.
  • a template RNA comprises a gRNA scaffold (e.g., that binds a gene modifying polypeptide, e.g., a Cas polypeptide) that comprises a sequence of a gRNA scaffold of Table 12.
  • a gRNA scaffold comprises a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to a gRNA scaffold of Table 12.
  • a gRNA scaffold comprises a sequence of a scaffold region of Table 12 that corresponds to the RT template sequence, the spacer sequence, or both, or a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity thereto.
  • a system further comprises a second strand-targeting gRNA that directs a nick to the second strand of the human SERPINA1 gene.
  • a second strand-targeting gRNA comprises a left gRNA spacer sequence or a right gRNA spacer sequence from Table 17.
  • a gRNA spacer additionally comprises one or more (c.g., 2, 3, or all) consecutive nucleotides starting with the 3’ end of the flanking nucleotides of the left gRNA spacer sequence or right gRNA spacer sequence.
  • a second strand-targeting gRNA comprises a sequence comprising the core nucleotides of a second nick gRNA sequence from Table 18, or a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity thereto.
  • a second nick gRNA sequence additionally comprises one or more consecutive nucleotides starting with the 3’ end of the flanking nucleotides of the second nick gRNA sequence.
  • a second nick gRNA comprises a gRNA scaffold sequence that is orthogonal to a Cas domain of a gene modifying polypeptide. In some embodiments, a second nick gRNA comprises a gRNA scaffold sequence of Table 14.
  • Table 17 provides exemplary second strand-targeting gRNA sequences for optional use for correcting the pathogenic E342K mutation in SERPINA1.
  • gRNA spacers from Table 15 were filtered, e.g., filtered by occurrence within 15 nt of the desired editing location and use of a Tier 1 Cas enzyme.
  • Second strand-targeting gRNAs were generated by searching the opposite strand of DNA in the regions -40 to -140 (“left”) and +40 to +140 (“right”), relative to the first nick site defined by the first gRNA, and for the PAM utilized by the corresponding Cas variant.
  • One exemplary spacer is shown for each side of the target nick site.
  • RNA sequence may (and frequently does) comprise uracil (U) in place of T.
  • T thymine
  • U uracil
  • an RNA sequence may comprise U at every position shown as T in the sequence in Table 17.
  • the present disclosure provides an RNA sequence according to every gRNA spacer sequence shown in Table 17, wherein the RNA sequence has a U in place of each T in the sequence in Table 17.
  • systems and methods provided herein may comprise a template sequence listed in Table 18.
  • Table 18 provides exemplary template RNA sequences (column 4) and optional second strand-targeting gRNA sequences (column 5) designed to be paired with a gene modifying polypeptide to correct a mutation in the SERPINA1 gene. Templates in Table 18 exemplify a total sequence including: (1) a gRNA spacer (c.g., for targeting a first strand nick), (2) a gRNA scaffold, (3) a heterologous object sequence, and (4) a PBS sequence (e.g., for initiating TPRT at the first strand nick).
  • Table 18 provides RNA components of gene modifying systems for correcting the pathogenic E342K mutation in SERPINA1.
  • gRNA spacers from Table 15 were filtered, e.g., filtered by occurrence within 15 nt of a desired editing location and use of a Tier 1 Cas enzyme.
  • Table 18 describes a sequence of a complete template RNA, an optional second strand-targeting gRNA, and a Cas variant for use in a Cas-RT fusion gene modifying polypeptide.
  • PBS sequences and post-edit homology regions are set to 12 nt and 30 nt, respectively.
  • a second strandtargeting gRNA is selected with preference for a distance near 100 nt from a first nick and a first preference for a design resulting in a PAM-in system, as described elsewhere in this application.
  • RNA sequence e.g., a template RNA sequence
  • a particular sequence e.g., a sequence of Table 18 or a portion thereof
  • T thymine
  • U uracil
  • the RNA sequence may comprise U at every position shown as T in the sequence in Table 18.
  • the present disclosure provides an RNA sequence according to every template sequence shown in Table 4, wherein the RNA sequence has a U in place of each T in the sequence of Table 18.
  • Table 19 provides select sequences from Table 18, with annotation illustrating inactivation of PAM sites.
  • Column “ID” contains a unique identifier for a template RNA that corresponds to the ID used in Tables 15-18 and can be used, e.g., to identify a corresponding gRNA spacer sequence in Table 15.
  • Column “Cas species” indicates a type of Cas domain suitable for inclusion in a gene modifying polypeptide for use with the template RNA.
  • Column “consensus” indicates a consensus PAM motif recognized by the Cas.
  • Column “PAM sequence” indicates a particular PAM sequence recognized by the Cas, e.g., in the SERPINA1 gene.
  • PAM mutation indicates a mutation that can be produced in the PAM by a template RNA described on the same row of the table; mutated nucleotides are indicated with bold and underlining.
  • strand indicates the + or 1 strand of the target nucleic acid.
  • distance indicates the number of nucleotides in the pre-edit homology region.
  • PBS sequence indicates a PBS sequence for partial or full inclusion in the template RNA, wherein core nucleotides are capitalized and flanking nucleotides are lower case.
  • RT template sequence indicates a heterologous object sequence for partial or full inclusion in the template RNA, wherein core nucleotides are capitalized, flanking nucleotides are lower case, and nucleotide differences from the target nucleic acid are shown in bold and underline.
  • Table 19 Exemplary template RNA sequences comprising PAM-inactivating sites
  • RNA sequence e.g., a template RNA sequence
  • a particular sequence e.g., a sequence of Table 19 or a portion thereof
  • RNA sequence may (and frequently does) comprise uracil
  • the RNA sequence may comprise U at every position shown as T in the sequence in Table 19. More specifically, the present disclosure provides an RNA sequence according to every template sequence shown in Table 19, wherein the RNA sequence has a U in place of each T in the sequence of Table 19.
  • a gRNA scaffold described herein comprises a nucleic acid sequence comprising, in the 5’ to 3’ direction, a crRNA of Table 20, a tetraloop from the same row of Table 20, and a tracrRNA from the same row of Table 20, or a sequence having at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity thereto.
  • a gRNA or template RNA having a sequence according to Table 20 is comprised by a system that further comprises a gene modifying polypeptide, and a spacer, wherein the spacer comprises a gRNA spacer described in the same row of Table 20.
  • Table 20 Exemplary spacer and scaffold pairs.
  • systems and methods provided herein may comprise a template sequence, or component thereof, listed in Table 21, or a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, or 100% identity thereto.
  • Table 21 provides exemplary template RNA sequences designed to be paired with a gene modifying polypeptide to correct a mutation in the SERPINA1 gene.
  • s stems and methods provided herein may comprise a template sequence, or component thereof, listed in any one of Tables 39-62 , or a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, or 100% identity thereto.
  • Table 22 and Table 23 provide exemplary template RNA sequences.
  • a gRNA scaffold of a template RNA according to Table 22 or Table 23 is replaced with a variant gRNA scaffold described herein, e.g., a gRNA scaffold of Table 26 or a sequence having no more than 1, no more than 2, or no more than 3 sequence alterations (e.g., substitutions) relative thereto.
  • Table 23 shows the sequences of Table 22 without modifications. In some embodiments, sequences can be used without chemical modifications.
  • Table 24 provides exemplary template RNA sequences.
  • the names of the template RNAs provided have the following nomenclature: the first set of characters indicates the compatible Cas (e.g., Stl indicates StlCas9), the second set of characters indicates the name of the variant gRNA scaffold (e.g., dSL2), the third set of characters indicates the target gene or protein encoded by the target gene (e.g., A1AT), the fourth set of characters indicates the name of the spacer (e.g., ED4), the fifth set of characters indicates the length of the PBS and heterologous object sequence (e.g., P17R5 indicates a PBS of length 17 and a heterologous object sequence of length 5), and the sixth set of characters indicates the edit (e.g., TtoC).
  • Column 2 shows the unmodified sequence corresponding to the chemically modified sequence of column 3.
  • Table 21 Exemplary template RNA sequences
  • Table 24 Exemplary template RNAs.

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Abstract

The disclosure provides, c.g., compositions, systems, and methods for targeting, editing, modifying, or manipulating a host cell's genome at one or more locations in a DNA sequence in a cell, tissue, or subject. Improved gRNA scaffolds compatible with StlCas9 are described. For instance, certain patterns of chemical modifications on gRNA scaffolds are provided.

Description

MODIFIED ST1CAS9 GUIDE NUCLEIC ACIDS
CROSS-REFERENCE TO RELATED APPLICATIONS
This application claims priority to U.S. Provisional Patent Application No. 63/565,379, filed March 14, 2024; the title of which is “MODIFIED ST1CAS9 GUIDE NUCLEIC ACIDS” and the content of which is incorporated herein by reference in its entirety.
SEQUENCE LISTING
The instant application contains a Sequence Listing which has been submitted electronically in XML format compliant with WIPO Standard ST.26 and is hereby incorporated by reference in its entirety. Said XML copy, created on March 14, 2025, is named 2017469_0038.XML and is 15,726,980 bytes in size.
BACKGROUND
Integration of a nucleic acid of interest into a genome occurs at low frequency and with little site specificity, in the absence of a specialized protein to promote the insertion event. Some existing approaches, like CRISPR/Cas9, are more suited for small edits that rely on host repair pathways and are less effective at integrating longer sequences. Other existing approaches, like Cre/loxP, require a first step of inserting a loxP site into the genome and then a second step of inserting a sequence of interest into the loxP site. There is a need in the art for improved compositions (e.g., proteins and nucleic acids) and methods for inserting, altering, or deleting sequences of interest in a genome.
Alpha- 1 antitrypsin deficiency (AATD) is characterized by low circulating levels of alpha- 1 antitrypsin (AAT). AAT is produced primarily in liver cells and secreted into the blood, but it is also made by other cell types including lung epithelial cells and certain white blood cells. AAT inhibits several serine proteases secreted by inflammatory cells (most notably neutrophil elastase [NE], proteinase 3, and cathepsin G) and thus protects organs, such as the lung, from protease-induced damage, especially during periods of inflammation.
The two most common clinical variants of AAT are E264V (PiS) and E342K (PiZ). The clinical E342K (PiZ) mutation (also referred to as the Z mutation) is caused by a single base-pair substitution in the SERPINA gene (referred to as the Z allele) and results in a glutamic acid to lysine mutation at position 342 of AAT. Inheritance of the Z allele is autosomal codominant and more than half of AATD patients harbor at least one copy of the Z allele. The E342K mutation leads to structurally unstable and/or inactive AAT-Z protein that causes toxicity in the liver and is inactive in the lungs. The E342K mutation is located at the hinge between the beta sheet and the Reactive Center Loop (RCL) of the AAT protein and causes a loop-sheet dimer that can extend to form long chains of loop-sheet polymers. These polymers form aggregates that accumulate inside the rough endoplasmic reticulum of hepatocytes during translation and are therefore not secreted into the bloodstream. Consequently, circulating AAT levels in individuals homozygous for the Z allele (PiZZ) are markedly reduced; only approximately 15% of mutant AAT-Z protein folds correctly and is secreted by the cell. An additional consequence of the Z mutation is that the secreted AAT-Z protein has reduced activity compared to wild-type protein, with 40% to 80% of normal antiprotease activity (American thoracic society /European respiratory society, Am J Respir Crit Care Med. 2003; 168(7):818-900; and Ogushi et al. J Clin Invest. 1987; 80(5): 1366-74, herein incorporated by reference in their entirety).
There are two disease phenotypes associated with the PiZZ genotype. A gain-of - function phenotype presents as the accumulation of polymerized AAT-Z protein in hepatocytes results in a gain-of-function cytotoxicity that can result in cellular stress, inflammation, fibrosis, cirrhosis, hepatocellular carcinoma (HCC), and neonatal liver disease in 12% of patients. This accumulation may spontaneously remit but can be fatal in a small number of children. A loss-of- function phenotype results from the reduced systemic levels of AAT that lead to increased protease digestion of connective tissue in the lower airway. Excess protease-digestion of the connective tissues and alveolar linings deteriorates lung elasticity and pulmonary function, leading to emphysema, a hallmark of Chronic Obstructive Pulmonary Disease (COPD). This effect is severe in PiZZ individuals and typically manifests in middle age, resulting in a decline in quality of life and shortened lifespan (mean 68 years of age) (Tanash et al. I nt J Chron Obstruct Pulm Dis. 2016; 11:1663-9, herein incorporated by reference in its entirety). The effect is more pronounced in PiZZ individuals who smoke, resulting in an even further shortened lifespan (58 years) (Piitulainen and Tanash, COPD 2015; 12( l):36-41 , herein incorporated by reference in its entirety). PiZZ individuals account for the majority of patients with clinically relevant AATD lung disease.
A milder form of AATD is associated with the SZ genotype in which a patient has a Z allele and an S allele. The S allele is associated with somewhat reduced levels of circulating AAT but the AAT S-protein is not hepatotoxic. Accordingly, the SZ genotype is associated with clinically significant lung disease but not liver disease. Frcgoncsc and Stolk, Orphanct J Rare Dis. 2008; 33:16. As with the PiZZ genotype, the deficiency of circulating AAT in subjects with the SZ genotype results in dysregulated protease activity that degrades lung tissue over time and can result in emphysema, particularly in smokers.
While limited treatment options for AATD exist, there is currently no cure. A small fraction of newborn patients and patients having advanced stage liver disease undergo liver transplant. The current standard of care for AATD patients who have or show signs of significant or developing lung disease is augmentation therapy or protein replacement therapy. Augmentation therapy involves administration (weekly infuction) of a human AAT protein concentrate purified from pooled from healthy donor plasma. Although infusions of plasma protein have been shown to improve survival or slow the rate of emphysema progression, augmentation therapy is often insufficient under challenging conditions (e.g., active lung infection). Augmentation therapy also fails to restore the normal physiological regulation of AAT in patients and efficacy has been difficult to demonstrate. In addition, augmentation therapy does not remedy liver disease driven by the toxic gain-of-function of the Z allele. Accordingly, there is a need for new and more effective treatments for AATD.
SUMMARY OF THE INVENTION
This disclosure relates to novel compositions, systems, and methods for altering a genome at one or more locations in a host cell, tissue, or subject, in vivo or in vitro. The disclosure provides, for instance, gene modifying systems that comprise a gene modifying polypeptide comprising a reverse transcriptase (RT) domain and a StlCas9 domain, and a template RNA comprising a variant gRNA scaffold that has been engineered for improved performance, e.g., when used in concert with the StlCas9 domain. The disclosure also provides gene modifying systems that are capable of modulating (e.g., inserting, altering, or deleting sequences of interest) alpha- 1 antitrypsin (AAT) activity and methods of treating alpha- 1 antitrypsin deficiency (AATD) by administering one or more such systems to alter a genomic sequence at a single nucleotide to correct the SERPINA1 PiZ mutation that causes AATD.
In some embodiments, the present disclosure provides a system for modifying DNA to correct a human SERPINA1 gene mutation that causes AATD comprising (a) a nucleic acid encoding a gene modifying polypeptide capable of target primed reverse transcription, the polypeptide comprising (i) a reverse transcriptase domain and (ii) a St1Cas9 nickase that binds DNA and has endonuclease activity, and (b) a template RNA comprising (i) a gRNA spacer that is complementary to a first portion of the human SERPINA1 gene, (ii) a gRNA scaffold that binds the polypeptide, (iii) a heterologous object sequence comprising a mutation region to correct the SERPINA1 gene mutation, and (iv) a primer binding site (PBS) sequence comprising at least 3, 4, 5, 6, 7, or 8 bases of 100% homology to a target DNA strand at the 3' end of the template RNA. The SERPINA1 gene may comprise an E342K mutation (also referred to as a PiZ mutation). A template RNA sequence may comprise a sequence described herein, e.g., in Table 15, 16, 18-29, or 39-62.
A gRNA spacer may comprise at least 15 bases of 100% homology to a target DNA at the 5' end of the template RNA. A template RNA may further comprise a PBS sequence comprising at least 5 bases of at least 80% homology to a target DNA strand. A template RNA may comprise one or more chemical modifications.
Domains of a gene modifying polypeptide may be joined by a peptide linker. A polypeptide may comprise one or more peptide linkers. A gene modifying polypeptide may further comprise a nuclear localization signal. A polypeptide may comprise more than one nuclear localization signal, e.g., multiple adjacent nuclear localization signals or one or more nuclear localization signals in different regions of the polypeptide, e.g., one or more nuclear localization signals in the N-terminus of the polypeptide and one or more nuclear localization signals in the C-terminus of the polypeptide. A nucleic acid encoding a gene modifying polypeptide may encode one or more intein domains.
Introduction of a system of the present disclosure into a target cell may result in insertion of at least 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 500, or 1000 base pairs of exogenous DNA. Introduction of a system of the present disclosure into a target cell may result in a deletion, wherein the deletion is less than 2, 3, 4, 5, 10, 50, or 100 base pairs of genomic DNA upstream or downstream of an insertion. Introduction of a system of the present disclosure into a target cell may result in substitution, e.g., substitution of 1, 2, or 3 nucleotides, e.g., consecutive nucleotides.
A heterologous object sequence may be at least 5, 10, 25, 50, 100, 150, 200, 250, 300, 400, 500, 600, or 700 base pairs. In some embodiments, the present disclosure provides a pharmaceutical composition comprising a system described herein and a pharmaceutically acceptable excipient or carrier, wherein the pharmaceutically acceptable excipient or carrier is selected from the group consisting of a plasmid vector, a viral vector, a vesicle, and a lipid nanoparticle. In some embodiments, the present disclosure provides a pharmaceutical composition comprising a system described herein and multiple pharmaceutically acceptable excipients or carriers, wherein the pharmaceutically acceptable excipients or carriers are selected from the group consisting of a plasmid vector, a viral vector, a vesicle, and a lipid nanoparticle, e.g., where the system described above is delivered by two distinct excipients or carriers, e.g., two lipid nanoparticles, two viral vectors, or one lipid nanoparticle and one viral vector. A viral vector may be an adeno- associated virus (AAV).
In some embodiments, aspect, the disclosure relates to a host cell (e.g., a mammalian cell, e.g., a human cell) comprising the system described above.
In some emdodiments, the present disclosure provides a method of correcting a mutation in the human SERPINA1 gene in a cell, tissue or subject, the method comprising administering a system described herein to the cell, tissue or subject, wherein optionally the correction of the mutant SERPINA1 gene comprises an amino acid substitution of K342E (i.e., reversing the pathogenic E342K mutation). A system described herein may be introduced in vivo, in vitro, ex vivo, or in situ. A nucleic acid of a system described herein may be integrated into the genome of a host cell. In some embodiments, a nucleic acid of a system described herein is not integrated into the genome of a host cell. In some embodiments, a heterologous object sequence is inserted at only one target site in a host cell genome. A heterologous object sequence may be inserted at two or more target sites in a host cell genome, e.g., at the same corresponding site in two homologous chromosomes or at two different sites on the same or different chromosomes. A heterologous object sequence may encode a mammalian polypeptide, or a fragment or a variant thereof. Components of a system described herein may be delivered on 1, 2, 3, 4, or more distinct nucleic acid molecules. A system of the present disclosure may be introduced into a host cell by electroporation or by using at least one vehicle selected from a plasmid vector, a viral vector, a vesicle, and a lipid nanoparticle.
Features of the compositions or methods can include one or more of the following enumerated embodiments. Enumerated Embodiments
1. A nucleic acid comprising a Repeat: anti-repeat (RAR) region which comprises a chemically modified nucleotide.
2. The nucleic acid of embodiment 1, which further comprises a stem-loop 1 (SL1) region (e.g., wherein the SL1 region is 3’ of the RAR region), wherein optionally the SL1 region comprises a chemically modified nucleotide.
3. The nucleic acid of embodiment 1 or 2, which further comprises a stem loop 2 (SL2) region (e.g., wherein the SL2 region is 3’ of the SL1 region), wherein optionally the SL2 region comprises a chemically modified nucleotide.
4. A nucleic acid comprising a stem-loop 1 (SL1) region which comprises a chemically modified nucleotide.
5. The nucleic acid of embodiment 4, which further comprises an RAR region (e.g., wherein the RAR region is 5’ of the SL1 region), wherein optionally the RAR region comprises a chemically modified nucleotide.
6. A nucleic acid molecule comprising: an StlCas9 scaffold; wherein the StlCas9 scaffold comprises a chemically modified nucleotide.
7. A nucleic acid molecule comprising: an StlCas9 scaffold; wherein the StlCas9 scaffold comprises a chemically modified nucleotide at one or more of (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38 or all of) positions 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, and 42 relative to SEQ ID NO: 25999.
8. A nucleic acid molecule comprising: an StlCas9 scaffold; wherein at least 15-20%, 20-30%, 30-40%, 40-50%, 50-60%, 60-70%, or 70-75% of nucleotides in the StlCas9 scaffold are chemically modified.
9. A nucleic acid molecule comprising: an StlCas9 scaffold comprising: a) a Repeat: anti-repeat (RAR) region, wherein optionally the RAR region comprises a RAR lower stem, a RAR upper stem, and an RAR loop (c.g., a tctraloop); b) a stem- loop 1 (SL1) region that is optionally 3’ of the RAR region, and c) optionally, a stem loop 2 (SL2) region that is optionally 3’ of the SL1 region; wherein the StlCas9 scaffold comprises a chemically modified nucleotide in one or both of the RAR region or the SL1 region.
10. The nucleic acid of embodiment 7 or 9, wherein at least 15-20%, 20-30%, 30-40%, 40-50%, 50-60%, 60-70, or 70-75% of nucleotides in the StlCas9 scaffold are chemically modified.
11. The nucleic acid of embodiment 7 or 8, wherein the StlCas9 scaffold comprises: a) a Repeat: anti-repeat (RAR) region, wherein optionally the RAR region comprises a RAR lower stem, a RAR upper stem, and an RAR loop (e.g., a tetraloop); b) a stem-loop 1 (SL1) region that is 3’ of the RAR region, and c) optionally, a stem loop 2 (SL2) region that is 3’ of the SL1 region; wherein the StlCas9 scaffold comprises a chemically modified nucleotide in one or both of the RAR region or the SL1 region.
12. The nucleic acid of any of the preceding embodiments, wherein the StlCas9 scaffold comprises a chemically modified nucleotide at one or more of (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or all of) positions 4, 5, 6, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 34, 35, 36, 37, 38, 39, 40, 41, and 42.
13. The nucleic acid of any of the preceding embodiments, wherein the StlCas9 scaffold comprises a chemically modified nucleotide at one or more of (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10,
11, 12, 13, 14, or all of) positions 7, 8, 9, 10, 11, 12, 25, 26, 27, 28, 29, 30, 31, 32, and 33.
14. The nucleic acid of any of the preceding embodiments, wherein the StlCas9 scaffold comprises a RAR region, wherein the RAR region comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,
12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or 31 chemically modified nucleotides (e.g., wherein the chemically modified nucleotides have the same chemical modification).
15. The nucleic acid of any of the preceding embodiments, wherein the StlCas9 scaffold comprises a SL1 region, wherein the SL1 region comprises 1, 2, 3, 4, 5, 6, 7, or 8 chemically modified nucleotides (e.g., wherein the chemically modified nucleotides have the same chemical modification). 16. The nucleic acid of any of the preceding embodiments, wherein the St 1 Cas9 scaffold comprises a chemically modified nucleotide in the RAR region and a chemically modified nucleotide in the SL1 region.
17. The nucleic acid of any of the preceding embodiments, wherein the StlCas9 scaffold comprises between 10-21 (e.g., 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or 21) chemically modified nucleotides in the RAR region.
18. The nucleic acid of any of the preceding embodiments, wherein the StlCas9 scaffold comprises between 0-8 (e.g., 0, 1, 2, 3, 4, 5, 6, 7, or 8) chemically modified nucleotides in the SL1 region.
19. The nucleic acid of any of the preceding embodiments, wherein positions 1, 2, and 3 (if present) do not comprise a 2’-O-methyl chemically modified nucleotide.
20. The nucleic acid of any of the preceding embodiments, wherein positions 1, 2, and 3 (if present) are not chemically modified.
21. The nucleic acid of any of the preceding embodiments, wherein positions 43 through 54 (if present) do not comprise a 2’-O-methyl chemically modified nucleotide.
22. The nucleic acid of any of the preceding embodiments, wherein positions 43 through 54 (if present) are not chemically modified.
23. The nucleic acid of any of the preceding embodiments, wherein the StlCas9 scaffold comprises a chemically modified nucleotide at each of positions 4 through 6 (if present).
24. The nucleic acid of any of the preceding embodiments, wherein the StlCas9 scaffold comprises a chemically modified nucleotide at each of positions 13 through 15 (if present).
25. The nucleic acid of any of the preceding embodiments, wherein the StlCas9 scaffold comprises a chemically modified nucleotide at each of positions 16 through 18 (if present).
26. The nucleic acid of any of the preceding embodiments, wherein the StlCas9 scaffold comprises a chemically modified nucleotide at each of positions 19 through 21 (if present).
27. The nucleic acid of any of the preceding embodiments, wherein the StlCas9 scaffold comprises a chemically modified nucleotide at each of positions 22 through 24 (if present).
28. The nucleic acid of any of the preceding embodiments, wherein the StlCas9 scaffold comprises a chemically modified nucleotide at each of positions 34 through 36 (if present).
29. The nucleic acid of any of the preceding embodiments, wherein the StlCas9 scaffold comprises a chemically modified nucleotide at each of positions 37 through 39 (if present). 30. The nucleic acid of any of the preceding embodiments, wherein the St 1 Cas9 scaffold comprises a chemically modified nucleotide at each of positions 40 through 42 (if present).
31. The nucleic acid of any of the preceding embodiments, wherein the StlCas9 scaffold comprises a chemically modified nucleotide at each of positions 13 through 24 (if present).
32. The nucleic acid of any of the preceding embodiments, wherein the StlCas9 scaffold comprises a chemically modified nucleotide at each of positions 34 through 42 (if present).
33. The nucleic acid of any of the preceding embodiments, wherein the StlCas9 scaffold comprises a chemically modified nucleotide at each of positions 12 through 21 (if present).
34. The nucleic acid of embodiment 33, wherein the StlCas9 scaffold comprises a lengthened RAR upper stem.
35. The nucleic acid of embodiment 33 or 34, wherein: the StlCas9 scaffold comprises a first insertion (e.g., of 4 nucleotides) between positions
14 and 15, and a second insertion (e.g., of 4 nucleotides) between positions 18 and 19; or the StlCas9 scaffold comprises an insertion (e.g., of 10 nucleotides) between positions 15 and 18, and a deletion of positions 16 and 17, wherein optionally the insertion has a sequence according to GACUUCGGUC.
36. The nucleic acid of any of embodiments 33-35, wherein the StlCas9 scaffold comprises a mutation in the tetraloop, e.g., wherein the tetraloop comprises a sequence of UUCG.
37. The nucleic acid of any of the preceding embodiments, wherein the StlCas9 scaffold comprises a chemically modified nucleotide (e.g., 2’-O-methyl) at one or more of (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or all of) positions 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, and 25.
38. The nucleic acid of any of the preceding embodiments, wherein the StlCas9 scaffold comprises a chemically modified nucleotide (e.g., 2’-O-methyl) at one or more of (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or all of) positions 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 50, 51, 52, 53, and 54.
39. The nucleic acid of any of the preceding embodiments, wherein the StlCas9 scaffold comprises a chemically modified nucleotide (e.g., 2’-O-methyl) at one or more of (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or all of) positions 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 51, 52, 53, and 54. 40. The nucleic acid of any of the preceding embodiments, wherein the St 1 Cas9 scaffold comprises a chemically modified nucleotide (c.g., 2’-O-mcthyl) at one or more of (c.g., 2, 3, 4, 5, 6, 7, 8, 9, or all of) positions 12, 13, 14, 15, 16, 17, 18, 19, 20, and 21.
41. The nucleic acid of any of the preceding embodiments, wherein the StlCas9 scaffold comprises a chemically modified nucleotide (e.g., 2’-O-methyl) at one or more of (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or all of) positions 4, 5, 6, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 34, 35, 36, 37, 38, 39, 40, 41, and 42.
42. The nucleic acid of any of the preceding embodiments, wherein the StlCas9 scaffold comprises a chemically modified nucleotide (e.g., 2’-O-methyl) at one or more of (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, or all of) positions 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, and 54.
43. The nucleic acid of any of the preceding embodiments, wherein the StlCas9 scaffold comprises a chemically modified nucleotide (e.g., 2’-O-methyl) at one or more of (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, or all of) positions 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, and 53.
44. The nucleic acid of any of the preceding embodiments, wherein the StlCas9 scaffold comprises a chemically modified nucleotide (e.g., 2’-O-methyl) at one or more of (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, or all of) positions 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, and 52.
45. The nucleic acid of any of the preceding embodiments, wherein the StlCas9 scaffold comprises a chemically modified nucleotide (e.g., 2’-O-methyl) at one or more of (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or all of) positions 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 53, and 54.
46. The nucleic acid of any of the preceding embodiments, wherein the StlCas9 scaffold comprises a chemically modified nucleotide (e.g., 2’-O-methyl) at one or more of (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or all of) positions 4,
6, 8, 10, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, and 42.
47. The nucleic acid of any of the preceding embodiments, wherein the StlCas9 scaffold comprises a chemically modified nucleotide (e.g., 2’-O-methyl) at one or more of (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or all of) positions 5, 7, 9, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, and 41.
48. The nucleic acid of any of the preceding embodiments, wherein the StlCas9 scaffold comprises a chemically modified nucleotide (e.g., 2’-O-methyl) at one or more of (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or all of) positions 5,
7, 9, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, and 42. 49. The nucleic acid of any of the preceding embodiments, wherein the St 1 Cas9 scaffold comprises a chemically modified nucleotide (c.g., 2’-O-mcthyl) at one or more of (c.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or all of) positions 4, 5, 6, 12, 13, 14, 15, 16, 17, 18, 19, 20, and 21.
50. The nucleic acid of any of the preceding embodiments, wherein the StlCas9 scaffold comprises a chemically modified nucleotide (e.g., 2’-O-methyl) at one or more of (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, or all of) positions 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, and 24.
51. The nucleic acid of any of the preceding embodiments, wherein the StlCas9 scaffold comprises a chemically modified nucleotide (e.g., 2’-O-methyl) at one or more of (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, or all of) positions 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 25, 26, 27, 28, 29, 30, 31, 32, and 33.
52. The nucleic acid of any of the preceding embodiments, wherein the StlCas9 scaffold comprises a chemically modified nucleotide (e.g., 2’-O-methyl) at one or more of (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, or all of) positions 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 34, 35, 36, 37, 38, 39, 40, 41, and 42.
53. The nucleic acid of any of the preceding embodiments, wherein the StlCas9 scaffold comprises a chemically modified nucleotide (e.g., 2’-O-methyl) at one or more of (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, or all of) positions 4, 5, 6, 12,
13, 14, 15, 16, 17, 18, 19, 20, 21, 34, 35, 36, 37, 38, 39, 40, 41, and 42.
54. The nucleic acid of any of the preceding embodiments, wherein the StlCas9 scaffold comprises a chemically modified nucleotide (e.g., 2’-O-methyl) at one or more of (e.g., 2, 3,
4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or all of) positions 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 34, 35, 36, 37, 38, 39, 40, 41, and 42.
55. The nucleic acid of any of the preceding embodiments, wherein the StlCas9 scaffold comprises a chemically modified nucleotide (e.g., 2’-O-methyl) at one or more of (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or all of) positions 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 43, and 44.
56. The nucleic acid of any of the preceding embodiments, wherein the StlCas9 scaffold comprises a chemically modified nucleotide (e.g., 2’-O-methyl) at one or more of (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, or all of) positions 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, and 43. 57. The nucleic acid of any of the preceding embodiments, wherein the St 1 Cas9 scaffold comprises a chemically modified nucleotide (c.g., 2’-O-mcthyl) at one or more of (c.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, or all of) positions 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, and 44.
58. The nucleic acid of any of the preceding embodiments, wherein the StlCas9 scaffold comprises a chemically modified nucleotide (e.g., 2’-O-methyl) at one or more of (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, or all of) positions 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, and 45.
59. The nucleic acid of any of the preceding embodiments, wherein the StlCas9 scaffold comprises a chemically modified nucleotide (e.g., 2’-O-methyl) at one or more of (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, or all of) positions 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, and 46.
60. The nucleic acid of any of the preceding embodiments, wherein the StlCas9 scaffold comprises a chemically modified nucleotide (e.g., 2’-O-methyl) at one or more of (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, or all of) positions 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, and 47.
61. The nucleic acid of any of the preceding embodiments, wherein the StlCas9 scaffold comprises a chemically modified nucleotide (e.g., 2’-O-methyl) at one or more of (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, or all of) positions 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, and 49.
62. The nucleic acid of any of the preceding embodiments, wherein; the StlCas9 scaffold comprises a chemically modified nucleotide (e.g., 2’-O-methyl) at one or more of (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, or all of) positions 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, and 50, or the StlCas9 scaffold comprises a chemically modified nucleotide (e.g., 2’-O-methyl) at one or more of (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, or all of) positions 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, and 51.
63. The nucleic acid of any of the preceding embodiments, wherein: a) the StlCas9 scaffold comprises a chemically modified nucleotide (e.g., 2’-O-methyl) at each of positions 12 through 21; b) the StlCas9 scaffold comprises a chemically modified nucleotide (e.g., 2’-O-methyl) at each of positions 34 through 36; c) the StlCas9 scaffold comprises a chemically modified nucleotide (e.g., 2’-Fluoro) at one or more of positions 37 through 42; d) the StlCas9 scaffold comprises a chemically modified nucleotide (e.g., 2’-Fluoro) at each of positions 37 through 42; e) the StlCas9 scaffold comprises a chemically modified nucleotide (e.g., 2’-Fluoro) at one or more of positions 45 through 47; f) the StlCas9 scaffold comprises a chemically modified nucleotide (e.g., 2’-Fluoro) at each of positions 45 through 47; g) the StlCas9 scaffold comprises a chemically modified nucleotide (e.g., 2’-Fluoro) at one or more of positions 52 through 54; h) the StlCas9 scaffold comprises a chemically modified nucleotide (e.g., 2’-Fluoro) at each of positions 52 through 54; i) the StlCas9 scaffold comprises a 2’-O-methyl chemically modified nucleotide at each of positions 12 through 21, a 2’-O-methyl chemically modified nucleotide at each of positions 34 through 36, and a 2’ -Fluoro chemically modified nucleotide at each of positions 37 through 42; j) the StlCas9 scaffold comprises a 2’-O-methyl chemically modified nucleotide at each of positions 12 through 21, a 2’-O-methyl chemically modified nucleotide at each of positions 34 through 36, a 2’ -Fluoro chemically modified nucleotide at each of positions 37 through 42, and a 2’ -Fluoro chemically modified nucleotide at each of positions 45 through 47 ; or k) the StlCas9 scaffold comprises a 2’-O-methyl chemically modified nucleotide at each of positions 12 through 21, a 2’-O-methyl chemically modified nucleotide at each of positions 34 through 36, a 2’ -Fluoro chemically modified nucleotide at each of positions 37 through 42, a 2’-Fluoro chemically modified nucleotide at each of positions 45 through 47 and a 2’ -Fluoro chemically modified nucleotide at each of positions 52 through 54.
64. The nucleic acid of any of the preceding embodiments, which comprises a plurality of chemically modified nucleotides in the StlCas9 scaffold, wherein the plurality of chemically modified nucleotides in the StlCas9 scaffold have the same chemical modification.
65. The nucleic acid of any of embodiments 1-63, which comprises a plurality of chemically modified nucleotides in the StlCas9 scaffold, wherein the plurality of chemically modified nucleotides in the StlCas9 scaffold have two or more different chemical modifications. 66. The nucleic acid of any of the preceding embodiments, wherein the St 1 Cas9 scaffold comprises a sequence having the chemically modified nucleotides set out in Table 22, 21, 20, E3, E7, E8, E9, E10, E12, or E13.
67. The nucleic acid of any of the preceding embodiments, wherein the chemically modified nucleotide is a modification to a sugar group, e.g., a modification to the 2’-0 of ribose, e.g., a 2’-O-Methyl chemically modified nucleotide.
68. The nucleic acid of any of the preceding embodiments, wherein the chemically modified nucleotide is a modification to a sugar group, e.g., the ribose ring contains a bridging moiety, e.g., a “locked” nucleic acid (LNA).
69. The nucleic acid of any of the preceding embodiments, which further comprises a second chemically modified nucleotide.
70. The nucleic acid of any of the preceding embodiments, which comprises a sequence according to SEQ ID NO: 26000, or a sequence having at least 70%, 80%, 90%, 95%, 97%, 98%, or 99% identity thereto.
71. The nucleic acid of any of the preceding embodiments, which comprises a sequence of Tables 42, 43, or 44, or a sequence having at least 70%, 80%, 90%, 95%, 97%, 98%, or 99% identity thereto.
72. The nucleic acid of any of the preceding embodiments, which comprises a sequence according to SEQ ID NO: 25999, or a sequence having at least 70%, 80%, 90%, 95%, 97%, 98%, or 99% identity thereto.
73. The nucleic acid of any of the preceding embodiments, which comprises one or more (e.g., at least 1, 2, or 3, and optionally no more than 10, 20, or 30) sequence differences (e.g., insertions, deletions, or substitutions) relative to SEQ ID NO: 25999.
74. The nucleic acid of any of the preceding embodiments, wherein the scaffold has a length of 50-60, 60-70, 70-80, or 80-90 nucleotides.
75. The nucleic acid of any of the preceding embodiments, wherein the RAR region has a length of 30-40 nucleotides (e.g. 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 nucleotides).
76. The nucleic acid of any of the preceding embodiments, wherein the SL1 region has a length of 6-15 nucleotides (e.g., 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 nucleotides).
77. The nucleic acid of any of the preceding embodiments, wherein the SL2 region has a length of 20-30 nucleotides (e.g., 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides). 78. The nucleic acid of any of embodiments 1-69, which comprises an SL2 region.
79. The nucleic acid of any of embodiments 1-69, which docs not comprise an SL2 region.
80. The nucleic acid of any of the preceding embodiments, wherein the StlCas9 scaffold binds to an StlCas9 protein having an amino acid sequence of SEQ ID NO: 23818.
81. The nucleic acid of any of the preceding embodiments, which further comprises a gRNA spacer situated 5’ of the StlCas9 scaffold region.
82. The nucleic acid of embodiment 81, wherein the gRNA spacer is complementary to a first portion of the human SERPINA1 gene.
83. The nucleic acid of any of the preceding embodiments, which further comprises a heterologous object sequence situated 3’ of the StlCas9 scaffold region.
84. The nucleic acid of any of the preceding embodiments, which further comprises a primer binding site (PBS) sequence situated 3’ of the StlCas9 scaffold region, e.g., wherein the PBS sequence is situated 3’ of the heterologous object sequence.
85. A template RNA comprising (tgRNA) comprising, from 5’ to 3’:
(1) a gRNA spacer;
(2) a chemically modified StlCas9 scaffold comprising a nucleic acid of any of claims 1-
80;
(3) a heterologous object sequence; and
(4) a primer binding site (PBS) sequence.
86. The template RNA of embodiment 85, which further comprises a chemical modification outside of the chemically modified StlCas9 scaffold.
87. The template RNA of embodiment 86, wherein the chemical modification outside of the chemically modified StlCas9 scaffold is situated in the gRNA spacer, optionally wherein the chemical modification is a phosphorothioate linkage or a 2’-O-Methyl nucleotide.
88. The template RNA of embodiment 86, wherein the chemical modification outside of the chemically modified StlCas9 scaffold is situated in the PBS sequence, optionally wherein the chemical modification is a phosphorothioate linkage or a 2’-O-Methyl nucleotide.
89. The template RNA of embodiment 86, wherein the chemical modification outside of the chemically modified StlCas9 scaffold is situated in the heterologous object sequence, optionally wherein the chemical modification is a 2’-fluoro nucleotide.
90. A template RNA comprising (tgRNA) comprising, from 5’ to 3’: (1) a gRNA spacer;
(2) a gRNA scaffold, c.g., a StlCas9 scaffold, c.g., a chemically modified StlCas9 scaffold of any of claims 1-80;
(3) a heterologous object sequence comprising a chemically modified nucleotide; and
(4) a primer binding site (PBS) sequence.
91. The template RNA of embodiment 90, wherein the chemically modified nucleotide in the heterologous object sequence is a 2’-Fluoro nucleotide.
92. The template RNA of embodiment 90 of 91, wherein the heterologous object sequence further comprises one or more (e.g., 2) additional chemically modified nucleotides, e.g., 2’- Fluoro nucleotides.
93. The template RNA of any of embodiments 90-92, wherein: position +4 of the heterologous object sequence is a chemically modified nucleotide (e.g., a 2’-Fluoro nucleotide); position +5 of the heterologous object sequence is a chemically modified nucleotide (e.g., a 2’-Fluoro nucleotide); position +6 of the heterologous object sequence is a chemically modified nucleotide (e.g., a 2’-Fluoro nucleotide); positions +4, +5, and +6 of the heterologous object sequence are each a chemically modified nucleotide (e.g., each is a 2’-Fluoro nucleotide);
94. The template RNA of any of the preceding embodiments, wherein: the 3’ most nucleotide in the template RNA is a chemically modified nucleotide (e.g., a 2’-O-methyl nucleotide); the second nucleotide from the 3’ end of the template RNA is a chemically modified nucleotide (e.g., a 2’-O-methyl nucleotide); the third nucleotide from the 3’ end of the template RNA is a chemically modified nucleotide (e.g., a 2’-O-methyl nucleotide); the 3’ most nucleotide and the second and third nucleotides from the 3’ end of the template RNA are each a chemically modified nucleotide (e.g., each is a 2’-O-methyl nucleotide); the twelfth nucleotide from the 3’ end of the template RNA is a chemically modified nucleotide (e.g., a 2’-Fluoro nucleotide); the thirteenth nucleotide from the 3’ end of the template RNA is a chemically modified nucleotide (e.g., a 2’-Fluoro nucleotide); the fourteenth nucleotide from the 3’ end of the template RNA is a chemically modified nucleotide (e.g., a 2 ’-Fluoro nucleotide); or the twelth, thirteenth, and fourteenth nucleotides from the 3’ end of the template RNA are each a chemically modified nucleotide (e.g., each is a 2’-Fluoro nucleotide); the nineteenth nucleotide from the 3’ end of the template RNA is a chemically modified nucleotide (e.g., a 2’-Fluoro nucleotide); the twentieth nucleotide from the 3’ end of the template RNA is a chemically modified nucleotide (e.g., a 2’-Fluoro nucleotide); the twenty-first nucleotide from the 3’ end of the template RNA is a chemically modified nucleotide (e.g., a 2’-Fluoro nucleotide); the nineteenth, twentieth, and twenty-first nucleotides from the 3’ end of the template RNA are each a chemically modified nucleotide (e.g., each is a 2’-Fluoro nucleotide); the twenty-sixth nucleotide from the 3’ end of the template RNA is a chemically modified nucleotide (e.g., a 2’-Fluoro nucleotide); the twenty- seventh nucleotide from the 3’ end of the template RNA is a chemically modified nucleotide (e.g., a 2’-Fluoro nucleotide); the twenty-eighth nucleotide from the 3’ end of the template RNA is a chemically modified nucleotide (e.g., a 2’-Fluoro nucleotide); the twenty-sixth, twenty- seventh, and twenty-eighth nucleotides from the 3’ end of the template RNA are each a chemically modified nucleotide (e.g., each is a 2’ -Fluoro nucleotide); the thirty-first nucleotide from the 3’ end of the template RNA is a chemically modified nucleotide (e.g., a 2’-Fluoro nucleotide); the thirty-first nucleotide from the 3’ end of the template RNA is a chemically modified nucleotide (e.g., a 2’-Fluoro nucleotide); the thirty-second nucleotide from the 3’ end of the template RNA is a chemically modified nucleotide (e.g., a 2’-Fluoro nucleotide); the thirty-third nucleotide from the 3’ end of the template RNA is a chemically modified nucleotide (e.g., a 2’-Fluoro nucleotide); the thirty-fourth nucleotide from the 3’ end of the template RNA is a chemically modified nucleotide (c.g., a 2’-Fluoro nucleotide); the thirty-fifth nucleotide from the 3’ end of the template RNA is a chemically modified nucleotide (e.g., a 2 ’-Fluoro nucleotide); the thirty-sixth nucleotide from the 3’ end of the template RNA is a chemically modified nucleotide (e.g., a 2’-Fluoro nucleotide); the thirty-first, thirty- second, thirty-third, thirty-fourth, thirty-fifth, and thirty-sixth nucleotide from the 3’ end of the template RNA each each a chemically modified nucleotide (e.g., each is a 2’ -Fluoro nucleotide); the thirty-seventh nucleotide from the 3’ end of the template RNA is a chemically modified nucleotide (e.g., a 2’-O-methyl nucleotide); the fifty- second nucleotide from the 3’ end of the template RNA is a chemically modified nucleotide (e.g., a 2’-O-methyl nucleotide); the fifty-third nucleotide from the 3’ end of the template RNA is a chemically modified nucleotide (e.g., a 2’-O-methyl nucleotide); the fifty-fourth nucleotide from the 3’ end of the template RNA is a chemically modified nucleotide (e.g., a 2’-O-methyl nucleotide); the fifty-fifth nucleotide from the 3’ end of the template RNA is a chemically modified nucleotide (e.g., a 2’-O-methyl nucleotide); the fifty- sixth nucleotide from the 3’ end of the template RNA is a chemically modified nucleotide (e.g., a 2’-O-methyl nucleotide); the fifty-seventh nucleotide from the 3’ end of the template RNA is a chemically modified nucleotide (e.g., a 2’-O-methyl nucleotide); the fifty-eighth nucleotide from the 3’ end of the template RNA is a chemically modified nucleotide (e.g., a 2’-O-methyl nucleotide); the fifty-ninth nucleotide from the 3’ end of the template RNA is a chemically modified nucleotide (e.g., a 2’-O-methyl nucleotide); the sixtieth nucleotide from the 3’ end of the template RNA is a chemically modified nucleotide (e.g., a 2’-O-methyl nucleotide); the sixty-first nucleotide from the 3’ end of the template RNA is a chemically modified nucleotide (e.g., a 2’-O-methyl nucleotide); the sixty-second nucleotide from the 3’ end of the template RNA is a chemically modified nucleotide (e.g., a 2’-O-mcthyl nucleotide); the sixty-third nucleotide from the 3’ end of the template RNA is a chemically modified nucleotide (e.g., a 2’-O-methyl nucleotide); the sixty-fourth nucleotide from the 3’ end of the template RNA is a chemically modified nucleotide (e.g., a 2’-O-methyl nucleotide); the sixty-fifth nucleotide from the 3’ end of the template RNA is a chemically modified nucleotide (e.g., a 2’-O-methyl nucleotide); the sixty-sixth nucleotide from the 3’ end of the template RNA is a chemically modified nucleotide (e.g., a 2’-O-methyl nucleotide); the sixty-seventh nucleotide from the 3’ end of the template RNA is a chemically modified nucleotide (e.g., a 2’-O-methyl nucleotide); the sixty-eighth nucleotide from the 3’ end of the template RNA is a chemically modified nucleotide (e.g., a 2’-O-methyl nucleotide); the sixty-ninth nucleotide from the 3’ end of the template RNA is a chemically modified nucleotide (e.g., a 2’-O-methyl nucleotide); or the fifty- second through the sixty-first nucleotides from the 3’ end of the template RNA are each a chemically modified nucleotide (e.g., each is a 2’-O-methyl nucleotide).
95. The template RNA or the nucleic acid of any of the preceding embodiments, wherein the chemically modified StlCas9 scaffold comprises a variant StlCas9 scaffold having a deletion of part or all of Stem loop 2.
96. The template RNA or nucleic acid of embodiment 95, wherein the deletion is between 1-32 (e.g., 2-29, 2-20, 2-10, or 10-20) nucleotides in length.
97. The template RNA or nucleic acid of embodiment 95, wherein the deletion is of all of Stem loop 2.
98. The template RNA or nucleic acid of embodiment 95, wherein the deletion is of positions 55 through 84.
99. The template RNA or nucleic acid of embodiment 95, wherein the StlCas9 scaffold comprises a deletion of part of the second single stranded region (e.g., 1, 2, 3, or 4 nucleotides at the 3’ end of the single stranded region). 100. The template RNA or the nucleic acid of any of the preceding embodiments, wherein the chemically modified StlCas9 scaffold comprises a variant StlCas9 scaffold having one or both of a lengthened RAR upper stem or a substitution resulting in a G-C base pair in the RAR upper stem.
101. The template RNA or nucleic acid of any of the preceding embodiments, wherein the RAR upper stem is lengthened by 1-8 base pairs (e.g., 1, 2, 3, 4, 5, 6, 7, or 8 base pairs) relative to the wild-type sequence of SEQ ID NO: 25999.
102. The template RNA or nucleic acid of embodiment 101, wherein at least 50%, 60%, 70%, 80%, or 90% of the base pairs that are new relative to SEQ ID NO: 25999 are G-C base pairs.
103. The template RNA or nucleic acid of embodiment 101 or 102, wherein the lengthened RAR upper stem comprises one or more base pairs that are new relative to SEQ ID NO: 25999, wherein the one or more new base pairs comprise one or more chemically modified nucleotides.
104. The template RNA or the nucleic acid of any of the preceding embodiments, wherein the chemically modified StlCas9 scaffold comprises a variant StlCas9 scaffold having a mutation in the tetraloop.
105. The template RNA or nucleic acid of any of the preceding embodiments, wherein one or more nucleotides in the tetraloop are substituted.
106. The template RNA or nucleic acid of any of the preceding embodiments, wherein the tetraloop comprises a sequence chosen from: AACA, AAUA, ACCA, ACUA, AGUA, AGCA, AUCA, AUUA, CAAC, CUCG, CUUG, GAAA, GAGA, GCAA, GCGA, GGAA, GGAG, GGGA, GUAA, GUGA, UAAC, UACG, UCAC, UCCG, UGAA, UGAC, UGCG, UUAC, or UUCG.
107. The template RNA or nucleic acid of any of the preceding embodiments, wherein the tetraloop is lengthened, e.g., to 5 nucleotides.
108. The template RNA or nucleic acid of embodiment On, wherein the lengthened tetraloop comprises a sequence chosen from: GAAGA or GACAA.
109. The template RNA or nucleic acid of embodiment 107 or 108, wherein the lengthened tetraloop comprises one or more chemically modified nucleotides (e.g., comprises 1, 2, 3, 4, or 5 chemically modified nucleotides). 110. The template RNA or nucleic acid of any of the preceding embodiments, wherein the variant gRNA scaffold comprises a sequence according to Tabic 23, or a sequence having no more than 1, 2, or 3 sequence alterations (e.g., substitutions) relative thereto.
111. The template RNA or nucleic acid of any of the preceding embodiments, which comprises a sequence according to any of Tables 39, 24, 40, 57, 41, 42, 45, 56, or 12-62 or a sequence having at least 80%, 85%, 90%, 95%, 98%, or 99% identity thereto.
112. The template RNA or nucleic acid of any of the preceding embodiments, which comprises a sequence according to SEQ ID NO: 27131, or a sequence having at least 80%, 85%, 90%, 95%, 98%, or 99% identity thereto.
113. The template RNA or nucleic acid of any of the preceding embodiments, which comprises a sequence according to SEQ ID NO: 27132, or a sequence having at least 80%, 85%, 90%, 95%, 98%, or 99% identity thereto.
114. The template RNA or nucleic acid of any of the preceding embodiments, which comprises a sequence according to SEQ ID NO: 27133, or a sequence having at least 80%, 85%, 90%, 95%, 98%, or 99% identity thereto.
115. The template RNA or nucleic acid of any of the preceding embodiments, which comprises a sequence according to SEQ ID NO: 27134, or a sequence having at least 80%, 85%, 90%, 95%, 98%, or 99% identity thereto.
116. The template RNA or nucleic acid of any of the preceding embodiments, wherein the variant StlCas9 scaffold has a length of 50-60, 60-70, 70-80, or 80-84 nucleotides.
117. The template RNA or the nucleic acid of any of the preceding embodiments, wherein the chemically modified StlCas9 scaffold comprises a variant gRNA scaffold comprising a sequence according to Table 23, or a sequence having no more than 1, 2, or 3 sequence alterations (e.g., substitutions) relative thereto.
118. A system comprising: a nucleic acid or template RNA of any of the preceding embodiments; and a polypeptide comprising a StlCas9 domain, or a nucleic acid encoding the polypeptide.
119. The system of embodiment 118, wherein the polypeptide further comprises a RT domain, and optionally comprises a linker situated between the RT domain and the StlCas9 domain.
120. A gene modifying system comprising: a template RNA of any of claims 85-117; and a gene modifying polypeptide, or a nucleic acid encoding the gene modifying polypeptide, the gene modifying polypeptide comprising:
(1) a Cas9 domain, e.g., a StlCas9 domain;
(2) a linker; and
(3) a reverse transcriptase (RT) domain.
121. The gene modifying system of embodiment 120, wherein the Cas9 nickase domain comprises an StlCas9 nickase domain.
122. The gene modifying system of embodiment 120 or 121, wherein the gene modifying polypeptide comprises, in an N-terminal to C-terminal direction, a first NLS, the StlCas9 nickase domain, a linker, an RT domain, and a second NLS.
123. The gene modifying system of embodiment 122, wherein one or both of: the first NLS comprises a sequence of SEQ ID NO: 11,095, and the second NLS comprises a sequence of SEQ ID NO: 11,099.
124. The gene modifying system of any of embodiments 120-123, wherein the linker comprises a sequence according to SEQ ID NO: 5006.
125. The gene modifying system of embodiment 120, wherein the StlCas9 domain has a sequence according to SEQ ID NO: 23818, or a sequence having at least 70%, 80%, 90%, 95%, 97%, 98%, or 99% identity thereto.
126. The gene modifying system of embodiment 120, wherein the linker has a sequence according to SEQ ID NO: 5006, or a sequence having at least 70%, 80%, 90%, 95%, 97%, 98%, or 99% identity thereto.
127. The gene modifying system of embodiment 120, wherein the RT domain has a sequence according to SEQ ID NO: 26006, or a sequence having at least 70%, 80%, 90%, 95%, 97%, 98%, or 99% identity thereto.
128. The gene modifying system of embodiment 120, wherein the gene modifying polypeptide has a sequence according to SEQ ID NO: 26002, or a sequence having at least 70%, 80%, 90%, 95%, 97%, 98%, or 99% identity thereto.
129. The system of any of embodiments 118-128, which is capable of specifically cleaving a target nucleic acid.
130. The system of any of embodiments 118-128, which is capable of producing indels in a target nucleic acid. 131 . The system of any of embodiments 118-128, which is capable of introducing an edit specified by the heterologous object sequence in a target nucleic acid.
132. A pharmaceutical composition, comprising the template RNA of nucleic acid of any one of embodiments 1-117 or the system of any one of embodiments 118-131, and a pharmaceutically acceptable excipient or carrier.
133. The pharmaceutical composition of embodiment 132, wherein the pharmaceutically acceptable excipient or carrier is selected from the group consisting of a plasmid vector, a viral vector, a vesicle, and a lipid nanoparticle (LNP).
134. A host cell (e.g., a mammalian cell, e.g., a human cell) comprising the gene modifying system, template RNA, or nucleic acid of any one of the preceding claims.
135. A method of making the nucleic acid or template RNA of any one of embodiments 118- 131, the method comprising synthesizing the template RNA in vitro (e.g., by in vitro transcription or solid state synthesis).
136. A method for modifying a target site (e.g., a target site in the human SERPINA1 gene) in a cell, the method comprising contacting the cell with the gene modifying system of any one of embodiments 118-131, or DNA encoding the same, or the pharmaceutical composition of embodiment 132 or 133, thereby modifying the target site.
137. A method for treating a subject having a disease or condition associated with a mutation in a gene (e.g., the human SERPINA1 gene), the method comprising administering to the subject the gene modifying system of any one of embodiments 118-131, or DNA encoding the same, or the pharmaceutical composition of embodiment 132 or 133, thereby treating the subject having a disease or condition.
138. The method of claim 137, wherein the disease or condition is alpha-1 antitrypsin deficiency (AATD).
139. The method of claim 137 or 138, wherein the subject has a E342K mutation.
140. A method for treating a subject having AATD, the method comprising administering to the subject the gene modifying system of any one of embodiments 118-131, or DNA encoding the same, or the pharmaceutical composition of embodiment 132 or 133, thereby treating the subject having AATD. BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 is a diagram depicting components of a gene modifying system as described herein. FIG. 1A is a diagram showing a gene modifying polypeptide comprising a Cas nickase domain (e.g., spCas9 N863A) and a reverse transcriptase domain (RT domain) which are linked by a linker. FIG. IB is a diagram showing a template RNA comprising, from 5’ to 3’, a gRNA spacer, a gRNA scaffold, a heterologous object sequence, and a primer binding site sequence (PBS sequence). A heterologous object sequence can comprise a mutation region that comprises one or more sequence differences relative to a target site. A heterologous object sequence can also comprise a pre-edit homology region and a post-edit homology region, which flank a mutation region. Without wishing to be bound by theory, it is thought that a gRNA spacer of a template RNA binds to a second strand of a target site in the genome, and a gRNA scaffold of the template RNA binds to a gene modifying polypeptide, e.g., localizing the gene modifying polypeptide to a target site in the genome. It is thought that a Cas domain of a gene modifying polypeptide nicks a target site (e.g., a first strand of the target site), e.g., allowing a PBS sequence to bind to a sequence adjacent to the target site to be altered on the first strand of the target site. It is thought that an RT domain of a gene modifying polypeptide uses a first strand of a target site that is bound to a complementary sequence comprising a PBS sequence of a template RNA as a primer and a heterologous object sequence of the template RNA as a template to, e.g., polymerize a sequence complementary to the heterologous object sequence. Without wishing to be bound by theory, it is thought that reverse transcription can then proceed through a pre-edit homology region, then through a mutation region, and then through a post-edit homology region, thereby producing a DNA strand comprising a mutation specified by a heterologous object sequence.
FIG. 2 is a diagram illustrating the hypothesized secondary structure of a wild-type StlCas9 gRNA scaffold and is overlaid with description of valiants described herein.
FIG. 3A is a bar graph showing the rewriting performance of exemplary StlCas9-based gene modifying systems comprising exemplary template RNAs comprising various scaffolds truncated in the stem loop 2 region as depicted in FIG. 2.
FIG. 3B is a bar graph showing the rewriting performance of exemplary StlCas9-based gene modifying systems comprising exemplary template RNAs comprising various scaffolds further engineered in the tetraloop (TL) region as depicted in FIG. 2. Bars without asterisk = 1 pmol of exemplary gene modifying system, bars with asterisk = 0.01 pmol of exemplary gene modifying system.
FIG. 3C is a bar graph showing the rewriting performance of exemplary StlCas9-based gene modifying systems comprising exemplary template RNAs comprising various scaffolds further engineered to elongate and/or stabilize the TL and/or RAR regions as depicted in FIG. 2. Bars without asterisk = 1 pmol of exemplary gene modifying system, bars with asterisk = 0.01 pmol of exemplary gene modifying system.
FIG. 3D is a bar graph showing the rewriting performance of exemplary StlCas9-based gene modifying systems comprising exemplary template RNAs comprising various lengths of spacers. Bars without asterisk = 1 pmol of exemplary gene modifying system, bars with asterisk = 0.01 pmol of exemplary gene modifying system.
FIG. 4A is a bar graph showing the rewriting efficiency of exemplary gene modifying systems comprising different StlCas9-compatible template RNAs comprising modified scaffold sequences.
FIG. 4B is a bar graph showing the % indel levels introduced by exemplary gene modifying systems evaluated in FIG. 4A.
FIG. 4C is a bar graph showing the rewriting efficiency of additional exemplary gene modifying systems comprising different StlCas9-compatible template RNAs comprising modified scaffold sequences.
FIG. 5 is a bar graph showin the rewriting efficiency of exemplary gene modifying systems comprising StlCas9-based gene modifying polypeptide.
FIG. 6A is a bar graph showing the rewriting efficiency of exemplary gene modifying systems comprising StlCas9-based gene modifying polypeptide, with and without ngRNA.
FIG. 6B is a bar graph showing the % indel levels introduced by exemplary gene modifying systems comprising StlCas9-based gene modifying polypeptide, with (open bars) and without ngRNA (closed bars).
FIG. 7 A is a bar graph showing the rewriting efficiency of exemplary StlCas9-based gene modifying systems comprising exemplary template RNAs containing a dSL2 variant gRNA scaffold, various lengths of PBS sequences and heterologous object sequences in primary hepatocytes. FIG. 7B is a bar graph showing the % indel levels introduced by exemplary StlCas9-based gene modifying systems comprising exemplary template RNAs containing a dSL2 variant gRNA scaffold, various lengths of PBS sequences and heterologous object sequences in primary hepatocytes.
FIG. 8A is a bar graph showing percent rewriting achieved using an exemplary gene modifying system comprising different StlCas9-compatible template RNAs comprising variant scaffolds containing various exemplary variant tetraloop structures in primary hepatocytes.
FIG. 8B is a bar graph showing percent rewriting achieved using an exemplary gene modifying system comprising different StlCas9-compatible template RNAs comprising variant scaffolds containing various exemplary variant tetraloop structures in HEK293T cells treated with a high dose of exemplary gene modifying system.
FIG. 8C is a bar graph showing percent rewriting achieved using an exemplary gene modifying system comprising different StlCas9-compatible template RNAs comprising variant scaffolds containing various exemplary variant tetraloop structures in HEK293T cells treated with a low dose of exemplary gene modifying system.
FIG. 8D is a diagram illustrating the hypothesized secondary structure of a dSL2 truncated StlCas9 gRNA scaffold and is overlaid with description of valiants described herein.
FIG. 9A is a bar graph showing the rewriting activity of exemplary StlCas9-based gene modifying systems comprising variant template RNAs having the nucleotide sequence of exemplary template RNA RNACS6681 with various 2-O'-methyl chemical modifications in the gRNA scaffold region.
FIGs. 9B-9K are schematic diagrams of tested chemical modification pattern designs of exemplary StlCas9-based gene modifying systems comprising variant template RNAs having the nucleotide sequence of exemplary template RNA RNACS6681, with 2’-O-methyl chemical modifications shown in bold.
FIG. 10A is a bar graph showing the rewriting activity of exemplary StlCas9-based gene modifying systems comprising variant template RNAs having the nucleotide sequence of exemplary template RNA RNACS9201 (containing a dSL2 variant gRNA scaffold) with various 2-O'-methyl chemical modifications in the gRNA scaffold region. FIG. 10B is a bar graph showing the rewriting activity of exemplary StlCas9-based gene modifying systems comprising variant template RNAs having a scaffold comprising three modified nucleotides at a time with 2’-O-methyl chemical modifications.
FIG. IOC is a diagram illustrating the positions of poorly tolerated (lowercase,), somewhat tolerated positions (capital), and tolerated positions (italic) to 2’-O-methyl chemical modification in the dSL2 StlCas9 scaffold sequence.
FIGs. 10D-10L are diagrams illustrating exemplary design patterns of 2’-O-methyl chemical modified nucleotides in the dSL2 StlCas9 scaffold sequence in FIG. 10A. Bold bases represent2'-O-methyl modified nucleotide positions.
FIG. 11 is a bar graph showing the rewriting efficiency of exemplary gene modifying systems comprising different StlCas9-compatible template RNAs comprising different patterns of 2’-O-methyl chemical modifications in the dSL2 StlCas9 scaffold in primary hepatocytes.
FIG. 12A is a bar graph showing the rewriting activity in the livers of mice administered with exemplary gene modifying systems comprising different StlCas9-compatible template RNAscontaining a dSL2 variant gRNA scaffold and formulated in lipid nanoparticles (LNP).
FIG. 12B is a bar graph showing the % indel levels in the livers of mice administered with exemplary gene modifying systems comprising different StlCas9-compatible template RNAscontaining a dSL2 variant gRNA scaffold and formulated in LNP.
FIG. 12C is a bar graph showing the the concentration of hAlAT in the serum of mice administered with exemplary gene modifying systems comprising different StlCas9-compatible template RNAscontaining a dSL2 variant gRNA scaffold and formulated in LNP.
FIG. 13A is a bar graph showing of the percent of perfect rewriting in the liver of mice administered with exemplary gene modifying systems that comprises different StlCas9- compatible template RNAs comprising different patterns of 2’-O-methyl chemical modifications in the dSL2 StlCas9 scaffold and are formulated in LNP.
FIG. 13B is a bar graph showing of the % indel levels in the liver of mice administered with exemplary gene modifying systems that comprises different StlCas9-compatible template RNAs comprising different patterns of 2’-O-methyl chemical modifications in the dSL2 StlCas9 scaffold and are formulated in LNP.
FIG. 13C is a bar graph showing the concentration of hAlAT in the serum of mice administered with exemplary gene modifying systems that comprises different StlCas9- compatible template RNAs comprising different patterns of 2’-O-methyl chemical modifications in the dSL2 StlCas9 scaffold and arc formulated in LNP.
FIG. 14A is a diagram illustrating the positions of the reference dSL2 StlCas9 scaffold sequence.
FIG. 14B is a diagram illustrating the positions of the reference wild-type StlCas9 scaffold sequence.
FIG. 14C is a diagram illustrating the hypothesized structure of RNACS 13597, having RAR+4_UUCG mutations relative to dSL2.
FIG. 14D is a diagram illustrating the hypothesized structure of RNACS17210, having RAR+4_AGCA mutations relative to dSL2.
FIG. 15 are schematics of three exemplary template RNAs comprising chemical modifications. Closed circles are 2’F modifications, open circles are 2’0Me modifications, diamonds are phosphorothioate modifications.
FIG. 16A is a bar graph showing the rewriting efficiency in the livers of mice administered with exemplary gene modifying systems comprising different template RNAs comprising scaffold chemical modifications.
FIG. 16B is a bar graph showing the % indel levels in the livers of mice administered with the exemplary gene modifying systems evaluated in FIG. 16A.
FIG. 17A is a bar graph showing the rewriting efficiency in the livers of mice administered with exemplary gene modifying systems comprising different template RNAs comprising scaffold chemical modifications in combination with fluoro modifications at the heterologous object sequence.
FIG. 17B is a bar graph showing the % indel levels in the livers of mice administered with the exemplary gene modifying systems evaluated in FIG. 17A.
FIG. 18A is a bar graph showing the rewriting efficiency in the livers of mice administered with exemplary gene modifying systems comprising different gene modifying polypeptides and different template RNAs comprising scaffold chemical modifications in combination with fluoro modifications at the heterologous object sequence.
FIG. 18B is a bar graph showing the % indel levels in the livers of mice administered with the exemplary gene modifying systems evaluated in FIG. 18A. FIG. 19A is a bar graph showing the % corrected genomic DNA in the livers of hSERPINAl E342K mice by administered with exemplary gene modifying systems comprising RNAIVT6241 or RNAIVT6898 polypeptides over evaluated dosages.
FIG. 19B is a bar graph showing the % corrected genomic DNA in the livers of NSG-PiZ mice by exemplary RNAIVT6898 systems over evaluated dosages at 7 days post-administration.
FIG. 19C is a bar graph showing the % corrected genomic DNA in the livers of NSG-PiZ mice by exemplary RNAIVT6898 systems over evaluated dosages at 21 days postadministration.
FIG. 19D is a bar graph showing the % edited mRNA in the livers of hSERPINAl E342K and NSG-PiZ mice by exemplary RNAIVT6898 systems over evaluated dosages at 21 days postadministration.
FIG. 19E is a bar graph showing the % indels introduced into the livers of hSERPINAl E342K by exemplary gene modifying polypeptides RNAVT6241 and RNAIVT6838 over evaluated dosages.
FIG. 19F is a bar graph showing the % indels introduced into the livers of NSG-PiZ mice by exemplary RNAIVT6898 systems over evaluated dosages at 7 days post-administration.
FIG. 19G is a bar graph showing the % indels introduced into the livers of NSG-PiZ mice by exemplary RNAIVT6898 systems over evaluated dosages at 21 days post-administration.
FIG. 20A is a bar graph showing the serum concentration of human Al AT in hSERPINAl E342K mice administered with exemplary gene modifying systems comprising RNAIVT6241 or RNAIVT6898 polypeptides over evaluated dosages.
FIG. 20B is a bar graph showing the serum concentration of human Al AT in NSG-PiZ mice administered with exemplary RNAIVT6898 systems over evaluated dosages at 7 days postadministration.
FIG. 20C is a bar graph showing the serum concentration of human Al AT in NSG-PiZ mice administered with exemplary RNAIVT6898 systems over evaluated dosages at 21 days postadministration.
FIG. 21A is a bar graph showing the % liver area occupied by globules in NSG-PiZ mice administered with exemplary RNAIVT6898 systems over evaluated dosages at 7 days postadministration. FIG. 21B is a bar graph showing the % liver area occupied by globules in NSG-PiZ mice administered with exemplary RNAIVT6898 systems over evaluated dosages at 21 days postadministration.
FIG. 22A is a bar graph showing the % corrected genomic DNA in the livers of NSG-PiZ mice by exemplary gene modifying systems comprising RNAIVT9315 gene modifying polypeptide and RNACS22230 template RNA over evaluated dosages.
FIG. 22B is a bar graph showing the % edited mRNA in the livers of NSG-PiZ mice by exemplary gene modifying systems comprising RNAIVT9315 gene modifying polypeptide and RNACS22230 template RNA over evaluated dosages.
FIG. 22C is a bar graph showing the % indels introduced into the livers of NSG-PiZ mice by exemplary gene modifying systems comprising RNAIVT9315 gene modifying polypeptide and RNACS22230 template RNA over evaluated dosages.
FIG. 23A is a bar graph showing the serum concentration of human Al AT in NSG-PiZ mice administered with exemplary gene modifying systems comprising RNAIVT9315 gene modifying polypeptide and RNACS22230 template RNA over evaluated dosages.
FIG. 23B is a line graph showing the serum concentration of human Al AT as a function of % genomic DNA rewriting in NSG-PiZ mice administered with exemplary gene modifying systems comprising RNAIVT9315 gene modifying polypeptide and RNACS22230 template RNA over evaluated dosages.
FIG. 24A is a bar graph showing the % corrected genomic DNA in the livers of hSERPINAl E342K mice by exemplary gene modifying systems comprising RNAIVT9315 or RNAIVT9318 gene modifying polypeptides and RNACS22230 template RNA over evaluated dosages.
FIG. 24B is a bar graph showing the % indels introduced in the livers of hSERPINAl E342K mice by exemplary gene modifying systems comprising RNAIVT9315 or RNAIVT9318 gene modifying polypeptides and RNACS22230 template RNA over evaluated dosages.
FIGs. 25A to 25D show bar graphs of the rewriting performance in the livers of hSERPINAl E342K mice administered with exemplary StlCas9-based gene modifying systems comprising exemplary RNAIVT9315 gene modifying polypeptide and exemplary template RNAs RNACS24756 or RNACS24757 at 0.012 mg/kg (FIG. 25A), 0.025 mg/kg (FIG. 25B), 0.05 mg/kg (FIG. 25C) and 0.1 mg/kg (FIG. 25D). FIGs. 26A to 26D show bar graphs of the % indel levels in the livers of hSERPINA l E342K mice administered with exemplary StlCas9-bascd gene modifying systems comprising exemplary RNAIVT9315 gene modifying polypeptide and exemplary template RNAs RNACS24756 or RNACS24757 at 0.012 mg/kg (FIG. 26A), 0.025 mg/kg (FIG. 26B), 0.05 mg/kg (FIG. 26C) and 0.1 mg/kg (FIG. 26D).
FIG. 27A is a bar graph showing the % corrected genomic DNA in the livers of hSERPINAl E342K mice by exemplary gene modifying systems comprising RNAIVT9315 gene modifying polypeptide and RNACS27457 template RNA over evaluated dosages (mpk = mg/kg) and formulated in LNP.
FIG. 27B is a bar graph showing the % edited mRNA in the livers of hSERPINAl E342K mice by exemplary gene modifying systems comprising RNAIVT9315 gene modifying polypeptide and RNACS27457 template RNA over evaluated dosages (mpk = mg/kg) and formulated in LNP.
FIG. 27C is a bar graph showing the % indels introduced in the livers of hSERPINAl E342K mice by exemplary gene modifying systems comprising RNAIVT9315 gene modifying polypeptide and RNACS27457 template RNA over evaluated dosages (mpk = mg/kg) and formulated in LNP.
FIG. 28A is a bar graph showing the serum concentration of human Al AT in hSERPINAl E342K mice administered with exemplary gene modifying systems comprising RNAIVT9315 gene modifying polypeptide and RNACS27457 template RNA over evaluated dosages (mpk = mg/kg) and formulated in LNP.
FIG. 28B is a line graph showing the serum concentration of human Al AT as a function of % genomic DNA rewriting in hSERPINAl E342K mice administered with exemplary gene modifying systems comprising RNAIVT9315 gene modifying polypeptide and RNACS27457 template RNA over evaluated dosages (mpk = mg/kg) and formulated in LNP.
Definitions
The term '‘expression cassette,” as used herein, refers to a nucleic acid construct comprising nucleic acid elements sufficient for the expression of the nucleic acid molecule of the instant invention. A “gRNA spacer,” as used herein, refers to a portion of a nucleic acid that has complementarity to a target nucleic acid and can, together with a gRNA scaffold, target a Cas protein to the target nucleic acid.
A “gRNA scaffold,” as used herein, refers to a portion of a nucleic acid that can bind a Cas protein and can, together with a gRNA spacer, target the Cas protein to the target nucleic acid. In some embodiments, the gRNA scaffold comprises a crRNA sequence, tetraloop, and tracrRNA sequence.
The term “StlCas9 scaffold,” as used herein, refers to a gRNA scaffold that can bind an StlCas9 protein and can, together with a gRNA spacer, target the StlCas9 protein to the target nucleic acid. In some embodiments, an StlCas9 scaffold comprises a crRNA sequence, tetraloop, and tracerRNA sequence. An exemplary position of StlCas9 scaffold within an exemplary template RNA is illustrated in FIG. 1.
In some embodiments, an StlCas9 scaffold comprises a full length wild type sequence. In some embodiments, an StlCas9 scaffold comprises a sequence with at least 80%, 85%. 90%, 95%, 96%, 97%, 98%, or 99% identity to the sequence of embodiments, an StlCas9 scaffold comprises a sequence identical to SEQ ID NO: 25999. In some embodiments, an StlCas9 scaffold comprises a truncation mutant. In some embodiments, an StlCas9 scaffold comprises a sequence with at least 80%, 85%. 90%, 95%, 96%, 97%, 98%, or 99% identity to the sequence of
A “variant gRNA scaffold,” as used herein, refers to gRNA scaffold having a non- naturally occurring sequence. In some embodiments, a variant gRNA scaffold sequence comprises one or more substitutions relative to the closest naturally occurring sequence. In some embodiments, a variant gRNA scaffold sequence comprises one or more insertions relative to the closest naturally occurring sequence. In some embodiments, a variant gRNA scaffold sequence comprises one or more deletions relative to the closest naturally occurring sequence.
A “gene modifying polypeptide,” as used herein, refers to a polypeptide comprising a retroviral reverse transcriptase, or a polypeptide comprising an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% amino acid sequence identity to a retroviral reverse transcriptase, which is capable of integrating a nucleic acid sequence (e.g., a sequence provided on a template nucleic acid) into a target DNA molecule (e.g., in a mammalian host cell, such as a genomic DNA molecule in the host cell). In some embodiments, a gene modifying polypeptide is capable of integrating a sequence substantially without relying on host machinery. In some embodiments, a gene modifying polypeptide integrates a sequence into a random position in a genome. In some embodiments, a gene modifying polypeptide integrates a sequence into a specific target site. In some embodiments, a gene modifying polypeptide includes one or more domains that, collectively, facilitate 1) binding a template nucleic acid, 2) binding a target DNA molecule, and 3) integration of at least a portion of the template nucleic acid into the target DNA. Gene modifying polypeptides include both naturally occurring polypeptides as well as engineered variants of the foregoing, e.g., having one or more amino acid substitutions to a naturally occurring sequence. Gene modifying polypeptides also include heterologous constructs, e.g., where one or more of the domains are heterologous to each other, whether through a heterologous fusion (or other conjugate) of otherwise wild-type domains, as well as fusions of modified domains, e.g., by way of replacement or fusion of a heterologous subdomain or other substituted domain. Exemplary gene modifying polypeptides, and systems comprising the same can be used in methods provided herein and described, e.g., in PCT/US2021/020948, which is incorporated herein by reference with respect to gene modifying polypeptides that comprise a retroviral reverse transcriptase domain. In some embodiments, a gene modifying polypeptide integrates a sequence into a gene. In some embodiments, a gene modifying polypeptide integrates a sequence into a sequence outside of a gene. A “gene modifying system,” as used herein, refers to a system comprising a gene modifying polypeptide and a template nucleic acid.
The term “domain,” as used herein, refers to a structure of a biomolecule that contributes to a specified function of the biomolecule. A domain may comprise a contiguous region (e.g., a contiguous sequence) or distinct, non-contiguous regions (e.g., non-contiguous sequences) of a biomolcculc. Examples of protein domains include, but arc not limited to, an endonuclease domain, a DNA binding domain, a reverse transcription domain; an example of a domain of a nucleic acid is a regulatory domain, such as a transcription factor binding domain. In some embodiments, a domain (e.g., a Cas domain) can comprise two or more smaller domains (e.g., a DNA binding domain and an endonuclease domain).
As used herein, the term “exogenous,” when used with reference to a biomolecule (such as a nucleic acid sequence or polypeptide) means that the biomolecule was introduced into a host genome, cell or organism by the hand of man. For example, a nucleic acid that is as added into an existing genome, cell, tissue or subject using recombinant DNA techniques or other methods is exogenous to the existing nucleic acid sequence, cell, tissue or subject.
As used herein, “first strand” and “second strand,” as used to describe the individual DNA strands of target DNA, distinguish the two DNA strands based upon which strand a reverse transcriptase domain initiates polymerization, e.g., based upon where target primed synthesis initiates. A “first strand” refers to the strand of a target DNA upon which a reverse transcriptase domain initiates polymerization, e.g., where target primed synthesis initiates. A “second strand” refers to the other strand of the target DNA. First and second strand designations do not describe a target site DNA strands in other respects; for example, in some embodiments the first and second strands are nicked by a polypeptide described herein, but the designations ‘first’ and ‘second’ strand have no bearing on the order in which such nicks occur.
The term “heterologous,” as used herein to describe a first element in reference to a second element means that a first element and second element do not exist in nature disposed as described. For example, a heterologous polypeptide, nucleic acid molecule, construct or sequence refers to (a) a polypeptide, nucleic acid molecule or portion of a polypeptide or nucleic acid molecule sequence that is not native to a cell in which it is expressed, (b) a polypeptide or nucleic acid molecule or portion of a polypeptide or nucleic acid molecule that has been altered or mutated relative to its native state, or (c) a polypeptide or nucleic acid molecule with an altered expression as compared to the native expression levels under similar conditions. For example, a heterologous regulatory sequence (e.g., promoter, enhancer) may be used to regulate expression of a gene or a nucleic acid molecule in a way that is different than the gene or a nucleic acid molecule is normally expressed in nature. In another example, a heterologous domain of a polypeptide or nucleic acid sequence (e.g., a DNA binding domain of a polypeptide or nucleic acid encoding a DNA binding domain of a polypeptide) may be disposed relative to other domains or may be a different sequence or from a different source, relative to other domains or portions of a polypeptide or its encoding nucleic acid. In certain embodiments, a heterologous nucleic acid molecule may exist in a native host cell genome but may have an altered expression level or have a different sequence or both. In other embodiments, heterologous nucleic acid molecules may not be endogenous to a host cell or host genome but instead may have been introduced into a host cell by transformation (e.g., transfection, electroporation), wherein the added molecule may integrate into the host genome or can exist as extra-chromosomal genetic material either transiently (e.g., mRNA) or semi-stably for more than one generation (e.g., episomal viral vector, plasmid or other self-replicating vector).
As used herein, “insertion” of a sequence into a target site refers to the net addition of DNA sequence at a target site, e.g., where there are new nucleotides in a heterologous object sequence with no cognate positions in the unedited target site. In some embodiments, a nucleotide alignment of a PBS sequence and heterologous object sequence to a target nucleic acid sequence would result in an alignment gap in the target nucleic acid sequence.
As used herein, a “deletion” generated by a heterologous object sequence in a target site refers to the net deletion of DNA sequence at the target site, e.g., where there are nucleotides in the unedited target site with no cognate positions in the heterologous object sequence. In some embodiments, a nucleotide alignment of the PBS sequence and heterologous object sequence to the target nucleic acid sequence would result in an alignment gap in the molecule comprising the PBS sequence and heterologous object sequence.
The term “inverted terminal repeats” or “ITRs” as used herein refers to AAV viral ciselements named so because of their symmetry. These elements promote efficient multiplication of an AAV genome. It is hypothesized that the minimal elements for ITR function are a Repbinding site (RBS; 5'-GCGCGCTCGCTCGCTC-3' for AAV2; SEQ ID NO: 4601) and a terminal resolution site (TRS; 5'-AGTTGG-3' for AAV2; SEQ ID NO: 4602) plus a variable palindromic sequence allowing for hairpin formation. According to the present invention, an ITR comprises at least these three elements (RBS, TRS, and sequences allowing the formation of a hairpin). In addition, in the present invention, the term “ITR” refers to ITRs of known natural AAV serotypes (e.g., ITR of a serotype 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or 11 AAV), to chimeric ITRs formed by the fusion of ITR elements derived from different serotypes, and to functional variants thereof. “Functional variant” refers to a sequence presenting a sequence identity of at least 80%, 85%, 90%, preferably of at least 95% with a known ITR and allowing multiplication of the sequence that includes said ITR in the presence of Rep proteins.
The term “mutation region,” as used herein, refers to a region in a template RNA having one or more sequence difference relative to the corresponding sequence in a target nucleic acid. The one or more sequence difference may comprise, for example, a substitution, insertion, frameshift, or deletion.
The term “mutated” when applied to nucleic acid sequences means that nucleotides in a nucleic acid sequence are inserted, deleted, or changed compared to a reference (e.g., native) nucleic acid sequence. A single alteration may be made at a locus (a point mutation), or multiple nucleotides may be inserted, deleted, or changed at a single locus. In addition, one or more alterations may be made at any number of loci within a nucleic acid sequence. A nucleic acid sequence may be mutated by any method known in the ail.
“Nucleic acid molecule” refers to both RNA and DNA molecules including, without limitation, complementary DNA (“cDNA”), genomic DNA (“gDNA”), and messenger RNA (“mRNA”), and also includes synthetic nucleic acid molecules, such as those that are chemically synthesized or recombinantly produced, such as RNA templates, as described herein. A nucleic acid molecule can be double- stranded or single-stranded, circular’, or linear. If single- stranded, a nucleic acid molecule can be a sense strand or an antisense strand. Unless otherwise indicated, and as an example for all sequences described herein under the general format “SEQ ID NO:,” or “nucleic acid comprising SEQ ID NO: 1” refers to a nucleic acid, at least a portion which has either (i) the sequence of SEQ ID NO: 1, or (ii) a sequence complimentary to SEQ ID NO: 1. The choice between the two is dictated by the context in which SEQ ID NO: 1 is used. For instance, if a nucleic acid is used as a probe, the choice between the two is dictated by the requirement that the probe be complementary to a desired target. Nucleic acid sequences of the present disclosure may be modified chemically or biochemically or may contain non-natural or derivatized nucleotide bases, as will be readily appreciated by those of skill in the art. Such modifications include, for example, labels, methylation, substitution of one or more naturally occurring nucleotides with an analog, inter-nucleotide modifications such as uncharged linkages (for example, methyl phosphonates, phosphotriesters, phosphoramidates, carbamates, etc.), charged linkages (for example, phosphorothioates, phosphorodithioates, etc.), pendant moieties, (for example, polypeptides), intcrcalators (for example, acridine, psoralen, etc.), chelators, alkylators, and modified linkages (for example, alpha anomeric nucleic acids, etc.). Also included are chemically modified bases (see, for example, Table 32), backbones (see, for example, Table 33), and modified caps (see, for example, Table 34). Also included are synthetic molecules that mimic polynucleotides in their ability to bind to a designated sequence via hydrogen bonding and other chemical interactions. Such molecules are known in the art and include, for example, those in which peptide linkages substitute for phosphate linkages in the backbone of a molecule, e.g., peptide nucleic acids (PNAs). Other modifications can include, for example, analogs in which the ribose ring contains a bridging moiety or other structure such as modifications found in “locked” nucleic acids (LNAs). In some embodiments, nucleic acids are in operative association with additional genetic elements, such as tissue-specific expressioncontrol sequence(s) (e.g., tissue- specific promoters and tissue-specific microRNA recognition sequences), as well as additional elements, such as inverted repeats (e.g., inverted terminal repeats, such as elements from or derived from viruses, e.g., AAV ITRs) and tandem repeats, inverted repeats/direct repeats, homology regions (segments with various degrees of homology to a target DNA), untranslated regions (UTRs) (5k 3k or both 5' and 3" UTRs), and various combinations of the foregoing. Nucleic acid elements of systems disclosed in the present application may be provided in a variety of topologies, including single-stranded, doublestranded, circular, linear, linear with open ends, linear with closed ends, and particular versions of these, such as doggybone DNA (dbDNA), and closed-ended DNA (ceDNA).
The term “chemically modified nucleotide,” as used herein, refers to a nucleotide comprising one or more structural differences relative to the canonical ribonucleotides (i.e., G, U, C, and .A). A chemically modified nucleotide may have (relative to a canonical nucleotide) a chemically modified nudeobase, a chemically modified sugar, a chemically modified phosphodiester linkage, or a combination thereof. In some embodiments, a chemically modified nucleotide is a 2'-O-methyl nucleotide, e.g., 2'-O-methyl- Adenosine, 2'-O-methyl-Cytidine, 2'-O- methyl-Guanosine, or 2'-O-methyl-Uridine. No particular process of making is implied: for instance, a chemically modified nucleotide can be produced directly by chemical synthesis, or by covalently modifying a canonical nucleotide. The term “chemical modification,” as used herein, refers to a structural difference of a chemical modified nucleotide relative to die canonical ribonucleotides (i.c., G, U, C. and A). A chemical modification may comprise a modification resulting in a chemically modified nucleobase, a chemically modified sugar, a chemically modified phosphodiester linkage, or a combination thereof. In some embodiments, a chemical modification is 2'-O-methylation or 2’- fluoro modification. No particular process of making is implied; for instance, a chemical modification can be produced directly by chemical synthesis, or by covalently modifying a canonical nucleotide.
As used herein, the term “position” with respect to an StlCas9 scaffold refers to anucleotide of the StlCas9 scaffold that aligns with a corresponding nucleotide of a reference sequence of SEQ ID NO: 25999. The positions of the reference sequence are illustrated in FIG, 14. Alignments of nucleic acid or polypeptide sequences can be performed by using a routine sequence analysis tool such as Basic Local Alignment Search Tool (BLAST), for instance NIH megablast using default parameters.
In some embodiments, a position of an StlCas9 scaffold can be identified by providing an alignment of an StlCas9 scaffold (query sequence) to a reference sequence of SEQ ID NO: 25999 (a full length wild-type sequence, see e.g., FIG 14B) or SEQ ID NO: 26000 (a truncation mutant, see e.g., FIG. 14A), and identifying the position in the query sequence that corresponds to the position in the reference sequence. For example, in an StlCas9 scaffold consisting of the sequence of SEQ ID NO: 25999 except that the 5’ most G is substituted with a single nucleotide other than G, the substituted position is position 1.
As another example, in an StlCas9 scaffold consisting of the sequence of SEQ ID NO: 25999 except that a single new nucleotide is inserted just 5’ of the 5’ most G, the G is still position 1.
As yet another example, in an StICas9 scaffold consisting of the sequence of SEQ ID NO: 25999 except that a sequence of n nucleotides is inserted between the G of position 1 and the U of position 2, nucleotides 3’ of the insert maintain their original position number. For example, the U of position 2 is still position 2 rather than position n+2. A nucleotide that is inserted relative to the reference sequence need not be assigned a position number. A range of nucleotides includes all nucleotides in that range regardless of whether they are assigned a number; for example, if a scaffold comprises a chemically modified nucleotide at each of positions 12 through 21 , and the scaffold comprises inserted nucleotides anywhere between positions 12 and 21, then the scaffold comprises chemically modified nucleotides at each of the inserted nucleotides situated anywhere between positions 12 and 21 (which inserted nucleotides do not have a position number in this example), as well as chemically modified nucleotides at positions 12, 13, 14, 15, 16, 17, 18, 19, 20, and 21.
As used herein, a “gene expression unit” is a nucleic acid sequence comprising at least one regulatory nucleic acid sequence operably linked to at least one effector sequence. A first nucleic acid sequence is operably linked with a second nucleic acid sequence when the first nucleic acid sequence is placed in a functional relationship with the second nucleic acid sequence. For instance, a promoter or enhancer is operably linked to a coding sequence if the promoter or enhancer affects the transcription or expression of the coding sequence. Operably linked DNA sequences may be contiguous or non-contiguous. Where necessary to join two protein-coding regions, operably linked sequences may be in the same reading frame.
The terms “host genome” or “host cell,” as used herein, refer to a cell and/or its genome into which protein and/or genetic material has been introduced. It should be understood that such terms are intended to refer not only to the particular subject cell and/or genome, but to the progeny of such a cell and/or the genome of the progeny of such a cell. Because certain modifications may occur in succeeding generations due to either mutation or environmental influences, such progeny may not, in fact, be identical to the parent cell, but are still included within the scope of the term “host cell” as used herein. A host genome or host cell may be an isolated cell or cell line grown in culture, or genomic material isolated from such a cell or cell line, or may be a host cell or host genome which composing living tissue or an organism. In some embodiments, a host cell may be an animal cell or a plant cell, e.g., as described herein. In some embodiments, a host cell may be a mammalian cell, a human cell, avian cell, reptilian cell, bovine cell, horse cell, pig cell, goat cell, sheep cell, chicken cell, or turkey cell. In some embodiments, a host cell may be a corn cell, soy cell, wheat cell, or rice cell.
As used herein, “operative association” describes a functional relationship between two nucleic acid sequences, such as a 1) promoter and 2) a heterologous object sequence, and means, in such example, the promoter and heterologous object sequence (e.g., a gene of interest) are oriented such that, under suitable conditions, the promoter drives expression of the heterologous object sequence. For instance, a template nucleic acid carrying a promoter and a heterologous object sequence may be single-stranded, e.g., either the (+) or (-) orientation. An “operative association” between the promoter and the heterologous object sequence in this template means that, regardless of whether the template nucleic acid will be transcribed in a particular state, when it is in the suitable state (e.g., is in the (+) orientation, in the presence of required catalytic factors, and NTPs, etc.), it is accurately transcribed. Operative association applies analogously to other pairs of nucleic acids, including other tissue- specific expression control sequences (such as enhancers, repressors and microRNA recognition sequences), IR/DR, ITRs, UTRs, or homology regions and heterologous object sequences or sequences encoding a retroviral RT domain.
The term “primer binding site sequence” or “PBS sequence,” as used herein, refers to a portion of a template RNA capable of binding to a region in a target nucleic acid sequence, hi some embodiments, a PBS sequence is a nucleic acid sequence comprising at least 3, 4, 5, 6, 7, or 8 bases with 100% identity to a region in a target nucleic acid sequence. In some embodiments, a primer region comprises at least 5, 6, 7, 8 bases with 100% identity to a region in a target nucleic acid sequence. Without wishing to be bound by theory, in some embodiments, when a template RNA comprises a PBS sequence and a heterologous object sequence, the PBS sequence binds to a region in a target nucleic acid sequence, allowing a reverse transcriptase domain to use that region as a primer for reverse transcription, and to use the heterologous object sequence as a template for reverse transcription.
As used herein, a “stem-loop sequence” refers to a nucleic acid sequence (e.g., RNA sequence) with sufficient self-complementarity to form a stem-loop, e.g., having a stem comprising at least two (e.g., 3, 4, 5, 6, 7, 8, 9, or 10) base pairs, and a loop with at least three (e.g., four) base pairs. The stem may comprise mismatches or bulges.
As used herein, a “tissue- specific expression-control sequence” means nucleic acid elements that increase or decrease the level of a transcript comprising the heterologous object sequence in a target tissue in a tissue-specific manner, e.g., preferentially in on-target tissue(s), relative to off-target tissue(s). In some embodiments, a tissue- specific expression-control sequence preferentially drives or represses transcription, activity, or the half-life of a transcript comprising the heterologous object sequence in the target tissue in a tissue- specific manner, e.g., preferentially in an on-target tissue(s), relative to an off-target tissue(s). Exemplary tissuespecific expression-control sequences include tissue-specific promoters, repressors, enhancers, or combinations thereof, as well as tissue-specific microRNA recognition sequences. Tissue specificity refers to on-target (tissuc(s) where expression or activity of the template nucleic acid is desired or tolerable) and off-target (tissue(s) where expression or activity of the template nucleic acid is not desired or is not tolerable). For example, a tissue-specific promoter drives expression preferentially in on-target tissues, relative to off-target tissues. In contrast, a microRNA that binds the tissue- specific microRNA recognition sequences is preferentially expressed in off-target tissues, relative to on-target tissues, thereby reducing expression of a template nucleic acid in off-target tissues. Accordingly, a promoter and a microRNA recognition sequence that are specific for the same tissue, such as the target tissue, have contrasting functions (promote and repress, respectively, with concordant expression levels, i.e., high levels of the microRNA in off-target tissues and low levels in on-target tissues, while promoters drive high expression in on-target tissues and low expression in off-target tissues) with regard to the transcription, activity, or half-life of an associated sequence in that tissue.
As used herein, “indel” refers to a mutation resulting from an insertion, deletion, or a combination thereof. As will be appreciated by those skilled in the art, an indel in a coding region of a genomic sequence will result in a frameshift mutation, unless the length of the indel is a multiple of three. In some embodiments, a genetic modification is a point mutation. As used herein, "point mutation" refers to a substitution that replaces one of the nucleotides. A system of the present disclosure can be used to induce an indel of any length or a point mutation in a target polynucleotide sequence.
DETAILED DESCRIPTION
This disclosure provides methods for treating alpha- 1 antitrypsin deficiency (AATD) and compositions for targeting, editing, modifying or manipulating a DNA sequence (e.g., inserting a heterologous object sequence into a target site of a mammalian genome) at one or more locations in a DNA sequence in a cell, tissue or subject, e.g., in vivo or in vitro. A heterologous object DNA sequence may include, e.g., a substitution.
In some embdoients, the disclosure provides methods for treating AATD using reverse transcriptase-based systems for altering a genomic DNA sequence of interest, e.g., by inserting, deleting, or substituting one or more nucleotides into/from a sequence of interest.
In some embodiments, the disclosure provides methods for treating AATD using a gene modifying system comprising a gene modifying polypeptide component and a template nucleic acid (e.g., template RNA) component. Tn some embodiments, a gene modifying system can be used to introduce an alteration into a target site in a genome. In some embodiments, a gene modifying polypeptide component comprises a writing domain (e.g., a reverse transcriptase domain), a DNA-binding domain, and an endonuclease domain (e.g., a nickase domain). In some embodiments, a template nucleic acid (e.g., template RNA) comprises a sequence (e.g., a gRNA spacer) that binds a target site in the genome (e.g., that binds to a second strand of a target site), a sequence (e.g., a gRNA scaffold) that binds a gene modifying polypeptide component, a heterologous object sequence, and a PBS sequence. Without wishing to be bound by theory, it is thought that a template nucleic acid (e.g., template RNA) binds to the second strand of a target site in the genome and binds to a gene modifying polypeptide component (e.g., localizing the gene modifying polypeptide component to the target site in the genome). It is thought that the endonuclease (e.g., nickase) of a gene modifying polypeptide component cuts a target site (e.g., the first strand of the target site), e.g., allowing a PBS sequence to bind to a sequence adjacent to a site to be altered on the first strand of the target site. It is thought that a writing domain (e.g., reverse transcriptase domain) of a gene modifying polypeptide component uses the first strand of a target site that is bound to a complementary sequence comprising a PBS sequence of a template nucleic acid as a primer and a heterologous object sequence of the template nucleic acid as a template to, e.g., polymerize a sequence complementary to the heterologous object sequence. Without wishing to be bound by theory, it is thought that selection of an appropriate heterologous object sequence can result in substitution, deletion, and/or insertion of one or more nucleotides at the target site.
Gene modifying systems
In some embodiments, a gene modifying system described herein comprises: (A) a gene modifying polypeptide or a nucleic acid encoding the gene modifying polypeptide, wherein the gene modifying polypeptide comprises (i) a reverse transcriptase domain, and either (x) an endonuclease domain that contains DNA binding functionality or (y) an endonuclease domain and separate DNA binding domain; and (B) a template RNA. A gene modifying polypeptide, in some embodiments, acts as a substantially autonomous protein machine capable of integrating a template nucleic acid sequence into a target DNA molecule (e.g., in a mammalian host cell, such as a genomic DNA molecule in the host cell), substantially without relying on host machinery. For example, a gene modifying polypeptide may comprise a DNA-binding domain, a reverse transcriptase domain, and an endonuclease domain. In some embodiments, a DNA-binding function may involve an RNA component that directs a gene modifying polypeptide to a DNA sequence, e.g., a gRNA spacer. In other embodiments, a gene modifying polypeptide may comprise a reverse transcriptase domain and an endonuclease domain. An RNA template element of a gene modifying system may be heterologous to a gene modifying polypeptide element and provides an object sequence to be inserted (reverse transcribed) into a host genome. In some embodiments, a gene modifying polypeptide is capable of target primed reverse transcription. In some embodiments, a gene modifying polypeptide is capable of second-strand synthesis.
In some embodiments, a gene modifying system is combined with a second polypeptide. In some embodiments, a second polypeptide may comprise an endonuclease domain. In some embodiments, a second polypeptide may comprise a polymerase domain, e.g., a reverse transcriptase domain. In some embodiments, a second polypeptide may comprise a DNA- dependent DNA polymerase domain. In some embodiments, a second polypeptide aids in completion of a genome edit, e.g., by contributing to second-strand synthesis or DNA repair resolution.
A functional gene modifying polypeptide can be made up of unrelated DNA binding, reverse transcription, and endonuclease domains. This modular structure allows combining of functional domains, e.g., dCas9 (DNA binding), MMLV reverse transcriptase (reverse transcription), FokI (endonuclease). In some embodiments, multiple functional domains may arise from a single protein, e.g., Cas9 or Cas9 nickase (DNA binding, endonuclease).
In some embodiments, a gene modifying polypeptide includes one or more domains that, collectively, facilitate 1) binding a template nucleic acid, 2) binding a target DNA molecule, and 3) integration of at least a portion of the template nucleic acid into the target DNA. In some embodiments, a gene modifying polypeptide is an engineered polypeptide that comprises one or more amino acid substitutions to a corresponding naturally occurring sequence. In some embodiments, a gene modifying polypeptide comprises two or more domains that are heterologous relative to each other, e.g., through a heterologous fusion (or other conjugate) of otherwise wild-type domains, or well as fusions of modified domains, e.g., by way of replacement or fusion of a heterologous sub-domain or other substituted domain. For instance, in some embodiments, one or more of: an RT domain is heterologous to a DNA-binding domain (DBD); a DBD is heterologous to an endonuclease domain; or an RT domain is heterologous to an endonuclease domain.
In some embodiments, a template RNA molecule for use in a system of the present disclosure comprises, from 5' to 3' (1) a gRNA spacer; (2) a gRNA scaffold; (3) a heterologous object sequence; and (4) a primer binding site (PBS) sequence. In some embodiments, a gRNA spacer is about!8 to -22 nucleotides in length (e.g., about 20 nucleotides in length). In some embodiments, a gRNA scaffold comprises one or more hairpin loops, e.g., 1, 2, or 3 loops for associating a template RNA with a Cas domain, e.g., a nickase Cas9 domain. In some embodiments, a gRNA scaffold comprises the sequence, from 5' to 3', GTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAA AGTGGGACCGAGTCGGTCC (SEQ ID NO: 5008). In some embodiments, a heterologous object sequence is, e.g., 7-74, e.g., 10-20, 20-30, 30-40, 40-50, 50-60, 60-70, or 70-80 nucleotides or, 80-90 nucleotdies in length. In some embodiments, a first (i.e., 5 '-most) base of a heterologous object sequence is not C. In some embodiments, a PBS sequence that binds a target priming sequence after nicking occurs is e.g., 3-20 nucleotides, e.g., 7-15 nucleotides, e.g., 12-14 nucleotides in length. In some embodiments, a PBS sequence has 40-60% GC content.
In some embodiments, a second gRNA associated with a system of the present disclosure may help drive complete integration. In some embodiments, a second gRNA may target a location that is 0-200 nucleotides away from a first-strand nick, e.g., 0-50, 50-100, 100-200 nucleotides away from the first-strand nick. In some embodiments, a second gRNA can only bind its target sequence after an edit is made, e.g., the gRNA binds a sequence present in a heterologous object sequence, but not in the initial target sequence.
In some embodiments, a gene modifying system described herein is used to make an edit in HEK293, K562, U2OS, or HeLa cells. In some embodiment, a gene modifying system is used to make an edit in primary cells, e.g., primary liver cells or primary lung cells.
In some embodiments, a gene modifying polypeptide as described herein comprises a reverse transcriptase or RT domain (e.g., as described herein) that comprises a MoMLV RT sequence or variant thereof. In embodiments, a MoMLV RT sequence comprises one or more mutations selected from D200N, L603W, T330P, T306K, W313F, D524G, E562Q, D583N, P51L, S67R, E67K, T197A, H204R, E302K, F309N, L435G, N454K, H594Q, D653N, R110S, and K103L. In some embodiments, a MoMLV RT sequence comprises a combination of mutations, such as D200N, L603W, and T33OP, optionally further including T306K and/or W313F.
In some embodiments, an endonuclease domain (e.g., as described herein) is Cas9. In some embodiments, an endonuclease domain is nCas9. In some embodiments, an endonuclease domain comprises an N863A mutation (e.g., in spCas9). In some embodiments, an endonuclease domain comprises a H840A mutation.
In some embodiments, a heterologous object sequence (e.g., of a system as described herein) is about 1-50, 50-100, 100-200, 200-300, 300-400, 400-500, 500-600, 600-700, 700-800, 800-900, 900-1000, or more, nucleotides in length.
In some embodiments, RT and endonuclease domains are joined by a flexible linker. In some embdoiments, a linker comprises the amino acid sequence SGGSSGGSSGSETPGTSESATPESSGGSSGGSS (SEQ ID NO: 5006).
In some embodiments, an endonuclease domain is N-terminal relative to an RT domain. In some embodiments, an endonuclease domain is C-terminal relative to an RT domain.
In some embodiments, a system of the present disclosure incorporates a heterologous object sequence into a target site by target primed reverse transcription (TPRT), e.g., as described herein.
In some embodiments, a gene modifying polypeptide comprises a DNA binding domain (DBD). In some embodiments, a gene modifying polypeptide comprises an RNA binding domain. In some embodiments, an RNA binding domain comprises an RNA binding domain of B-box protein, MS2 coat protein, dCas, or an element of a sequence of a Table herein. In some embodiments, an RNA binding domain is capable of binding to a template RNA with greater affinity than a reference RNA binding domain.
In some embodiments, a gene modifying system is capable of producing an insertion of at least 45, at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, or at least 100 nucleotides (and optionally no more than 500, no more than 400, no more than 300, no more than 200, or no more than 100 nucleotides) in a target site. In some embodiments, a gene modifying system is capable of producing an insertion of at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, or at least 100 nucleotides (and optionally no more than 500, no more than 400, no more than 300, no more than 200, or no more than 100 nucleotides) in a target site. In some embodiments, a gene modifying system is capable of producing an insertion of at least 0.2, at least 0.3, at least 0.4, at least 0.5, at least 0.6, at least 0.7, at least 0.8, at least 0.9, at least 1, at least 1.5, at least 2, at least 2.5, at least 3, at least 3.5, at least 4, at least 4.5, at least 5, at least 5.5, at least 6, at least 6.5, at least 7, at least 7.5, at least 8, at least 8.5, at least 9, at least 9.5 or at least 10 kilobases (and optionally no more than 1, 5, 10, or 20 kilobases) into a target site. In some embodiments, a gene modifying system is capable of producing a deletion of at least 81, at least 85, at least 90, at least 95, at least 100, at least 110, at least 120, at least 130, at least 140, at least 150, at least 160, at least 170, at least 180, at least 190, or at least 200 nucleotides (and optionally no more than 500, no more than 400, no more than 300, or no more than 200 nucleotides). In some embodiments, a gene modifying system is capable of producing a deletion of at least 81, at least 85, at least 90, at least 95, at least 100, at least 110, at least 120, at least 130, at least 140, at least 150, at least 160, at least 170, at least 180, at least 190, or at least 200 nucleotides (and optionally no more than 500, 400, 300, or 200 nucleotides). In some embodiments, a gene modifying system is capable of producing a deletion of at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, or at least 100 nucleotides (and optionally no more than 500, no more than 400, no more than 300, no more than 200, or no more than 100 nucleotides). In some embodiments, a gene modifying system is capable of producing a deletion of at least 0.2, at least 0.3, at least 0.4, at least 0.5, at least 0.6, at least 0.7, at least 0.8, at least 0.9, at least 1, at least 1.5, at least 2, at least 2.5, at least 3, at least 3.5, at least 4, at least 4.5, at least 5, at least 5.5, at least 6, at least 6.5, at least 7, at least 7.5, at least 8, at least 8.5, at least 9, at least 9.5 or at least 10 kilobases (and optionally no more than 1, 5, 10, or 20 kilobases). In some embodiments, a gene modifying system is capable of producing a substitution of at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, or at least 100, or more nucleotides in a target site. In some embodiments, a gene modifying system is capable of producing a substitution of 1 -2, 2-3, 3-4, 4-5, 5-10, 10-15, 15-20, 20-30, 30-40, 40- 50, 50-60, 60-70, 70-80, 80-90, or 90-100 nucleotides in a target site.
In some embodiments, a substitution is a transition mutation. In some embodiments, a substitution is a transversion mutation. In some embodiments, a substitution converts an adenine to a thymine, an adenine to a guanine, an adenine to a cytosine, a guanine to a thymine, a guanine to a cytosine, a guanine to an adenine, a thymine to a cytosine, a thymine to an adenine, a thymine to a guanine, a cytosine to an adenine, a cytosine to a guanine, or a cytosine to a thymine.
In some embodiments, an insertion, deletion, substitution, or combination thereof, increases or decreases expression (e.g., transcription or translation) of a gene. In some embodiments, an insertion, deletion, substitution, or combination thereof, increases or decreases expression (e.g,. transcription or translation) of a gene by altering, adding, or deleting sequences in a promoter or enhancer, e.g., sequences that bind transcription factors. In some embodiments, an insertion, deletion, substitution, or combination thereof alters translation of a gene (e.g., alters an amino acid sequence), inserts or deletes a start or stop codon, or alters or fixes the translation frame of a gene. In some embodiments, an insertion, deletion, substitution, or combination thereof alters splicing of a gene, e.g., by inserting, deleting, or altering a splice acceptor or donor site. In some embodiments, an insertion, deletion, substitution, or combination thereof alters transcript or protein half-life. In some embodiments, an insertion, deletion, substitution, or combination thereof alters protein localization in the cell (e.g., from the cytoplasm to a mitochondria, from the cytoplasm into the extracellular space (e.g., adds a secretion tag)). In some embodiments, an insertion, deletion, substitution, or combination thereof alters (e.g., improves) protein folding (e.g., to prevent accumulation of misfolded proteins). In some embodiments, an insertion, deletion, substitution, or combination thereof, alters, increases, decreases the activity of a gene, e.g., a protein encoded by the gene.
Exemplary gene modifying polypeptides, systems comprising the same, and methods of using the same are described, e.g., in PCT/US2021/020948, which is incorporated herein by reference with respect to retroviral RT domains, including the amino acid and nucleic acid sequences therein.
Exemplary gene modifying polypeptides and retroviral RT domain sequences are also described, e.g., in International Application No. PCT/US21/20948, filed March 4, 2021, e.g., at Table 30, Table 31 , and Table 44 therein; the entire application is incorporated by reference herein with respect to retroviral RTs, e.g., in said sequences and Tables. Accordingly, a gene modifying polypeptide described herein may comprise an amino acid sequence according to any of the Tables mentioned in this paragraph, or a domain thereof (e.g., a retroviral RT domain), or a functional fragment or variant of any of the foregoing, or an amino acid sequence having at least 70%, 80%, 85%, 90%, 95%, or 99% identity thereto.
In some embodiments, a polypeptide for use in any of the systems described herein can be a molecular reconstruction or ancestral reconstruction based upon the aligned polypeptide sequence of multiple homologous proteins. In some embodiments, a reverse transcriptase domain for use in any of the systems described herein can be a molecular reconstruction or an ancestral reconstruction, or can be modified at particular residues, based upon alignments of reverse transcriptase domains from the same or different sources. A skilled artisan can, based on the Accession numbers provided herein, align polypeptides or nucleic acid sequences, e.g., by using routine sequence analysis tools as Basic Local Alignment Search Tool (BLAST) or CD- Search for conserved domain analysis. Molecular reconstructions can be created based upon sequence consensus, e.g., using approaches described in Ivies et al., Cell 1997, 501 - 510; Wagstaff et al., Molecular Biology and Evolution 2013, 88-99.
Polypeptide components of gene modifying systems
In some embodiments, a gene modifying polypeptide possesses the functions of DNA target site binding, template nucleic acid (e.g., template RNA) binding, DNA target site cleavage, and template nucleic acid (e.g., template RNA) writing (e.g., reverse transcription). In some embodiments, each function is contained within a distinct domain. In some embodiments, a function may be attributed to two or more domains (e.g., two or more domains, together, exhibit the functionality). In some embodiments, two or more domains may have the same or similar function (e.g., two or more domains each independently have DNA-binding functionality, e.g., for two different DNA sequences). In some embodiments, one or more domains may be capable of enabling one or more functions, e.g., a Cas9 domain enabling both DNA binding and target site cleavage. In some embodiments, domains are all located within a single polypeptide. In some embodiments, a first domain is in one polypeptide and a second domain is in a second polypeptide. For example, in some embodiments, sequences may be split between a first polypeptide and a second polypeptide, e.g., wherein the first polypeptide comprises a reverse transcriptase (RT) domain and wherein the second polypeptide comprises a DNA-binding domain and an endonuclease domain, c.g., a nickase domain. As a further example, in some embodiments, a first polypeptide and a second polypeptide each comprise a DNA binding domain (e.g., a first DNA binding domain and a second DNA binding domain). In some embodiments, a first polypeptides and a second polypeptide may be brought together post- translationally via a split-intein to form a single gene modifying polypeptide.
In some embodiments, a gene modifying polypeptide described herein comprises an StlCas9 domain. An StlCas9 domain can comprise a naturally occurring StlCas9 amino acid sequence, or a variant thereof. In some embodiments, an StlCas9 domain is a nickase. In some embodiments, an StlCas9 domain comprises a sequence according to SEQ ID NO: 23818, or a sequence having at least 80%, 85%, 90%, 95%, 98%, or 99% identity thereto. In some embodiments, a gene modifying polypeptide comprising an StlCas9 domain is used together with a compatible template RNA comprising a variant gRNA scaffold described herein.
In some embodiments, a gene modifying polypeptide described herein comprises (e.g., a system described herein comprises a gene modifying polypeptide that comprises): 1) a Cas domain (e.g., a Cas nickase domain, e.g., a Cas9 nickase domain); 2) a reverse transcriptase (RT) domain of Table 7 or Table 8, or a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% identity thereto, wherein the RT domain is C-terminal of the Cas domain; and a linker is disposed between the RT domain and the Cas domain, wherein the linker has a sequence from the same row of Table 7 or Table 8 as the RT domain, or a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% identity thereto.
In some embodiments, an RT domain has a sequence with 100% identity to an RT domain of Table 7 or Table 8and a linker has a sequence with 100% identity to the linker sequence from the same row of Table 7 or Table 8as the RT domain. In some embodiments, a Cas domain comprises a sequence of Table 4, or a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% identity thereto. In some embodiments, a gene modifying polypeptide comprises an amino acid sequence according to any one of SEQ ID NOs: 1-3332 in the sequence listing, or a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% identity thereto.
In some embodiments, a gene modifying polypeptide comprises a GG amino acid sequence between a Cas domain and a linker, an AG amino acid sequence between an RT domain and a second nuclear localization seqeuence (NLS), and/or a GG amino acid sequence between the linker and the RT domain. In some embodiments, a gene modifying polypeptide comprises a sequence of SEQ ID NO: 4000 which comprises a first NLS and a Cas domain, or a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% identity thereto. In some embodiments, a gene modifying polypeptide comprises a sequence of SEQ ID NO: 4001 which comprises a second NLS, or a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% identity thereto.
Writing domain (RT Domain)
In some embodiments, a writing domain of a gene modifying system of the present disclosure possesses reverse transcriptase activity and is also referred to as a reverse transcriptase domain (an RT domain). In some embodiments, an RT domain comprises an RT catalytic portion and an RNA-binding region (c.g., a region that binds a template RNA).
In some embodiments, a nucleic acid encoding a reverse transcriptase is altered from its natural sequence to have altered codon usage, e.g., improved for human cells. In some embodiments a reverse transcriptase domain is a heterologous reverse transcriptase from a retrovirus. In some embodiments, an RT domain has been mutated from its original amino acid sequence, e.g., has at least about 1, at least about 2, at least about 3, at least about 4, at least about 5, at least about 6, at least about 7, at least about 8, at least about 9, at least about 10, at least about 20, at least about 30, at least about 40, at least about 50, at least about 60, at least about 70, at least about 80, at least about 90, or at least about 100 substitutions. In some embodiments, an RT domain is derived from an RT of a retrovirus, e.g., HIV-1 RT, Moloney Murine Leukemia Virus (MMLV) RT, avian myeloblastosis virus (AMV) RT, or Rous Sarcoma Virus (RSV) RT.
In some embodiments, a retroviral reverse transcriptase (RT) domain exhibits enhanced stringency of target-primed reverse transcription (TPRT) initiation, e.g., relative to an endogenous RT domain. In some embodiments, an RT domain initiates TPRT when the 3 nucleotides in a target site immediately upstream of a first strand nick, e.g., genomic DNA priming of an RNA template, have at least 66% or 100% complementarity to 3 nucleotides of homology in the RNA template. In some embodiments, an RT domain initiates TPRT when there are less than 5 nucleotides mismatched (e.g., less than 1, less than 2, less than 3, less than 4, or less than 5 nt mismatched) between an RNA template and a target DNA priming reverse transcription. In some embodiments, an RT domain is modified such that the stringency for mismatches in priming a TPRT reaction is increased, e.g., wherein the RT domain does not tolerate any mismatches or tolerates fewer mismatches in a priming region relative to a wild-type (e.g., unmodified) RT domain. In some embodiments, an RT domain comprises a HIV-1 RT domain. In embodiments, an HIV-1 RT domain initiates lower levels of synthesis even with three nucleotide mismatches relative to an alternative RT domain (e.g., as described by Jamburuthugoda and Eickbush J Mol Biol 407(5):661-672 (2011); incorporated herein by reference in its entirety). In some embodiments, an RT domain forms a dimer (e.g., a heterodimer or homodimer). In some embodiments, an RT domain is monomeric. In some embodiments, an RT domain naturally functions as a monomer or as a dimer (e.g., heterodimer or homodimer). In some embodiments, an RT domain naturally functions as a monomer, e.g., is derived from a virus wherein it functions as a monomer. In embodiments, an RT domain is selected from an RT domain from murine leukemia virus (MLV ; sometimes referred to as MoMLV) (e.g., P03355), porcine endogenous retrovirus (PERV) (e.g., UniProt Q4VFZ2), mouse mammary tumor virus (MMTV) (e.g., UniProt P03365), Avian reticuloendotheliosis vims (AVIRE) (e.g., UniProtKB accession: P03360); Feline leukemia virus (FLV or FeLV) (e.g., e.g., UniProtKB accession: P10273); Mason-Pfizer monkey vims (MPMV) (e.g., UniProt P07572), bovine leukemia virus (BLV) (e.g., UniProt P03361), human T-cell leukemia virus-1 (HTLV-1) (e.g., UniProt P03362), human foamy virus (HFV) (e.g., UniProt P14350), simian foamy vims (SFV) (e.g., SFV3L) (e.g., UniProt P23074 or P27401), or bovine foamy/syncytial virus (BFV/BSV) (e.g., UniProt 041894), or a functional fragment or variant thereof (e.g., an amino acid sequence having at least 70%, at least 80%, at least 90%, at least 95%, or at least 99% identity thereto). In some embodiments, an RT domain is dimeric in its natural functioning. In some embodiments, an RT domain is derived from a virus wherein it functions as a dimer. In embodiments, an RT domain is selected from an RT domain from avian sarcoma/leukemia vims (ASLV) (e.g., UniProt A0A142BKH1), Rous sarcoma virus (RSV) (e.g., UniProt P03354), avian myeloblastosis vims (AMV) (e.g., UniProt Q83133), human immunodeficiency vims type I (HIV-1) (e.g., UniProt P03369), human immunodeficiency virus type II (HIV-2) (e.g., UniProt P15833), simian immunodeficiency virus (SIV) (e.g., UniProt P05896), bovine immunodeficiency vims (BIV) (e.g., UniProt P19560), equine infectious anemia virus (EIAV) (e.g., UniProt P03371), or feline immunodeficiency vims (FIV) (e.g., UniProt P16088) (Herschhom and Hizi Cell Mol Life Sci 67(16):2717-2747 (2010)), or a functional fragment or variant thereof (e.g., an amino acid sequence having at least 70%, at least 80%, at least 90%, at least 95%, or at least 99% identity thereto). Naturally heterodimeric RT domains may, in some embodiments, also be functional as homodimers. In some embodiments, dimeric RT domains are expressed as fusion proteins, e.g., as homodimeric fusion proteins or heterodimeric fusion proteins. In some embodiments, an RT function ofa system of the present disclosure is fulfilled by multiple RT domains (e.g., as described herein). In further embodiments, multiple RT domains are fused or separate, e.g., may be on the same polypeptide or on different polypeptides.
In some embodiments, a gene modifying system described herein comprises an integrase domain, e.g., wherein the integrase domain may be part of an RT domain. In some embodiments, an RT domain (e.g., as described herein) comprises an integrase domain. In some embodiments, an RT domain (c.g., as described herein) lacks an integrase domain, or comprises an integrase domain that has been inactivated by mutation or deleted. In some embodiment, a gene modifying system described herein comprises an RNase H domain, e.g., wherein the RNase H domain may be part of an RT domain. In some embodiments, an RNase H domain is not part of an RT domain and is covalently linked via a flexible linker. In some embodiments, an RT domain (e.g., as described herein) comprises an RNase H domain, e.g., an endogenous RNAse H domain or a heterologous RNase H domain. In some embodiments, an RT domain (e.g., as described herein) lacks an RNase H domain. In some embodiments, an RT domain (e.g., as described herein) comprises an RNase H domain that has been added, deleted, mutated, or swapped for a heterologous RNase H domain. In some embodiments, a gene modifying polypeptide comprises an inactivated endogenous RNase H domain. In some embodiments, an endogenous RNase H domain of a polypeptide is genetically removed such that it is not included in the polypeptide, e.g., the endogenous RNase H domain is partially or completely truncated from the polypeptide. In some embodiments, one or more mutations of an RNase H domain yields a polypeptide exhibiting lower RNase activity, e.g., as determined by the methods described in Kotewicz et al. Nucleic Acids Res 16( 1 ) :265-277 (1988) (incorporated herein by reference in its entirety), e.g., lower by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% compared to an otherwise similar domain without the one or more mutations. In some embodiments, RNase H activity of a gene modifying polypeptide is abolished.
In some embodiments, an RT domain is mutated to increase fidelity compared to an otherwise similar domain without the mutation. In some embodiments, a YADD or YMDD motif in an RT domain (e.g., in a reverse transcriptase) is replaced with YVDD. In some embodiments, replacement of a YADD, YMDD, or YVDD motif results in higher fidelity in retroviral reverse transcriptase activity (e.g., as described in Jamburuthugoda and Eickbush J Mol Biol 2011; incorporated herein by reference in its entirety).
In some embodiments, a gene modifying polypeptide described herein comprises an RT domain having an amino acid sequence according to any RT domain described in Table 1, or a sequence having at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, or at least 99% identity thereto. In some embodiments, a nucleic acid described herein encodes an RT domain having an amino acid sequence according to any RT domain described in Table 1, or a sequence having at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity thereto.
Table 1: Exemplary reverse transcriptase domains from retroviruses
In some embodiments, an RT domain described herein is modified, for example, by sitespecific mutation. In some embodiments, an RT domain is engineered to have improved properties, e.g. SuperScript IV (SSIV) RT derived from the MMLV RT. In some embodiments, an RT domain may be engineered to have lower error rates as compared to a reference RT domain, e.g., as described in W02001068895, incorporated herein by reference. In some embodiments, an RT domain may be engineered to be more thermostable as compared to a reference RT domain. In some embodiments, an RT domain may be engineered to be more processive as compared to a reference RT domain. In some embodiments, an RT domain may be engineered to have improved tolerance to inhibitors as compared to a reference RT domain. In some embodiments, an RT domain may be engineered to be faster as compared to a reference RT domain. In some embodiments, an RT domain may be engineered to better tolerate modified nucleotides in an RNA template as compared to a reference RT domain. In some embodiments, an RT domain may be engineered to be capable of inserting modified DNA nucleotides. In some embodiments, an RT domain is engineered to bind a template RNA. In some embodiments, one or more mutations are chosen from D200N, L603W, T33OP, D524G, E562Q, D583N, P51L, S67R, E67K, T197A, H204R, E302K, F309N, W313F, L435G, N454K, H594Q, L671P, E69K, H8Y, T306K, or D653N in an RT domain of murine leukemia virus reverse transcriptase or a corresponding mutation at a corresponding position of another RT domain.
In some embodiments, a gene modifying polypeptide comprises an RT domain from a retroviral reverse transcriptase, e.g., a wild-type M-MLV RT, e.g., comprising the following sequence:
In some embodiments, a gene modifying polypeptide comprises an RT domain from a retroviral reverse transcriptase comprising the sequence of amino acids 659-1329 of NP_057933. In some embodiments, a gene modifying polypeptide further comprises one additional amino acid at the N-terminus of the sequence of amino acids 659-1329 of NP_057933, e.g., as shown below:
In some embodiments, a gene modifying polypeptide further comprises one additional amino acid at the C-terminus of the sequence of amino acids 659-1329 of NP_057933. In embodiments, a gene modifying polypeptide comprises an RNaseHl domain (e.g., amino acids 1178-1318 of NP_057933).
In some embodiments, a retroviral reverse transcriptase domain, e.g., M-MLV RT, may comprise one or more mutations from a wild-type sequence that may improve features of the RT, e.g., thermostability, processivity, and/or template binding. In some embodiments, an M-MLV RT domain comprises, relative to the M-MLV (WT) sequence above, one or more mutations, e.g., selected from D200N, L603W, T330P, T306K, W313F, D524G, E562Q, D583N, P51L, S67R, E67K, T197A, H204R, E302K, F309N, L435G, N454K, H594Q, D653N, R110S, K103L, or a combination thereof. In some embodiments, an M-MLV RT domain comprises, relative to the M-MLV (WT) sequence above, a combination of mutations including D200N, L603W, and T330P, and optionally further including T306K and W313F. In some embodiments, an M-MLV RT used herein comprises D200N, L603W, T33OP, T306K and W313F mutations. In some embodiments, a mutant M-MLV RT comprises the following amino acid sequence:
In some embodiments, a writing domain (e.g., an RT domain) comprises an RNA-binding domain, e.g., that specifically binds to an RNA sequence. In some embodiments, a template
RNA comprises an RNA sequence that is specifically bound by an RNA-binding domain of a writing domain (e.g., an RT domain).
In some embodiments, an RT domain only recognizes and reverse transcribes a specific template, e.g., a template RNA of a system of the preset disclosure. In some embodiments, a template RNA comprises a sequence or structure that enables recognition and reverse transcription by a reverse transcription domain. In some embodiments, a template RNA comprises a sequence or structure that enables association with an RNA-binding domain of a gene modifying polypeptide component of a system described herein. In some embodiments, a system of the present disclosure preferably reverse transcribes a template comprising an association sequence over a template lacking an association sequence.
In some embodiments, a writing domain (e.g., and RT domain) may also comprise DNA- dependent DNA polymerase activity, e.g., comprise enzymatic activity capable of writing DNA into the genome from a template DNA sequence. In some embodiments, DNA-dependent DNA polymerization is employed to complete second-strand synthesis of a target site edit. In some embodiments, DNA-dependent DNA polymerase activity is provided by a DNA polymerase domain in a gene modifying polypeptide. In some embodiments, DNA-dependent DNA polymerase activity is provided by an RT domain that is also capable of DNA-dependent DNA polymerization, e.g., second-strand synthesis. In some embodiments, DNA-dependent DNA polymerase activity is provided by a second polypeptide of a system of the present disclosure. In some embodiments, DNA-dependent DNA polymerase activity is provided by an endogenous host cell polymerase that is optionally recruited to a target site by a component of a system of the present disclosure.
In some embodiments, an RT domain has a lower probability of premature termination rate (Poff) in vitro relative to a reference RT domain. In some embodiments, a reference RT domain is a viral RT domain, e.g., the RT domain from M-MLV.
In some embodiments, an RT domain has a lower probability of premature termination rate (Poff) in vitro of less than about 5 x 10-3 /nucleotides, less than about 5 x 10-4/ nucleotides, or less than about 5 x 10-6/ nucleotides, e.g., as measured on a 1094 nucleotide RNA. In some embodiments, an in vitro premature termination rate is determined as described in Bibillo and Eickbush (2002) J Biol Chem 277(38):34836-34845 (incorporated by reference herein its entirety).
In some embodiments, an RT domain is able to complete at least about 30% or 50% of integrations in cells. The percent of complete integrations can be measured by dividing the number of substantially full-length integration events (e.g., genomic sites that comprise at least 98% of the expected integrated sequence) by the number of total (including substantially full- length and partial) integration events in a population of cells. In some embodiments, the integrations in cells is determined (e.g., across the integration site) using long-read amplicon sequencing, e.g., as described in Karst et al. (2020) bioRxiv doi.org/10.1101/645903 (incorporated by reference herein in its entirety).
In some embodiments, quantifying integrations in cells comprises counting the fraction of integrations that contain at least about 75%, about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99%, or 100% of the DNA sequence corresponding to a template RNA (e.g., a template RNA having a length of at least 0.05, at least 0.1, at least 0.5, at least 0.6, at least 0.7, at least 0.8, at least 0.9, at least 1, at least 1.5, at least 2, at least 3, at least 4, or at least 5 kb, e.g., a length between 0.5-0.6, 0.6-0.7, 0.7-0.8, 0.8-0.9, 1.0-1.2, 1.2-1.4, 1.4- 1.6, 1.6-1.8, 1.8-2.0, 2-3, 3-4, or 4-5 kb).
In some embodiments, an RT domain is capable of polymerizing dNTPs in vitro. In some embodiments, an RT domain is capable of polymerizing dNTPs in vitro at a rate between 0.1 - 50 nucleotides/sec (e.g., between 0.1-1 , 1- 10, or 10-50 nucleotides/sec). In some embodiments, polymerization of dNTPs by an RT domain is measured by a single-molecule assay, e.g., as described in Schwartz and Quake (2009) PNAS 106(48):20294- 20299 (incorporated by reference in its entirety).
In some embodiments, an RT domain has an in vitro error rate (e.g., misincorporation of nucleotides) of between 1 x 10’3 - l x 10-4 or 1 x 10'4 - 1 x 10'5 substitutions/nucleotide, e.g., as described in Yasukawa et al. (2017) Biochem Biophys Res Commun 492(2): 147- 153 (incorporated herein by reference in its entirety). In some embodiments, an RT domain has an error rate (e.g., misincorporation of nucleotides) in cells (e.g., HEK293T cells, primary liver, or primary lung cells) of between 1 x 10-3 - l x 10-4 or 1 x 10'4 - l 10-5 substitutions/nucleotides, e.g., by long-read amplicon sequencing, e.g., as described in Karst et al. (2020) bioRxiv doi.org/10.1101/645903 (incorporated by reference herein in its entirety).
In some embodiments, an RT domain is capable of performing reverse transcription of a target RNA in vitro. In some embodiments, an RT domain requires a primer of at least 3 nucleotides to initiate reverse transcription of a template. In some embodiments, reverse transcription of a target RNA is determined by detection of cDNA from the target RNA (e.g., when provided with a ssDNA primer, e.g., which anneals to a target with at least 3, 4, 5, 6, 7, 8, 9, or 10 nt at the 3" end), e.g., as described in Bibillo and Eickbush (2002) J Biol Chem 277(38):34836-34845 (incorporated herein by reference in its entirety).
In some embodiments, an RT domain performs reverse transcription at least 5 or 10 times more efficiently (e.g., by cDNA production), e.g., when converting its RNA template to cDNA, for example, as compared to an RNA template lacking a protein binding motif (e.g., a 3' UTR). In some embodiments, efficiency of reverse transcription is measured as described in Yasukawa et al. (2017) Biochem Biophys Res Commun 492(2): 147- 153 (incorporated by reference herein in its entirety).
In some embodiments, an RT domain specifically binds a specific RNA template with higher frequency (e.g., about 5 or 10-fold higher frequency) than any endogenous cellular RNA, e.g., when expressed in cells (e.g., HEK293T cells, primary liver cells, or primary lung cells). In some embodiments, frequency of specific binding between an RT domain and a template RNA is measured by CLIP-seq, e.g., as described in Lin and Miles (2019) Nucleic Acids Res 47(l l):5490-5501 (incorporated herein by reference in its entirety). Template nucleic acid binding domain
In some embodiments, a gene modifying polypeptide contains regions capable of associating with a template nucleic acid (e.g., template RNA). In some embodiments, a template nucleic acid binding domain is an RNA binding domain. In some embodiments, an RNA binding domain is a modular domain that can associate with RNA molecules containing specific signatures, e.g., structural motifs. In some embodiments, a template nucleic acid binding domain (e.g., RNA binding domain) is contained within an RT domain, e.g., the reverse transcriptase- derived component has a known signature for RNA preference.
In some embodiments, a template nucleic acid binding domain (e.g., RNA binding domain) is contained within a target DNA binding domain. For example, in some embodiments, a DNA binding domain is a CRIS PR-associated protein that recognizes the structure of a template nucleic acid (e.g., a template RNA) comprising a gRNA. In some embodiments, a gene modifying polypeptide comprises a DNA-binding domain comprising a CRISPR-associated protein that associates with a gRNA scaffold that allows the DNA-binding domain to bind a target genomic DNA sequence. In some embodiments, a gRNA scaffold and a gRNA spacer is comprised within a template nucleic acid (e.g., template RNA), thus, in some embodiments, a DNA-binding domain is also a template nucleic acid binding domain. In some embodiments, a gene modifying polypeptide possesses RNA binding function in multiple domains, e.g., can bind a gRNA structure in a CRISPR-associated DNA binding domain and an additional sequence or structure in an RT domain.
In some embodiments, an RNA binding domain is capable of binding to a template RNA with greater affinity than a reference RNA binding domain. In some embodiments, a reference RNA binding domain is an RNA binding domain from Cas9 of S. pyogenes. In some embodiments, an RNA binding domain is capable of binding to a template RNA with an affinity between 100 pM - 10 nM (e.g., between 100 pM-I nM or I nM - 10 nM). In some embodiments, the affinity of an RNA binding domain for a template RNA is measured in vitro, e.g., by thermophoresis, e.g., as described in Asmari et al. Methods 146:107-119 (2018) (incorporated by reference herein in its entirety). In some embodiments, the affinity of an RNA binding domain for a template RNA is measured in cells (e.g., by FRET or CLIP-Seq).
In some embodiments, an RNA binding domain is associated with a template RNA in vitro at a frequency at least about 5-fold higher or at least about 10-fold higher than with a scrambled RNA. In some embodiments, the frequency of association between an RNA binding domain and a template RNA or scrambled RNA is measured by CLIP-scq, c.g., as described in Lin and Miles (2019) Nucleic Acids Res 47(11 ) :5490-5501 (incorporated by reference herein in its entirety). In some embodiments, an RNA binding domain is associated with a template RNA in cells (e.g., in HEK293T cells, primary liver cells, or primary lung cells) at a frequency at least about 5-fold or at least about 10-fold higher than with a scrambled RNA. In some embodiments, the frequency of association between an RNA binding domain and a template RNA or scrambled RNA is measured by CLIP-seq, e.g., as described in Lin and Miles (2019), supra.
In some embodiments, an RT domain (e.g., as listed in Table 1) comprises one or more mutations as listed in Table 2A below. In some embodiment, an RT domain as listed in Table 1 comprises one, two, three, four, five, or six of the mutations listed in the corresponding row of Table 2 below.
Table 2: Exemplary RT domain mutations (relative to corresponding wild-type sequences as listed in the corresponding row of Table 1)
Endonuclease domains and DNA binding domains In some embodiments, a gene modifying polypeptide possesses the function of DNA target site cleavage via an endonuclease domain. In some embodiments, a gene modifying polypeptide comprises a DNA binding domain, e.g., for binding to a target nucleic acid. In some embodiments, a domain (e.g., a Cas domain) of a gene modifying polypeptide comprises two or more smaller domains, e.g., a DNA binding domain and an endonuclease domain. It is understood that when a DNA binding domain (e.g., a Cas domain) is said to bind to a target nucleic acid sequence, in some embodiments, the binding is mediated by a gRNA.
In some embodiments, a domain has two or more functions. For example, in some embodiments, the endonuclease domain is also a DNA-binding domain. In some embodiments, an endonuclease domain is also a template nucleic acid (e.g., a template RNA) binding domain. For example, in some embodiments, a gene modifying polypeptide comprises a CRISPR- associated endonuclease domain that binds a template RNA comprising a gRNA, binds a target DNA sequence (e.g., with complementarity to a portion of the gRNA), and cuts the target DNA sequence. In some embodiments, an endonuclease domain or endonuclease/DNA-binding domain from a heterologous source can be used or can be modified (e.g., by insertion, deletion, or substitution of one or more residues) in a gene modifying system described herein.
In some embodiments, a nucleic acid encoding an endonuclease domain or endonuclease/DNA binding domain is altered from its natural sequence to have altered codon usage, e.g. improved for human cells. In some embodiments, the endonuclease element is a heterologous endonuclease element, such as a Cas endonuclease (e.g., Cas9), a type-II restriction endonuclease (e.g., Fokl), a meganuclease (e.g., I-Scel), or other endonuclease domain.
In some embodiments, a DNA-binding domain of a gene modifying polypeptide described herein is selected, designed, or constructed for binding to a desired host DNA target sequence. In some embodiments, a DNA-binding domain of a gene modifying polypeptide is a heterologous DNA-binding element. In some embodiments a heterologous DNA binding element is a zinc-finger element or a TAL effector element, e.g., a zinc-finger or TAL polypeptide or functional fragment thereof. In some embodiments a heterologous DNA binding element is a sequence-guided DNA binding element, such as Cas9, Cpfl, or other CRISPR- related protein that has been altered to have no endonuclease activity. In some embodiments, a heterologous DNA binding element retains endonuclease activity. In some embodiments, a heterologous DNA binding element retains partial endonuclease activity to cleave ssDNA, e.g., possesses nickase activity. In some embodiments, a heterologous DNA-binding domain comprises a Cas9 domain, a TAL domain, a ZF domain, a Myb domain, a combination thereof, or multiples thereof.
In some embodiments, a DNA-binding domain is modified, for example by site-specific mutation, increasing or decreasing DNA-binding elements (for example, number and/or specificity of zinc fingers), etc., to alter DNA-binding specificity and affinity. In some embodiments a nucleic acid sequence encoding a DNA binding domain is altered from its natural sequence to have altered codon usage, e.g., improved for human cell expression. In some embodiments, a DNA binding domain comprises one or more modifications relative to a wildtype DNA binding domain, e.g., a modification via directed evolution, e.g., phage-assisted continuous evolution (PACE).
In some embodiments, a DNA binding domain comprises a meganuclease domain (e.g., as described herein), or a functional fragment thereof. In some embodiments, a meganuclease domain possesses endonuclease activity, e.g., double-strand cleavage and/or nickase activity. In some embodiments, a meganuclease domain has reduced activity, e.g., lacks endonuclease activity, e.g., the meganuclease is catalytically inactive. In some embodiments, a catalytically inactive meganuclease is used as a DNA binding domain, e.g., as described in Fonfara et al. Nucleic Acids Res 40(2):847-860 (2012), incorporated herein by reference in its entirety.
In some embodiments, a gene modifying polypeptide comprises a modification to a DNA-binding domain, e.g., relative to the wild-type polypeptide. In some embodiments, a DNA-binding domain comprises an addition, deletion, replacement, or modification to the amino acid sequence of the original DNA-binding domain. In some embodiments, a DNA-binding domain is modified to include a heterologous functional domain that binds specifically to a target nucleic acid (e.g., DNA) sequence of interest. In some embodiments, a functional domain replaces at least a portion (e.g., the entirety of) the prior DNA-binding domain of the gene modifying polypeptide. In some embodiments, a functional domain comprises a zinc finger (e.g., a zinc finger that specifically binds to the target nucleic acid (e.g., DNA) sequence of interest. In some embodiments, a functional domain comprises a Cas domain (e.g., a Cas domain that specifically binds to a target nucleic acid (e.g., DNA) sequence of interest. In some embodiments, a Cas domain comprises a Cas9 or a mutant or a variant thereof (e.g., as described herein). In some embodiments, a Cas domain is associated with a guide RNA (gRNA), e.g., as described herein. In some embodiments, a Cas domain is directed to a target nucleic acid (e.g., DNA) sequence of interest by a gRNA. In embodiments, a Cas domain is encoded by the same nucleic acid (e.g., RNA) molecule as a gRNA. In some embodiments, a Cas domain is encoded by a different nucleic acid (e.g., RNA) molecule from the gRNA.
In some embodiments, a DNA binding domain is capable of binding to a target sequence (e.g., a dsDNA target sequence) with greater affinity than a reference DNA binding domain. In some embodiments, a reference DNA binding domain is a DNA binding domain from Cas9 of S. pyogenes. In some embodiments, a DNA binding domain is capable of binding to a target sequence (e.g., a dsDNA target sequence) with an affinity between 100 pM - 10 nM (e.g., between 100 pM-1 nM or 1 nM - 10 nM).
In some embodiments, the affinity of a DNA binding domain for a target sequence (e.g., dsDNA target sequence) is measured in vitro, e.g., by thermophoresis, e.g., as described in Asmari et al. Methods 146:107-119 (2018) (incorporated by reference herein in its entirety).
In some embodiments, a DNA binding domain is capable of binding to a target sequence (e.g., dsDNA target sequence), e.g, with an affinity between 100 pM - 10 nM (e.g., between 100 pM-1 nM or 1 nM - 10 nM) in the presence of a molar excess of scrambled sequence competitor dsDNA, e.g., of about 100-fold molar excess.
In some embodiments, a DNA binding domain is found associated with a target sequence (e.g., a dsDNA target sequence) more frequently than any other sequence in the genome of a target cell, e.g., human target cell, e.g., as measured by ChlP-seq (e.g., in HEK293T cells), e.g., as described in He and Pu (2010) Curr. Protoc Mol Biol Chapter 21 (incorporated herein by reference in its entirety). In some embodiments, a DNA binding domain is found associated with a target sequence (e.g., a dsDNA target sequence) at least about 5-fold or at least about 10-fold more frequently than any other sequence in the genome of a target cell, e.g., as measured by ChlP-seq (e.g., in HEK293T cells), e.g., as described in He and Pu (2010), supra.
In some embodiments, an endonuclease domain has nickase activity and cleaves one strand of a target DNA. In some embodiments, nickase activity reduces the formation of doublestranded breaks at a target site. In some embodiments, an endonuclease domain creates a staggered nick structure in the first and second strands of a target DNA. In some embodiments, a staggered nick structure generates free 3’ overhangs at a target site. In some embodiments, free 3’ overhangs at a target site improve editing efficiency, e.g., by enhancing access and annealing of a 3’ homology region of a template nucleic acid. In some embodiments, a staggered nick structure reduces the formation of double-stranded breaks at a target site.
In some embodiments, an endonuclease domain cleaves both strands of a target DNA, e.g., results in blunt-end cleavage of a target with no ssDNA overhangs on either side of the cutsite. The amino acid sequence of an endonuclease domain of a gene modifying system described herein may be at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% identical to the amino acid sequence of an endonuclease domain described herein, e.g., an endonuclease domain described in Table 4.
In some embodiments, a heterologous endonuclease is Fokl or a functional fragment thereof. In some embodiments, a heterologous endonuclease is a Holliday junction resolvase or homolog thereof, such as the Holliday junction resolving enzyme from Sulfolobus solfataricus — Ssol Hje (Govindaraju et al., Nucleic Acids Research 44:7, 2016). In some embodiments, a heterologous endonuclease is an endonuclease of a large fragment of a spliceosomal protein, such as Prp8 (Mahbub et al., Mobile DNA 8:16, 2017). In some embodiments, a heterologous endonuclease is derived from a CRIS PR-associated protein, e.g., Cas9. In some embodiments, a heterologous endonuclease is engineered to have only ssDNA cleavage activity, e.g., only nickase activity, e.g., be a Cas9 nickase, e.g., SpCas9 with D10A, H840A, or N863A mutations. Table 4 provides exemplary Cas proteins and mutations associated with nickase activity. In some embodiments, an endonuclease domain is modified, for example by site-specific mutation, to alter DNA endonuclease activity. In some embodiments, an endonuclease domain is modified to reduce DNA-sequence specificity, e.g., by truncation to remove domains that confer DNA- sequence specificity or mutation to inactivate regions conferring DNA-sequence specificity.
In some embodiments, an endonuclease domain has nickase activity and does not form double-stranded breaks. In some embodiments, an endonuclease domain forms single-stranded breaks at a higher frequency than double-stranded breaks, e.g., at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% of the breaks are single-stranded breaks, or less than 10%, less than 5%, less than 4%, less than 3%, less than 2%, or less than 1% of the breaks are double-stranded breaks. In some embodiments, an endonuclease domain forms substantially no double- stranded breaks. In some embodiments, an endonuclease domain does not form detectable levels of double- stranded breaks. In some embodiments, an endonuclease domain has nickase activity that nicks the first strand of a target site DNA. In some embodiments, an endonuclease domain cuts the genomic DNA of a target site near to the site of alteration on the strand that will be extended by a writing domain (e.g., an RT domain). In some embodiments, an endonuclease domain has nickase activity that nicks the first strand of a target site DNA and does not nick the second strand of the target site DNA. For example, when a gene modifying polypeptide comprises a CRISPR- associated endonuclease domain having nickase activity, in some embodiments, said CRISPR- associated endonuclease domain nicks a target site DNA strand containing a PAM site (e.g., and does not nick the target site DNA strand that does not contain the PAM site). As a further example, when a gene modifying polypeptide comprises a CRISPR-associated endonuclease domain having nickase activity, in some embodiments, said CRISPR-associated endonuclease domain nicks a target site DNA strand that does not containa PAM site (e.g., and does not nick the target site DNA strand that contains the PAM site).
In some embodiments, an endonuclease domain has nickase activity that nicks the first strand and the second strand of a target site DNA. Without wishing to be bound by any particular theory, after a writing domain (e.g., an RT domain) of a gene modifying polypeptide described herein polymerizes (e.g., reverse transcribes) from a heterologous object sequence of a template nucleic acid (e.g., a template RNA), the cellular DNA repair machinery must repair the nick on the first DNA strand. The target site DNA now contains two different sequences for the first DNA strand: one corresponding to the original genomic DNA (e.g., having a free 5' end) and a second corresponding to that polymerized from the heterologous object sequence (e.g., having a free 3' end). It is thought that the two different sequences equilibrate with one another, first one hybridizing the second strand, then the other, and which sequence the cellular DNA repair apparatus incorporates into its repaired target site may be a stochastic process. Without wishing to be bound by any particular theory, it is thought that introducing an additional nick to the second-strand may bias the cellular DNA repair machinery to adopt the heterologous object sequence-based sequence more frequently than the original genomic sequence (Anzalone et al. Nature 576:149-157 (2019)). In some embodiments, an additional nick is positioned at least 10, at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 100, at least 105, at least 110, at least 115, at least 120, at least 125, at least 130, at least 135, at least 140, at least 145, or at least 150 nucleotides 5' or 3' of a target site modification (e.g., an insertion, deletion, or substitution) or to a nick on the first strand.
Alternatively or additionally, without wishing to be bound by theory, it is thought that an additional nick to the second strand may promote second- strand synthesis. In some embodiments, where a system of the present disclosure has inserted or substituted a portion of a first strand, synthesis of a new sequence corresponding to the insertion/substitution in the second strand is necessary.
In some embodiments, a gene modifying polypeptide comprises a single domain having endonuclease activity (e.g., a single endonuclease domain) and said domain nicks both the first strand and the second strand. For example, in some embodiments an endonuclease domain may be a CRISPR-associated endonuclease domain, and a template nucleic acid (e.g., template RNA) comprises a gRNA spacer that directs nicking of the first strand and an additional gRNA spacer that directs nicking of the second strand. In some embodiments, a gene modifying polypeptide comprises a plurality of domains having endonuclease activity, and a first endonuclease domain nicks the first strand and a second endonuclease domain nicks the second strand (optionally, the first endonuclease domain does not (e.g., cannot) nick the second strand and the second endonuclease domain does not (e.g., cannot) nick the first strand).
In some embodiments, an endonuclease domain is capable of nicking a first strand and a second strand. In some embodiments, first and second strand nicks occur at the same position in a target site but on opposite strands. In some embodiments, a second strand nick occurs in a staggered location, e.g., upstream or downstream, from a first nick. In some embodiments, an endonuclease domain generates a target site deletion if a second strand nick is upstream of a first strand nick. In some embodiments, an endonuclease domain generates a target site duplication if a second strand nick is downstream of a first strand nick. In some embodiments, an endonuclease domain generates no duplication and/or deletion if a first and second strand nicks occur in the same position of a target site. In some embodiments, an endonuclease domain has altered activity depending on protein conformation or RNA-binding status, e.g., which promotes the nicking of the first or second strand (e.g., as described in Christensen et al. PNAS 2006; incorporated by reference herein in its entirety).
In some embodiments, an endonuclease domain comprises a meganuclease, or a functional fragment thereof. In some embodiments, an endonuclease domain comprises a homing endonuclease, or a functional fragment thereof. In some embodiments, an endonuclease domain comprises a mcganuclcasc from the LAGLID ADG, GIY-YIG, HNH, His-Cys Box, or PD-(D/E) XK families, or a functional fragment or variant thereof, e.g., which possess conserved amino acid motifs, e.g., as indicated in the family names. In some embodiments, an endonuclease domain comprises a meganuclease, or fragment thereof, chosen from, e.g., I- SmaMI (Uniprot F7WD42), I-Scel (Uniprot P03882), I- Anil (Uniprot PO388O), I-Dmol (Uniprot P21505), I-Crel (Uniprot P05725), I-TevI (Uniprot P13299), LOnuI (Uniprot Q4VWW5), or I- Bmol (Uniprot Q9ANR6). In some embodiments, a meganuclease is naturally monomeric, e.g., I-Scel, I-TevI, or dimeric, e.g., I-Crel, in its functional form. For example, LAGLID ADG meganucleases with a single copy of the LAGLID ADG motif generally form homodimers, whereas members with two copies of the LAGLID ADG motif arc generally found as monomers. In some embodiments, a meganuclease that normally forms as a dimer is expressed as a fusion, e.g., two subunits are expressed as a single open reading frame (ORF) and, optionally, connected by a linker, e.g., an I-Crel dimer fusion (Rodriguez-Fomes et al. Gene Therapy 2020; incorporated by reference herein in its entirety). In some embodiments, a meganuclease, or a functional fragment thereof, is altered to favor nickase activity for one strand of a doublestranded DNA molecule, e.g., I-Scel (K122I and/or K223I) (Niu et al. J Mol Biol 2008), I-Anil (K227M) (McConnell Smith et al. PNAS 2009), I-Dmol (Q42A and/or K120M) (Molina et al. J Biol Chem 2015). In some embodiments, a meganuclease or functional fragment thereof possessing this preference for single-strand cleavage is used as an endonuclease domain, e.g., with nickase activity. In some embodiments, an endonuclease domain comprises a meganuclease, or a functional fragment thereof, which naturally targets or is engineered to target a safe harbor site, e.g., an I-Crel targeting SH6 site (Rodriguez-Fomes et al., supra). In some embodiments, an endonuclease domain comprises a meganuclease, or a functional fragment thereof, with a sequence-tolerant catalytic domain, e.g., I-TevI recognizing the minimal motif CNNNG (Kleinstiver et al. PNAS 2012). In some embodiments, a target sequence-tolerant catalytic domain is fused to a DNA binding domain, e.g., to direct activity, e.g., by fusing I-TevI to: (i) zinc fingers to create Tev-ZFEs (Kleinstiver et al. PNAS 2012), (ii) other meganucleases to create MegaTevs (Wolfs et al. Nucleic Acids Res 2014), and/or (iii) Cas9 to create TevCas9 (Wolfs et al. PNAS 2016). In some embodiments, an endonuclease domain comprises a restriction enzyme, e.g., a Type IIS or Type IIP restriction enzyme. In some embodiments, an endonuclease domain comprises a Type IIS restriction enzyme, e.g., FokI, or a fragment or variant thereof. In some embodiments, an endonuclease domain comprises a Type IIP restriction enzyme, e.g., PvuII, or a fragment or variant thereof. In some embodiments, a dimeric restriction enzyme is expressed as a fusion such that it functions as a single chain, e.g., a FokI dimer fusion (Minczuk et al. Nucleic Acids Res 36(12):3926-3938 (2008)).
The use of additional endonuclease domains is described, for example, in Guha and Edgell Int J Mol Sci 18(22):2565 (2017), which is incorporated herein by reference in its entirety.
In some embodiments, a gene modifying polypeptide comprises a modification to an endonuclease domain, e.g., relative to a wild-type Cas protein. In some embodiments, an endonuclease domain comprises an addition, deletion, replacement, or modification to the amino acid sequence of a wild-type Cas protein. In some embodiments, an endonuclease domain is modified to include a heterologous functional domain that binds specifically to and/or induces endonuclease cleavage of a target nucleic acid (e.g., DNA) sequence of interest. In some embodiments, an endonuclease domain comprises a zinc finger. In embodiments, an endonuclease domain comprising a Cas domain is associated with a guide RNA (gRNA), e.g., as described herein. In some embodiments, an endonuclease domain is modified to include a functional domain that does not target a specific target nucleic acid (e.g., DNA) sequence. In some embodiments, an endonuclease domain comprises a FokI domain.
In some embodiments, an endonuclease domain is associated with a target dsDNA in vitro at a frequency at least about 5-fold or at least about 10-fold higher than with a scrambled dsDNA. In some embodiments, an endonuclease domain is associated with a target dsDNA in vitro at a frequency at least about 5-fold or at least about 10-fold higher than with a scrambled dsDNA, e.g., in a cell (e.g., a HEK293T cell, a primary liver cell, or a primary lung cell). In some embodiments, the frequency of association between an endonuclease domain and a target DNA or scrambled DNA is measured by ChlP-seq, e.g., as described in He and Pu (2010) Curr. Protoc Mol Biol Chapter 21 (incorporated by reference herein in its entirety).
In some embodiments, an endonuclease domain can catalyze the formation of a nick at a target sequence, e.g., to an increase of at least about 5-fold or at least about 10-fold relative to a non-target sequence (e.g., relative to any other genomic sequence in the genome of the target cell). In some embodiments, the level of nick formation is determined using NickScq, e.g., as described in Elacqua et al. (2019) bioRxiv doi.org/10.1101/867937 (incorporated herein by reference in its entirety).
In some embodiments, an endonuclease domain is capable of nicking DNA in vitro. In some embodiments, a nick results in an exposed base. In embodiments, an exposed base can be detected using a nuclease sensitivity assay, e.g., as described in Chaudhry and Weinfeld (1995) Nucleic Acids Res 23(19):3805-3809 (incorporated by reference herein in its entirety). In some embodiments, the level of exposed bases (e.g., detected by the nuclease sensitivity assay) is increased by at least about 10%, at least about 50%, or more relative to a reference endonuclease domain. In some embodiments, a reference endonuclease domain is an endonuclease domain from Cas9 of S. pyogenes.
In some embodiments, an endonuclease domain is capable of nicking DNA in a cell. In some embodiments, an endonuclease domain is capable of nicking DNA in a HEK293T cell. In some embodiments, an unrepaired nick that undergoes replication in the absence of Rad51 results in increased NHEJ rates at the site of the nick, which can be detected, e.g., by using a Rad51 inhibition assay, e.g., as described in Bothmer et al. (2017) Nat Commun 8:13905 (incorporated by reference herein in its entirety). In some embodiments, NHEJ rates are increased above 0-5%. In some embodiments, NHEJ rates are increased to 20-70% (e.g., between 30%-60% or 40-50%), e.g., upon Rad51 inhibition.
In some embodiments, an endonuclease domain releases a target after cleavage. In some embodiments, release of a target is indicated indirectly by assessing for multiple turnovers by an enzyme, e.g., as described in Yourik at al. RNA 25( 1 ):35-44 (2019) (incorporated herein by reference in its entirety) and shown in FIG. 2 therein. In some embodiments, the keXp of an endonuclease domain is 1 x 10’3 - 1 x 10’5 min’1 as measured by such methods.
In some embodiments, an endonuclease domain has a catalytic efficiency (fcCat/ m) greater than about 1 x 108 s’1 M’1 in vitro. In some embodiments, an endonuclease domain has a catalytic efficiency greater than about 1 x 105, greater than about 1 x 106, greater than about 1 x 107, or greater than about 1 x 108, s’1 M’1 in vitro. In some embodiments, catalytic efficiency is determined as described in Chen et al. (2018) Science 360(6387):436-439 (incorporated herein by reference in its entirety). In some embodiments, an endonuclease domain has a catalytic efficiency (fcat/^m) greater than about 1 x 108 s"1 M'1 in cells. In some embodiments, an endonuclease domain has a catalytic efficiency greater than about 1 x 105, greater than about 1 x 106, greater than about 1 x 107, or greater than about 1 x 108 s 1 M 1 in cells.
Gene modifying polypeptides comprising Cas domains
In some embodiments, a gene modifying polypeptide described herein comprises a Cas domain. In some embodiments, a Cas domain can direct a gene modifying polypeptide to a target site specified by a gRNA spacer, thereby modifying a target nucleic acid sequence in “cis”. In some embodiments, a gene modifying polypeptide is fused to a Cas domain. In some embodiments, a gene modifying polypeptide comprises a CRISPR/Cas domain (also referred to herein as a CRISPR-associated protein). In some embodiments, a CRISPR/Cas domain comprises a protein involved in the clustered regulatory interspaced short palindromic repeat (CRISPR) system, e.g., a Cas protein, and optionally binds a guide RNA, e.g., single guide RNA (sgRNA).
CRISPR systems are adaptive defense systems originally discovered in bacteria and archaea. CRISPR systems use RNA-guided nucleases termed CRISPR-associated or “Cas” endonucleases (e. g., Cas9 or Cpfl) to cleave foreign DNA. For example, in a typical CRISPR- Cas system, an endonuclease is directed to a target nucleotide sequence (e. g., a site in the genome that is to be sequence-edited) by sequence-specific, non-coding “guide RNAs” that target single- or double-stranded DNA sequences. Three classes (I-III) of CRISPR systems have been identified. The class II CRISPR systems use a single Cas endonuclease (rather than multiple Cas proteins). One class II CRISPR system includes a type II Cas endonuclease such as Cas9, a CRISPR RNA (“crRNA”), and a trans-activating crRNA (“tracrRNA”). The crRNA contains a “spacer” sequence, a typically about 20-nucleotide RNA sequence that corresponds to a target DNA sequence (“protospacer”). In the wild-type system, and in some engineered systems, crRNA also contains a region that binds to the tracrRNA to form a partially doublestranded structure that is cleaved by RNase III, resulting in a crRNA/tracrRNA hybrid molecule. A crRNA/tracrRNA hybrid then directs the Cas endonuclease to recognize and cleave a target DNA sequence. A target DNA sequence is generally adjacent to a “protospacer adjacent motif’ (“PAM”) that is specific for a given Cas endonuclease and required for cleavage activity at a target site matching the spacer of the crRNA. CRISPR endonucleases identified from various prokaryotic species have unique PAM sequence requirements, e.g., as listed for exemplary Cas enzymes in Table 3; examples of PAM sequences include 5'-NGG (Streptococcus pyogenes; SEQ ID NO: 11,019), 5'-NNAGAA (Streptococcus thermophilus CRISPR1; SEQ ID NO: 11,020), 5 '-NGGNG (Streptococcus thermophilus CRISPR3; SEQ ID NO: 11,021), and 5'- NNNGATT (Neisseria meningiditis; SEQ ID NO: 11,022). Some endonucleases, e.g., Cas9 endonucleases, are associated with G-rich PAM sites, e. g., 5'-NGG (SEQ ID NO: 11,023), and perform blunt-end cleaving of the target DNA at a location 3 nucleotides upstream from (5' from) the PAM site. Another class II CRISPR system includes the type V endonuclease Cpfl, which is smaller than Cas9; examples include AsCpfl (from Acidaminococcus sp.) and LbCpfl (from Lachnospiraceae sp.). Cpfl -associated CRISPR arrays are processed into mature crRNAs without the requirement of a tracrRNA; in other words, a Cpfl system, in some embodiments, comprises only Cpfl nuclease and a crRNA to cleave a target DNA sequence. Cpfl endonucleases, are typically associated with T-rich PAM sites, e. g., 5'-TTN. Cpfl can also recognize a 5'-CTA PAM motif. Cpfl typically cleaves a target DNA by introducing an offset or staggered double-strand break with a 4- or 5-nucleotide 5' overhang, for example, cleaving a target DNA with a 5-nucleotide offset or staggered cut located 18 nucleotides downstream from (3' from) a PAM site on the coding strand and 23 nucleotides downstream from the PAM site on the complimentary strand; the 5-nucleotide overhang that results from such offset cleavage allows more precise genome editing by DNA insertion by homologous recombination than by insertion at blunt-end cleaved DNA. See, e.g., Zetsche et al. (2015) Cell, 163:759 - 771.
A variety of CRISPR associated (Cas) genes or proteins can be used in the technologies provided by the present disclosure and the choice of Cas protein will depend upon the particular conditions of the method. Specific examples of Cas proteins include class II systems including Casl, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9, CaslO, Cpfl, C2C1, or C2C3. In some embodiments, a Cas protein, e.g., a Cas9 protein, may be from any of a variety of prokaryotic species. In some embodiments a Cas protein, e.g., a Cas9 protein, is selected to recognize a particular protospacer-adjacent motif (PAM) sequence. In some embodiments, a DNA-binding domain or endonuclease domain includes a sequence targeting polypeptide, such as a Cas protein, e.g., Cas9. In some embodiments a Cas protein, e.g., a Cas9 protein, may be obtained from a bacteria or archaea or synthesized using known methods. In some embodiments, a Cas protein may be from a gram-positive bacteria or a gram-negative bacteria. In some embodiments, a Cas protein may be from a Streptococcus (e.g., a S. pyogenes, or a S. thermophilus), a Francisella (e.g., an F. novicida), a Staphylococcus (e.g., an S. aureus), an Acidaminococcus (e.g., an Acidaminococcus sp. BV3L6), a Neisseria (e.g., an N. meningitidis), a Cryptococcus, a Corynebacterium, a Haemophilus, a Eubacterium, a Pasteurella, a Prevotella, a Veillonella, or a Marinobacter.
In some embodiments, a gene modifying polypeptide may comprise the amino acid sequence of SEQ ID NO: 4000, or a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity thereto. In some embodiments, the amino acid sequence of SEQ ID NO: 4000, or a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity thereto, is positioned at the N- terminal end of a gene modifying polypeptide. In some embodiments, the amino acid sequence of SEQ ID NO: 4000, or a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identity thereto, is positioned within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, or 30 amino acids of the N-terminal end of a gene modifying polypeptide.
In some embodiments, a gene modifying polypeptide may comprise the amino acid sequence of SEQ ID NO: 4001, or a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity thereto. In some embodiments, the amino acid sequence of SEQ ID NO: 4001, or a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity thereto, is positioned at the C- terminal end of a gene modifying polypeptide. In some embodiments, an amino acid sequence of SEQ ID NO: 4001 below, or a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity thereto, is positioned within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, or 30 amino acids of the C -terminal end of a gene modifying polypeptide.
Exemplary benchmarking sequence
In some embodiments, a gene modifying polypeptide may comprise a Cas domain as listed in Table 3 or Table 4, or a functional fragment thereof, or a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity thereto.
Table 3: CRISPR/Cas Proteins, Species, and Mutations
Table 4: Amino Acid Sequences of StlCas9 Proteins, Species, and Mutations
In some embodiments, a Cas protein requires a protospacer adjacent motif (PAM) to be present in or adjacent to a target DNA sequence for the Cas protein to bind and/or function. In some embodiments, a PAM is or comprises, from 5' to 3', NGG (SEQ ID NO: 11,024), YG (SEQ ID NO: 11,025), NNGRRT (SEQ ID NO: 11,026), NNNRRT (SEQ ID NO: 11,027), NGA (SEQ ID NO: 11,029), TYCV (SEQ ID NO: 11,030), TATV (SEQ ID NO: 11,031), NTTN (SEQ ID NO: 11,032), or NNNGATT (SEQ ID NO: 11,033), where N stands for any nucleotide, Y stands for C or T, R stands for A or G, and V stands for A or C or G. In some embodiments, a Cas protein is a protein listed in Table 3 or Table 4. In some embodiments, a Cas protein comprises one or more mutations altering its PAM. In some embodiments, a Cas protein comprises E1369R, E1449H, and R1556A mutations or analogous substitutions to the amino acids corresponding to said positions. In some embodiments, a Cas protein comprises E782K, N968K, and R1015H mutations or analogous substitutions to the amino acids corresponding to said positions. Tn some embodiments, a Cas protein comprises DI 135V, R1335Q, and T1337R mutations or analogous substitutions to the amino acids corresponding to said positions. In some embodiments, a Cas protein comprises S542R and K607R mutations or analogous substitutions to the amino acids corresponding to said positions. In some embodiments, a Cas protein comprises S542R, K548V, and N552R mutations or analogous substitutions to the amino acids corresponding to said positions. Exemplary advances in the engineering of Cas enzymes to recognize altered PAM sequences are reviewed in Collias et al Nature Communications 12:555 (2021), incorporated herein by reference in its entirety.
In some embodiments, a Cas protein is catalytically active and cuts one or both strands of a target DNA site. In some embodiments, cutting a target DNA site is followed by formation of an alteration, e.g., an insertion or deletion, e.g., by the cellular repair machinery.
In some embodiments, a Cas protein is modified to deactivate or partially deactivate its nuclease, e.g., a nuclease-deficient Cas9. Whereas wild-type Cas9 generates double-strand breaks (DSBs) at specific DNA sequences targeted by a gRNA, a number of CRISPR endonucleases having modified functionalities are available, for example: a “nickase” version of Cas9 that has been partially deactivated generates only a single-strand break; a catalytically inactive Cas9 (“dCas9”) does not cut target DNA. In some embodiments, dCas9 binding to a DNA sequence may interfere with transcription at that site by steric hindrance. In some embodiments, dCas9 binding to an anchor sequence may interfere with (e.g., decrease or prevent) genomic complex (e.g., ASMC) formation and/or maintenance. In some embodiments, a DNA-binding domain comprises a catalytically inactive Cas9, e.g., dCas9. Many catalytically inactive Cas9 proteins arc known in the art. In some embodiments, dCas9 comprises mutations in each endonuclease domain of the Cas protein, e.g., D10A and H840A or N863A mutations. In some embodiments, a catalytically inactive or partially inactive CRISPR/Cas domain comprises a Cas protein comprising one or more mutations, e.g., one or more of the mutations listed in Table 3. In some embodiments, a Cas protein described on a given row of Table 3 comprises one, two, three, or all of the mutations listed in the same row of Table 3. In some embodiments, a Cas protein, e.g., not described in Table 3, comprises one, two, three, or all of the mutations listed in a row of Table 3 or a corresponding mutation at a corresponding site in that Cas protein.
In some embodiments, a catalytically inactive, e.g., dCas9, or partially deactivated Cas9 protein comprises a Dl l mutation (e.g., Dl l A mutation) or an analogous substitution to the amino acid corresponding to said position. In some embodiments, a catalytically inactive Cas9 protein, c.g., dCas9, or partially deactivated Cas9 protein comprises a H969 mutation (c.g., H969A mutation) or an analogous substitution to the amino acid corresponding to said position. In some embodiments, a catalytically inactive Cas9 protein, e.g., dCas9, or partially deactivated Cas9 protein comprises a N995 mutation (e.g., N995A mutation) or an analogous substitution to the amino acid corresponding to said position. In some embodiments, a catalytically inactive Cas9 protein, e.g., dCas9, comprises mutations at one, two, or three of positions Dl l, H969, and N995 (e.g., D11A, H969A, and N995A mutations) or analogous substitutions to the amino acids corresponding to said positions.
In some embodiments, a catalytically inactive Cas9 protein, e.g., dCas9, or partially deactivated Cas9 protein comprises a DIO mutation (e.g., a D10A mutation) or an analogous substitution to the amino acid corresponding to said position. In some embodiments, a catalytically inactive Cas9 protein, e.g., dCas9, or partially deactivated Cas9 protein comprises a H557 mutation (e.g., a H557A mutation) or an analogous substitution to the amino acid corresponding to said position. In some embodiments, a catalytically inactive Cas9 protein, e.g., dCas9, comprises a DIO mutation (e.g., a D10A mutation) and a H557 mutation (e.g., a H557A mutation) or analogous substitutions to the amino acids corresponding to said positions.
In some embodiments, a catalytically inactive Cas9 protein, e.g., dCas9, or partially deactivated Cas9 protein comprises a D839 mutation (e.g., a D839A mutation) or an analogous substitution to the amino acid corresponding to said position. In some embodiments, a catalytically inactive Cas9 protein, e.g., dCas9, or partially deactivated Cas9 protein comprises a H840 mutation (e.g., a H840A mutation) or an analogous substitution to the amino acid corresponding to said position. In some embodiments, a catalytically inactive Cas9 protein, e.g., dCas9, or partially deactivated Cas9 protein comprises a N863 mutation (e.g., a N863A mutation) or an analogous substitution to the amino acid corresponding to said position. In some embodiments, a catalytically inactive Cas9 protein, e.g., dCas9, comprises a DIO mutation (e.g., D10A), a D839 mutation (e.g., D839A), a H840 mutation (e.g., H840A), and a N863 mutation (e.g., N863A) or analogous substitutions to the amino acids corresponding to said positions.
In some embodiments, a catalytically inactive Cas9 protein, e.g., dCas9, or partially deactivated Cas9 protein comprises a E993 mutation (e.g., a E993A mutation) or an analogous substitution to the amino acid corresponding to said position. In some embodiments, a catalytically inactive Cas9 protein, e.g., dCas9, or partially deactivated Cas9 protein comprises a D917 mutation (e.g., a D917A mutation) or an analogous substitution to the amino acid corresponding to said position. In some embodiments, a catalytically inactive Cas9 protein, e.g., dCas9, or partially deactivated Cas9 protein comprises a a E1006 mutation (e.g., a E1006A mutation) or an analogous substitution to the amino acid corresponding to said position. In some embodiments, a catalytically inactive Cas9 protein, e.g., dCas9, or partially deactivated Cas9 protein comprises a D1255 mutation (e.g., a D1255A mutation) or an analogous substitution to the amino acid corresponding to said position. In some embodiments, a catalytically inactive Cas9 protein, e.g., dCas9, comprises a D917 mutation (e.g., D917A), a E1006 mutation (e.g., E1006A), and a D1255 mutation (e.g., D1255A) or analogous substitutions to the amino acids corresponding to said positions.
In some embodiments, a catalytically inactive Cas9 protein, e.g., dCas9, or partially deactivated Cas9 protein comprises a D16 mutation (e.g., a D16A mutation) or an analogous substitution to the amino acid corresponding to said position. In some embodiments, a catalytically inactive Cas9 protein, e.g., dCas9, or partially deactivated Cas9 protein comprises a D587 mutation (e.g., a D587A mutation) or an analogous substitution to the amino acid corresponding to said position. In some embodiments, a partially deactivated Cas domain has nickase activity. In some embodiments, a partially deactivated Cas9 domain is a Cas9 nickase domain. In some embodiments, the catalytically inactive Cas domain or dead Cas domain produces no detectable double strand break formation. In some embodiments, a catalytically inactive Cas9 protein, e.g., dCas9, or partially deactivated Cas9 protein comprises a H588 mutation (e.g., a H588A mutation) or an analogous substitution to the amino acid corresponding to said position. In some embodiments, a catalytically inactive Cas9 protein, e.g., dCas9, or partially deactivated Cas9 protein comprises a N611 mutation (e.g., a N611A mutation) or an analogous substitution to the amino acid corresponding to said position. In some embodiments, a catalytically inactive Cas9 protein, e.g., dCas9, comprises a D16 mutation (e.g., D16A), a D587 mutation (e.g., D587A), a H588 mutation (e.g., H588A), and a N611 mutation (e.g., N611A) or analogous substitutions to the amino acids corresponding to said positions.
In some embodiments, a DNA-binding domain or endonuclease domain may comprise a Cas molecule comprising or linked (e.g., covalently) to a gRNA (e.g., a template nucleic acid, e.g., template RNA, comprising a gRNA). In some embodiments, an endonuclease domain or DNA binding domain comprises a Streptococcus pyogenes Cas9 (SpCas9) or a functional fragment or variant thereof. In some embodiments, an endonuclease domain or DNA binding domain comprises a modified SpCas9. In some embodiments, a modified SpCas9 comprises a modification that alters protospacer- adjacent motif (PAM) specificity. In some embodiments, a PAM has specificity for the nucleic acid sequence 5'-NGT-3'. In some embodiments, a modified SpCas9 comprises one or more amino acid substitutions, e.g., at one or more of positions LI 111, DI 135, G1218, E1219, A1322, or R1335, e.g., selected from Li l HR, DI 135V, G1218R, E1219F, A1322R, or R1335V. In some embodiments, a modified SpCas9 comprises the amino acid substitution T1337R and one or more additional amino acid substitutions, e.g., selected from Li l HR, D1135L, S1136R, G1218S, E1219V, D1332A, D1332S, D1332T, D1332V, D1332L, D1332K, D1332R, R1335Q, T1337, T1337L, T1337Q, T1337I, T1337V, T1337F, T1337S, T1337N, T1337K, T1337H, T1337Q, and T1337M, or corresponding amino acid substitutions thereto. In some embodiments, a modified SpCas9 comprises: (i) one or more amino acid substitutions selected from D1135L, S1136R, G1218S, E1219V, A1322R, R1335Q, and T1337; and (ii) one or more amino acid substitutions selected from Li l HR, G1218R, E1219F, D1332A, D1332S, D1332T, D1332V, D1332L, D1332K, D1332R, T1337L, T1337I, T1337V, T1337F, T1337S, T1337N, T1337K, T1337R, T1337H, T1337Q, and T1337M, or corresponding amino acid substitutions thereto.
In some embodiments, an endonuclease domain or DNA binding domain comprises a Cas domain, e.g., a Cas9 domain. In some embodiments, an endonuclease domain or DNA binding domain comprises a nuclease-active Cas domain, a Cas nickase (nCas) domain, or a nucleaseinactive Cas (dCas) domain. In some embodiments, an endonuclease domain or DNA binding domain comprises a nuclease-active Cas9 domain, a Cas9 nickase (nCas9) domain, or a nuclease-inactive Cas9 (dCas9) domain. In some embodiments, an endonuclease domain or DNA binding domain comprises a Cas9 domain of Cas9 (e.g., dCas9 and nCas9), Casl2a/Cpfl, Casl2b/C2cl, Casl2c/C2c3, Casl2d/CasY, Casl2e/CasX, Casl2g, Casl2h, or Casl2i. In some embodiments, an endonuclease domain or DNA binding domain comprises a Cas9 (e.g., dCas9 and nCas9), Casl2a/Cpfl, Casl2b/C2cl, Casl2c/C2c3, Casl2d/CasY, Casl2e/CasX, Casl2g, Casl2h, or Casl2i. In some embodiments, an endonuclease domain or DNA binding domain comprises an S. pyogenes or an S. thermophilus Cas9, or a functional fragment thereof. In some embodiments, an endonuclease domain or DNA binding domain comprises a Cas9 sequence, e.g., as described in Chylinski, Rhun, and Charpentier (2013) RNA Biology 10:5, 726-737; incorporated herein by reference. In some embodiments, an endonuclease domain or DNA binding domain comprises the HNH nuclease subdomain and/or the RuvCl subdomain of a Cas, e.g., Cas9, e.g., as described herein, or a variant thereof. In some embodiments, an endonuclease domain or DNA binding domain comprises Casl2a/Cpfl, Casl2b/C2cl, Casl2c/C2c3, Casl2d/CasY, Casl2e/CasX, Casl2g, Casl2h, or Casl2i. In some embodiments, an endonuclease domain or DNA binding domain comprises a Cas polypeptide (e.g., enzyme), or a functional fragment thereof. In some embodiments, a Cas polypeptide (e.g., enzyme) is selected from Cast, CaslB, Cas2, Cas3, Cas4, Cas5, Cas5d, Cas5t, Cas5h, Cas5a, Cas6, Cas7, Cas8, Cas8a, Cas8b, Cas8c, Cas9 (e.g., Csnl or Csxl2), CaslO, CaslOd, Casl2a/Cpfl, Casl2b/C2cl, Casl2c/C2c3, Casl2d/CasY, Casl2e/CasX, Casl2g, Casl2h, Casl2i, Csyl , Csy2, Csy3, Csy4, Csel, Cse2, Cse3, Cse4, Cse5e, Cscl, Csc2, Csa5, Csnl, Csn2, Csml, Csm2, Csm3, Csm4, Csm5, Csm6, Cmrl, Cmr3, Cmr4, Cmr5, Cmr6, Csbl, Csb2, Csb3, Csxl7, Csxl4, CsxlO, Csxl6, CsaX, Csx3, Csxl, CsxlS, Csxl l, Csfl, Csf2, CsO, Csf4, Csdl, Csd2, Cstl, Cst2, Cshl, Csh2, Csal, Csa2, Csa3, Csa4, Csa5, Type II Cas effector proteins, Type V Cas effector proteins, Type VI Cas effector proteins, CARF, DinG, Cpfl, Casl2b/C2cl, Casl2c/C2c3, Casl2b/C2cl, Casl2c/C2c3, SpCas9(K855A), eSpCas9(l.l), SpCas9-HFl, hyper accurate Cas9 variant (HypaCas9), homologues thereof, modified or engineered versions thereof, and/or functional fragments thereof. In some embodiments, a Cas9 comprises one or more substitutions, e.g., selected from H840A, D10A, P475A, W476A, N477A, DI 125 A, W1126A, and D1127A. In some embodiments, a Cas9 comprises one or more mutations at positions selected from: DIO, G12, G17, E762, H840, N854, N863, H982, H983, A984, D986, and/or A987, e.g., one or more substitutions selected from D10A, G12A, G17A, E762A, H840A, N854A, N863A, H982A, H983A, A984A, and/or D986A. In some embodiments, an endonuclease domain or DNA binding domain comprises a Cas (e.g., Cas9) sequence from Corynebacterium ulcerans, Corynebacterium diphtheria, Spiroplasma syrphidicola, Prevotella intermedia, Spiroplasma taiwanense, Streptococcus iniae, Belliella baltica, Psychroflexus torquis, Streptococcus thermophilus, Listeria innocua, Campylobacter jejuni, Neisseria meningitidis, Streptococcus pyogenes, or Staphylococcus aureus, or a fragment or variant thereof.
In some embodiments, an endonuclease domain or DNA binding domain comprises a Cpfl domain, e.g., comprising one or more substitutions, e.g., at position D917, E1006A, D1255 or any combination thereof, e.g., selected from D917A, E1006A, D1255A, D917A/E1006A,
D917A/D1255A, E1006A/D1255A, and D917A/E1006A/D1255A.
In some embodiments, an endonuclease domain or DNA binding domain comprises spCas9, spCas9-VRQR (SEQ ID NO: 5019), spCas9- VRER (SEQ ID NO: 5020), xCas9 (sp), saCas9, saCas9-KKH, spCas9-MQKSER (SEQ ID NO: 5021), spCas9-LRKIQK (SEQ ID NO: 5022), or spCas9- LRVSQL (SEQ ID NO: 5023).
In some embodiments, a gene modifying polypeptide has an endonuclease domain comprising a Cas9 nickase, e.g., Cas9 H840A. In some embodiments, the Cas9 H840A has the following amino acid sequence:
In some embodiments, a gene modifying polypeptide comprises a dCas9 sequence comprising a D10A and/or H840A mutation, e.g., the following sequence:
TAL Effectors and Zinc Finger Nucleases
In some embodiments, an endonuclease domain or DNA-binding domain comprises a TAL effector molecule. A TAL effector molecule, e.g., a TAL effector molecule that specifically binds a DNA sequence, typically comprises a plurality of TAL effector domains or fragments thereof, and optionally one or more additional portions of naturally occurring TAL effectors (e.g., N- and/or C-terminal of the plurality of TAL effector domains). Many TAL effectors are known to those of skill in the art and are commercially available, e.g., from Thermo Fisher Scientific.
Naturally occurring TALEs are natural effector proteins secreted by numerous species of bacterial pathogens including the plant pathogen Xanlhomonas which modulates gene expression in host plants and facilitates bacterial colonization and survival. The specific binding of TAL effectors is based on a central repeat domain of tandemly arranged nearly identical repeats of typically 33 or 34 amino acids (the repeat-variable di-residues, RVD domain). Members of the TAL effectors family differ mainly in the number and order of their repeats. The number of repeats typically ranges from 1.5 to 33.5 repeats and the C-tcrminal repeat is usually shorter in length (e.g., about 20 amino acids) and is generally referred to as a “half-repeat.” Each repeat of the TAL effector generally features a one-repeat-to-one-base-pair correlation with different repeat types exhibiting different base-pair specificity (one repeat recognizes one base-pair on the target gene sequence). Generally, the smaller the number of repeats, the weaker the protein-DNA interactions. A number of 6.5 repeats has been shown to be sufficient to activate transcription of a reporter gene (Scholze et al., 2010).
Repeat to repeat variations occur predominantly at amino acid positions 12 and 13, which have therefore been termed “hypervariable” and which are responsible for the specificity of the interaction with the target DNA promoter sequence, as shown in Table 5 listing exemplary repeat variable diresidues (RVD) and their correspondence to nucleic acid base targets.
Table 5: RVDs and Nucleic Acid Base Specificity
Accordingly, it is possible to modify the repeats of a TAL effector to target specific DNA sequences. Further studies have shown that the RVD NK can target G. Target sites of TAL effectors also tend to include a T flanking the 5' base targeted by the first repeat, but the exact mechanism of this recognition is not known. More than 113 TAL effector sequences are known to date. Non-limiting examples of TAL effectors from Xanthomonas include, Hax2, Hax3, Hax4, AvrXa7, AvrXalO and AvrBs3.
Accordingly, a TAL effector domain of a TAL effector molecule described herein may be derived from a TAL effector from any bacterial species (e.g., Xanthomonas species such as the African strain of Xanthomonas oryzae pv. Oryzae (Yu et al. 2011), Xanthomonas campestris pv. raphani strain 756C and Xanthomonas oryzae pv. Oryzicola strain BLS256 (Bogdanove et al. 2011). In some embodiments, a TAL effector domain comprises an RVD domain as well as flanking sequence(s) (sequences on the N-terminal and/or C-terminal side of the RVD domain) also from the naturally occurring TAL effector. It may comprise more or fewer repeats than the RVD of the naturally occurring TAL effector. A TAL effector molecule can be designed to target a given DNA sequence based on the above code and others known in the art. The number of TAL effector domains (e.g., repeats (monomers or modules)) and their specific sequence can be selected based on the desired DNA target sequence. For example, TAL effector domains, e.g., repeats, may be removed or added in order to suit a specific target sequence. In someembodiments, a TAL effector molecule comprises between 6.5 and 33.5 TAL effector domains, e.g., repeats. In some embodiments, a TAL effector molecule of the present invention comprises between 8 and 33.5 TAL effector domains, e.g., repeats, e.g., between 10 and 25 TAL effector domains, e.g., repeats, e.g., between 10 and 14 TAL effector domains, e.g., repeats.
In some embodiments, a TAL effector molecule comprises TAL effector domains that correspond to a perfect match to a DNA target sequence. In some embodiments, a mismatch between a repeat and a target base-pair on a DNA target sequence is permitted as along as it allows for the function of the polypeptide comprising the TAL effector molecule. Wihtout wishing to be bound by any particular theory, TALE binding is inversely correlated with the number of mismatches. In some embodiments, a TAL effector molecule of a gene modfiying polypeptide comprises no more than 7 mismatches, no more than 6 mismatches, no more than 5 mismatches, no more than 4 mismatches, no more than 3 mismatches, no more than 2 mismatches, or no more than 1 mismatch, and optionally no mismatch, with a target DNA sequence. Without wishing to be bound by any particular theory, the smaller the number of TAL effector domains in a TAL effector molecule, the smaller the number of mismatches will be tolerated and still allow for the function of a gene modifying polypeptide comprising the TAL effector molecule. Binding affinity is thought to depend on the sum of matching repeat-DNA combinations. For example, TAL effector molecules having 25 TAL effector domains or more may be able to tolerate up to 7 mismatches.
In addition to TAL effector domains, a TAL effector molecule may comprise additional sequences derived from a naturally occurring TAL effector. The length of C-terminal and/or N- terminal sequence(s) included on each side of a TAL effector domain portion of a TAL effector molecule can vary and be selected by one skilled in the art, for example based on the studies of Zhang et al. (2011). Zhang et al., have characterized a number of C-terminal and N-terminal truncation mutants in Hax3 derived TAL-effector based proteins and have identified key elements, which contribute to optimal binding to the target sequence and thus activation of transcription. Generally, it was found that transcriptional activity is inversely correlated with the length of N-terminus. Regarding the C-terminus, an important element for DNA binding residues within the first 68 amino acids of the Hax 3 sequence was identified. Accordingly, in some embodiments, the first 68 amino acids on the C-terminal side of a TAL effector domains of the naturally occurring TAL effector is included in a TAL effector molecule. Accordingly, in some embodiments, a TAL effector molecule comprises 1) one or more TAL effector domains derived from a naturally occurring TAL effector; 2) at least 70, at least 80, at least 90, at least 100, at least 110, at least 110, at least 120, at least 130, at least 140, at least 150, at least 160, at least 170, at least 180, at least 190, at least 200, at least 210, at least 220, at least 230, at least 240, at least 250, at least 260, at least 270, at least 280 or more amino acids from the naturally occurring TAL effector on the N-terminal side of the TAL effector domains; and/or 3) at least 68, at least 80, at least 90, at least 100, at least 110, at least 120, at least 130, at least 140, at least 150, at least 160, at least 170, at least 180, at least 190, at least 200, at least 210, at least 220, at least 230, at least 240, at least 250, at least 260 or more amino acids from the naturally occurring TAL effector on the C-terminal side of the TAL effector domains.
In some embodiments, an endonuclease domain or DNA-binding domain is or comprises a Zn finger molecule. A Zn finger molecule comprises a Zn finger protein, e.g., a naturally occurring Zn finger protein or engineered Zn finger protein, or fragment thereof. Many Zn finger proteins are known to those of skill in the art and are commercially available, e.g., from Sigma-Aldrich.
In some embodiments, a Zn finger molecule comprises a non-naturally occurring Zn finger protein that is engineered to bind to a target DNA sequence of choice. See, for example, Beerli, et al. (2002) Nature Biotechnol. 20:135-141; Pabo, et al. (2001) Ann. Rev. Biochem. 70:313-340; Isalan, et al. (2001) Nature Biotechnol. 19:656-660; Segal, et al. (2001) Curr. Opin. Biotechnol. 12:632-637; Choo, et al. (2000) Curr. Opin. Struct. Biol. 10:411-416; U.S. Pat. Nos. 6,453,242; 6,534,261; 6,599,692; 6,503,717; 6,689,558; 7,030,215; 6,794,136; 7,067,317; 7,262,054; 7,070,934; 7,361,635; 7,253,273; and U.S. Patent Publication Nos. 2005/0064474; 2007/0218528; 2005/0267061, all incorporated herein by reference in their entireties.
An engineered Zn finger protein may have a novel binding specificity, compared to a naturally-occurring Zn finger protein. Engineering methods include, but are not limited to, rational design and various types of selection. Rational design includes, for example, using databases comprising triplet (or quadruplet) nucleotide sequences and individual Zn finger amino acid sequences, in which each triplet or quadruplet nucleotide sequence is associated with one or more amino acid sequences of zinc fingers which bind the particular triplet or quadruplet sequence. See, for example, U.S. Pat. Nos. 6,453,242 and 6,534,261, incorporated by reference herein in their entireties.
Exemplary selection methods, including phage display and two-hybrid systems, are disclosed in U.S. Pat. Nos. 5,789,538; 5,925,523; 6,007,988; 6,013,453; 6,410,248; 6,140,466; 6,200,759; and 6,242,568; as well as International Patent Publication Nos. WO 98/37186; WO 98/53057; WO 00/27878; WO 01/88197; and GB 2,338,237. In addition, enhancement of binding specificity for zinc finger proteins has been described, for example, in International Patent Publication No. WO 02/077227.
In addition, as disclosed in these and other references, zinc finger domains and/or multifingered zinc finger proteins may be linked together using any suitable linker sequences, including for example, linkers of 5 or more amino acids in length. See, also, U.S. Pat. Nos. 6,479,626; 6,903,185; and 7,153,949 for exemplary linker sequences 6 or more amino acids in length. The proteins described herein may include any combination of suitable linkers between the individual zinc fingers of the protein. In addition, enhancement of binding specificity for zinc finger binding domains has been described, for example, in co-owned International Patent Publication No. WO 02/077227.
Zn finger proteins and methods for design and construction of fusion proteins (and polynucleotides encoding same) are known to those of skill in the art and described in detail in U.S. Pat. Nos. 6,140,0815; 789,538; 6,453,242; 6,534,261; 5,925,523; 6,007,988; 6,013,453; and 6,200,759; International Patent Publication Nos. WO 95/19431; WO 96/06166; WO 98/53057; WO 98/54311; WO 00/27878; WO 01/60970; WO 01/88197; WO 02/099084; WO 98/53058; WO 98/53059; WO 98/53060; WO 02/016536; and WO 03/016496.
In addition, as disclosed in these and other references, Zn finger proteins and/or multifingered Zn finger proteins may be linked together, e.g., as a fusion protein, using any suitable linker sequences, including for example, linkers of 5 or more amino acids in length. See, also, U.S. Pat. Nos. 6,479,626; 6,903,185; and 7,153,949 for exemplary linker sequences 6 or more amino acids in length. The Zn finger molecules described herein may include any combination of suitable linkers between the individual zinc finger proteins and/or multi-fingered Zn finger proteins of the Zn finger molecule.
In some embodiments, a DNA-binding domain or endonuclease domain comprises a Zn finger molecule comprising an engineered zinc finger protein that binds (in a sequence-specific manner) to a target DNA sequence. In some embodiments, a Zn finger molecule comprises one Zn finger protein or fragment thereof. In some embodiments, a Zn finger molecule comprises a plurality of Zn finger proteins (or fragments thereof), e.g., 2, 3, 4, 5, 6 or more Zn finger proteins (and optionally no more than 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, or 2 Zn finger proteins). In some embodiments, a Zn finger molecule comprises at least three Zn finger proteins. In some embodiments, a Zn finger molecule comprises four, five or six Zn finger proteins. In some embodiments, a Zn finger molecule comprises 8, 9, 10, 11 or 12 finger proteins. In some embodiments, a Zn finger molecule comprising three Zn finger proteins recognizes a target DNA sequence comprising 9 or 10 nucleotides. In some embodiments, a Zn finger molecule comprising four Zn finger proteins recognizes a target DNA sequence comprising 12 to 14 nucleotides. In some embodiments, a Zn finger molecule comprising six Zn finger proteins recognizes a target DNA sequence comprising 18 to 21 nucleotides.
In some embodiments, a Zn finger molecule comprises a two-handed Zn finger protein. Two handed zinc finger proteins are those proteins in which two clusters of zinc finger proteins are separated by intervening amino acids so that the two zinc finger domains bind to two discontinuous target DNA sequences. An example of a two-handed type of zinc finger binding protein is SIP1, where a cluster of four zinc finger proteins is located at the amino terminus of the protein and a cluster of three Zn finger proteins is located at the carboxyl terminus (see Remade, et al. (1999) EMBO Journal 18(18):5073-5084). Each cluster of zinc fingers in these proteins is able to bind to a unique target sequence and the spacing between the two target sequences can comprise many nucleotides.
Linkers
In some embodiments, a gene modifying polypeptide may comprise a linker, e.g., a peptide linker, e.g., a linker as described in Table 6. In some embodiments, a gene modifying polypeptide comprises, in an N-terminal to C-terminal direction, a Cas domain (e.g., a Cas domain of Table 4), a linker of Table 6 (or a sequence having at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity thereto), and an RT domain (e.g., an RT domain of Table 1). In some embodiments, a gene modifying polypeptide comprises a flexible linker between an endonuclease and ab RT domain, c.g., a linker comprising the amino acid sequence SGGSSGGSSGSETPGTSESATPESSGGSSGGSS (SEQ ID NO: 11,002). In some embodiments, an RT domain of a gene modifying polypeptide may be located C-terminal to an endonuclease domain. In some embodiments, an RT domain of a gene modifying polypeptide may be located N-terminal to the endonuclease domain.
Table 6: Exemplary linker sequences
In some embodiments, a linker of a gene modifying polypeptide comprises a motif chosen from: (SGGS)n(SEQ ID NO: 5025), (GGGS)„ (SEQ ID NO: 5026), (GGGGS)n (SEQ ID NO: 5027), (G)n, (EAAAK)U (SEQ ID NO: 5028), (GGS)U, or (XP)U. Gene modifying polypeptide selection by pooled screening
Candidate gene modifying polypeptides may be screened to evaluate a candidate’s gene editing ability. For example, an RNA gene modifying system designed for the targeted editing of a coding sequence in the human genome may be used. In some embodiments, such a gene modifying system may be used in conjunction with a pooled screening approach. For example, a library of gene modifying polypeptide candidates and a template guide
RNA (tgRNA) may be introduced into mammalian cells to test the candidates’ gene editing abilities by a pooled screening approach. In some embodiments, a library of gene modifying polypeptide candidates is introduced into mammalian cells followed by introduction of tgRNA into the cells. Representative, non-limiting examples of mammalian cells that may be used in screening include HEK293T cells, U2OS cells, HeLa cells, HepG2 cells, Huh7 cells, K562 cells, or iPS cells. A gene modifying polypeptide candidate may comprise 1 ) a Cas-nuclease, for example a wild-type Cas nuclease, c.g., a wild-type Cas9 nuclease, a mutant Cas nuclease, c.g., a Cas nickase, for example, a Cas9 nickase such as a Cas9 N863A nickase, or a Cas nuclease selected from Table 3 or Table 4, 2) a peptide linker, e.g., a sequence from Table 6, Table 7, or Table 8, that may exhibit varying degrees of length, flexibility, hydrophobicity, and/or secondary structure; and 3) a reverse transcriptase (RT), e.g. an RT domain from Table 7, Table 8, or Table 1. A gene modifying polypeptide candidate library comprises: a plurality of different gene modifying polypeptide candidates that differ from each other with respect to one, two or all three of a Cas nuclease, a peptide linker or an RT domain component, or a plurality of nucleic acid expression vectors that encode such gene modifying polypeptide candidates.
For screening of gene modifying polypeptide candidates, a two-component system may be used that comprises a gene modifying polypeptide component and a tgRNA component. A gene modifying component may comprise, for example, an expression vector, e.g., an expression plasmid or lentiviral vector, that encodes a gene modifying polypeptide candidate, for example, comprises a human codon-optimized nucleic acid that encodes a gene modifying polypeptide candidate, e.g., a Cas-linker-RT fusion as described above. In some embodiments, a lentiviral cassette is utilized that comprises: (i) a promoter for expression in mammalian cells, e.g., a CMV promoter; (ii) a gene modifying library candidate, e.g. a Cas-linker-RT fusion comprising a Cas nuclease of Table 3 or Table 4, a peptide linker of Table 6, and an RT of Table 1, for example a Cas-linker-RT fusion as in Table 7 or Table 8; (iii) a self-cleaving polypeptide, e.g., a T2A peptide; (iv) a marker enabling selection in mammalian cells, e.g., a puromycin resistance gene; and (v) a termination signal, e.g., a poly A tail.
A tgRNA component may comprise a tgRNA or expression vector, e.g., an expression plasmid, that produces the tgRNA, for example, utilizes a U6 promoter to drive expression of the tgRNA, wherein the tgRNA is a non-coding RNA sequence that is recognized by Cas and localizes it to the genomic locus of interest, and that also templates reverse transcription of a desired edit into the genome by an RT domain.
To prepare a pool of cells expressing gene modifying polypeptide library candidates, mammalian cells, e.g., HEK293T or U2OS cells, may be transduced with pooled gene modifying polypeptide candidate expression vector preparations, e.g., lentiviral preparations, of the gene modifying candidate polypeptide library. In some embodiments, lentiviral plasmids are utilized, and HEK293 Lenti-X cells are seeded in 15 cm plates (~12xl06 cells) prior to lentiviral plasmid transfection. In some embodiments, lentiviral plasmid transfection may be performed using the Lentiviral Packaging Mix (Biosettia) and transfection of the plasmid DNA for the gene modifying candidate library is performed the following day using Lipofectamine 2000 and Opti-MEM media according to the manufacturer’s protocol. In some embodiments, extracellular DNA may be removed by a full media change the next day and virus-containing media may be harvested 48 hours after. Lentiviral media may be concentrated using Lenti-X Concentrator (TaKaRa Biosciences) and 5 mL lentiviral aliquots may be made and stored at -80°C. Lentiviral titering is performed by enumerating colony forming units post-selection, e.g., post Puromycin selection.
For monitoring gene editing of a target DNA, mammalian cells, e.g., HEK293T or U2OS cells, carrying a target DNA may be utilized. In some embodiments for monitoring gene editing of a target DNA, mammalian cells, e.g., HEK293T or U2OS cells, carrying a target DNA genomic landing pad may be utilized. In some embodiments, a target DNA genomic landing pad may comprise a gene to be edited for treatment of a disease or disorder of interest. In some embodiments, a target DNA is a gene sequence that expresses a protein that exhibits detectable characteristics that may be monitored to determine whether gene editing has occurred. For example, in some embodiments, a blue fluorescence protein (BFP)- or green fluorescence protein (GFP)-expressing genomic landing pad is utilized. In some embodiments, mammalian cells, e.g., HEK293T or U2OS cells, comprising a target DNA, e.g., a target DNA genomic landing pad, are seeded in culture plates at 500x - 3000x cells per gene modifying library candidate and transduced at a 0.2 - 0.3 multiplicity of infection (MOI) to minimize multiple infections per cell. Puromycin (2.5 ug/mL) may be added 48 hours post infection to allow for selection of infected cells. In such an embodiment, cells may be kept under puromycin selection for at least 7 days and then scaled up for tgRNA introduction, e.g., tgRNA electroporation.
To ascertain whether gene editing occurs, mammalian cells containing a target DNA to be edited may be infected with gene modifying polypeptide library candidates then transfected with tgRNA designed for use in editing of the target DNA. Subsequently, the cells may be analyzed to determine whether editing of the target locus has occurred according to the designed outcome, or whether no editing or imperfect editing has occurred, e.g., by using cell sorting and sequence analysis. In some embodiments, to ascertain whether genome editing occurs, BFP- or GFP- cxprcssing mammalian cells, e.g., HEK293T or U2OS cells, may be infected with gene modifying library candidates and then transfected or electroporated with tgRNA plasmid or RNA, e.g., by electroporation of 250,000 cells/well with 200 ng of a tgRNA plasmid designed to convert BFP- to-GFP or GFP-to-BFP, at a cell count ensuring >250x - lOOOx coverage per library candidate. In such an embodiment, the genome-editing capacity of the various constructs in this assay may be assessed by sorting the cells by Fluorescence-Activated Cell Sorting (FACS) for expression of the color-converted fluorescent protein (FP) at 4-10 days post-electroporation. Cells are sorted and harvested as distinct populations of unedited cells (exhibiting original florescence protein signal), edited cells (exhibiting converted fluorescence protein signal), and imperfect edit (exhibiting no florescence protein signal) cells. A sample of unsorted cells may also be harvested as the input population to determine candidate enrichment during analysis.
To determine which gene modifying library candidates exhibit genome-editing capacity in an assay, genomic DNA (gDNA) is harvested from the sorted cell populations, and analyzed by sequencing the gene modifying library candidates in each population. Briefly, gene modifying candidates may be amplified from the genome using primers specific to the gene modifying polypeptide expression vector, e.g., the lentiviral cassette, amplified in a second round of PCR to dilute genomic DNA, and then sequenced, for example, sequenced by a next-generation sequencing platform. After quality control of sequencing reads, reads of at least about 1500 nucleotides and generally no more than about 3200 nucleotides are mapped to the gene modifying polypeptide library sequences and those containing a minimum of about an 80% match to a library sequence are considered to be successfully aligned to a given candidate for purposes of this pooled screen. In order to identify candidates capable of performing gene editing in the assay, e.g., the BFP-to-GFP or GFP-to-BFP edit, the read count of each library candidate in the edited population is compared to its read count in the initial, unsorted population.
For purposes of pooled screening, gene modifying candidates with genome-editing capacity are identified based on enrichment in the edited (converted FP) population relative to unsorted (input) cells. In some embodiments, an enrichment of at least 1.0, at least 1.5, at least 2.0, at least 2.5, at least 3.0, at least 4.0, at least 5.0, at least 6.0, at least 7.0, at least 8.0, at least 9.0, at least 10, at least 15, at least 20, at least 25, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, or at least 100-fold over the input indicates potentially useful gene editing activity, e.g., at least 2-fold enrichment. In some embodiments, the enrichment is converted to a log-value by taking the log base 2 of the enrichment ratio. In some embodiments, a log2 enrichment score of at least 0, at least 1, at least 2, at least 3, at least 4, at least 5, at least 5.5, at least 6.0, at least 6.1, at least 6.2, at least 6.3, at least 6.4, at least 6.5, or at least 6.6 indicates potentially useful gene editing activity, e.g., a log2 enrichment score of at least 1.0. In particular embodiments, enrichment values observed for gene modifying candidates may be compared to enrichment values observed under similar conditions utilizing a reference, e.g., Element ID No: 17380.
In some embodiments, multiple tgRNAs may be used to screen a gene modifying candidate library. In some embodiments, a plurality of tgRNAs may be utilized to optimize template/Cas- linker-RT fusion pairs, e.g., for gene editing of particular target genes, for example, gene targets for the treatment of disease. In some embodiments, a pooled approach to screening gene modifying candidates may be performed using a multiplicity of different tgRNAs in an arrayed format.
In some embodiments, multiple types of edits, e.g., insertions, substitutions, and/or deletions of different lengths, may be used to screen the gene modifying candidate library.
In some embodiments, multiple target sequences, e.g., different fluorescent proteins, may be used to screen the gene modifying candidate library. In some embodiments, multiple target sequences, e.g., different fluorescent proteins, may be used to screen the gene modifying candidate library. In some embodiments, multiple cell types, e.g., HEK293T or U2OS, may be used to screen a gene modifying candidate library. A person of ordinary skill in the art will appreciate that a given candidate may exhibit altered editing capacity or even the gain or loss of any observable or useful activity across different conditions, including tgRNA sequence (e.g., nucleotide modifications, PBS length, RT template length), target sequence, target location, type of edit, location of mutation relative to the first-strand nick of the gene modifying polypeptide, or cell type. Thus, in some embodiments, gene modifying library candidates are screened across multiple parameters, e.g., with at least two distinct tgRNAs in at least two cell types, and gene editing activity is identified by enrichment in any single condition. In some embodiments, a candidate with more robust activity across different tgRNA and cell types is identified by enrichment in at least two conditions, e.g., in all conditions screened. For clarity, candidates found to exhibit little to no enrichment under any given condition are not assumed to be inactive across all conditions and may be screened with different parameters or reconfigured at the polypeptide level, e.g., by swapping, shuffling, or evolving domains (e.g., RT domain), linkers, or other signals (e.g., NLS).
Sequences of exemplary Cas9-linker-RT fusions
In some embodiments, a gene modifying polypeptide comprises a linker sequence and an RT sequence. In some embodiments, a gene modifying polypeptide comprises a linker sequence as listed in Table 7 or Table 8, or an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity thereto. In some embodiments, a gene modifying polypeptide comprises the amino acid sequence of an RT domain as listed in Table 7 or Table 8, or an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity thereto. In some embodiments, a gene modifying polypeptide comprises a linker sequence as listed in Table 7 or Table 8, or an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity thereto; and the amino acid sequence of an RT domain as listed in Table 7 or Table 8, or an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity thereto. In some embodiments, a gene modifying polypeptide comprises: (i) a linker sequence as listed in a row of Table 7 or Table 8, or an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity thereto; and (ii) the amino acid sequence of an RT domain as listed in the same row of Table 7 or Table 8, or an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity thereto.
Exemplary Gene Modifying Polypeptides
In some embodiments, a gene modifying polypeptide (e.g., a gene modifying polypeptide that is part of a system described herein) comprises an amino acid sequence of any one of SEQ ID NOs: 1-7743 of the sequence listing, or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity thereto. In some embodiments, a gene modifying polypeptide comprises an amino acid sequence of any one of SEQ ID NOs: 1 -7743, or an amino acid sequence having at least 80% identity thereto. In some embodiments, a gene modifying polypeptide comprises an amino acid sequence of any one of SEQ ID NOs: 1 -7743, or an amino acid sequence having at least 90% identity thereto. In some embodiments, a gene modifying polypeptide comprises an amino acid sequence of any one of SEQ ID NOs: 1-7743, or an amino acid sequence having at least 95% identity thereto. In some embodiments, a gene modifying polypeptide comprises an amino acid sequence of any one of SEQ ID NOs: 1-7743, or an amino acid sequence having at least 99% identity thereto. In some embodiments, a gene modifying polypeptide comprises an amino acid sequence of any one of SEQ ID NOs: 1-7743. In some embodiments, a gene modifying polypeptide comprises an amino acid sequence of any one of SEQ ID NOs: 6001-7743, or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity thereto. In some embodiments, a gene modifying polypeptide comprises an amino acid sequence of any one of SEQ ID NOs: 4501-4541, or an amino acid sequence having at least at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity thereto. In some embodiments, a gene modifying polypeptide described herein comprises an RT and linker sequence from any of SEQ ID NOs: 1-7743, or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity thereto and a StlCas9 domain described herein. In some embodiments, a gene modifying polypeptide described herein comprises an RT and linker sequence from any of SEQ ID NOs: 1-7743, and a StlCas9 domain described herein.
In some embodiments, a gene modifying polypeptide comprises an amino acid sequence as listed in Table 12, or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity thereto.
In some embodiments, a gene modifying polypeptide comprises an amino acid sequence as listed in Table 7, or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity thereto. In some embodiments, a gene modifying polypeptide comprises a linker comprising a linker sequence as listed in Table 7, or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity thereto. In some embodiments, a gene modifying polypeptide comprises an RT domain comprising an RT domain sequence as listed in Table 7, or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity thereto. In some embodiments, a gene modifying polypeptide comprises: (i) a linker comprising a linker sequence as listed in a row of Table 7, or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity thereto; and (ii) an RT domain comprising an RT domain sequence as listed in the same row of Table 7, or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity thereto.
Table 7: Selection of exemplary gene modifying polypeptides
In some embodiments, a gene modifying polypeptide comprises an amino acid sequence as listed in Table 8, or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity thereto. In some embodiments, a gene modifying polypeptide comprises a linker comprising a linker sequence as listed in Table 8, or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity thereto. In some embodiments, a gene modifying polypeptide comprises an RT domain comprising an RT domain sequence as listed in Table 8, or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity thereto. In some embodiments, a gene modifying polypeptide comprises: (i) a linker comprising a linker sequence as listed in a row of Table 8, or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity thereto; and (ii) an RT domain comprising an RT domain sequence as listed in the same row of Table 8, or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity thereto.
Table 8: Selection of exemplary gene modifying polypeptides
Subsequences of Exemplary Gene Modifying Polypeptides
In some embodiments, a gene modifying polypeptide comprises, in N-terminal to C- terminal order, one or more (e.g., 1, 2, 3, 4, 5, or all 6) of an N-terminal methionine residue, a first nuclear localization signal (NLS), a DNA binding domain, a linker, an RT domain, and/or a second NLS. In some embodiments, a gene modifying polypeptide comprises, in N-terminal to C-terminal order, a NLS (e.g., a first NLS), a DNA binding domain, a linker, and an RT domain, wherein the linker and RT domain are the linker and RT domain of a gene modifying polypeptide of any one of SEQ ID NOs: 1-7743, or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity to said linker and RT domain. In some embodiments, a gene modifying polypeptide comprises, in N-terminal to C-terminal order, a DNA binding domain, a linker, an RT domain, and an NLS (e.g., a second NLS) wherein the linker and RT domain are the linker and RT domain of a gene modifying polypeptide of any one of SEQ ID NOs: 1-7743, or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity to said linker and RT domain. In some embodiments, a gene modifying polypeptide comprises, in N-terminal to C-terminal order, a first NLS, a DNA binding domain, a linker, an RT domain, and a second NLS, wherein the linker and RT domain are the linker and RT domain of a gene modifying polypeptide of any one of SEQ ID NOs: 1-7743, or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity to said linker and RT domain. In some embodimetns, the gene modifying polypeptide further comprises an N-terminal methionine residue.
In some embodiments, a gene modifying polypeptide comprises, in N-terminal to C- terminal order, one or more (e.g., 1, 2, 3, 4, 5, or all 6) of an N-terminal methionine residue, a first nuclear localization signal (NLS) (e.g., of a gene modifying polypeptide of any one of SEQ ID NOs: 1-7743 and/or as listed in any of Table 12, Table 7, or Table 8, or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity thereto), a DNA binding domain (e.g., a Cas domain, e.g., a SpyCas9 domain, e.g., as listed in Table 4, or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity thereto; or a DNA binding domain of a gene modifying polypeptide of any one of SEQ ID NOs: 1-7743 and/or as listed in any of Table 12, Table 7, or Table 8, or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity thereto), a linker (e.g., of a gene modifying polypeptide of any one of SEQ ID NOs: 1-7743 and/or as listed in any of Table 12, Table 7, or Table 8, or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity thereto), an RT domain (e.g., of a gene modifying polypeptide of any one of SEQ ID NOs: 1-7743 and/or as listed in any of Table 12, Table 7, or Table 8, or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity thereto), and a second NLS (e.g., of a gene modifying polypeptide of any one of SEQ ID NOs: 1- 7743 and/or as listed in any of Table 12, Table 7, or Table 8, or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity thereto). In some embodiments, the gene modifying polypeptide further comprises (e.g., C-terminal to the second NLS) a T2A sequence and/or a puromycin sequence (e.g., of a gene modifying polypeptide of any one of SEQ ID NOs: 1-7743 and/or as listed in any of Table 12, Table 7, or Table 8, or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity thereto). In some embodiments, a nucleic acid encoding a gene modifying polypeptide (e.g., as described herein) encodes a T2A sequence, e.g., wherein the T2A sequence is situated between a region encoding the gene modifying polypeptide and a second region, wherein the second region optionally encodes a selectable marker, e.g., puromycin.
In some embodiments, the first NLS comprises a first NLS sequence of a gene modifying polypeptide having an amino acid sequence of any one of SEQ ID NOs: 1-7743, or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity thereto. In certain embodiments, the first NLS comprises a first NLS sequence of a gene modifying polypeptide as listed in any of Table 12, Table 7, or Table 8, or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99%idcntity thereto. In some embodiments, a first NLS sequence comprises a C-myc NLS. In some embodiments, a first NLS comprises the amino acid sequence PAAKRVKLD (SEQ ID NO: 11,095), or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity thereto.
In some embodiments, a gene modifying polypeptide further comprises a spacer sequence between a first NLS and a DNA binding domain. In some embodiments, a spacer sequence between a first NLS and a DNA binding domain comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids. In some embodiments, a spacer sequence between a first NLS and a DNA binding domain comprises the amino acid sequence GG.
In some embodiments, a DNA binding domain comprises a DNA binding domain of a gene modifying polypeptide of any one of SEQ ID NOs: 1-7743, or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity thereto. In some embodiments, a DNA binding domain comprises a DNA binding domain of a gene modifying polypeptide as listed in any of Table 12, Table 7, or Table 8, or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity thereto. In some embodiments, a DNA binding domain comprises a Cas domain (e.g., as listed in Table 4). In some embodiments, a DNA binding domain comprises the amino acid sequence of a SpyCas9 polypeptide (e.g., as listed in Table 4, e.g., a Cas9 N863A polypeptide), or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity thereto. In some embodiments, a DNA binding domain comprises the amino acid sequence:
DKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSG ETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDK KHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRG HFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRL ENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDN LLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLT LLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEE LLVKLNREDLLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILT FRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDK NLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFK TNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDN EENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQLKRRRYTGWGRLSR KLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDS LHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMARENQTTQKG QKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQE LDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKARGKSDNVPSEEVVKKMKN YWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILD SRMNTKYDENDKL1REVKV1TLKSKLVSDFRKDFQFYKVRE1NNYHHAHDAYLN AVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMN FFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEV QTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKS KKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRK RMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKH YLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAP AAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDLSQLGGD (SEQ ID NO: 11,096) or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity thereto.
In some embodiments, a gene modifying polypeptide further comprises a spacer sequence between a DNA binding domain and a linker. In some embodiments, a spacer sequence between a DNA binding domain and a linker comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids. In some embodiments, a spacer sequence between a DNA binding domain and a linker comprises the amino acid sequence GG.
In some embodiments, a linker comprises a linker sequence of a gene modifying polypeptide of any one of SEQ ID NOs: 1-7743, or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity thereto. In certain embodiments, the linker comprises a linker sequence of a gene modifying polypeptide as listed in any of Table 12, Table 7, or Table 8, or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity thereto. In some embodiments, the linker comprises an amino acid sequence as listed in Table 6, Table 7 or Table 8 , or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity thereto.
In some embodiments, a gene modifying polypeptide further comprises a spacer sequence between a linker and an RT domain. In some embodiments, a spacer sequence between a linker and an RT domain comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids. In some embodiments, a spacer sequence between a linker and an RT domain comprises the amino acid sequence GG. In some embodiments, an RT domain comprises an RT domain sequence of a gene modifying polypeptide of any one of SEQ ID NOs: 1-7743, or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity thereto. In some embodiments, an RT domain comprises a RT domain sequence of a gene modifying polypeptide as listed in any of Table 12, Table 7, or Table 8, or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity thereto. In some embodiments, an RT domain comprises an amino acid sequence as listed in Table 7, Table 8, or Table 1, or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity thereto. In some embodiments, an RT domain has a length of about 400-500, about 500-600, about 600-700, about 700-800, about 800-900, or about 900-1000 amino acids.
In some embodiments, a gene modifying polypeptide further comprises a spacer sequence between an RT domain and a second NLS. In some embodiments, a spacer sequence between an RT domain and a second NLS comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids. In some embodiments, a spacer sequence between an RT domain and a second NLS comprises the amino acid sequence AG.
In some embodiments, a second NLS comprises a second NLS sequence of a gene modifying polypeptide of any one of SEQ ID NOs: 1-7743. In some embodiments, a second NLS comprises a second NLS sequence of a gene modifying polypeptide as listed in any of Table 12, Table 7, or Table 8. In some embodiments, a second NLS sequence comprises a plurality of partial NLS sequences. In some embodiments, an NLS sequence, e.g., a second NLS sequence, comprises a first partial NLS sequence, e.g., comprising the amino acid sequence KRTADGSEFE (SEQ ID NO: 11,097), or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity thereto. In embodiments, an NLS sequence, e.g., a second NLS sequence, comprises a second partial NLS sequence. In some embodiments, an NLS sequence, e.g., a second NLS sequence, comprises an SV40A5 NLS, e.g., a bipartite SV40A5 NLS, e.g., comprising the amino acid sequence KRTADGSEFESPKKKAKVE (SEQ ID NO: 11,098), or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity thereto. In some embodiments, an NLS sequence, e.g., a second NLS sequence, comprises the amino acid sequence KRTADGSEFEKRTADGSEFESPKKKAKVE (SEQ ID NO: 11,099), or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity thereto.
In some embodiments, a gene modifying polypeptide further comprises a spacer sequence between a second NLS and a T2A sequence and/or puromycin sequence. In some embodiments, a spacer sequence between a second NLS and a T2A sequence and/or puromycin sequence comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids. In some embodiments, a spacer sequence between a second NLS and a T2A sequence and/or puromycin sequence comprises the amino acid sequence GSG.
Linkers and RT domains
In some embodiments, a gene modifying polypeptide comprises a linker (e.g., as described herein) and an RT domain (e.g., as described herein). In some embodiments, a gene modifying polypeptide comprises, in N-tcrminal to C-tcrminal order, a linker (e.g., as described herein) and an RT domain (e.g., as described herein).
In some embodiments, a linker comprises a linker sequence as listed in Table 6, or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity thereto. In certain embodiments, the linker comprises a linker sequence of any one of SEQ ID NOs: 1-7743, or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity thereto. In some embodiments, a linker comprises a linker sequence of any one of SEQ ID NOs: 6001-7743, or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity thereto. In some embodiments, a linker comprises a linker sequence present in any one of SEQ ID NOs: 4501-4541, or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity thereto. In some embodiments, a linker comprises a linker sequence of an exemplary gene modifying polypeptide listed in any of Table 12, Table 7, or Table 8, or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity thereto. In some embodiments, an RT domain comprises an RT domain sequence as listed in Table 1, or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity thereto. In some embodiments, an RT domain comprises an RT domain sequence of an exemplary gene modifying polypeptide listed in any of Table 12, Table 7, or Table 8, or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity thereto.
In some embodiments, a gene modifying polypeptide comprises a portion of a gene modifying polypeptide of any one of SEQ ID NOs: 1-7743, wherein the portion comprises a linker and RT domain, or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity to said portion.
In some embodiments, a gene modifying polypeptide comprises a linker of a gene modifying polypeptide of any one of SEQ ID NOs: 1-7743, or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity to said linker. In some embodiments, a gene modifying polypeptide comprises a linker of a gene modifying polypeptide of any one of SEQ ID NOs: 6001-7743, or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity to said linker. In some embodiments, a gene modifying polypeptide comprises a linker of a gene modifying polypeptide of any one of SEQ ID NOs: 4501-4541, or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity to said linker. In some embodiments, a gene modifying polypeptide comprises a linker of a gene modifying polypeptide as listed in any of Table 12, Table 7, or Table 8, or a linker comprising an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity thereto.
In some embodiments, a gene modifying polypeptide comprises an RT domain of a gene modifying polypeptide of any one of SEQ ID NOs: 1-7743, or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity to said RT domain. In some embodiments, a gene modifying polypeptide comprises an RT domain of a gene modifying polypeptide of any one of SEQ ID NOs: 6001-7743, or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity said RT domain. In some embodiments, a gene modifying polypeptide comprises an RT domain of a gene modifying polypeptide of any one of SEQ ID NOs: 4501-4541, or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity said RT domain. In some embodiments, a gene modifying polypeptide comprises an RT domain of a gene modifying polypeptide as listed in any of Table 12, Table 7, or Table 8, or an RT domain comprising an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity thereto.
In some embodiments, a linker and an RT domain of a gene modifying polypeptide comprise the amino acid sequences of a linker and RT domain (or amino acid sequences having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity thereto) of a gene modifying polypeptide having the amino acid sequence of any one of SEQ ID NOs: 1-7743. In some embodiments, a linker and an RT domain of a gene modifying polypeptide comprise amino acid sequences of a linker and RT domain having at least 80% identity to the linker and RT domains of any one of SEQ ID NOs: 1-7743. In some embodiments, a linker and an RT domain of a gene modifying polypeptide comprise amino acid sequences of a linker and RT domain having at least 90% identity to the linker and RT domains of any one of SEQ ID NOs: 1-7743. In some embodiments, a linker and an RT domain of a gene modifying polypeptide comprise amino acid sequences of a linker and RT domain having at least 95% identity to the linker and RT domains of any one of SEQ ID NOs: 1-7743. In some embodiments, a linker and an RT domain of a gene modifying polypeptide comprise amino acid sequences of a linker and RT domain having at least 99% identity to the linker and RT domains of any one of SEQ ID NOs: 1-7743. In some embodiments, a linker and an RT domain of a gene modifying polypeptide comprise the amino acid sequences of a linker and RT domain (or amino acid sequences having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity thereto) of a gene modifying polypeptide having the amino acid sequence of any one of SEQ ID NOs: 6001-7743. In some embodiments, a linker and an RT domain of a gene modifying polypeptide comprise the amino acid sequences of a linker and RT domain (or amino acid sequences having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity thereto) of a gene modifying polypeptide having the amino acid sequence of any one of SEQ ID NOs: 4501-4541. In some embodiments, a linker and am RT domain of a gene modifying polypeptide comprise the amino acid sequences of a linker and RT domain (or amino acid sequences having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity thereto) from a single row of any of Table 12, Table 7, or Table 8 (e.g., from a single exemplary gene modifying polypeptide as listed in any of Table 12, Table 7, or Table 8). Insome embodiments, a linker and an RT domain of a gene modifying polypeptide comprise the amino acid sequences of a linker and RT domain (or amino acid sequences having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity thereto) from two different amino acid sequences selected from SEQ ID NOs: 1-7743. In some embodiments, a linker and an RT domain of a gene modifying polypeptide comprise the amino acid sequences of a linker and RT domain (or amino acid sequences having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity thereto) from different rows of any of Table 12, Table 7, or Table 8.
In some embodiments, a gene modifying polypeptide further comprises a first NLS (e.g., a 5’ NLS), e.g., as described herein. In some embodiments, a gene modifying polypeptide further comprises a second NLS (e.g., a 3’ NLS), e.g., as described herein. In some embodiments, a gene modifying polypeptide further comprises an N-terminal methionine residue.
RT Families and Mutants
In some embodiments, a gene modifying polypeptide comprises the amino acid sequence of an RT domain sequence from a family selected from: AVIRE, BAEVM, FFV, FLV, FOAMV, GALV, KORV, MLVAV, MLVBM, MLVCB, MLVFF, ML VMS, PERV, SFV1, SFV3L, WMSV, XMRV6, BLVAU, BLVJ, HTL1A, HTL1C, HTL1L, HTL32, HTL3P, HTLV2, JSRV, MLVF5, MLVRD, MMTVB, MPMV, SFVCP, SMRVH, SRV1, SRV2, and WDSV. In some embodiments, a gene modifying polypeptide comprises the amino acid sequence of an RT domain sequence from a family selected from: AVIRE, BAEVM, FFV, FLV, FOAMV, GALV, KORV, MLVAV, MLVBM, MLVCB, MLVFF, MLVMS, PERV, SFV1, SFV3L, WMSV, and XMRV6.
In some embodiments, a gene modifying polypeptide comprises the amino acid sequence of an RT domain sequence from an MLVMS RT domain. In some embodiments, the amino acid sequence of an RT domain sequence comprises one or more point mutations as listed in column 1 of Table 9, or a point mutation corresponding thereto. In some embodiments, the amino acid sequence of an RT domain sequence comprises one or more point mutations as listed in column 3 of Table 9 (Genl MLVMS), or a point mutation corresponding thereto. In some embodiments, the amino acid sequence of an RT domain sequence comprises one or more point mutations at an amino acid position of the RT domain as listed in columns 1 and 2 of Table 10, or an amino acid position corresponding thereto.
In some embodiments, a gene modifying polypeptide comprises the amino acid sequence of an RT domain sequence from an AVIRE RT domain. In some embodiments, the amino acid sequence of an RT domain sequence comprises one or more point mutations as listed in column 2 of Table 9, or a point mutation corresponding thereto. In some embodiments, the amino acid sequence of an RT domain sequence comprises one or more point mutations as listed in column 4 of Table 9 (Gen2 AVIRE), or a point mutation corresponding thereto. In some embodiments, the amino acid sequence of an RT domain sequence comprises one or more point mutations at an amino acid position of the RT domain as listed in columns 3 and 4 of Table 10, or an amino acid position corresponding thereto. In some embodiments, an RT domain comprises an IENSSP (e.g., at the C-terminus).
Table 9: Exemplary point mutations in ML VMS and AVIRE RT domains
Table 10: Positions that can be mutated in exemplary ML VMS and AVIRE RT domains
In some embodiments, a gene modifying polypeptide comprises a gamma retrovirus derived RT domain. In some embodiments, a gamma retrovirus-derived RT domain of a gene modifying polypeptide comprises the amino acid sequence of an RT domain sequence from a family selected from: AVIRE, BAEVM, FFV, FLV, FOAMV, GALV, KORV, MLVAV, MLVBM, MLVCB, MLVFF, ML VMS, PERV, SFV1 , SFV3L, WMSV, and XMRV6. In some embodiments, a gamma retrovirus-derived RT domain of a gene modifying polypeptide is not derived from PERV. In some embodiments, an RT domain includes one, two, three, four, five, six or more mutations shown in Table 2 and corresponding to mutations D200N, L603W, T330P, D524G, E562Q, D583N, P51L, S67R, E67K, T197A, H204R, E302K, F309N, W313F, L435G, N454K, H594Q, L67 IP, E69K, or D653N in the RT domain of murine leukemia virus reverse transcriptase. In some embodiments, a gene modifying polypeptide further comprises a linker having at least 99% identity to a linker domains of any one of SEQ ID NOs: 1-7743. In some embodiments, the gene modifying polypeptide further comprises a linker having at least 99% or 100% identity to SEQ ID NO: 5217 or SEQ ID NO: 11,041.
In some embodiments, an RT domain comprises the amino acid sequence of an RT domain of an AVIRE RT (e.g., an AVIRE_P03360 sequence, e.g., SEQ ID NO: 8001), or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity thereto. In some embodiments, an RT domain comprises the amino acid sequence of an AVIRE RT further comprising one, two, three, four, or five mutations selected from the group consisting of D200N, G33OP, L605W, T306K, and W313F, or a corresponding position in a homologous RT domain. In some embodiments, an RT domain comprises the amino acid sequence of an AVIRE RT further comprising one, two, or three mutations selected from the group consisting of D200N, G33OP, and L605W, or a corresponding position in a homologous RT domain.
In some embodiments, an RT domain comprises the amino acid sequence of an RT domain of a BAEVM RT (e.g., an BAEVM_P10272 sequence, e.g., SEQ ID NO: 8004), or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity thereto. In some embodiments, an RT domain comprises the amino acid sequence of a BAEVM RT further comprising one, two, three, four, or five mutations selected from the group consisting of D198N, E328P, L602W, T304K, and W311F, or a corresponding position in a homologous RT domain. In some embodiments, an RT domain comprises the amino acid sequence of a BAEVM RT further comprising one, two, or three mutations selected from the group consisting of D198N, E328P, and L602W, or a corresponding position in a homologous RT domain. In some embodiments, an RT domain comprises the amino acid sequence of an RT domain of an FFV RT (c.g., an FFV_O93209 sequence, c.g., SEQ ID NO: 8012), or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity thereto. In some embodiments, an RT domain comprises the amino acid sequence of an FFV RT further comprising one, two, three, or four mutations selected from the group consisting of D21N, T293N, T419P, and L393K, or a corresponding position in a homologous RT domain. In some embodiments, an RT domain comprises the amino acid sequence of an FFV RT further comprising one, two, or three mutations selected from the group consisting of D21N, T293N, and T419P, or a corresponding position in a homologous RT domain. In some embodiments, an RT domain comprises the amino acid sequence of an FFV RT further comprising the mutation D21N. In some embodiments, an RT domain comprises the amino acid sequence of an FFV RT further comprising one, two, or three mutations selected from the group consisting of T207N, T333P, and L307K, or a corresponding position in a homologous RT domain. In some embodiments, an RT domain comprises the amino acid sequence of an FFV RT further comprising one or two mutations selected from the group consisting of T207N and T333P, or a corresponding position in a homologous RT domain.
In some embodiments, an RT domain comprises the amino acid sequence of an RT domain of an FLV RT (e.g., an FLV_P10273 sequence, e.g., SEQ ID NO: 8019), or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity thereto. In some embodiments, an RT domain comprises the amino acid sequence of an FLV RT further comprising one, two, three, or four mutations selected from the group consisting of D199N, L602W, T305K, and W312F, or a corresponding position in a homologous RT domain. In some embodiments, an RT domain comprises the amino acid sequence of an FLV RT further comprising one or two mutations selected from the group consisting of D199N and L602W, or a corresponding position in a homologous RT domain.
In some embodiments, an RT domain comprises the amino acid sequence of an RT domain of a FOAMV RT (e.g., a FOAMV_P14350 sequence, e.g., SEQ ID NO: 8021), or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity thereto. In some embodiments, an RT domain comprises the amino acid sequence of a FOAMV RT further comprising one, two, three, or four mutations selected from the group consisting of D24N, T296N, S420P, and L396K, or a corresponding position in a homologous RT domain. In some embodiments, an RT domain comprises the amino acid sequence of a FOAMV RT further comprising one, two, or three mutations selected from the group consisting of D24N, T296N, and S420P, or a corresponding position in a homologous RT domain. In some embodiments, an RT domain comprises the amino acid sequence of a FOAMV RT further comprising the mutation D24N, or a corresponding position in a homologous RT domain. In some embodiments, an RT domain comprises the amino acid sequence of a FOAMV RT further comprising one, two, or three mutations selected from the group consisting of T207N, S331P, and L307K, or a corresponding position in a homologous RT domain. In some embodiments, an RT domain comprises the amino acid sequence of a FOAMV RT further comprising one or two mutations selected from the group consisting of T207N and S331P, or a corresponding position in a homologous RT domain.
In some embodiments, an RT domain comprises the amino acid sequence of an RT domain of a GALV RT (e.g., an GALV_P21414 sequence, e.g., SEQ ID NO: 8027), or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity thereto. In some embodiments, an RT domain comprises the amino acid sequence of a GALV RT further comprising one, two, three, four, or five mutations selected from the group consisting of D198N, E328P, L600W, T304K, and W31 IF, or a corresponding position in a homologous RT domain. In some embodiments, an RT domain comprises the amino acid sequence of a GALV RT further comprising one, two, or three mutations selected from the group consisting of D198N, E328P, and L600W, or a corresponding position in a homologous RT domain.
In embodiments, an RT domain comprises the amino acid sequence of an RT domain of a KORV RT (e.g., an KORV_Q9TTC1 sequence, e.g., SEQ ID NO: 8047), or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity thereto. In some embodiments, an RT domain comprises the amino acid sequence of a GALV RT further comprising one, two, three, four, five, or six mutations selected from the group consisting of D32N, D322N, E452P, L274W, T428K, and W435F, or a corresponding position in a homologous RT domain. In some embodiments, an RT domain comprises the amino acid sequence of a GALV RT further comprising one, two, three, or four mutations selected from the group consisting of D32N, D322N, E452P, and L274W, or a corresponding position in a homologous RT domain. In some embodiments, an RT domain comprises the amino acid sequence of a GALV RT further comprising the mutation D32N. Tn some embodiments, an RT domain comprises the amino acid sequence of a KORV RT further comprising one, two, three, four, or five mutations selected from the group consisting of D231N, E361P, L633W, T337K, and W344F, or a corresponding position in a homologous RT domain. In some embodiments, an RT domain comprises the amino acid sequence of a KORV RT further comprising one, two, or three mutations selected from the group consisting of D231N, E361P, and L633W, or a corresponding position in a homologous RT domain.
In some embodiments, an RT domain comprises the amino acid sequence of an RT domain of a MLVAV RT (e.g., an MLVAV_P03356 sequence, e.g., SEQ ID NO: 8053), or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity thereto. In some embodiments, an RT domain comprises the amino acid sequence of a MLVAV RT further comprising one, two, three, four, or five mutations selected from the group consisting of D200N, T33OP, L603W, T306K, and W313F, or a corresponding position in a homologous RT domain. In some embodiments, an RT domain comprises the amino acid sequence of a MLVAV RT further comprising one, two, or three mutations selected from the group consisting of D200N, T33OP, and L603W, or a corresponding position in a homologous RT domain.
In some embodiments, an RT domain comprises the amino acid sequence of an RT domain of a MLVBM RT (e.g., an MLVBM_Q7SVK7 sequence, e.g., SEQ ID NO: 8056), or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity thereto. In some embodiments, an RT domain comprises the amino acid sequence of a MLVBM RT further comprising one, two, three, four, or five mutations selected from the group consisting of D199N, T329P, L602W, T305K, and W312F, or a corresponding position in a homologous RT domain. In some embodiments, an RT domain comprises the amino acid sequence of a MLVBM RT further comprising one, two, and three mutations selected from the group consisting of D200N, T33OP, and L603W, or a corresponding position in a homologous RT domain.
In some embodiments, an RT domain comprises the amino acid sequence of an RT domain of a MLVCB RT (e.g., an MLVCB_P08361 sequence, e.g., SEQ ID NO: 8062), or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity thereto. In some embodiments, an RT domain comprises the amino acid sequence of a MLVCB RT further comprising one, two, three, four, or five mutations selected from the group consisting of D200N, T33OP, L603W, T306K, and W313F, or a corresponding position in a homologous RT domain. In some embodiments, an RT domain comprises the amino acid sequence of a MLVCB RT further comprising one, two, and three mutations selected from the group consisting of D200N, T33OP, and L603W, or a corresponding position in a homologous RT domain.
In some embodiments, an RT domain comprises the amino acid sequence of an RT domain of a MLVFF RT, or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity thereto. In some embodiments, an RT domain comprises the amino acid sequence of a MLVFF RT further comprising one, two, three, four, or five mutations selected from the group consisting of D200N, T330P, L603W, T306K, and W313F, or a corresponding position in a homologous RT domain. In some embodiments, an RT domain comprises the amino acid sequence of a MLVFF RT further comprising one, two, and three mutations selected from the group consisting of D200N, T330P, and L603W, or a corresponding position in a homologous RT domain.
In embodiments, an RT domain comprises the amino acid sequence of an RT domain of a MLVMS RT (e.g., an MLVMS_reference sequence, e.g., SEQ ID NO: 8137; or an MLVMS_P03355 sequence, e.g., SEQ ID NO: 8070), or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity thereto. In some embodiments, an RT domain comprises the amino acid sequence of a MLVMS RT further comprising one, two, three, four, five, or six mutations selected from the group consisting of D200N, T330P, L603W, T306K, W313F, and H8Y, or a corresponding position in a homologous RT domain. In some embodiments, an RT domain comprises the amino acid sequence of a MLVMS RT further comprising one, two, three, four, or five mutations selected from the group consisting of D200N, T330P, L603W, T306K, and W313F, or a corresponding position in a homologous RT domain. In some embodiments, an RT domain comprises the amino acid sequence of a MLVMS RT further comprising one, two, or three mutations selected from the group consisting of D200N, T330P, and L603W, or a corresponding position in a homologous RT domain.
In some embodiments, an RT domain comprises the amino acid sequence of an RT domain of a PERV RT (e.g., an PERV_Q4VFZ2 sequence, e.g., SEQ ID NO: 8099), or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity thereto. In some embodiments, an RT domain comprises the amino acid sequence of a PERV RT further comprising one, two, three, four, or five mutations selected from the group consisting of D196N, E326P, L599W, T302K, and W309F, or a corresponding position in a homologous RT domain. In some embodiments, an RT domain comprises the amino acid sequence of a PERV RT further comprising one, two, or three mutations selected from the group consisting of D196N, E326P, and L599W, or a corresponding position in a homologous RT domain.
In some embodiments, an RT domain comprises the amino acid sequence of an RT domain of a SFV1 RT (e.g., an SFV1_P23O74 sequence, e.g., SEQ ID NO: 8105), or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity thereto. In some embodiments, an RT domain comprises the amino acid sequence of a SFV 1 RT further comprising one, two, three, or four mutations selected from the group consisting of D24N, T296N, N420P, and L396K, or a corresponding position in a homologous RT domain. In some embodiments, an RT domain comprises the amino acid sequence of a SFV 1 RT further comprising one, two, or three mutations selected from the group consisting of D24N, T296N, and N420P, or a corresponding position in a homologous RT domain. In some embodiments, an RT domain comprises the amino acid sequence of a SFV1 RT further comprising the D24N, or a corresponding position in a homologous RT domain.
In some embodiments, an RT domain comprises the amino acid sequence of an RT domain of a SFV3L RT (e.g., an SFV3L_P27401 sequence, e.g., SEQ ID NO: 8111), or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity thereto. In some embodiments, an RT domain comprises the amino acid sequence of a SFV3L RT further comprising one, two, three, or four mutations selected from the group consisting of D24N, T296N, N422P, and L396K, or a corresponding position in a homologous RT domain. In some embodiments, an RT domain comprises the amino acid sequence of a SFV3L RT further comprising one, two, or three mutations selected from the group consisting of D24N, T296N, and N422P, or a corresponding position in a homologous RT domain. In some embodiments, an RT domain comprises the amino acid sequence of a SFV3L RT further comprising the mutation D24N, or a corresponding position in a homologous RT domain. In some embodiments, an RT domain comprises the amino acid sequence of a SFV3L RT further comprising one, two, or three mutations selected from the group consisting of T307N, N333P, and L307K, or a corresponding position in a homologous RT domain. In some embodiments, an RT domain comprises the amino acid sequence of a SFV3L RT further comprising one or two mutations selected from the group consisting of T307N and N333P, or a corresponding position in a homologous RT domain.
In embodiments, an RT domain comprises the amino acid sequence of an RT domain of a WMSV RT (e.g., an WMSV_P03359 sequence, e.g., SEQ ID NO: 8131), or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity thereto. In some embodiments, an RT domain comprises the amino acid sequence of a WMSV RT further comprising one, two, three, four, or five mutations selected from the group consisting of D198N, E328P, L600W, T304K, and W31 IF, or a corresponding position in a homologous RT domain. In some embodiments, an RT domain comprises the amino acid sequence of a WMSV RT further comprising one, two, or three mutations selected from the group consisting of D198N, E328P, and L600W, or a corresponding position in a homologous RT domain.
In some embodiments, an RT domain comprises the amino acid sequence of an RT domain of a XMRV6 RT (e.g., an XMRV6_A1Z651 sequence, e.g., SEQ ID NO: 8134), or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity thereto. In some embodiments, an RT domain comprises the amino acid sequence of a XMRV6 RT further comprising one, two, three, four, or five mutations selected from the group consisting of D200N, T33OP, L603W, T306K, and W313F, or a corresponding position in a homologous RT domain. In some embodiments, an RT domain comprises the amino acid sequence of a XMRV6 RT further comprising one, two, or three mutations selected from the group consisting of D200N, T33OP, and L603W, or a corresponding position in a homologous RT domain.
In some embodiments, an RT domain of a gene modifying polypeptide comprises the amino acid sequence of an RT domain of an AVIRE RT, or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity thereto. In embodiments, an RT domain comprises the amino acid sequence of an RT domain comprised in a sequence listed in column 1 of Table 11, or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity thereto. In some embodiments, a gene modifying polypeptide further comprises a linker having at least 99% or 100% identity to SEQ ID NO: 5217 or SEQ ID NO:11,041.
In some embodiments, an RT domain of a gene modifying polypeptide comprises the amino acid sequence of an RT domain of an MLVMS RT, or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity thereto. In some embodiments, an RT domain comprises the amino acid sequence of an RT domain comprised in a sequence listed in any of columns 2-6 of Table 11, or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity thereto. In some embodiments, a gene modifying polypeptide further comprises a linker having at least 99% or 100% identity to SEQ ID NO: 5217 or SEQ ID NO:11,041.
Table 11: Exemplary gene modifying polypeptides comprising an AVIRE RT domain or an MLVMS RT domain.
Systems
In some embodiments, the present disclosure provides a system comprising a nucleic acid molecule encoding a gene modifying polypeptide (e.g., as described herein) and a template nucleic acid (e.g., a template RNA, e.g., as described herein). In some embodiments, a nucleic acid molecule encoding a gene modifying polypeptide comprises one or more silent mutations in the coding region (e.g., in the sequence encoding an RT domain) relative to a nucleic acid molecule as described herein. In some embodiments, a system further comprises a gRNA (e.g., a gRNA that binds to a polypeptide that induces a nick, e.g., in the opposite strand of a target DNA bound by a gene modifying polypeptide).
In some embodiments, a nucleic acid molecule encoding a gene modifying polypeptide encodes a polypeptide having an amino acid sequence selected from SEQ ID NOs: 1-7743, or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity thereto. In some embodiments, a nucleic acid molecule encoding a gene modifying polypeptide encodes a polypeptide having an amino acid sequence selected from SEQ ID NOs: 6001-7743, or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity thereto. In some embodiments, a nucleic acid molecule encoding a gene modifying polypeptide encodes a polypeptide having an amino acid sequence selected from SEQ ID NOs: 4501-4541, or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity thereto. In some embodiments, a nucleic acid molecule encoding a gene modifying polypeptide encodes a polypeptide as listed in any of Tables 12, 7, or 8, or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity thereto.
In some embodiments, the nucleic acid molecule encoding the gene modifying polypeptide comprises a sequence encoding a portion of an amino acid sequence selected from SEQ ID NOs: 1-7743, wherein the portion comprises a linker and RT domain, or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity to said portion. In some embodiments, the nucleic acid molecule encoding the gene modifying polypeptide comprises a sequence encoding a portion of an amino acid sequence selected from SEQ ID NOs: 6001-7743, wherein the portion comprises a linker and RT domain, or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity to said portion. In some embodiments, the nucleic acid molecule encoding the gene modifying polypeptide comprises a sequence encoding a portion of an amino acid sequence selected from SEQ ID NOs: 4501-4541, wherein the portion comprises a linker and RT domain, or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity to said portion. In some embodiments, the nucleic acid molecule encoding the gene modifying polypeptide comprises a sequence encoding a portion of a polypeptide listed in any of Table 12, Table 7, or Table 8, wherein the portion comprises a linker and RT domain, or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity to said portion.
In some embodiments, a nucleic acid molecule encoding a gene modifying polypeptide comprises a sequence encoding a linker of an amino acid sequence selected from SEQ ID NOs: 1-7743, or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity thereto. In some embodiments, a nucleic acid molecule encoding a gene modifying polypeptide comprises a sequence encoding a linker of a polypeptide having an amino acid sequence selected from SEQ ID NOs: 6001-7743, or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity thereto. In some embodiments, a nucleic acid molecule encoding a gene modifying polypeptide comprises a sequence encoding a linker of a polypeptide having an amino acid sequence selected from SEQ ID NOs: 4501-4541, or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity thereto. In some embodiments, a nucleic acid molecule encoding a gene modifying polypeptide comprises a sequence encoding a linker of a polypeptide as listed in any of Table 12, Table 7, or Table 8, or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity thereto.
In some embodiments, a nucleic acid molecule encoding a gene modifying polypeptide comprises a sequence encoding an RT domain of an amino acid sequence selected from SEQ ID NOs: 1-7743, or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity thereto. In some embodiments, a nucleic acid molecule encoding a gene modifying polypeptide comprises a sequence encoding an RT domain of a polypeptide having an amino acid sequence selected from SEQ ID NOs: 6001- 7743, or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity thereto. In some embodiments, a nucleic acid molecule encoding a gene modifying polypeptide comprises a sequence encoding an RT domain of a polypeptide having an amino acid sequence selected from SEQ ID NOs: 4501-4541 , or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity thereto. In some embodiments, a nucleic acid molecule encoding a gene modifying polypeptide comprises a sequence encoding an RT domain of a polypeptide as listed in any of Table 12, Table 7, or Table 8, or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity thereto.
In some embodiments, the present disclosure provides a system comprising a gene modifying polypeptide (e.g., as described herein) and a template nucleic acid (e.g., a template RNA, e.g., as described herein).
In some embodiments, a gene modifying polypeptide comprises a polypeptide having an amino acid sequence selected from any one of SEQ ID NOs: 1-7743, or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity thereto. In some embodiments, a gene modifying polypeptide comprises a polypeptide having an amino acid sequence selected from any one of SEQ ID NOs: 6001-7743, or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity thereto. In some embodiments, a gene modifying polypeptide comprises a polypeptide having an amino acid sequence selected from any one of SEQ ID NOs: 4501-4541, or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity thereto. In some embodiments, a gene modifying polypeptide comprises a polypeptide as listed in any of Table 12, Table 7, or Table 8, or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity thereto.
In some embodiments, a gene modifying polypeptide comprises a portion of an amino acid sequence selected from SEQ ID NOs: 1-7743, wherein the portion comprises a linker and RT domain, or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity to said portion. In some embodiments, a gene modifying polypeptide comprises a portion of an amino acid sequence selected from SEQ ID NOs: 6001-7743, wherein the portion comprises a linker and RT domain, or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity to said portion. In some embodiments, a gene modifying polypeptide comprises a portion of an amino acid sequence selected from SEQ ID NOs: 4501-4541 , wherein the portion comprises a linker and RT domain, or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity to said portion. In some embodiments, a gene modifying polypeptide comprises a portion of a polypeptide listed in any of Table 12, Table 7, or Table 8, wherein the portion comprises a linker and RT domain, or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity to said portion.
In some embodiments, a gene modifying polypeptide comprises a linker of an amino acid sequence selected from SEQ ID NOs: 1-7743, or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity thereto. In some embodiments, a gene modifying polypeptide comprises a sequence encoding the linker of a polypeptide having an amino acid sequence selected from SEQ ID NOs: 6001-7743, or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity thereto. In some embodiments, a gene modifying polypeptide comprises a sequence encoding the linker of a polypeptide having an amino acid sequence selected from SEQ ID NOs: 4501-4541, or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity thereto. In some embodiments, a gene modifying polypeptide comprises the linker of a polypeptide as listed in any of Table 12, Table 7, or Table 8, or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity thereto.
In some embodiments, a gene modifying polypeptide comprises an RT domain of an amino acid sequence selected from SEQ ID NOs: 1-7743, or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity thereto. In some embodiments, a gene modifying polypeptide comprises a sequence encoding an RT domain of a polypeptide having an amino acid sequence selected from SEQ ID NOs: 6001-7743, or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity thereto. In some embodiments, a gene modifying polypeptide comprises a sequence encodingan RT domain of a polypeptide having an amino acid sequence selected from SEQ ID NOs: 4501-4541, or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity thereto. In some embodiments, a gene modifying polypeptide comprises an RT domain of a polypeptide as listed in any of Table 12, Table 7, or Table 8, or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity thereto. Table 12: Exemplary amino acid sequences for gene modifying polypeptides
In some embodiments, a gene modifying polypeptide comprises an amino acid sequence selected from SEQ ID NOs: 26002, 26004, 10001, 10011, 10118, 10119, 10120, 31453, 31454, 31455, 31458, or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity thereto. Localization sequences for gene modifying systems
In some embodiments, a gene modifying system RNA further comprises an intracellular localization sequence, c.g., a nuclear localization sequence (NLS). In some embodiments, a gene modifying polypeptide comprises an NLS as comprised in SEQ ID NO: 4000 and/or SEQ ID NO: 4001, or an NLS having an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity thereto.
A nuclear localization sequence may be an RNA sequence that promotes the import of an RNA into the nucleus. In some embodiments, a nuclear localization signal is located on a template RNA. In some embodiments, a gene modifying polypeptide is encoded on a first RNA, and a template RNA is a second, separate, RNA, and a nuclear localization signal is located on the template RNA and not on an RNA encoding the gene modifying polypeptide. While not wishing to be bound by theory, in some embodiments, an RNA encoding a gene modifying polypeptide is targeted primarily to the cytoplasm to promote its translation, while a template RNA is targeted primarily to the nucleus to promote insertion into the genome. In some embodiments a nuclear localization signal is at the 3' end, 5' end, or in an internal region of a template RNA. In some embodiments, a nuclear localization signal is 3' of a heterologous sequence (e.g., is directly 3' of the heterologous sequence) or is 5' of the heterologous sequence (e.g., is directly 5' of the heterologous sequence). In some embodiments, a nuclear localization signal is placed outside of a 5' UTR or outside of a 3' UTR of a template RNA. In some embodiments, a nuclear localization signal is placed between a 5' UTR and a 3' UTR, wherein optionally the nuclear localization signal is not transcribed with a transgene (e.g., the nuclear localization signal is in an anti-sense orientation or is downstream of a transcriptional termination signal or polyadenylation signal). In some embodiments, a nuclear localization sequence is situated inside of an intron. In some embodiments, a plurality of the same or different nuclear localization signals are in an RNA, e.g., in a template RNA. In some embodiments, a nuclear localization signal is less than 5, less than 10, less than 25, less than 50, less than 75, less than 100, less than 150, less than 200, less than 250, less than 300, less than 350, less than 400, less than 450, less than 500, less than 600, less than 700, less than 800, less than 900 or less than 1000 bp in length. Various RNA nuclear localization sequences can be used. For example, Lubelsky and Ulitsky, Nature 555 (107-111), 2018 describe RNA sequences which drive RNA localization into the nucleus. In some embodiments, a nuclear localization signal is a SINE-derived nuclear RNA localization (SIRLOIN) signal. In some embodiments, a nuclear localization signal binds a nuclear-enriched protein. In some embodiments, a nuclear localization signal binds the HNRNPK protein. In some embodiments, a nuclear localization signal is rich in pyrimidines, e.g., is a C/T rich, C/U rich, C rich, T rich, or U rich region. In some embodiments, a nuclear localization signal is derived from a long non-coding RNA. In some embodiments, a nuclear localization signal is derived from MALAT1 long non-coding RNA or is the 600 nucleotide M region of MALAT 1 (described in Miyagawa et al., RNA 18, (738-751), 2012). In some embodiments a nuclear localization signal is derived from BORG long non-coding RNA or is a AGCCC motif (described in Zhang et al., Molecular and Cellular Biology 34, 2318-2329 (2014). In some embodiments, a nuclear localization sequence is described in Shukla et al., The EMBO Journal c98452 (2018). In some embodiments, a nuclear localization signal is derived from a retrovirus.
In some embodiments, a gene modifying polypeptide described herein comprises one or more (e.g., 2, 3, 4, 5) nuclear targeting sequences, for example a nuclear localization sequence (NLS). In some embodiments, an NLS is a bipartite NLS. In some embodiments, an NLS facilitates the import of a protein comprising an NLS into the cell nucleus. In some embodiments, an NLS is fused to the N-terminus of a gene modifying polypeptide as described herein. In some embodiments, an NLS is fused to the C-terminus of a gene modifying polypeptide. In some embodiments, an NLS is fused to the N-terminus or the C-terminus of a Cas domain. In some embodiments, a linker sequence is disposed between an NLS and a neighboring domain of a gene modifying polypeptide.
In some embodiments, an NLS comprises the amino acid sequence MDSLLMNRRKFLYQFKNVRWAKGRRETYLC (SEQ ID NO: 5009), PKKRKVEGADKRTADGSEFESPKKKRKV(SEQ ID NO: 5010), RKSGKIAAIWKRPRKPKKKRKV (SEQ ID NO: 5011) KRTADGSEFESPKKKRKV(SEQ ID NO: 5012), KKTELQTTNAENKTKKL (SEQ ID NO: 5013), or KRGINDRNFWRGENGRKTR (SEQ ID NO: 5014), KRPAATKKAGQAKKKK (SEQ ID NO: 5015), PAAKRVKLD (SEQ ID NO:4644), KRTADGSEFEKRTADGSEFESPKKKAKVE (SEQ ID NO: 4649), KRTADGSEFE (SEQ ID NO: 4650), KRTADGSEFESPKKKAKVE (SEQ ID NO: 4651), AGKRTADGSEFEKRTADGSEFESPKKKAKVE (SEQ ID NO: 4001), or a functional fragment or variant thereof. Exemplary NLS sequences are also described in PCT/EP2000/011690, the contents of which are incorporated herein by reference for their disclosure of exemplary nuclear localization sequences. In some embodiments, an NLS comprises an amino acid sequence as disclosed in Table 13. An NLS of Table 13 may be utilized with one or more copies in a polypeptide in one or more locations in a polypeptide, e.g., 1, 2, 3 or more copies of an NLS in an N-terminal domain, between peptide domains, in a C- terminal domain, or in a combination of locations, in order to improve subcellular localization to the nucleus. Multiple unique sequences may be used within a single polypeptide. Sequences may be naturally monopartite or bipartite, e.g., having one or two stretches of basic amino acids, or may be used as chimeric bipartite sequences. Sequence references correspond to UniProt accession numbers, except where indicated as SeqNLS for sequences mined using a subcellular localization prediction algorithm (Lin et al BMC Bioinformat 13:157 (2012), incorporated herein by reference in its entirety).
Table 13: Exemplary nuclear localization signals for use in gene modifying systems
In some embodiments, an NLS is a bipartite NLS. A bipartite NLS typically comprises two basic amino acid clusters separated by a spacer sequence (which may be, e.g., about 10 amino acids in length). A monopartite NLS typically lacks a spacer. An example of a bipartite NLS is the nucleoplasmin NLS, having the sequence KR[PAATKKAGQA]KKKK (SEQ ID NO: 5015), wherein the spacer is indicated in square brackets. Another exemplary bipartite NLS has the sequence PKKKRKVEGADKRTADGSEFESPKKKRKV (SEQ ID NO: 5016). Exemplary NLSs are described in International Application W02020051561, which is herein incorporated by reference in its entirety, including for its disclosures regarding nuclear localization sequences.
In some embodiments, a gene editor system polypeptide (e.g., a gene modifying polypeptide as described herein) further comprises an intracellular localization sequence, e.g., a nuclear localization sequence and/or a nucleolar localization sequence. A nuclear localization sequence and/or nucleolar localization sequence may be an amino acid sequences that promotes the import of the protein into the nucleus and/or nucleolus, where it can promote integration of heterologous sequence into the genome. In some embodiments, a gene editor system polypeptide (e.g., (e.g., a gene modifying polypeptide as described herein) further comprises a nucleolar localization sequence. In some embodiments, a gene modifying polypeptide is encoded on a first RNA, and a template RNA is a second, separate, RNA, and a nucleolar localization signal is encoded on the RNA encoding the gene modifying polypeptide and not on the template RNA. In some embodiments, a nucleolar localization signal is located at the N-terminus, C- terminus, or in an internal region of a gene modifying polypeptide. In some embodiments, a plurality of the same or different nucleolar localization signals are used. In some embodiments, a nuclear localization signal is less than 5, less than 10, less than 25, less than 50, less than 75, or less than 100 amino acids in length. Various polypeptide nucleolar localization signals can be used. For example, Yang et al., Journal of Biomedical Science 22, 33 (2015), describe a nuclear localization signal that also functions as a nucleolar localization signal. In some embodiments, a nucleolar localization signal may also be a nuclear localization signal. In some embodiments, a nucleolar localization signal may overlap with a nuclear localization signal. In some embodiments, a nucleolar localization signal may comprise a stretch of basic residues. In some embodiments, a nucleolar localization signal may be rich in arginine and lysine residues. In some embodiments, a nucleolar localization signal may be derived from a protein that is enriched in the nucleolus. In some embodiments, a nucleolar localization signal may be derived from a protein enriched at ribosomal RNA loci. In some embodiments, a nucleolar localization signal may be derived from a protein that binds rRNA. In some embodiments, a nucleolar localization signal may be derived from MSP58. In some embodiments, a nucleolar localization signal may be a monopartite motif. In some embodiments, a nucleolar localization signal may be a bipartite motif. In some embodiments, a nucleolar localization signal may comprise multiple monopartite or bipartite motifs. In some embodiments, a nucleolar localization signal may comprise a mix of monopartite and bipartite motifs. In some embodiments, a nucleolar localization signal may be a dual bipartite motif. In some embodiments, a nucleolar localization motif may be a KRASSQALGTIPKRRSSSRFIKRKK (SEQ ID NO: 5017). In some embodiments, a nucleolar localization signal may be derived from nuclear factor-KB-inducing kinase. In some embodiments, a nucleolar localization signal may be an RKKRKKK motif (SEQ ID NO: 5018) (described in Birbach et al., Journal of Cell Science, 117 (3615-3624), 2004).
Evolved Variants of Gene Modifying Polypeptides and Systems
In some embodiments, the present disclosure provides evolved variants of gene modifying polypeptides as described herein. Evolved variants can, in some embodiments, be produced by mutagenizing a reference gene modifying polypeptide, or one of the fragments or domains comprised therein. In some embodiments, one or more domains (e.g., a reverse transcriptase domain) is evolved. One or more of such evolved variant domains can, in some embodiments, be evolved alone or together with other domains. An evolved variant domain or domains may, in some embodiments, be combined with unevolved cognate component(s) or evolved variants of the cognate component(s), e.g., which may have been evolved in either a parallel or serial manner.
In some embodiments, a process of mutagenizing a reference gene modifying polypeptide, or fragment or domain thereof, comprises mutagenizing a reference gene modifying polypeptide or fragment or domain thereof. In some embodiments, mutagenesis comprises a continuous evolution method (e.g., PACE) or non-continuous evolution method (e.g., PANCE), e.g., as described herein. In some embodiments, an evolved gene modifying polypeptide, or a fragment or domain thereof, comprises one or more amino acid variations introduced into its amino acid sequence relative to the amino acid sequence of a reference gene modifying polypeptide, or fragment or domain thereof. In some embodiments, amino acid sequence variations may include one or more mutated residues (e.g., conservative substitutions, nonconservative substitutions, or a combination thereof) within the amino acid sequence of a reference gene modifying polypeptide, e.g., as a result of a change in the nucleotide sequence encoding the gene modifying polypeptide that results in, e.g., a change in the codon at any particular position in the coding sequence, the deletion of one or more amino acids (e.g., a truncated protein), the insertion of one or more amino acids, or any combination of the foregoing. An evolved variant gene modifying polypeptide may include variants in one or more components or domains of the gene modifying polypeptide (e.g., variants introduced into a reverse transcriptase domain).
In some embodiments, the present disclosure provides gene modifying polypeptides, systems, kits, and methods using or comprising an evolved variant of a gene modifying polypeptide, e.g., employs an evolved variant of a gene modifying polypeptide or a gene modifying polypeptide produced or producible by PACE or PANCE. In some embodiments, an unevolved reference gene modifying polypeptide is a gene modifying polypeptide as disclosed herein.
The term “phage-assisted continuous evolution (PACE),”as used herein, generally refers to continuous evolution that employs phage as viral vectors. Examples of PACE technology have been described, for example, in International PCT Application No. PCT/US 2009/056194, filed September 8, 2009, published as WO 2010/028347 on March 11, 2010; International PCT Application, PCT/US2011/066747, filed December 22, 2011, published as WO 2012/088381 on June 28, 2012; U.S. Patent No. 9,023,594, issued May 5, 2015; U.S. Patent No. 9,771,574, issued September 26, 2017; U.S. Patent No. 9,394,537, issued July 19, 2016; International PCT Application, PCT/US2015/012022, filed January 20, 2015, published as WO 2015/134121 on September 11, 2015; U.S. Patent No. 10,179,911, issued January 15, 2019; and International PCT Application, PCT/US2016/027795, filed April 15, 2016, published as WO 2016/168631 on October 20, 2016, the entire contents of each of which are incorporated herein by reference.
The term “phage-assisted non-continuous evolution (PANCE),” as used herein, generally refers to non-continuous evolution that employs phage as viral vectors. Examples of PANCE technology have been described, for example, in Suzuki T. et al, Crystal structures reveal an elusive functional domain of pyrrolysyl-tRNA synthetase, Nat Chem Biol. 13(12): 1261-1266 (2017), incorporated herein by reference in its entirety. Briefly, PANCE is a technique for rapid in vivo directed evolution using serial flask transfers of evolving selection phage (SP), which contain a gene of interest to be evolved, across fresh host cells (e.g., E. coli cells). Genes inside the host cell may be held constant while genes contained in the SP continuously evolve. Following phage growth, an aliquot of infected cells may be used to transfect a subsequent flask containing host E. coli. This process can be repeated and/or continued until the desired phenotype is evolved, e.g., for as many transfers as desired.
Methods of applying PACE and PANCE to gene modifying polypeptides may be readily appreciated by askilled artisan by reference to, inter alia, the foregoing references. Additional exemplary methods for directing continuous evolution of genome-modifying proteins or systems, e.g., in a population of host cells, e.g., using phage particles, can be applied to generate evolved variants of gene modifying polypeptides, or fragments or subdomains thereof. Non-limiting examples of such methods are described in International PCT Application, PCT/US2009/056194, filed September 8, 2009, published as WO 2010/028347 on March 11, 2010; International PCT Application, PCT/US2011/066747, filed December 22, 2011, published as WO 2012/088381 on June 28, 2012; U.S. Patent No. 9,023,594, issued May 5, 2015; U.S. Patent No. 9,771,574, issued September 26, 2017; U.S. Patent No. 9,394,537, issued July 19, 2016; International PCT Application, PCT/US2015/012022, filed January 20, 2015, published as WO 2015/134121 on September 11, 2015; U.S. Patent No. 10,179,911, issued January 15, 2019; International Application No. PCT/US2019/37216, filed June 14, 2019, International Patent Publication WO 2019/023680, published January 31, 2019, International PCT Application, PCT/US2016/027795, filed April 15, 2016, published as WO 2016/168631 on October 20, 2016, and International Patent Publication No. PCT/US2019/47996, filed August 23, 2019, each of which is incorporated herein by reference in its entirety.
In some non-limiting illustrative embodiments, a method of evolution of an evolved variant gene modifying polypeptide, of a fragment or domain thereof, comprises: (a) contacting a population of host cells with a population of viral vectors comprising the gene of interest (the starting gene modifying polypeptide or fragment or domain thereof), wherein: (1) the host cell is amenable to infection by the viral vector; (2) the host cell expresses viral genes required for the generation of viral particles; (3) the expression of at least one viral gene required for the production of an infectious viral particle is dependent on a function of the gene of interest; and/or (4) the viral vector allows for expression of the protein in the host cell, and can be replicated and packaged into a viral particle by the host cell. In some embodiments, the method comprises (b) contacting the host cells with a mutagen, using host cells with mutations that elevate mutation rate (e.g., either by carrying a mutation plasmid or some genome modification — e.g., proofing- impaired DNA polymerase, SOS genes, such as UmuC, UmuD’, and/or RecA, which mutations, if plasmid-bound, may be under control of an inducible promoter), or a combination thereof. In some embodiments, the method additionally comprises (c) incubating the population of host cells under conditions allowing for viral replication and the production of viral particles, wherein host cells are removed from the host cell population, and fresh, uninfected host cells are introduced into the population of host cells, thus replenishing the population of host cells and creating a flow of host cells. In some embodiments, the cells are incubated under conditions allowing for the gene of interest to acquire a mutation. In some embodiments, the method further comprises (d) isolating a mutated version of the viral vector, encoding an evolved gene product (e.g., an evolved variant gene modifying polypeptide, or fragment or domain thereof), from the population of host cells.
A skilled artisan will appreciate a variety of features employable within the abovedescribed framework. For example, in some embodiments, a viral vector or a phage is a filamentous phage, for example, an M13 phage, e.g., an M13 selection phage. In some embodiments, a gene required for the production of infectious viral particles is the M13 gene III (gill). In embodiments, a phage may lack a functional gill, but otherwise comprise gl, gll, gIV, gV, gVI, gVII, gVIII, glX, and a gX. In some embodiments, generation of infectious VSV particles involves the envelope protein VSV-G. Various embodiments can use different retroviral vectors, for example, Murine Leukemia Virus vectors, or Lentiviral vectors. In some embodiments, retroviral vectors can efficiently be packaged with VSV-G envelope protein, e.g., as a substitute for the native envelope protein of the virus.
In some embodiments, host cells are incubated according to a suitable number of viral life cycles, e.g., at least 10, at least 20, at least 30, at least 40, at least 50, at least 100, at least 200, at least 300, at least 400, at least, 500, at least 600, at least 700, at least 800, at least 900, at least 1000, at least 1250, at least 1500, at least 1750, at least 2000, at least 2500, at least 3000, at least 4000, at least 5000, at least 7500, at least 10000, or more consecutive viral life cycles, which in on illustrative and non-limiting examples of M13 phage is 10-20 minutes per virus life cycle. Similarly, conditions can be modulated to adjust the time a host cell remains in a population of host cells, e.g., about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, about 25, about 30, about 35, about 40, about 45, about 50, about 55, about 60, about 70, about 80, about 90, about 100, about 120, about 150, or about 180 minutes. Host cell populations can be controlled in part by density of the host cells, or, in some embodiments, the host cell density in an inflow, e.g., 103 cells/ml, about 104 cells/ml, about IO3 cells/ml, about 5 - 105 cells/ml, about 106 cells/ml, about 5 - 106 cells/ml, about 107 cells/ml, about 5 - 107 cells/ml, about 108 cells/ml, about 5 - 108 cells/ml, about 109 cells/ml, about 5-- 109 cells/ml, about 1010 cells/ml, or about 5-- 1010 cells/ml.
Inteins
In some embodiments, as described in more detail below, an intein-N (intN) domain may be fused to the N-terminal portion of a first domain of a gene modifying polypeptide described herein, and an intein-C (intC) domain may be fused to the C-terminal portion of a second domain of a gene modifying polypeptide described herein for the joining of the N-terminal portion to the C-terminal portion, thereby joining the first and second domains. In some embodiments, the first and second domains are each independently chosen from a DNA binding domain, an RNA binding domain, an RT domain, and an endonuclease domain.
Inteins can occur as self-splicing protein intron (e.g., peptide), e.g., which ligates flanking N-terminal and C-terminal exteins (e.g., fragments to be joined). An intein may, in some embodiments, comprise a fragment of a protein that is able to excise itself and join the remaining fragments (the exteins) with a peptide bond in a process known as protein splicing. Inteins are also referred to as “protein introns.” The process of an intein excising itself and joining the remaining portions of the protein is herein termed “protein splicing” or “intcin-mcdiatcd protein splicing.”
In some embodiments, an intein of a precursor protein (an intein containing protein prior to intein-mediated protein splicing) comes from two genes. Such intein is referred to herein as a split intein (e.g., split intein-N and split intein-C). Accordingly, an intein-based approach may be used to join a first polypeptide sequence and a second polypeptide sequence together. For example, in cyanobacteria, DnaE, the catalytic subunit a of DNA polymerase III, is encoded by two separate genes, dnaE-n and dnaE-c. An intein-N domain, such as that encoded by the dnaE- n gene, when situated as part of a first polypeptide sequence, may join the first polypeptide sequence with a second polypeptide sequence, wherein the second polypeptide sequence comprises an intein-C domain, such as that encoded by the dnaE-c gene. Accordingly, in some embodiments, a protein can be made by providing nucleic acid encoding the first and second polypeptide sequences (e.g., wherein a first nucleic acid molecule encodes the first polypeptide sequence and a second nucleic acid molecule encodes the second polypeptide sequence), and the nucleic acid is introduced into the cell under conditions that allow for production of the first and second polypeptide sequences, and for joining of the first to the second polypeptide sequence via an intein-based mechanism.
Use of inteins for joining heterologous protein fragments is described, for example, in Wood et al., J. Biol. Chem.289(21); 14512-9 (2014) (incorporated herein by reference in its entirety). For example, when fused to separate protein fragments, the inteins IntN and IntC may recognize each other, splice themselves out, and/or simultaneously ligate the flanking N- and C- terminal exteins of the protein fragments to which they were fused, thereby reconstituting a full- length protein from the two protein fragments.
In some embodiments, a synthetic intein based on the dnaE intein, the Cfa-N (e.g., split intein-N) and Cfa-C (e.g., split intein-C) intein pair, is used. Examples of such inteins have been described, e.g., in Stevens et al., J Am Chem Soc. 2016 Feb. 24; 138(7):2162-5 (incorporated herein by reference in its entirety). Non-limiting examples of intein pairs that may be used in accordance with the present disclosure include: Cfa DnaE intein, Ssp GyrB intein, Ssp DnaX intein, Ter DnaE3 intein, Ter ThyX intein, Rma DnaB intein and Cne Prp8 intein (e.g., as described in U.S. Pat. No. 8,394,604, incorporated herein by reference. In some embodiments involving a split Cas9, an intein-N domain and an intein-C domain may be fused to the N-tcrminal portion of the split Cas9 and the C-tcrminal portion of a split Cas9, respectively, for the joining of the N-terminal portion of the split Cas9 and the C-terminal portion of the split Cas9. For example, in some embodiments, an intein-N is fused to the C- terminus of the N-terminal portion of the split Cas9, i.e., to form a structure of N — [N-terminal portion of the split Cas9] -[intein-N] ~ C. In some embodiments, an intein-C is fused to the N- terminus of the C-terminal portion of the split Cas9, i.e., to form a structure of N- [intein-C] ~ [C- tenninal portion of the split Cas9]-C. The mechanism of intein-mediated protein splicing for joining the proteins the inteins are fused to (e.g., split Cas9) is described in Shah et al., Chem Sci. 2014; 5(1):446-461, incorporated herein by reference. Methods for designing and using inteins are known in the ail and described, for example by W02020051561, W02014004336, WO2017132580, US20150344549, and US20180127780, each of which is incorporated herein by reference in their entirety.
In some embodiments, a split refers to a division into two or more fragments. In some embodiments, a split Cas9 protein or split Cas9 comprises a Cas9 protein that is provided as an N-terminal fragment and a C-terminal fragment encoded by two separate nucleotide sequences. The polypeptides corresponding to the N-terminal portion and the C-terminal portion of the Cas9 protein may be spliced to form a reconstituted Cas9 protein. In some embodiments, the Cas9 protein is divided into two fragments within a disordered region of the protein, e.g., as described in Nishimasu et al., Cell, Volume 156, Issue 5, pp. 935-949, 2014, or as described in Jiang et al. (2016) Science 351: 867-871 and PDB file: 5F9R (each of which is incorporated herein by reference in its entirety). A disordered region may be determined by one or more protein structure determination techniques known in the art, including, without limitation, X-ray crystallography, NMR spectroscopy, electron microscopy (e.g., cryoEM), and/or in silica protein modeling. In some embodiments, the protein is divided into two fragments at any C, T, A, or S, e.g., within a region of SpCas9 between amino acids A292- G364, F445-K483, or E565-T637, or at corresponding positions in any other Cas9, Cas9 variant (e.g., nCas9, dCas9), or other napDNAbp. In some embodiments, protein is divided into two fragments at SpCas9 T310, T313, A456, S469, or C574. In some embodiments, a process of dividing the protein into two fragments is referred to as splitting the protein. In some embodiments, a protein fragment ranges from about 2-1000 amino acids (e.g., between 2-10, 10-50, 50-100, 100-200, 200-300, 300-400, 400-500, 500-600, 600-700, 700-800, 800-900, or 900-1000 amino acids) in length. In some embodiments, a protein fragment ranges from about 5-500 amino acids (e.g., between 5-10, 10-50, 50-100, 100-200, 200-300, 300-400, or 400-500 amino acids) in length. In some embodiments, a protein fragment ranges from about 20- 200 amino acids (e.g., between 20-30, 30-40, 40-50, 50-100, or 100-200 amino acids) in length.
In some embodiments, a portion or fragment of a gene modifying polypeptide is fused to an intein. A nuclease can be fused to the N-terminus or the C-terminus of the intein. In some embodiments, a portion or fragment of a fusion protein is fused to an intein and fused to an AAV capsid protein. An intein, nuclease and capsid protein can be fused together in any arrangement (e.g., nuclease-intein-capsid, intein-nuclease-capsid, capsid-intein-nuclease, etc.). In some embodiments, the N-terminus of an intein is fused to the C-terminus of a fusion protein and the C-terminus of the intein is fused to the N-terminus of an AAV capsid protein.
In some embodiments, an endonuclease domain (e.g., a nickase Cas9 domain) is fused to intein-N and a polypeptide comprising an RT domain is fused to an intein-C.
Exemplary nucleotide and amino acid sequences of intein-N domains and compatible intein-C domains are provided below:
Additional domains
A gene modifying polypeptide can bind a target DNA sequence and template nucleic acid (e.g., template RNA), nick a target site, and write (e.g., reverse transcribe) the template into DNA, resulting in a modification of the target site. In some embodiments, additional domains may be added to a gene modifying polypeptide to enhance the efficiency of the process. In some embodiments, a gene modifying polypeptide may contain an additional DNA ligation domain to join reverse transcribed DNA to the DNA of a target site. In some embodiments, a gene modifying polypeptide may comprise a heterologous RNA-binding domain. In some embodiments, a gene 195ubstitut polypeptide may comprise a domain having 5' to 3' exonuclease activity (e.g., wherein the 5' to 3' exonuclease activity increases repair of the alteration of the target site, e.g., in favor of alteration over the original genomic sequence). In some embodiments, a gene modifying polypeptide may comprise a domain having 3' to 5' exonuclease activity, e.g., proof-reading activity. Tn some embodiments, a writing domain, e.g., an RT domain, has 3' to 5' exonuclease activity, e.g., proof-reading activity.
Template nucleic acids
Gene modifying systems described herein can modify a host target DNA site using a template nucleic acid sequence. In some embodiments, gene modifying systems described herein transcribe an RNA sequence template into host target DNA sites by target-primed reverse transcription (TPRT). By modifying DNA sequence(s) via reverse transcription of an RNA sequence template directly into the host genome, a gene modifying system can insert an object sequence into a target genome without the need for exogenous DNA sequences to be introduced into the host cell (unlike, for example, CRISPR systems), as well as eliminate an exogenous DNA insertion step. A gene modifying system can also delete a sequence from a target genome or introduce a substitution using an object sequence. Therefore, a gene modifying system provides a platform for the use of customized RNA sequence templates containing object sequences, e.g., sequences comprising heterologous gene coding and/or function information.
In some embodiments, a template nucleic acid comprises one or more sequence (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, or more sequences) that binds a gene modifying polypeptide.
In some embodiments, a system or method described herein comprises a single template nucleic acid (e.g., template RNA). In some embodiments, a system or method described herein comprises a plurality of template nucleic acids (e.g., template RNAs). For example, a system described herein comprises a first RNA comprising (e.g., from 5' to 3") a sequence that binds a gene modifying polypeptide (e.g., a DNA-binding domain and/or an endonuclease domain, e.g., a gRNA) and a sequence that binds a target site (e.g., a second strand of a site in a target genome), and a second RNA (e.g., a template RNA) comprising (e.g., from 5' to 3") optionally a sequence that binds the gene modifying polypeptide (e.g., that specifically binds an RT domain), a heterologous object sequence, and a PBS sequence. In some embodiments, when a system comprises a plurality of nucleic acids, each nucleic acid comprises a conjugating domain. In some embodiments, a conjugating domain enables association of nucleic acid molecules, e.g., by hybridization of complementary sequences. For example, in some embodiments a first RNA comprises a first conjugating domain and a second RNA comprises a second conjugating domain, and the first and second conjugating domains are capable of hybridizing to one another, e.g., under stringent conditions. In some embodiments, stringent conditions for hybridization include hybridization in 4x sodium chloride/sodium citrate (SSC), at about 65 C, followed by a wash in IxSSC, at about 65 C.
In some embodiments, a template nucleic acid comprises RNA. In some embodiments, a template nucleic acid comprises DNA (e.g., single stranded or double stranded DNA).
In some embodiments, a template nucleic acid comprises one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) homology domains that have homology to the target sequence. In some embodiments, a homology domain is about 10-20, 20-50, or 50-100 nucleotides in length.
In some embodiments, a template RNA can comprise a gRNA sequence, e.g., to direct a gene modifying polypeptide to a target site of interest. In some embodiments, a template RNA comprises (e.g., from 5' to 3') (i) optionally a gRNA spacer that binds a target site (e.g., a second strand of a site in a target genome), (ii) optionally a gRNA scaffold that binds a polypeptide described herein (e.g., a gene modifying polypeptide or a Cas polypeptide), (iii) a heterologous object sequence comprising a mutation region (optionally the heterologous object sequence comprises, from 5’ to 3’, a first homology region, a mutation region, and a second homology region), and (iv) a primer binding site (PBS) sequence comprising a 3' target homology domain.
A template nucleic acid (e.g., template RNA) component of a genome editing system described herein typically is able to bind a gene modifying polypeptide of a system described herein. In some embodiments, a template nucleic acid (e.g., template RNA) has a 3' region that is capable of binding a gene modifying polypeptide. A binding region, e.g., 3' region, may be a structured RNA region, e.g., having at least 1, 2 or 3 hairpin loops, capable of binding the gene modifying polypeptide of the system. A binding region may associate with a template nucleic acid (e.g., template RNA) with any of the polypeptide modules. In some embodiments, a binding region of a template nucleic acid (e.g., template RNA) may associate with an RNA-binding domain in a gene modifying polypeptide. In some embodiments, a binding region of a template nucleic acid (e.g., template RNA) may associate with a reverse transcription domain of a gene modifying polypeptide (e.g., specifically bind to the RT domain). In some embodiments, a template nucleic acid (e.g., a template RNA) may associate with a DNA binding domain of a gene modifying polypeptide, e.g., a gRNA associating with a Cas9-derived DNA binding domain. In some embodiments, a binding region may also provide DNA target recognition, e.g., a gRNA hybridizing to a target DNA sequence and binding a gene modifying polypeptide, e.g., a Cas9 domain. In some embodiments, a template nucleic acid (e.g., template RNA) may associate with multiple components of the polypeptide, e.g., DNA binding domain and reverse transcription domain.
In some embodiments, a template RNA has a poly-A tail at its 3' end. In some embodiments, a template RNA does not have a poly-A tail at its 3' end.
In some embodiments, a template nucleic acid is a template RNA. In some embodiments, a template RNA comprises one or more modified nucleotides. For example, in some embodiments, a template RNA comprises one or more deoxyribonucleotides. In some embodiments, regions of a template RNA are replaced by DNA nucleotides, e.g., to enhance stability of the molecule. For example, the 3' end of a template may comprise DNA nucleotides, while the rest of the template comprises RNA nucleotides that can be reverse transcribed. For instance, in some embodiments, a heterologous object sequence is primarily or wholly made up of RNA nucleotides (e.g., at least 90%, at least 95%, at least 98%, at least 99%, or 100% RNA nucleotides). In some embodiments, a PBS sequence is primarily or wholly made up of DNA nucleotides (e.g., at least 90%, at least 95%, at least 98%, at least 99%, or 100% DNA nucleotides). In some embodiments, a heterologous object sequence for writing into the genome may comprise DNA nucleotides. In some embodiments, DNA nucleotides in a template nucleic acid are copied into the genome by a domain capable of DNA-dependent DNA polymerase activity. In some embodiments, DNA-dependent DNA polymerase activity is provided by a DNA polymerase domain in a gene modifying polypeptide. In some embodiments, DNA- dependent DNA polymerase activity is provided by a reverse transcriptase domain that is also capable of DNA-dependent DNA polymerization, e.g., second strand synthesis. In some embodiments, a template nucleic acid molecule is composed of only DNA nucleotides.
In some embodiments, a system described herein comprises two nucleic acids which together comprise the sequences of a template RNA described herein. In some embodiments, two nucleic acids are associated with each other non-covalently, e.g., directly associated with each other (e.g., via base pairing), or indirectly associated as part of a complex comprising one or more additional molecule.
A template RNA described herein may comprise, from 5’ to 3’: (1) a gRNA spacer; (2) a gRNA scaffold; (3) heterologous object sequence (4) a primer binding site (PBS) sequence. Each of these components is now described in more detail. gRNA spacer and gRNA scaffold
A template RNA described herein may comprise a gRNA spacer that directs a gene modifying system to a target nucleic acid, and a gRNA scaffold that promotes association of a template RNA with a Cas domain of a gene modifying polypeptide. In some embodiments, a gRNA scaffold has been engineered for improved performance with StlCas9. Systems described herein can also comprise a gRNA that is not part of a template nucleic acid. For example, a gRNA that comprises a gRNA spacer and gRNA scaffold, but not a heterologous object sequence or a PBS sequence, can be used, e.g., to induce second strand nicking, e.g., as described in the section herein entitled “Second Strand Nicking”.
The present disclosure provides, variant gRNA scaffolds that are compatible with StlCas9. In some embodiments, variant gRNA scaffolds are used in a system comprising a gene modifying polypeptide that comprises an StlCas9 domain.
The wild-type StlCas9 gRNA scaffold has a hypothesized secondary structure, shown in FIG. 2. Generally, from 5’ to 3’, the gRNA scaffold comprises: a region comprising a lower stem, an upper stem, and tetraloop (also collectively referred to as Repeat: anti-repeat duplex or RAR); a first single stranded region; a Stem loop 1, a second single stranded region; a Stem loop 2; and a third single stranded region. More specifically, the upper stem comprises three paired bases (nt 12-14 pair with nt 19-21) and the 4-nucleotide tetraloop is nt 15-18. At the base of the three paired bases of the upper stem is a region with bulges (nt 22 and nt 25 bulge from the region), and at the base of the region with bulges is a lower stem (nt 1-9 pair with nt 26-34). Moving in a 3’ direction, the next region is the first single stranded region which contains nt 35 and 36. Following the first single stranded region is Stem loop 1 which comprises nucleotides 37-47. Next is the second single stranded region, comprising nucleotides 48-53. Next is Stem loop 2 which comprises nucleotides 54-82. 3’ of Stem loop 2 is a third single stranded region which comprises nucleotides 83-84. The hypothesized structure represents the likely secondary structure of the StlCas9 gRNA scaffold under physiogically relevant conditions. However, even if the StlCas9 gRNA scaffold were to adopt a different structure from the hypothesized structure shown herein, the named regions (such as Stem loop 1, Stem loop 2, RAR upper stem, RAR lower stem, and tetraloop) of variant scaffolds could still be readily identified based at least on sequence alignments to the wild-type reference sequence, and optionally using additional tools such as RNA folding algorithms. The spacer is typically situated at the 5’ end of the gRNA scaffold.
A variant gRNA scaffold herein can comprise mutations in different regions of the gRNA scaffold. For example, a variant gRNA scaffold may comprise a mutation in the upper stem that results in the thermodynamic strengthening of RAR. More specifically, the upper stem may be lengthened, e.g., by 1-8 base pairs (e.g., 1, 2, 3, 4, 5, 6, 7, or 8 base pairs).
As a second example, a variant gRNA scaffold may comprise a mutation in the tetraloop of RAR, which may optimize performance by improving the thermodynamic stability of RAR. More specifically, one or more nucleotides in the loop of the tetraloop may be substituted. In some embodiments, the loop region of the tetraloop may be lengthened, e.g., by 1 nucleotide, resulting in a loop 5 nucleotides in length.
Third, a variant gRNA scaffold may comprise a truncation in the stem of Stem loop 2 and/or in one or both single stranded regions at its base (i.e., the second and third single stranded regions). In some embodiments, the stem of Stem loop 2 comprises truncations in 3’-5’ direction end ranging from 1- 32 nt.
As another example, a variant gRNA scaffold may comprise one or more mutations that destabilize the upper RAR stem relative to the wild-type sequence. In some embodiments, a variant gRNA scaffold has a deletion of one or more nucleotides of the upper RAR stem. In some embodiments, a variant gRNA scaffold has a deletion of one or more nucleotides in the region with bulges that is situated between the upper RAR stem and lower RAR stem. In some embodiments, a variant gRNA scaffold has a substitution wherein a G-C base pair in the upper RAR stem is replaced with a base pair other than G-C (e.g., an A-U base pair).
The different mutations to a variant gRNA scaffold may be combined. For instance, in some embodiments, a variant gRNA scaffold comprises a mutation in the upper stem of the RAR and a mutation in the tetraloop of the RAR. More specifically, in some embodiments, the upper stem is lengthened, e.g., by 1-8 base pairs (e.g., 1, 2, 3, 4, 5, 6, 7, or 8 base pairs), and the tetraloop comprises a substitution or an insertion (e.g., a 1 nucleotide insertion).
In some embodiments, a variant gRNA scaffold comprises a mutation in the upper stem of the RAR and a truncation in the stem of Stem loop 2. More specifically, in some embodiments, the upper stem is lengthened, e.g., by 1-8 base pairs (e.g., 1, 2, 3, 4, 5, 6, 7, or 8 base pairs), and the stem of Stem loop 2 comprises a truncation of from 1- 32 nt. In some embodiments, a variant gRNA scaffold comprises a mutation in the tetraloop of the RAR and a truncation in the stem of Stem loop 2. More specifically, in some embodiments the tetraloop comprises a substitution or an insertion (e.g., a 1 nucleotide insertion) and the stem of Stem loop 2 comprises a truncation of from 1- 32 nt.
In some embodiments, a variant gRNA scaffold comprises: (1) a mutation in the upper stem of the RAR, (2) a mutation in the tetraloop of the RAR, and (3) a truncation in the stem of Stem loop 2. More specifically, in some embodiments, the upper stem is lengthened, e.g., by 1-8 base pairs (e.g., 1, 2, 3, 4, 5, 6, 7, or 8 base pairs), the tetraloop comprises a substitution or an insertion (e.g., a 1 nucleotide insertion), and the stem of Stem loop 2 comprises a truncation of from 1- 32 nt.
Exemplary variant gRNA scaffolds containing the alterations described in this section are provided in Table 26.
In some embodiments, an StlCas9 scaffold comprises an insertion (e.g., of 10 nucleotides) between positions 15 and 18, and a deletion of positions 16 and 17. In some embodiments, an insertion has a sequence according to GACUUCGGUC (SEQ ID N0:30006).
In some embodiments, an StlCas9 scaffold comprises an insertion (e.g., of 10 nucleotides) between positions 15 and 18, and a deletion of positions 16 and 17. In some embodiments, an insertion has a sequence according to CUAGAAAUAG (SEQ ID N0:30007).
In some embodiments, an StlCas9 scaffold comprises an insertion (e.g., of 12 nucleotides) between positions 14 and 19, and a deletion of positions 15-18. In some embodiments, an insertion has a sequence according to CGCGGUAACGCG (SEQ ID N0:30008).
In some embodiments, a variant StlCas9 scaffold has a substitution resulting in a G-C base pair in the RAR lower stem. In some embodiments, a substitution comprises a substitution of position 4 with a G and a template further comprises a substitution of position 31 with a C.
In some embodiments, a template RNA comprises a substitution in the second single stranded region. In some embodiments, a substitution is a substitution of position 51 with U or a substitution of position 54 with C.
In some embodiments, a gRNA is a short synthetic RNA composed of a scaffold sequence that participates in CRIS PR-associated protein binding and a user-defined ~20 nucleotide targeting sequence for a genomic target. The structure of a complete gRNA was described by Nishimasu et al. Cell 156, P935-949 (2014). A gRNA (also referred to as sgRNA for single-guide RNA) comprises crRNA- and tracrRNA-dcrivcd sequences connected by an artificial tetraloop. The crRNA sequence can be divided into guide (20 nt) and repeat (12 nt) regions, whereas the tracrRNA sequence can be divided into anti-repeat (14 nt) and three tracrRNA stem loops (Nishimasu et al. Cell 156, P935-949 (2014)). In practice, guide RNA sequences are generally designed to have a length of between 17 - 24 nucleotides (e.g., 19, 20, or 21 nucleotides) and be complementary to a targeted nucleic acid sequence. Custom gRNA generators and algorithms are available commercially for use in the design of effective guide RNAs. In some embodiments, a gRNA comprises two RNA components from the native CRISPR system, e.g., crRNA and tracrRNA. A gRNA may also comprise a chimeric, single guide RNA (sgRNA) containing sequence from both a tracrRNA (for binding the nuclease) and at least one crRNA (to guide the nuclease to the sequence targeted for editing/binding).
Chemically modified sgRNAs have also been demonstrated to be effective for use with CRISPR- associated proteins; see, for example, Hendel et al. (2015) Nature Biotechnol., 985 - 991. In some embodiments, a gRNA spacer comprises a nucleic acid sequence that is complementary to a DNA sequence associated with a target gene.
In some embodiments, the region of a template nucleic acid, e.g., template RNA, comprising a gRNA adopts an underwound ribbon-like structure of gRNA bound to target DNA (e.g., as described in Mulepati et al. Science 19 Sep 2014:Vol. 345, Issue 6203, pp. 1479-1484). Without wishing to be bound by any particular theory, this non-canonical structure is thought to be facilitated by rotation of every sixth nucleotide out of the RNA-DNA hybrid. Thus, in some embodiments, the region of a template nucleic acid, e.g., template RNA, comprising a gRNA may tolerate increased mismatching with a target site at some interval, e.g., every sixth base. In some embodiments, the region of a template nucleic acid, e.g., template RNA, comprising a gRNA comprising homology to a target site may possess wobble positions at a regular interval, e.g., every sixth base, that do not need to base pair with the target site.
In some embodiments, a Cas9 derivative with enhanced activity may be used in a gene modification polypeptide. In some embodiments, a Cas9 derivative may comprise mutations that improve activity of the HNH endonuclease domain, e.g., SpyCas9 R221K, N394K, or mutations that improve R-loop formation, e.g., SpyCas9 L1245V, or comprise a combination of such mutations, e.g., SpyCas9 R221K/N394K, SpyCas9 N394K/L1245V, SpyCas9 R221K/L1245V, or SpyCas9 R221K/N394K/L 1245V (see, e.g., Spencer and Zhang Sci Rep 7:16836 (2017), the Cas9 derivatives and mutations thereof which arc incorporated herein by reference). In some embodiments, a Cas9 derivative may comprise one or more types of mutations described herein, e.g., PAM-modifying mutations, protein stabilizing mutations, activity enhancing mutations, and/or mutations partially or fully inactivating one or two endonuclease domains relative to the parental enzyme (e.g., one or more mutations to abolish endonuclease activity towards one or both strands of a target DNA, e.g., a nickase or catalytically dead enzyme). In some embodiments, a Cas9 enzyme used in a system described herein may comprise mutations that confer nickase activity toward the enzyme (e.g., SpyCas9 N863A or H840A) in addition to mutations improving catalytic efficiency (e.g., SpyCas9 R221K, N394K, and/or L1245V). In some embodiments, a Cas9 enzyme used in a system described herein is a SpyCas9 enzyme or derivative that further comprises an N863A mutation to confer nickase activity in addition to R221K and N394K mutations to improve catalytic efficiency.
In some embodiments, a template nucleic acid (e.g., template RNA) has at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, or at least 24 bases of at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% homology to a target site, e.g., at the 5’ end, e.g., comprising a gRNA spacer sequence of length appropriate to the Cas9 domain of a gene modifying polypeptide (Table 4).
Table 14 provides parameters to define components for designing gRNA and/or template RNAs to apply Cas variants listed in Table 4 for gene modifying. The cut site indicates the validated or predicted protospacer adjacent motif (PAM) requirements, validated or predicted location of cut site (relative to the most upstream base of the PAM site). A gRNA for a given enzyme can be assembled by concatenating the crRNA, tetraloop, and tracrRNA sequences, and further adding a 5 ' spacer of a length within Spacer (min) and Spacer (max) that matches a protospacer at a target site. Further, the predicted location of a ssDNA nick at a target site is important for designing a PBS sequence of a template RNA that can anneal to the sequence immediately 5' of a nick in order to initiate target primed reverse transcription. In some embodiments, a gRNA scaffold described herein comprises a nucleic acid sequence comprising, in the 5’ to 3’ direction, a crRNA of Table 14, a tetraloop from the same row of Table 14, and a tracrRNA from the same row of Table 14, or a sequence having at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity thereto. In some embodiments, a gRNA or template RNA comprising a scaffold further comprises a gRNA spacer having a length within the Spacer (min) and Spacer (max) indicated in the same row of Table 14. In some embodiments, a gRNA or template RNA having a sequence according to Table 14 is incorporated in a system that further comprises a gene modifying polypeptide, wherein the gene modifying polypeptide comprises a Cas domain described in the same row of Table 14.
Table 14: Parameters to define components for designing gRNA and/or Template RNAs to apply Cas variants listed in Table 4 in gene modifying systems.
Herein, when an RNA sequence (e.g., a template RNA sequence) is said to comprise a particular sequence (e.g., a sequence of Table 14 or a portion thereof) that comprises thymine
(T), it is of course understood that the RNA sequence may (and frequently does) comprise uracil
(U) in place of T. For instance, the RNA sequence may comprise U at every position shown as T in the sequence in Table 14. More specifically, the present disclosure provides an RNA sequence according to every gRNA scaffold sequence of Table 14, wherein the RNA sequence has a U in place of each T in the sequence in Table 14. Additionally, it is understood that terminal Us and Ts may optionally be added or removed from tracrRNA sequences and may be modified or unmodified when provided as RNA. Without wishing to be bound by example, versions of gRNA scaffold sequences alternative to those exemplified in Table 14 may also function with the different Cas9 enzymes or derivatives thereof exemplified in Table 4, e.g., alternate gRNA scaffold sequences with nucleotide additions, substitutions, or deletions, e.g., sequences with stem-loop structures added or removed. It is contemplated herein that gRNA scaffold sequences represent a component of gene modifying systems that can be similarly optimized for a given system, Cas-RT fusion polypeptide, indication, target mutation, template RNA, or delivery vehicle.
Heterologous object sequence
A template RNA described herein may comprise a heterologous object sequence that a gene modifying polypeptide can use as a template for reverse transcription, to write a desired sequence into a target nucleic acid. In some embodiments, a heterologous object sequence comprises, from 5’ to 3’, a post-edit homology region, a mutation region, and a pre-edit homology region. Without wishing to be bound by any particular theory, an RT performing reverse transcription on a template RNA first reverse transcribes a pre-edit homology region, then a mutation region, and then a post-edit homology region, thereby creating a DNA strand comprising a desired mutation with a homology region on either side.
In some embodiments, a heterologous object sequence is at least 32, at least 33, at least 34, at least 35, at least 36, at least 37, at least 38, at least 39, at least 40, at least 41, at least 42, at least 43, at least 44, at least 45, at least 46, at least 47, at least 48, at least 49, at least 50, at least 51 , at least 52, at least 53, at least 54, at least 55, at least 56, at least 57, at least 58, at least 59, at least 60, at least 61, at least 62, at least 63, at least 64, at least 65, at least 66, at least 67, at least 68, at least 69, at least 70, at least 71, at least 72, at least 73, at least 74, at least 75, at least 76, at least 77, at least 78, at least 79, at least 80, at least 81 , at least 82, at least 83, at least 84, at least 85, at least 86, at least 87, at least 88, at least 89, at least 90, at least 91, at least 92, at least 93, at least 94, at least 95, at least 96, at least 97, at least 98, at least 99, at least 100, at least 120, at least 140, at least 160, at least 180, at least 200, at least 500, or at least 1,000 nucleotides (nts) in length, or at least 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, or 10 kilobases in length. In some embodiments, a heterologous object sequence is no more than 33, no more than 34, no more than 35, no more than 36, no more than 37, no more than 38, no more than 39, no more than 40, no more than 41, no more than 42, no more than 43, no more than 44, no more than 45, no more than 46, no more than 47, no more than 48, no more than 49, no more than 50, no more than 51, no more than 52, no more than 53, no more than 54, no more than 55, no more than 56, no more than 57, no more than 58, no more than 59, no more than 60, no more than 61, no more than 62, no more than 63, no more than 64, no more than 65, no more than 66, no more than 67, no more than 68, no more than 69, no more than 70, no more than 71, no more than 72, no more than 73, no more than 74, no more than 75, no more than 76, no more than 77, no more than 78, no more than 79, no more than 80, no more than 81, no more than 82, no more than 83, no more than 84, no more than 85, no more than 86, no more than 87, no more than 88, no more than 89, no more than 90, no more than 91, no more than 92, no more than 93, no more than 94, no more than 95, no more than 96, no more than 97, no more than 98, no more than 99, no more than 100, no more than 120, no more than 140, no more than 160, no more than 180, no more than 200, no more than 500, no more than 1,000, or no more than 2000 nucleotides (nts) in length, or no more than 20, no more than 15, no more than 10, no more than 9, no more than 8, no more than 7, no more than 6, no more than 5, no more than 4, or no more than 3 kilobases in length. In some embodiments, a heterologous object sequence is 30-1000, 40-1000, 50-1000, 60- 1000, 70-1000, 74-1000, 75-1000, 76-1000, 77-1000, 78-1000, 79-1000, 80-1000, 85-1000, 90- 1000, 100-1000, 120-1000, 140-1000, 160-1000, 180-1000, 200-1000, 500-1000, 30-500, 40- 500, 50-500, 60-500, 70-500, 74-500, 75-500, 76-500, 77-500, 78-500, 79-500, 80-500, 85-500, 90-500, 100-500, 120-500, 140-500, 160-500, 180-500, 200-500, 30-200, 40-200, 50-200, 60- 200, 70-200, 74-200, 75-200, 76-200, 77-200, 78-200, 79-200, 80-200, 85-200, 90-200, 100-200, 120-200, 140-200, 160-200, 180-200, 30-100, 40-100, 50-100, 60-100, 70-100, 74-100, 75-100, 76-100, 77-100, 78-100, 79-100, 80-100, 85-100, or 90-100 nucleotides (nts) in length, or 1-20, 1-15, 1-10, 1-9, 1-8, 1-7, 1-6, 1-5, 1-4, 1-3, 1-2, 2-20, 2-15, 2-10, 2-9, 2-8, 2-7, 2-6, 2-5, 2-4, 2-3, 3-20, 3-15, 3-10, 3-9, 3-8, 3-7, 3-6, 3-5, 3-4, 4-20, 4-15, 4- 10, 4-9, 4-8, 4-7, 4-6, 4-5, 5-20, 5-15, 5-10, 5-9, 5-8, 5-7, 5-6, 6-20, 6-15, 6-10, 6-9, 6-8, 6-7, 7-20, 7-15, 7-10, 7-9, 7-8, 8-20, 8-15, 8- 10, 8-9, 9-20, 9-15, 9-10, 10-15, 10-20, or 15-20 kilobases in length. In some embodiments, a heterologous object sequence is 10-100, 10-90, 10-80, 10-70, 10-60, 10-50, 10-40, 10-30, or 10- 20 nt in length, e.g., 10-80, 10-50, or 10-20 nt in length, e.g., aboutl0-20 nt in length. In some embodiments, a heterologous object sequence is 8-30, 9-25, 10-20, 11-16, or 12-15 nucleotides in length, e.g., is 11-16 nt in length. Without wishing to be bound by any particular theory, in some embodiments, a larger insertion size, larger region of editing (e.g., the distance between a first edit/substitution and a second edit/substitution in the target region), and/or greater number of desired edits (e.g., mismatches of the heterologous object sequence to the target genome), may result in a longer optimal heterologous object sequence.
In some embodiments, a template nucleic acid comprises a customized RNA sequence template which can be identified, designed, engineered and constructed to contain sequences altering or specifying host genome function, for example by introducing a heterologous coding region into a genome; affecting or causing exon structure/altemative splicing, e.g., leading to exon skipping of one or more exons; causing disruption of an endogenous gene, e.g., creating a genetic knockout; causing transcriptional activation of an endogenous gene; causing epigenetic regulation of an endogenous DNA; causing up-regulation of one or more operably linked genes, e.g., leading to gene activation or overexpression; causing down-regulation of one or more operably linked genes, e.g., creating a genetic knock-down; etc. In some embodiments, a customized RNA sequence template can be engineered to contain sequences coding for exons and/or transgenes, provide binding sites for transcription factor activators, repressors, enhancers, etc., and combinations thereof. In some embodiments, a customized template can be engineered to encode a nucleic acid or peptide tag to be expressed in an endogenous RNA transcript or endogenous protein operably linked to the target site. In other embodiments, the coding sequence can be further customized with splice donor sites, splice acceptor sites, or poly-A tails.
A template nucleic acid (e.g., a template RNA) of a system provided herin may comprise an object sequence (e.g., a heterologous object sequence) for writing a desired sequence into a target DNA. An object sequence (e.g., a heterologous object sequence) may be coding or noncoding. A template nucleic acid (e.g., template RNA) can be designed to result in insertions, mutations, or deletions at the target DNA locus. In some embodiments, a template nucleic acid (e.g., template RNA) may be designed to cause an insertion in a target DNA. For example, a template nucleic acid (e.g., template RNA) may contain a heterologous sequence, wherein reverse transcription will result in insertion of the heterologous sequence into a target DNA. In some embodiments, an RNA template may be designed to introduce a deletion into a target DNA. For example, a template nucleic acid (e.g., template RNA) may match a target DNA upstream and downstream of a desired deletion, wherein reverse transcription will result in the copying of the upstream and downstream sequences from the template nucleic acid (e.g., template RNA) without the intervening sequence, e.g., causing deletion of the intervening sequence. In some embodiments, a template nucleic acid (e.g., template RNA) may be designed to introduce an edit into a target DNA. For example, a template RNA may match a target DNA sequence with the exception of one or more nucleotides, wherein reverse transcription will result in the copying of these edits into the target DNA, e.g., resulting in mutations, e.g., transition or trans vers ion mutations.
In some embodiments, writing (e.g., reverse transcription) of an object sequence (e.g., a heterologous object sequence) into a target site results in the substitution of nucleotides, e.g., where the full length of the object sequence corresponds to a matching length of the target site with one or more mismatched bases. In some embodiments, a heterologous object sequence may be designed such that a combination of sequence alterations may occur, e.g., a simultaneous addition and deletion, addition and substitution, or deletion and substitution.
In some embodiments, a heterologous object sequence may contain an open reading frame or a fragment of an open reading frame. In some embodiments, a heterologous object sequence has a Kozak sequence. In some embodiments, a heterologous object sequence has an internal ribosome entry site. In some embodiments, a heterologous object sequence has a selfcleaving peptide such as a T2A or P2A site. In some embodiments, a heterologous object sequence has a start codon. In some embodiments, a template RNA has a splice acceptor site. In some embodiments, a template RNA has a splice donor site. Exemplary splice acceptor and splice donor sites are described in WO2016044416, incorporated herein by reference in its entirety. Exemplary splice acceptor site sequences are known to those of skill in the art. In some embodiments, a template RNA has a microRNA binding site downstream of a stop codon. In some embodiments, a template RNA has a polyA tail downstream of a stop codon of an open reading frame. In some embodiments, a template RNA comprises one or more exons. In some embodiments, a template RNA comprises one or more introns. In some embodiments, a template RNA comprises a eukaryotic transcriptional terminator. In some embodiments, a template RNA comprises an enhanced translation element or a translation enhancing element. In some embodiments, a termplate RNA comprises a human T-cell leukemia virus (HTLV-1) R region. In some embodiments, a template RNA comprises a posttranscriptional regulatory element that enhances nuclear export, such as that of Hepatitis B Virus (HPRE) or Woodchuck Hepatitis Virus (WPRE).
In some embodiments, a heterologous object sequence may contain a non-coding sequence. For example, a template nucleic acid (e.g., a template RNA) may comprise a regulatory element, e.g., a promoter or enhancer sequence or miRNA binding site. In some embodiments, integration of an object sequence (e.g., a heterologous object seqeucen) at a target site will result in upregulation of an endogenous gene. In some embodiments, integration of an object sequence (e.g., a heterologous object sequence)_at a target site will result in downregulation of an endogenous gene. In some embodiments, a template nucleic acid (e.g., template RNA) comprises a tissue specific promoter or enhancer, each of which may be unidirectional or bidirectional. In some embodiments, a promoter is an RNA polymerase I promoter, RNA polymerase II promoter, or RNA polymerase III promoter. In some embodiments, a promoter comprises a TATA element. In some embodiments, a promoter comprises a B recognition element. In some embodiments, a promoter has one or more binding sites for transcription factors.
In some embodiments, a template nucleic acid (e.g., a template RNA) comprises a site that coordinates epigenetic modification. In some embodiments, a template nucleic acid (e.g., a template RNA) comprises a chromatin insulator. For example, a template nucleic acid (e.g., a template RNA) may comprise a CTCF site or a site targeted for DNA methylation.
In some embodiments, a template nucleic acid (e.g., a template RNA) comprises a gene expression unit composed of at least one regulatory region operably linked to an effector sequence. An effector sequence may be a sequence that is transcribed into RNA (e.g., a coding sequence or a non-coding sequence such as a sequence encoding a micro RNA).
In some embodiments, a heterologous object sequence of a template nucleic acid (e.g., a template RNA) is inserted into a target genome in an endogenous intron. In some embodiments, a heterologous object sequence of a template nucleic acid (e.g., a template RNA) is inserted into a target genome and thereby acts as a new exon. In some embodiments, insertion of a heterologous object sequence into a target genome results in replacement of a natural exon or the skipping of a natural exon.
A template nucleic acid (e.g., a template RNA) can be designed to result in insertions, mutations, or deletions at a target DNA locus. In some embodiments, a template nucleic acid (e.g., a template RNA) may be designed to cause an insertion in a target DNA. For example, a template nucleic acid (e.g., a template RNA) may contain a heterologous object sequence, wherein reverse transcription will result in insertion of the heterologous object sequence into a target DNA. In some embodiments, an RNA template may be designed to write a deletion into a target DNA. For example, a template nucleic acid (e.g., a template RNA) may match a target DNA upstream and downstream of a desired deletion, wherein reverse transcription will result in copying of the upstream and downstream sequences from the template nucleic acid (e.g., the template RNA) without the intervening sequence, e.g., causing deletion of the intervening sequence. In some embodiments, a template nucleic acid (e.g., a template RNA) may be designed to write (e.g. reverse transcribe) an edit into a target DNA. For example, a template RNA may match a target DNA sequence with the exception of one or more nucleotides, wherein reverse transcription will result in copying of these edits into the target DNA, e.g., resulting in mutations, e.g., transition or transversion mutations.
In some embodiments, a pre-edit homology domain comprises a nucleic acid sequence having 100% sequence identity with a nucleic acid sequence comprised in a target nucleic acid molecule.
In some embodiments, a post-edit homology domain comprises a nucleic acid sequence having 100% sequence identity with a nucleic acid sequence comprised in a target nucleic acid molecule.
PBS sequence
In some embodiments, a template nucleic acid (e.g., a template RNA) comprises a primer binding site (PBS) sequence. In some embodiments, a PBS sequence is disposed 3' of a heterologous object sequence and is complementary to a sequence adjacent to a site to be modified by a system described herein, or comprises no more than 1, no more than 2, no more than 3, no more than 4, or no more than 5 mismatches to a sequence complementary to a sequence adjacent to a site to be modified by a system/gene modifying polypeptide. In some embodiments, a PBS sequence binds within 1 , 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides of a nick site in a target nucleic acid molecule. In some embodiments, binding of a PBS sequence to a target nucleic acid molecule permits initiation of target-primed reverse transcription (TPRT), e.g., with the 3' homology domain acting as a primer for TPRT. In some embodiments, a PBS sequence is 3-5, 5-10, 10-30, 10-25, 10-20, 10-19, 10-18, 10-17, 10-16, 10-15, 10-14, 10-13, 10- 12, 10-11, 11-30, 11-25, 11-20, 11-19, 11-18, 11-17, 11-16, 11-15, 11-14, 11-13, 11-12, 12-30, 12-25, 12-20, 12-19, 12-18, 12-17, 12-16, 12-15, 12-14, 12-13, 13-30, 13-25, 13-20, 13-19, 13- 18, 13-17, 13-16, 13-15, 13-14, 14-30, 14-25, 14-20, 14-19, 14-18, 14-17, 14-16, 14-15, 15-30, 15-25, 15-20, 15-19, 15-18, 15-17, 15-16, 16-30, 16-25, 16-20, 16-19, 16-18, 16-17, 17-30, 17- 25, 17-20, 17-19, 17-18, 18-30, 18-25, 18-20, 18-19, 19-30, 19-25, 19-20, 20-30, 20-25, or 25-30 nucleotides in length, e.g., 10-17, 12-16, or 12-14 nucleotides in length. In some embodiments, a PBS sequence is 5-20, 8-16, 8-14, 8-13, 9-13, 9-12, or 10-12 nucleotides in length, e.g., 9-12 nucleotides in length.
A template nucleic acid (e.g., a template RNA) may have some homology to a target DNA. In some embodiments, a template nucleic acid (e.g., a template RNA) PBS sequence domain may serve as an annealing region to a target DNA, such that the target DNA is positioned to prime the reverse transcription of the template nucleic acid (e.g., the template RNA). In some embodiments, a template nucleic acid (e.g., a template RNA) has at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 110, at least 120, at least 130, at least 140, at least 150, at least 175, at least 200 or more bases of exact homology to a target DNA at the 3' end of the RNA. In some embodiments, a template nucleic acid (e.g., a template RNA) has at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 110, at least 120, at least 130, at least 140, at least 150, at least 175, at least 200 or more bases of at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% homology to a target DNA, e.g., at the 5' end of the template nucleic acid (e.g., the template RNA). Exemplary template sequences
In some embodiments of systems and methods described herein, a template RNA comprises a gRNA spacer comprising the core nucleotides of a gRNA spacer sequence of Table 15. In some embodiments, a gRNA spacer additionally comprises one or more (e.g., 2, 3, or all) consecutive nucleotides starting with the 3’ end of the flanking nucleotides of the gRNA spacer.
In some embodiments, a template RNA comprising a sequence of Table 15 is comprised by a system that further comprises a gene modifying polypeptide having an RT domain listed in the same line of Table 15. RT domain amino acid sequences can be found, e.g., in Table 15 herein.
Table 15 provides exemplary gRNAs for correcting the pathogenic E342K mutation in SERPINA1. Also provided is a listing of spacers, PAMs, and Cas variants for generating a nick at an appropriate position to enable installation of a desired genomic edit with a gene modifying system of the present disclsourc. Spacers in Table 15 arc designed to be used with a gene modifying polypeptide comprising a nickase variant of the Cas species indicated in Table 15. Table 17, Table 16, and Table 18 detail other components of exemplary systems of the present disclosure and are organized such that the ID number shown in Column 1 (“ID”) of Table 15 is meant to correspond to the same ID number in Table 17, Table 16, and Table 18.
Table 15: Exemplary gRNA spacer Cas pairs In exemplary template sequences provided herein, capital letters indicate “core nucleotides” while lower case letters indicate “flanking nucleotides.” Herein, when an RNA sequence (e.g., a template RNA sequence) is said to comprise a particular sequence (e.g., a sequence of Table 15 or a portion thereof) that comprises thymine (T), it is of course understood that the RNA sequence may (and frequently does) comprise uracil (U) in place of T. For instance, the RNA sequence may comprise U at every position shown as T in the sequence in Table 15. More specifically, the present disclosure provides an RNA sequence according to every gRNA spacer sequence shown in Table 15, wherein the RNA sequence has a U in place of each T in the sequence in Table 15.
In some embodiments of systems and methods described herein, a heterologous object sequence comprises the core nucleotides of an RT template sequence from Table 16. In some embodiments, a heterologous object sequence additionally comprises one or more (e.g., 2, 3, 4, 5, 10, 20, 30, 40, or all) consecutive nucleotides starting with the 3’ end of the flanking nucleotides of an RT template sequence. In some embodiments, a heterologous object sequence comprises the core nucleotides of an RT template sequence of Table 16 that corresponds to a gRNA spacer sequence. In the context of the sequence tables, a first component “corresponds to” a second component when both components have the same ID number in the referenced table. For example, for a gRNA spacer of ID #1, the corresponding RT template would be the RT template also having ID #1. In some embodiments, a heterologous object sequence additionally comprises one or more consecutive nucleotides starting with the 3’ end of the flanking nucleotides of the RT template sequence.
In some embodiments, a primer binding site (PBS) sequence has a sequence comprising the core nucleotides of a PBS sequence from the same row of Table 16 as an RT template sequence. In some embodiments, a PBS sequence additionally comprises one or more (e.g., 1, 2, 3, 4, 5, 6, 7, or all) consecutive nucleotides starting with the 5’ end of the flanking nucleotides of a primer region.
Table 16 provides exemplary PBS sequences and heterologous object sequences (reverse transcription template regions) of a template RNA for correcting the pathogenic E342K mutation in SERPINA1. gRNA spacers from Table 15 were filtered, e.g., filtered by occurrence within 15 nt of the desired editing location and use of a Tier 1 Cas enzyme. PBS sequences and heterologous object sequences (reverse transcription template regions) were designed relative to a nick site directed by a cognate gRNA from Table 15. Tn some embodiments, these regions were designed to be 8-17 nt (priming) and 1-50 nt extended beyond the location of an edit (e.g., RT). Without wishing to be limited by example, given variability of length, sequences are provided that use the maximum length parameters and comprise all templates of shorter length within the given parameters. Sequences are shown with uppercase letters indicating core sequence and lowercase letters indicating flanking sequence that may be truncated within the described length parameters.
Table 16: Exemplary RT sequence (heterologous object sequence) and PBS sequence pairs
Herein, when an RNA sequence (e.g., a template RNA sequence) is said to comprise a particular sequence (e.g., a sequence of Table 16 or a portion thereof) that comprises thymine
(T), it is of course understood that the RNA sequence may (and frequently does) comprise uracil
(U) in place of T. For instance, an RNA sequence may comprise U at every position shown as T in the sequence in Table 16. More specifically, the present disclosure provides an RNA sequence according to every heterologous object sequence and PBS sequence shown in Table 16, wherein the RNA sequence has a U in place of each T in the sequence of Table 16.
In some embodiments ofsystems and methods described herein, a template RNA comprises a gRNA scaffold (e.g., that binds a gene modifying polypeptide, e.g., a Cas polypeptide) that comprises a sequence of a gRNA scaffold of Table 12. In some embodiments, a gRNA scaffold comprises a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to a gRNA scaffold of Table 12. In some embodiments, a gRNA scaffold comprises a sequence of a scaffold region of Table 12 that corresponds to the RT template sequence, the spacer sequence, or both, or a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity thereto.
In some embodiments of systems and methods described herein, a system further comprises a second strand-targeting gRNA that directs a nick to the second strand of the human SERPINA1 gene. In some embodiments, a second strand-targeting gRNA comprises a left gRNA spacer sequence or a right gRNA spacer sequence from Table 17. In some embodiments, a gRNA spacer additionally comprises one or more (c.g., 2, 3, or all) consecutive nucleotides starting with the 3’ end of the flanking nucleotides of the left gRNA spacer sequence or right gRNA spacer sequence. In some embodiments, a second strand-targeting gRNA comprises a sequence comprising the core nucleotides of a second nick gRNA sequence from Table 18, or a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity thereto. In some embodiments, a second nick gRNA sequence additionally comprises one or more consecutive nucleotides starting with the 3’ end of the flanking nucleotides of the second nick gRNA sequence. In some embodiments, a second nick gRNA comprises a gRNA scaffold sequence that is orthogonal to a Cas domain of a gene modifying polypeptide. In some embodiments, a second nick gRNA comprises a gRNA scaffold sequence of Table 14.
Table 17 provides exemplary second strand-targeting gRNA sequences for optional use for correcting the pathogenic E342K mutation in SERPINA1. gRNA spacers from Table 15 were filtered, e.g., filtered by occurrence within 15 nt of the desired editing location and use of a Tier 1 Cas enzyme. Second strand-targeting gRNAs were generated by searching the opposite strand of DNA in the regions -40 to -140 (“left”) and +40 to +140 (“right”), relative to the first nick site defined by the first gRNA, and for the PAM utilized by the corresponding Cas variant. One exemplary spacer is shown for each side of the target nick site.
Table 17: Exemplary left gRNA spacer and right gRNA spacer pairs comprise a particular sequence (e.g., a sequence of Table 17 or a portion thereof) that comprises thymine (T), it is of course understood that the RNA sequence may (and frequently does) comprise uracil (U) in place of T. For instance, an RNA sequence may comprise U at every position shown as T in the sequence in Table 17. More specifically, the present disclosure provides an RNA sequence according to every gRNA spacer sequence shown in Table 17, wherein the RNA sequence has a U in place of each T in the sequence in Table 17.
In some embodiments, systems and methods provided herein may comprise a template sequence listed in Table 18. Table 18 provides exemplary template RNA sequences (column 4) and optional second strand-targeting gRNA sequences (column 5) designed to be paired with a gene modifying polypeptide to correct a mutation in the SERPINA1 gene. Templates in Table 18 exemplify a total sequence including: (1) a gRNA spacer (c.g., for targeting a first strand nick), (2) a gRNA scaffold, (3) a heterologous object sequence, and (4) a PBS sequence (e.g., for initiating TPRT at the first strand nick).
Table 18 provides RNA components of gene modifying systems for correcting the pathogenic E342K mutation in SERPINA1. gRNA spacers from Table 15 were filtered, e.g., filtered by occurrence within 15 nt of a desired editing location and use of a Tier 1 Cas enzyme. For each gRNA ID, Table 18 describes a sequence of a complete template RNA, an optional second strand-targeting gRNA, and a Cas variant for use in a Cas-RT fusion gene modifying polypeptide. For exemplification, PBS sequences and post-edit homology regions (after the location of the edit) are set to 12 nt and 30 nt, respectively. Additionally, a second strandtargeting gRNA is selected with preference for a distance near 100 nt from a first nick and a first preference for a design resulting in a PAM-in system, as described elsewhere in this application.
Table 18. Exemplary template RNA sequences and second nick gRNA sequences
Capital letters indicate “core nucleotides” while lower case letters indicate “flanking nucleotides.” Herein, when an RNA sequence (e.g., a template RNA sequence) is said to comprise a particular sequence (e.g., a sequence of Table 18 or a portion thereof) that comprises thymine (T), it is of course understood that the RNA sequence may (and frequently does) comprise uracil (U) in place of T. For instance, the RNA sequence may comprise U at every position shown as T in the sequence in Table 18. More specifically, the present disclosure provides an RNA sequence according to every template sequence shown in Table 4, wherein the RNA sequence has a U in place of each T in the sequence of Table 18. Table 19 provides select sequences from Table 18, with annotation illustrating inactivation of PAM sites. Column “ID” contains a unique identifier for a template RNA that corresponds to the ID used in Tables 15-18 and can be used, e.g., to identify a corresponding gRNA spacer sequence in Table 15. Column “Cas species” indicates a type of Cas domain suitable for inclusion in a gene modifying polypeptide for use with the template RNA. Column “consensus” indicates a consensus PAM motif recognized by the Cas. Column “PAM sequence” indicates a particular PAM sequence recognized by the Cas, e.g., in the SERPINA1 gene. Column “PAM mutation” indicates a mutation that can be produced in the PAM by a template RNA described on the same row of the table; mutated nucleotides are indicated with bold and underlining. Column “strand” indicates the + or 1 strand of the target nucleic acid. Column “distance” indicates the number of nucleotides in the pre-edit homology region. Column “PBS sequence” indicates a PBS sequence for partial or full inclusion in the template RNA, wherein core nucleotides are capitalized and flanking nucleotides are lower case. Column “RT template sequence” indicates a heterologous object sequence for partial or full inclusion in the template RNA, wherein core nucleotides are capitalized, flanking nucleotides are lower case, and nucleotide differences from the target nucleic acid are shown in bold and underline.
Table 19: Exemplary template RNA sequences comprising PAM-inactivating sites
Herein, when an RNA sequence (e.g., a template RNA sequence) is said to comprise a particular sequence (e.g., a sequence of Table 19 or a portion thereof) that comprises thymine
(T), it is of course understood that the RNA sequence may (and frequently does) comprise uracil
(U) in place of T. For instance, the RNA sequence may comprise U at every position shown as T in the sequence in Table 19. More specifically, the present disclosure provides an RNA sequence according to every template sequence shown in Table 19, wherein the RNA sequence has a U in place of each T in the sequence of Table 19.
In some embodiments, a gRNA scaffold described herein comprises a nucleic acid sequence comprising, in the 5’ to 3’ direction, a crRNA of Table 20, a tetraloop from the same row of Table 20, and a tracrRNA from the same row of Table 20, or a sequence having at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity thereto. In some embodiments, a gRNA or template RNA having a sequence according to Table 20 is comprised by a system that further comprises a gene modifying polypeptide, and a spacer, wherein the spacer comprises a gRNA spacer described in the same row of Table 20.
Table 20: Exemplary spacer and scaffold pairs.
In some embodiments, systems and methods provided herein may comprise a template sequence, or component thereof, listed in Table 21, or a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, or 100% identity thereto. Table 21 provides exemplary template RNA sequences designed to be paired with a gene modifying polypeptide to correct a mutation in the SERPINA1 gene.
In some embodiments, s stems and methods provided herein may comprise a template sequence, or component thereof, listed in any one of Tables 39-62 , or a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, or 100% identity thereto.
Table 22 and Table 23 provide exemplary template RNA sequences. In some embodiments, a gRNA scaffold of a template RNA according to Table 22 or Table 23 is replaced with a variant gRNA scaffold described herein, e.g., a gRNA scaffold of Table 26 or a sequence having no more than 1, no more than 2, or no more than 3 sequence alterations (e.g., substitutions) relative thereto. Table 23 shows the sequences of Table 22 without modifications. In some embodiments, sequences can be used without chemical modifications.
Table 24 provides exemplary template RNA sequences. The names of the template RNAs provided have the following nomenclature: the first set of characters indicates the compatible Cas (e.g., Stl indicates StlCas9), the second set of characters indicates the name of the variant gRNA scaffold (e.g., dSL2), the third set of characters indicates the target gene or protein encoded by the target gene (e.g., A1AT), the fourth set of characters indicates the name of the spacer (e.g., ED4), the fifth set of characters indicates the length of the PBS and heterologous object sequence (e.g., P17R5 indicates a PBS of length 17 and a heterologous object sequence of length 5), and the sixth set of characters indicates the edit (e.g., TtoC). Column 2 shows the unmodified sequence corresponding to the chemically modified sequence of column 3. Table 21: Exemplary template RNA sequences
Table 22: Exemplary Template RNAs for Correcting PiZ Mutation
Table 23: Exemplary Template RNAs for Correcting PiZ Mutation (without modifications)
Table 24: Exemplary template RNAs.
Tables 25-28 below describe different sequences suitable for use in template RNAs described herein. In some embodiments, a template RNA described herein comprises (e.g., from 5’ to 3’) a spacer sequence of Table 25, a gRNA scaffold of Table 26, a heterologous object sequence of Table 27, and a PBS of Table 28. In some embodiments, a template RNA described herein comprises a spacer sequence of Table 25, or a sequence having or a sequence having no more than 1, no more than 2, or no more than 3 sequence alterations (e.g., substitutions) relative thereto. In some embodiments, a template RNA described herein comprises a gRNA scaffold of Table 26, or a sequence having or a sequence having no more than 1, no more than 2, or no more than 3 sequence alterations (e.g., substitutions) relative thereto. Table 26 provides gRNA scaffolds that have been engineered for improved performance, e.g., for use with StlCas9. In some embodiments, a template RNA described herein comprises a heterologous object sequence of Table 27, or a sequence having or a sequence having no more than 1, no more than 2, or no more than 3 sequence alterations (e.g., substitutions) relative thereto. In some embodiments, a template RNA described herein comprises a PBS sequence of Table 28, or a sequence having or a sequence having no more than 1, no more than 2, or no more than 3 sequence alterations (e.g., substitutions) relative thereto. In some embodiments, the template RNA is pail of a gene modifying system that further comprises a gene modifying polypeptide described herein (e.g., comprising a StlCas9 domain). In some embodiments, a template RNA is part of a gene modifying system that further comprises a second-nicking gRNA, e.g., according to Table 29. Table 25: Exemplary spacers for template RNAs and gRNAs described herein.
Table 26. Exemplary variant gRNA scaffolds for template RNAs and gRNAs described herein.
Table 27: Exemplary heterologous object sequences for template RNAs described herein.
Table 28. Exemplary PBS sequences for template RNAs described herein.
Table 29: Exemplary second nick gRNAs for systems described herein.
Template RNA sequences shown in Tables 15-21 may be customized depending on the cell being targeted. For example, in some embodiments it may be desired to inactivate a PAM sequence upon editing (e.g., using a “PAM-kill” modification) to decrease the potential for further gene editing (e.g., by Cas retargeting) following an initial edit. Consequently, certain template RNAs described herein are designed to write a mutation (e.g., a substitution) into the PAM of a target site, such that upon editing, the PAM site will be mutated to a sequence no longer recognized by a gene modifying polypeptide. Thus, a mutation region within a heterologous object sequence of a template RNA may comprise a PAM-kill sequence. Without wishing to be bound by any particular theory, in some embodiments, a PAM-kill sequence prevents re-engagement of a gene modifying polypeptide upon completion of a gene modification, or decreases re-engagement relative to a template RNA lacking a PAM-kill sequence. In some embodiments, a PAM-kill sequence does not alter the amino acid sequence encoded by a gene, e.g., the PAM-kill sequence results in a silent mutation. In some embodiments, it may be desired to leave a PAM sequence intact (no PAM-kill).
Similarly, in some embodiments, to decrease the potential for further gene editing (e.g., by Cas retargeting) following an initial edit, it may be desirable to alter the first three nucleotides of an RT template sequence via a “seed-kill” motif. Consequently, in some embodiments, template RNAs described herein are designed to write (e.g., reverse transcribe) a mutation (e.g., a substitution) into a portion of a target site corresponding to the first three nucleotides of an RT template sequence, such that upon editing, the target site will be mutated to a sequence with lower homology to the RT template sequence. Thus, a mutation region within a heterologous object sequence of a template RNA may comprise a seed-kill sequence. Without wishing to be bound by any particular theory, in some embodiments, a seed-kill sequence prevents reengagement of a gene modifying polypeptide upon completion of genetic modification, or decreases re-engagement relative to an otherwise similar template RNA lacking a seed-kill sequence. In some embodiments, a seed-kill sequence does not alter the amino acid sequence encoded by a gene, e.g., the seed-kill sequence results in a silent mutation. In other embodiments, it is desired to leave a seed region intact, and a seed-kill sequence is not used.
In some embodiments, to optimize or improve gene editing efficiency, it may be desirable to evade the target cell’s mismatch repair or nucleotide repair pathways or to bias the target cell’s repair pathways toward preservation of the edited strand. In some embodiments, multiple silent mutations (for example, silent substitutions) may be introduced within an RT template sequence to evade the target cell’s mismatch repair or nucleotide repair pathways or to bias the target cell’s repair pathways toward preservation of the edited strand.
Table 30 provides exemplary silent mutations for various positions within the SERPINA1 gene.
Table 30. Exemplary Silent Mutation Codons for the SERPINA1 Gene
* Counting initial Met.
In some embodiments, a template RNA comprises one or more silent mutations. It should be understood that the silent mutations illustrated in Table 30 may be used individually or combined in any manner in a template RNA sequence described herein. gRNAs with inducible activity
In some embodiments, a gRNA described herein (e.g., a gRNA that is pail of a template RNA or a gRNA used for second strand nicking) has inducible activity. Inducible activity may be achieved by a template nucleic acid, e.g., a template RNA, further comprising (in addition to a gRNA) a blocking domain, wherein the sequence of a portion of or all of the blocking domain is at least partially complementary to a portion or all of the gRNA. A blocking domain is thus capable of hybridizing or substantially hybridizing to a portion of or all of a gRNA. In some embodiments, a blocking domain and inducibly active gRNA are disposed on a template nucleic acid, e.g., a template RNA, such that a gRNA can adopt a first conformation where the blocking domain is hybridized or substantially hybridized to the gRNA, and a second conformation where the blocking domain is not hybridized or not substantially hybridized to the gRNA. In some embodiments, in a first conformation a gRNA is unable to bind to a gene modifying polypeptide (e.g., a template nucleic acid binding domain, DNA binding domain, or endonuclease domain (e.g., a CRISPR/Cas protein)) or binds with substantially decreased affinity compared to an otherwise similar template RNA lacking a blocking domain. In some embodiments, in a second conformation a gRNA is able to bind to a gene modifying polypeptide (e.g., a template nucleic acid binding domain, DNA binding domain, or endonuclease domain (e.g., a CRISPR/Cas protein)). In some embodiments, whether a gRNA is in a first or second conformation can influence whether DNA binding or endonuclease activities of a gene modifying polypeptide (e.g., of a CRISPR/Cas protein the gene modifying polypeptide comprises) are active.
In some embodiments, a gRNA that coordinates a second nick has inducible activity. In some embodiments, a gRNA that coordinates a second nick is induced after a template is reverse transcribed. In some embodiments, hybridization of a gRNA to a blocking domain can be disrupted using an opener molecule. In some embodiments, an opener molecule comprises an agent that binds to a portion or all of a gRNA or blocking domain and inhibits hybridization of the gRNA to the blocking domain. In some embodiments, an opener molecule comprises a nucleic acid, e.g., comprising a sequence that is partially or wholly complementary to a gRNA, blocking domain, or both. By choosing or designing an appropriate opener molecule, providing the opener molecule can promote a change in the conformation of a gRNA such that it can associate with a CRISPR/Cas protein and provide the382ssociateed functions of the CRISPR/Cas protein (e.g., DNA binding and/or endonuclease activity). Without wishing to be bound by any particular theory, providing an opener molecule at a selected time and/or location may allow for spatial and temporal control of the activity of a gRNA, CRISPR/Cas protein, or gene modifying system comprising the same. In some embodiments, an opener molecule is exogenous to the cell comprising a gene modifying polypeptide and or template nucleic acid. In some embodiments, an opener molecule comprises an endogenous agent (c.g., endogenous to the cell comprising a gene modifying polypeptide and or template nucleic acid comprising a gRNA and blocking domain). For example, an inducible gRNA, blocking domain, and opener molecule may be chosen such that the opener molecule is an endogenous agent expressed in a target cell or tissue, e.g., thereby ensuring activity of a gene modifying system in the target cell or tissue. As a further example, an inducible gRNA, blocking domain, and opener molecule may be chosen such that the opener molecule is absent or not substantially expressed in one or more non-target cells or tissues, e.g., thereby ensuring that activity of a gene modifying system does not occur or substantially occur in the one or more non-target cells or tissues, or occurs at a reduced level compared to a target cell or tissue. Exemplary blocking domains, opener molecules, and uses thereof are described in PCT App. Publication W02020044039A1, which is incorporated herein by reference in its entirety. In some embodiments, a template nucleic acid, e.g., template RNA, may comprise one or more sequences or structures for binding by one or more components of a gene modifying polypeptide, e.g., by a reverse transcriptase or RNA binding domain, and a gRNA. In some embodiments, a gRNA facilitates interaction with a template nucleic acid binding domain (e.g., an RNA binding domain) of a gene modifying polypeptide. In some embodiments, a gRNA directs a gene modifying polypeptide to a matching target sequence, e.g., in a target cell genome.
Circular RNAs and Ribozymes in Gene Modifying Systems
It is contemplated that it may be useful to employ circular and/or linear RNA states during the formulation, delivery, or gene modifying reaction within the target cell. Thus, in some embodiments, a gene modifying system comprises one or more circular RNAs (circRNAs). In some embodiments, a gene modifying system comprises one or more linear RNAs. In some embodiments, a nucleic acid as described herein (e.g., a template nucleic acid, a nucleic acid molecule encoding a gene modifying polypeptide, or both) is a circRNA. In some embodiments, a circular RNA molecule encodes a gene modifying polypeptide. In some embodiments, a circRNA molecule encoding a gene modifying polypeptide is delivered to a host cell. In some embodiments, a circular RNA molecule encodes a recombinase, e.g., as described herein. In some embodiments, a circRNA molecule encoding a recombinase is delivered to a host cell. In some embodiments, a circRNA molecule encoding a gene modifying polypeptide is linearized (c.g., in the host cell, c.g., in the nucleus of the host cell) prior to translation.
Circular RNAs (circRNAs) have been found to occur naturally in cells and have been found to have diverse functions, including both non-coding and protein coding roles in human cells. It has been shown that a circRNA can be engineered by incorporating a self-splicing intron into an RNA molecule (or DNA encoding the RNA molecule) that results in circularization of the RNA, and that an engineered circRNA can have enhanced protein production and stability (Wesselhoeft et al. Nature Communications 2018). In some embodiments, a gene modifying polypeptide is encoded by a circRNA. In some embodiments, a template nucleic acid is a DNA, such as a dsDNA or ssDNA. In some embodiments, a circDNA comprises a template RNA.
In some embodiments, a circRNA comprises one or more ribozyme sequences. In some embodiments, a ribozyme sequence is activated for autocleavage, e.g., in a host cell, e.g., thereby resulting in linearization of the circRNA. In some embodiments, a ribozyme is activated when the concentration of magnesium reaches a sufficient level for cleavage, e.g., in a host cell. In some embodiments, a circRNA is maintained in a low magnesium environment prior to delivery to the host cell. In some embodiments, a ribozyme is a protein-responsive ribozyme. In some embodiments, a ribozyme is a nucleic acid-responsive ribozyme. In some embodiments, a circRNA comprises a cleavage site. In some embodiments, a circRNA comprises two cleavage sites.
In some embodiments, a circRNA is linearized in the nucleus of a target cell. In some embodiments, linearization of a circRNA in the nucleus of a cell involves components present in the nucleus of the cell, e.g., to activate a cleavage event. In some embodiments, a ribozyme, e.g., a ribozyme from a B2 or ALU element, that is responsive to a nuclear element, e.g., a nuclear protein, e.g., a genome-interacting protein, e.g., an epigenetic modifier, e.g., EZH2, is incorporated into a circRNA, e.g., of a gene modifying system. In some embodiments, nuclear localization of a circRNA results in an increase in autocatalytic activity of the ribozyme and linearization of the circRNA.
In some embodiments, a ribozyme is heterologous to one or more of the other components of the gene modifying system. In some embodiments, an inducible ribozyme (e.g., in a circRNA as described herein) is created synthetically, for example, by utilizing a protein ligand-responsive aptamer design. A system for utilizing the satellite RNA of tobacco ringspot virus hammerhead ribozyme with an MS2 coat protein aptamer has been described (Kennedy et al. Nucleic Acids Res 42( 19): 12306- 12321 (2014), incorporated herein by reference in its entirety) that results in activation of the ribozyme activity in the presence of the MS2 coat protein. In some embodiments, such a system responds to protein ligand localized to the cytoplasm or the nucleus. In some embodiments the protein ligand is not MS2. Methods for generating RNA aptamers to target ligands have been described, for example, based on the systematic evolution of ligands by exponential enrichment (SELEX) (Tuerk and Gold, Science 249(4968):505-510 (1990); Ellington and Szostak, Nature 346(6287):818-822 (1990); the methods of each of which are incorporated herein by reference) and have, in some instances, been aided by in silico design (Bell et al. PNAS 117(15):8486-8493, the methods of which are incorporated herein by reference). Thus, in some embodiments, an aptamer for a target ligand is generated and incorporated into a synthetic ribozyme system, e.g., to trigger ribozyme- mediated cleavage and circRNA linearization, e.g., in the presence of the protein ligand. In some embodiments, circRNA linearization is triggered in the cytoplasm, e.g., using an aptamer that associates with a ligand in the cytoplasm. In some embodiments, circRNA linearization is triggered in the nucleus, e.g., using an aptamer that associates with a ligand in the nucleus. In some embodiments, a ligand in the nucleus comprises an epigenetic modifier or a transcription factor. In some embodiments, a ligand that triggers linearization is present at higher levels in on- target cells than off-target cells.
It is further contemplated that a nucleic acid-responsive ribozyme system can be employed for circRNA linearization. For example, biosensors that sense defined target nucleic acid molecules to trigger ribozyme activation are described, e.g., in Penchovsky (Biotechnology Advances 32(5): 1015-1027 (2014), incorporated herein by reference). By these methods, a ribozyme naturally folds into an inactive state and is only activated in the presence of a defined target nucleic acid molecule (e.g., an RNA molecule). In some embodiments, a circRNA of a gene modifying system comprises a nucleic acid-responsive ribozyme that is activated in the presence of a defined target nucleic acid, e.g., an RNA, e.g., an mRNA, miRNA, guide RNA, gRNA, sgRNA, ncRNA, IncRNA, tRNA, snRNA, or mtRNA. In some embodiments the nucleic acid that triggers linearization is present at higher levels in on-target cells than off-target cells.
In some embodiments, a gene modifying system incorporates one or more ribozymes with inducible specificity to a target tissue or target cell of interest, e.g., a ribozyme that is activated by a ligand or nucleic acid present at higher levels in a target tissue or target cell of interest. In some embodiments, a gene modifying system incorporates a ribozyme with inducible specificity to a subcellular compartment, e.g., the nucleus, nucleolus, cytoplasm, or mitochondria. In some embodiments, a ribozyme that is activated by a ligand or nucleic acid present at higher levels in the target subcellular compartment. In some embodiments, an RNA component of a gene modifying system is provided as circRNA, e.g., that is activated by linearization. In some embodiments, linearization of a circRNA encoding a gene modifying polypeptide activates the molecule for translation. In some embodiments, a signal that activates a circRNA component of a gene modifying system is present at higher levels in on-target cells or tissues, e.g., such that the system is specifically activated in these cells.
In some embodiments, an RNA component of a gene modifying system is provided as a circRNA that is inactivated by linearization. In some embodiments, a circRNA encoding a gene modifying polypeptide is inactivated by cleavage and degradation. In some embodiments, a circRNA encoding a gene modifying polypeptide is inactivated by cleavage that separates a translation signal from the coding sequence of the polypeptide. In some embodiments, a signal that inactivates a circRNA component of a gene modifying system is present at higher levels in off-target cells or tissues, such that the system is specifically inactivated in these cells. Target Nucleic Acid Site
In some embodiments, after gene modification, a target site surrounding an edited sequence contains a limited number of insertions or deletions, for example, in less than about 50% or 10% of editing events, e.g., as determined by long-read amplicon sequencing of the target site, e.g., as described in Karst et al. (2020) bioRxiv doi.org/10.1101/645903 (incorporated by reference herein in its entirety). In some embodiments, a target site does not show multiple consecutive editing events, e.g., head-to-tail or head-to-head duplications, e.g., as determined by long-read amplicon sequencing of the target site, e.g., as described in Karst et al. bioRxiv doi.org/10.1101/645903 (2020) (incorporated herein by reference in its entirety). In some embodiments, a target site contains an integrated sequence corresponding to a template RNA. In some embodiments, a target site does not contain insertions resulting from endogenous RNA in more than about 1% or 10% of events, e.g., as determined by long-read amplicon sequencing of the target site, e.g., as described in Karst et al. bioRxiv doi.org/10. 1101/645903 (2020) (incorporated herein by reference in its entirety). In some embodiments, a target site contains an integrated sequence corresponding to the template RNA.
In some embodiments, a host DNA-binding site integrated into by a gene modifying system of the present disclosure can be in a gene, in an intron, in an exon, an ORF, outside of a coding region of any gene, in a regulatory region of a gene, or outside of a regulatory region of a gene. In some embodiments, a gene modifying polypeptide may bind to one or more than one host DNA sequence.
In some embodiments, a gene modifying system is used to edit a target locus in multiple alleles. In some embodiments, a gene modifying system is designed to edit a specific allele. For example, a gene modifying polypeptide may be directed to a specific sequence that is only present on one allele, e.g., comprises a template RNA with homology to a target allele, e.g., a gRNA or annealing domain, but not to a second cognate allele. In some embodiments, a gene modifying system can alter a haplotype-specific allele. In some embodiments, a gene modifying system that targets a specific allele preferentially targets that allele, e.g., has at least a 2-fold, at least a 4-fold, at least a 6-fold, at least a 8-fold, or at least a 10-fold preference for a target allele. Second Strand Nicking
In some embodiments, a gene modifying system described herein comprises a nickase activity (e.g., in the gene modifying polypeptide) that nicks the first strand, and a nickase activity (e.g., in a polypeptide separate from the gene modifying polypeptide) that nicks the second strand of target DNA. As discussed herein, without wishing to be bound by any particular theory, nicking of the first strand of a target site DNA is thought to provide a 3' OH that can be used by an RT domain to reverse transcribe a sequence of a template RNA, e.g., a heterologous object sequence. Without wishing to be bound by any particular theory, it is thought that introducing an additional nick to the second strand may bias the cellular DNA repair machinery to adopt a heterologous object sequence-based sequence more frequently than the original genomic sequence. In some embodiments, the additional nick to the second strand is made by the same endonuclease domain (e.g., nickase domain) as the endonuclease domain that nicks the first strand. In some embodiments, the same gene modifying polypeptide performs both the nick to the first strand and the nick to the second strand. In some embodiments, a gene modifying polypeptide comprises a CRISPR/Cas domain and an additional nick to the second strand is directed by an additional nucleic acid, e.g., comprising a second gRNA directing the CRISPR/Cas domain to nick the second strand. Tn some embodiments, an additional second strand nick is made by a different endonuclease domain (c.g., nickase domain) than the endonuclease domain that nicks the first strand. In some embodiments, a second strand nicking endonuclease domain is situated in an additional polypeptide (e.g., a system of the invention further comprises the additional polypeptide), separate from a gene modifying polypeptide. In some embodiments, an additional polypeptide comprises an endonuclease domain (e.g., nickase domain) described herein. In some embodiments, an additional polypeptide comprises a DNA binding domain, e.g., described herein.
It is contemplated herein that the position at which a second strand nick occurs relative to a first strand nick may influence the extent to which one or more of: desired gene modifying DNA modifications are obtained, undesired double-strand breaks (DSBs) occur, undesired insertions occur, or undesired deletions occur. Without wishing to be bound by any particular theory, second strand nicking may occur in two general orientations: inward nicks and outward nicks.
In some embodiments, in an inward nick orientation, an RT domain polymerizes (e.g., using a template RNA (e.g., a heterologous object sequence)) away from a second strand nick. In some embodiments, in an inward nick orientation, the location of a nick to the first strand and the location of the nick to the second strand are positioned between the first PAM site and second PAM site (e.g., in a scenario wherein both nicks are made by a polypeptide (e.g., a gene modifying polypeptide) comprising a CRISPR/Cas domain). When there are two PAMs on the outside and two nicks on the inside, this inward nick orientation can also be referred to as “P AM- out”. In some embodiments, in an inward nick orientation, the location of a nick to the first strand and the location of a nick to the second strand are between the sites where the polypeptide and the additional polypeptide bind to a target DNA. In some embodiments, in an inward nick orientation, the location of a nick to the second strand is positioned between the binding sites of a gene modifying polypeptide and an additional polypeptide, and a nick to the first strand is also located between the binding sites of the gene modifying polypeptide and the additional polypeptide. In some embodiments, in an inward nick orientation, the location of a nick to the first strand and the location of a nick to the second strand are positioned between a PAM site and a binding site of a second polypeptide which is at a distance from a target site. An example of a gene modifying system that provides an inward nick orientation comprises a gene modifying polypeptide comprising a CRISPR/Cas domain, a template RNA comprising a gRNA that directs nicking of the target site DNA on the first strand, and an additional nucleic acid comprising an additional gRNA that directs nicking at a site a distance from the location of the first nick, wherein the location of the first nick and the location of the second nick are between the PAM sites of sequences to which two gRNAs direct the gene modifying polypeptide. In some embodiments, a gene modifying system that provides an inward nick orientation comprises a gene modifying polypeptide comprising a zinc finger molecule and a first nickase domain wherein the zinc finger molecule binds to a target DNA in a manner that directs a first nickase domain to nick the first strand of the target site; an additional polypeptide comprising a CRISPR/Cas domain, and an additional nucleic acid comprising a gRNA that directs the additional polypeptide to nick a site a distance from the target site DNA on the second strand, wherein the location of the first nick and the location of the second nick are between the PAM site and the site to which the zinc finger molecule binds. In some embodiments, a gene modifying system that provides an inward nick orientation comprises a gene modifying polypeptide comprising a zinc finger molecule and a first nickase domain wherein the zinc finger molecule binds to a target DNA in a manner that directs a first nickase domain to nick the first strand of the target site; an additional polypeptide comprising a TAL effector molecule and a second nickase domain wherein the TAL effector molecule binds to a site a distance from the target site in a manner that directs the additional polypeptide to nick the second strand, wherein the location of the first nick and the location of the second nick are between the site to which the TAL effector molecule binds and the site to which the zinc finger molecule binds.
In some embodiments, in an outward nick orientation, an RT domain polymerizes (e.g., using a template RNA (e.g., a heterologous object sequence)) toward a second strand nick. In some embodiments, in an outward nick orientation, when both first and second nicks are made by a polypeptide comprising a CRISPR/Cas domain (e.g., a gene modifying polypeptide), a first PAM site and second PAM site are positioned between the location of the nick to the first strand and the location of the nick to the second strand. When there are two PAMs on the inside and two nicks on the outside, this outward nick orientation also can be referred to as “PAM-in”. In some embodiments, in an outward nick orientation, a polypeptide (e.g., a gene modifying polypeptide) and an additional polypeptide bind to sites on the target DNA between the location of the nick to the first strand and the location of the nick to the second. In some embodiments, in an outward nick orientation, the location of a nick to the second strand is positioned on the opposite side of the binding sites of a gene modifying polypeptide and an additional polypeptide relative to the location of a nick to the first strand. In some embodiments, in an outward orientation, a PAM site and a binding site of a second polypeptide which is at a distance from a target site are positioned between the location of a nick to the first strand and the location of a nick to the second strand.
In some embodiments, a gene modifying system providing an outward nick orientation comprises a gene modifying polypeptide comprising a CRISPR/Cas domain, a template RNA comprising a gRNA that directs nicking of a target site DNA on the first strand, and an additional nucleic acid comprising an additional gRNA that directs nicking at a site a distance from the location of the first nick, wherein the location of the first nick and the location of the second nick are outside of the PAM sites of the sites to which the two gRNAs direct the gene modifying polypeptide (i.e., the PAM sites are between the location of the first nick and the location of the second nick). In some embodiments, a gene modifying system that provides an outward nick orientation comprises a gene modifying polypeptide comprising a zinc finger molecule and a first nickase domain wherein the zinc finger molecule binds to a target DNA in a manner that directs the first nickase domain to nick the first strand of the target site; an additional polypeptide comprising a CRISPR/Cas domain, and an additional nucleic acid comprising a gRNA that directs the additional polypeptide to nick a site a distance from the target site DNA on the second strand, wherein the location of the first nick and the location of the second nick are outside the PAM site and the site to which the zinc finger molecule binds (i.e., the PAM site and the site to which the zinc finger molecule binds are between the location of the first nick and the location of the second nick). In some embodiments, a gene modifying system that provides an outward nick orientation comprises a gene modifying polypeptide comprising a zinc finger molecule and a first nickase domain wherein the zinc finger molecule binds to a target DNA in a manner that directs the first nickase domain to nick the first strand of the target site; an additional polypeptide comprising a TAL effector molecule and a second nickase domain wherein the TAL effector molecule binds to a site a distance from the target site in a manner that directs the additional polypeptide to nick the second strand, wherein the location of the first nick and the location of the second nick are outside the site to which the TAL effector molecule binds and the site to which the zinc finger molecule binds (i.e., the site to which the TAL effector molecule binds and the site to which the zinc finger molecule hinds are between the location of the first nick and the location of the second nick).
Without wishing to be bound by any particular theory, it is thought that, for gene modifying systems where a second strand nick is provided, in some embodiments, an outward nick orientation is preferred. As is described herein, an inward nick may produce a higher number of double-strand breaks (DSBs) than an outward nick orientation. DSBs may be recognized by DSB repair pathways in the nucleus of a cell, which can result in undesired insertions and deletions. An outward nick orientation may provide a decreased risk of DSB formation, and a corresponding lower amount of undesired insertions and deletions. In some embodiments, undesired insertions and deletions are insertions and deletions not encoded by a heterologous object sequence, e.g., an insertion or deletion produced by the double-strand break repair pathway unrelated to a modification encoded by a heterologous object sequence. In some embodiments, a desired gene modification comprises a change to a target DNA (e.g., a substitution, insertion, or deletion) encoded by the heterologous object sequence (e.g., and achieved by gene modifying writing (e.g., reverese transcribing) the heterologous object sequence into the target site). In some embodiments, a first strand nick and a second strand nick are in an outward orientation.
In some embodiemnts, the distance between a first strand nick and a second strand nick may influence the extent to which one or more of: desired gene modifying system DNA modifications are obtained, undesired double-strand breaks (DSBs) occur, undesired insertions occur, or undesired deletions occur. Without wishing to be bound by any particular theory, it is thought that a second strand nick biases DNA repair toward incorporation of the heterologous object sequence into a target DNA, and that this bias increases as the distance between the first strand nick and second strand nick decreases. However, it is thought that the risk of DSB formation also increases as the distance between the first strand nick and second strand nick decreases. Correspondingly, it is thought that the number of undesired insertions and/or deletions may increase as the distance between the first strand nick and second strand nick decreases. In some embodiments, the distance between a first strand nick and a second strand nick is chosen to balance the benefit of biasing DNA repair toward incorporation of a heterologous object sequence into a target DNA and the risk of DSB formation and of undesired deletions and/or insertions. In some embodiments, a system where a first strand nick and a second strand nick are at least a threshold distance apart has an increased level of desired gene modifying system modification outcomes, a decreased level of undcsircd deletions, and/or a decreased level of undesired insertions relative to an otherwise similar- inward nick orientation system where the first nick and the second nick are less than the threshold distance apart. In some embodiments, threshold distance(s) is/are described below.
In some embodiments, a first nick and a second nick are at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 100, at least 110, at least 120, at least 130, at least 140, at least 150, at least 160, at least 170, at least 180, at least 190, or at least 200 nucleotides apart. In some embodiments, a first nick and a second nick are no more than 25, no more than 30, no more than 35, no more than 40, no more than 45, no more than 50, no more than 55, no more than 60, no more than 65, no more than 70, no more than 75, no more than 80, no more than 85, no more than 90, no more than 95, no more than 100, no more than 110, no more than 120, no more than 130, no more than 140, no more than 150, no more than 160, no more than 170, no more than 180, no more than 190, no more than 200, or no more than 250 nucleotides apart. In some embodiments, a first nick and a second nick are 20-200, 30-200, 40- 200, 50-200, 60-200, 70-200, 80-200, 90-200, 100-200, 110-200, 120-200, 130-200, 140-200, 150-200, 160-200, 170-200, 180-200, 190-200, 20-190, 30-190, 40-190, 50-190, 60-190, 70-190, 80-190, 90-190, 100-190, 110-190, 120-190, 130-190, 140-190, 150-190, 160-190, 170-190, 180-190, 20-180, 30-180, 40-180, 50-180, 60-180, 70-180, 80-180, 90-180, 100-180, 110-180, 120-180, 130-180, 140-180, 150-180, 160-180, 170-180, 20-170, 30-170, 40-170, 50-170, 60- 170, 70-170, 80-170, 90-170, 100-170, 110-170, 120-170, 130-170, 140-170, 150-170, 160-170, 20-160, 30-160, 40-160, 50-160, 60-160, 70-160, 80-160, 90-160, 100-160, 110-160, 120-160, 130-160, 140-160, 150-160, 20-150, 30-150, 40-150, 50-150, 60-150, 70-150, 80-150, 90-150, 100-150, 110-150, 120-150, 130-150, 140-150, 20-140, 30-140, 40-140, 50-140, 60-140, 70-140, 80-140, 90-140, 100-140, 110-140, 120-140, 130-140, 20-130, 30-130, 40-130, 50-130, 60-130, 70-130, 80-130, 90-130, 100-130, 110-130, 120-130, 20-120, 30-120, 40-120, 50-120, 60-120, 70-120, 80-120, 90-120, 100-120, 110-120, 20-110, 30-110, 40-110, 50-110, 60-110, 70-110, 80- 110, 90-110, 100-110, 20-100, 30-100, 40-100, 50-100, 60-100, 70-100, 80-100, 90-100, 20-90, 30-90, 40-90, 50-90, 60-90, 70-90, 80-90, 20-80, 30-80, 40-80, 50-80, 60-80, 70-80, 20-70, 30- 70, 40-70, 50-70, 60-70, 20-60, 30-60, 40-60, 50-60, 20-50, 30-50, 40-50, 20-40, 30-40, or 20-30 nucleotides apart. In some embodiments, a first nick and a second nick are 40-100 nucleotides apart.
Without wishing to be bound by any particular theory, it is thought that, for gene modifying systems where a second strand nick is provided and an inward nick orientation is selected, increasing the distance between the first strand nick and second strand nick may be preferred. As described herein, an inward nick orientation may produce a higher number of DSBs than an outward nick orientation, and may result in a higher amount of undesired insertions and deletions than an outward nick orientation, but increasing the distance between the nicks may mitigate that increase in DSBs, undesired deletions, and/or undesired insertions. In some embodiments, an inward nick orientation wherein a first nick and a second nick are at least a threshold distance apart has an increased level of desired gene modifying system modification outcomes, a decreased level of undesired deletions, and/or a decreased level of undesired insertions relative to an otherwise similar inward nick orientation system where the first nick and the second nick are less than the threshold distance apart. In some embodiments, threshold distance(s) is/are described below.
In some embodiments, a first strand nick and a second strand nick are in an inward orientation. In some embodiments, a first strand nick and a second strand nick are in an inward orientation and the first strand nick and second strand nick are at least 100, at least 110, at least 120, at least 130, at least 140, at least 150, at least 160, at least 170, at least 180, at least 190, at least 200, at least 220, at least 240, at least 260, at least 280, at least 300, at least 350, at least 400, at least 450, or at least 500 nucleotides apart, e.g., at least 100 nucleotides apart, (and optionally no more than 500, no more than 400, no more than 300, no more than 200, no more than 190, no more than 180, no more than 170, no more than 160, no more than 150, no more than 140, no more than 130, or no more than 120 nucleotides apart). In some embodiments, a first strand nick and a second strand nick are in an inward orientation and the first strand nick and second strand nick are 100-200, 110-200, 120-200, 130-200, 140-200, 150-200, 160-200, 170-200, 180-200, 190-200, 100-190, 110-190, 120-190, 130-190, 140-190, 150-190, 160-190,
170-190, 180-190, 100-180, 110-180, 120-180, 130-180, 140-180, 150-180, 160-180, 170-180,
100-170, 110-170, 120-170, 130-170, 140-170, 150-170, 160-170, 100-160, 110-160, 120-160,
130-160, 140-160, 150-160, 100-150, 110-150, 120-150, 130-150, 140-150, 100-140, 110-140,
120-140, 130-140, 100-130, 110-130, 120-130, 100-120, 110-120, or 100-110 nucleotides apart. Chemically modified nucleic acids and nucleic acid end features
A nucleic acid described herein (e.g., a template nucleic acid, e.g., a template RNA; or a nucleic acid (e.g., mRNA) encoding a gene modifying polypeptide; or a gRNA) can comprise unmodified or modified nucleobases. Naturally occurring RNAs are synthesized from four basic ribonucleotides: ATP, CTP, UTP and GTP, but may contain post-transcriptionally modified nucleotides. Further, approximately one hundred different nucleoside modifications have been identified in RNA (Rozenski, J, Crain, P, and McCloskey, J. (1999). The RNA Modification Database: 1999 update. Nucl Acids Res 27: 196-197). An RNA can also comprise wholly synthetic nucleotides that do not occur in nature.
Unless specified otherwise, the following numbering system will be adhered to for describing the position of nucleotides having chemical modifications in the PBS and/or heterologous object sequence of a template RNA. The positions of nucleotides in a heterologous object sequence are numbered +1, +2, +3, and so on, starting from the 3’-most end of the heterologous object sequence. The positions of nucleotides in a PBS sequence are numbered -1, -2, -3, and so on, starting from the 5’-most end of the PBS sequence. Thus, positions +1 and -1 are directly adjacent to each other.
Nucleotide positions in a heterologous object sequence can also be described by reference to the 3’ end of an entire template RNA. For example, the 3’ most nucleotide of a template RNA is typically part of a PBS. Moving in a 5’ direction from the 3’ most nucleotide of a template RNA, one next encounters the second nucleotide from the 3’ end of the template RNA, and then the third nucleotide from the 3’ end of the template RNA, and so on. As an example, if a PBS is 8 nucleotides in length, then the ninth nucleotide from the 3’ end of a template RNA would be position +1 of a heterologous object sequence.
Exemplary chemically modified template RNAs are provided below in Table 31, and the same sequences are shown in graphical form in FIG. 15. In some embodiments, a chemically modified template RNA may have a sequence of AAGGCUGUGCUGACCAUCGAGUCUUUGUACUCUGGGACUUCGGUCCCAGAAGCUA CAAAGAUAAGGCUUCAUGCCGAAAUCAUUUCUCGUCGAUGGUCAG (SEQ ID NO: 28143). Nucleotide modifications in Table 31 are noted as follows: phosphorothioate linkages denoted by an asterisk, 2’-O-methyl groups denoted by an ‘m’ preceding a nucleotide, 2’- Fluororibose denoted by an ‘i2F’ preceding a nucleotide. In some embodiments, a template RNA described herein has one or more (e.g., all) chemical modifications of a sequence shown in
Table 31.
Table 31. Exemplary chemically modified template RNAs. For instance, in some embodiments, a StlCas9 scaffold comprises a 2’-O-methyl chemically modified nucleotide at each of positions 12 through 21 , a 2’-O-methyl chemically modified nucleotide at each of positions 34 through 36, and a 2’ -Fluoro chemically modified nucleotide at each of positions 37 through 42. In some embodiments, a StlCas9 scaffold comprises a 2’-O-methyl chemically modified nucleotide at each of positions 12 through 21, a 2’-O-methyl chemically modified nucleotide at each of positions 34 through 36, a 2’ -Fluoro chemically modified nucleotide at each of positions 37 through 42, and a 2’ -Fluoro chemically modified nucleotide at each of positions 45 through 47. In some embodiments, a StlCas9 scaffold comprises a 2’-O-methyl chemically modified nucleotide at each of positions 12 through 21, a 2’-O-methyl chemically modified nucleotide at each of positions 34 through 36, a 2’-Fluoro chemically modified nucleotide at each of positions 37 through 42, a 2’ -Fluoro chemically modified nucleotide at each of positions 45 through 47 and a 2’-Fluoro chemically modified nucleotide at each of positions 52 through 54. In some embodiments, the 3’ most nucleotide and the second and third nucleotides from the 3’ end of a template RNA arc each a chemically modified nucleotide (e.g., each is a 2’-O- methyl nucleotide). In some embodiments, a backbone modification (e.g., phosphorothioate) is situated between: the 3’ most nucleotide and the second nucleotide from the 3’ end of a template RNA; the second nucleotide from the 3’ end of the template RNA and the third nucleotide from the 3’ end of the template RNA; and/or the third nucleotide from the 3’ end of the template RNA and the fourth nucleotide from the 3’ end of the template RNA.
In some embodiments, the 5’ most nucleotide and the second and third nucleotides from the 5’ end of a template RNA are each a chemically modified nucleotide (e.g., each is a 2’-O- methyl nucleotide). In some embodiments, a backbone modification (e.g., phosphorothioate) is situated between: the 5’ most nucleotide and the second nucleotide from the 5’ end of a template RNA; the second nucleotide from the 5’ end of the template RNA and the third nucleotide from the 3’ end of the template RNA; and/or the third nucleotide from the 5’ end of the template RNA and the fourth nucleotide from the 5’ end of the template RNA.
In some embodiments, a chemical modification is one provided in WO/2016/183482, US Pat. Pub. No. 20090286852, of International Application No. WO/2012/019168,
WO/2012/04507 , WO/2012/135805, WO/2012/158736, WO/2013/039857, WO/2013/039861, WO/2013/052523, WO/2013/090648, WO/2013/096709, WO/2013/101690, WO/2013/106496, WO/2013/130161, WO/2013/151669, WO/2013/151736, WO/2013/151672, WO/2013/151664, WO/2013/151665, WO/2013/151668, WO/2013/151671, WO/2013/151667, WO/2013/151670, WO/2013/151666, WO/2013/151663, WO/2014/028429, WO/2014/081507, WO/2014/093924, WO/2014/093574, WO/2014/113089, WO/2014/ 144711, WO/2014/144767, WO/2014/144039, WO/2014/152540, WO/2014/ 152030, WO/2014/152031, WO/2014/ 152027, WO/2014/152211, WO/2014/158795, WO/2014/159813, WO/2014/ 164253, WO/2015/006747, WO/2015/034928, WO/2015/034925, WO/2015/038892, WO/2015/048744, WO/2015/051214, WO/2015/051173, WO/2015/051169, WO/2015/058069, WO/2015/085318, WO/2015/089511, WO/2015/ 105926, WO/2015/164674, WO/2015/ 196130, WO/2015/ 196128, WO/2015/196118, WO/2016/011226, WO/2016/011222, WO/2016/011306, WO/2016/014846, WO/2016/022914, WO/2016/036902, WO/2016/077125, or WO/2016/077123, each of which is herein incorporated by reference in its entirety. It is understood that incorporation of a chemically modified nucleotide into a polynucleotide can result in the modification being incorporated into a nucleobase, the backbone, or both, depending on the location of the modification in the nucleotide. In some embodiments, a backbone modification is one provided in EP 2813570, which is herein incorporated by reference in its entirety. In some embodiments, a modified cap is one provided in US Pat. Pub. No. 20050287539, which is herein incorporated by reference in its entirety.
In some embodiments, a chemically modified nucleic acid (e.g., RNA, e.g., mRNA) comprises one or more of ARCA: anti-reverse cap analog (m27.3'-OGP3G), GP3G (Unmethylated Cap Analog), m7GP3G (Monomethylated Cap Analog), m32.2.7GP3G (Trimethylated Cap Analog), m5CTP (5 '-methyl-cy tidine triphosphate), m6ATP (N6-methyl- adenosine-5 '-triphosphate), s2UTP (2-thio-uridine triphosphate), and (pseudouridine triphosphate).
In some embodiments, a chemically modified nucleic acid comprises a 5' cap, e.g.: a 7- methylguanosine cap (e.g., a 0-Me-m7G cap); a hypermethylated cap analog; an NAD+-derived cap analog (e.g., as described in Kiledjian, Trends in Cell Biology 28, 454-464 (2018)); or a modified, e.g., biotinylated, cap analog (e.g., as described in Bednarek et al., Phil Trans R Soc B 373, 20180167 (2018)).
In some embodiments, a chemically modified nucleic acid comprises a 3 ' feature selected from one or more of: a polyA tail; a 16-nucleotide long stem-loop structure flanked by unpaired 5 nucleotides (e.g., as described by Mannironi et al., Nucleic Acid Research 17, 9113-9126 (1989)); a triple-helical structure (e.g., as described by Brown et al., PNAS 109, 19202-19207 (2012)); a tRNA, Y RNA, or vault RNA structure (e.g., as described by Labno et al., Biochemica et Biophysica Acta 1863, 3125-3147 (2016)); incorporation of one or more deoxyribonucleotide triphosphates (dNTPs), 2’0-Methylated NTPs, or phosphorothioate-NTPs; a single nucleotide chemical modification (e.g., oxidation of the 3' terminal ribose to a reactive aldehyde followed by conjugation of the aldehyde-reactive modified nucleotide); or chemical ligation to another nucleic acid molecule.
In some embodiments, a nucleic acid (e.g., a template nucleic acid) comprises one or more modified nucleotides, e.g., selected from dihydrouridine, inosine, 7-methylguanosine, 5- methylcytidine (5mC), 5' Phosphate ribothymidine, 2'-O-methyl ribothymidine, 2'-0-ethyl ribothymidine, 2'-fluoro ribothymidine, C-5 propynyl-deoxycytidine (pdC), C-5 propynyl- deoxyuridine (pdU), C-5 propynyl-cytidine (pC), C-5 propynyl-uridine (pU), 5-methyl cytidine, 5-methyl uridine, 5-methyl deoxycytidine, 5-methyl deoxyuridine methoxy, 2,6-diaminopurine, 5'-Dimethoxytrityl-N4-ethyl-2'-deoxycytidine, C-5 propynyl-f-cytidine (pfC), C-5 propynyl-f- uridinc (pfU), 5-mcthyl f-cytidinc, 5-mcthyl f-uridinc, C-5 propynyl-m-cytidinc (pmC), C-5 propynyl-f-uridine (pmU), 5-methyl m-cytidine, 5-methyl m-uridine, LNA (locked nucleic acid), MGB (minor groove binder) pseudouridine ( ), 1-N-methylpseudouridine (1-Me-T), or 5- methoxyuridine (5-MO-U).
In some embodiments, a nucleic acid comprises a backbone modification, e.g., a modification to a sugar or phosphate group in the backbone. In some embodiments, a nucleic acid comprises a nucleobase modification.
In some embodiments, a nucleic acid comprises one or more chemically modified nucleotides of Table 32, one or more chemical backbone modifications of Table 33, one or more chemically modified caps of Table 34. For instance, in some embodiments, a nucleic acid comprises two or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more) different types of chemical modifications. As an example, the nucleic acid may comprise two or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more) different types of modified nucleobases, e.g., as described herein, e.g., in Table 32. Alternatively or in combination, a nucleic acid may comprise two or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more) different types of backbone modifications, e.g., as described herein, e.g., in Table 33. Alternatively or in combination, a nucleic acid may comprise one or more modified cap, e.g., as described herein, e.g., in Table 34. For instance, in some embodiments, a nucleic acid comprises one or more type of modified nucleobase and one or more type of backbone modification; one or more type of modified nucleobase and one or more modified cap; one or more type of modified cap and one or more type of backbone modification; or one or more type of modified nucleobase, one or more type of backbone modification, and one or more type of modified cap.
In some embodiments, a nucleic acid comprises one or more (e.g., 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, 20 or more, 30 or more, 40 or more, 50 or more, 60 or more, 70 or more, 80 or more, 90 or more, 100 or more, 150 or more, 200 or more, 250 or more, 300 or more, 350 or more, 400 or more, 450 or more, 500 or more, 600 or more, 700 or more, 800 or more, 900 or more, or 1000 or more) modified nucleobases. In some embodiments, all nucleobases of a nucleic acid are modified. In some embodiments, a nucleic acid is modified at one or more (e.g., 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, 20 or more, 30 or more, 40 or more, 50 or more, 60 or more, 70 or more, 80 or more, 90 or more, 100 or more, 150 or more, 200 or more, 250 or more, 300 or more, 350 or more, 400 or more, 450 or more, 500 or more, 600 or more, 700 or more, 800 or more, 900 or more, or 1000 or more) positions in the backbone. In some embodiments, all backbone positions of the nucleic acid are modified.
Table 32. Modified nucleotides
Table 33. Backbone modifications
Table 34. Modified caps
Nucleotides of a template nucleic acid can be natural, modified, or a combination thereof. For example, a template nucleic acid may contain pseudouridine, dihydrouridine, inosine, 7- mcthylguanosinc, or other modified bases. In some embodiments, a template nucleic acid may contain locked nucleic acid nucleotides. In some embodiments, modified bases used in a template nucleic acid do not inhibit the reverse transcription of the template. In some embodiments, modified bases used in a template nucleic acid may improve reverse transcription, e.g., specificity or fidelity.
In some embodiments, an RNA component of a system of the present disclosure (e.g., a template RNA or a gRNA) comprises one or more nucleotide modifications. In some embodiments, the modification pattern of a gRNA can significantly affect in vivo activity compared to unmodified or end-modified guides (e.g., as shown in Figure ID from Finn et al. Cell Rep 22(9):2227-2235 (2018); incorporated herein by reference in its entirety). Without wishing to be bound by any particular theory, this process may be due, at least in part, to a stabilization of the RNA conferred by the modifications. Non-limiting examples of such modifications may include 2'-O-methyl (2'-O-Me), 2'-O-(2-methoxyethyl) (2'-O-MOE), 2'- fluoro (2'-F), phosphorothioate (PS) bond between nucleotides, G-C substitutions, and inverted abasic linkages between nucleotides and equivalents thereof.
In some embodiments, a template RNA (e.g., a portion thereof that binds a target site) or a guide RNA comprises a 5' terminus region. In some embodiments, a template RNA or a guide RNA does not comprise a 5' terminus region. In some embodiments, a 5' terminus region comprises a gRNA spacer region, e.g., as described with respect to sgRNA in Briner AE et al, Molecular Cell 56: 333-339 (2014) (incorporated herein by reference in its entirety; applicable herein, e.g., to all guide RNAs). In some embodiments, a 5' terminus region comprises a 5' end modification. In some embodiments, a 5' terminus region with or without a spacer region may be associated with a crRNA, trRNA, sgRNA and/or dgRNA. A gRNA spacer region can, in some instances, comprise a guide region, guide domain, or targeting domain.
In some embodiments, a template RNA (e.g., a portion thereof that binds a target site) or a guide RNA described herein comprises any of the sequences shown in Table 4 of W02018107028A1, incorporated herein by reference in its entirety. In some embodiments, where a sequence shows a guide and/or spacer region, a template RNA may comprise this region or not. In some embodiments, a guide RNA comprises one or more of the modifications of any of the sequences shown in Table 4 of W02018107028A1, e.g., as identified therein by a SEQ ID NO. In some embodiments, nucleotides may be the same or different, and/or the modification pattern shown may be the same or similar to a modification pattern of a guide sequence as shown in Table 4 of W02018107028A1. In some embodiments, a modification pattern includes the relative position and identity of modifications of a gRNA or a region of the gRNA (e.g., 5' terminus region, lower stem region, bulge region, upper stem region, nexus region, hairpin 1 region, hairpin 2 region, 3' terminus region). In some embodiments, a modification pattern contains at least 50%, at least 55%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% of the modifications of any one of the sequences shown in the sequence column of Table 4 of W02018107028A1, and/or over one or more regions of the sequence. In some embodiments, a modification pattern is at least 50%, at least 55%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the modification pattern of any one of the sequences shown in the sequence column of Table 4 of W02018107028A1. In some embodiments, a modification pattern is at least 50%, at least 55%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%identical over one or more regions of the sequence shown in Table 4 of W02018107028A1, e.g., in a 5 ' terminus region, lower stem region, bulge region, upper stem region, nexus region, hairpin 1 region, hairpin 2 region, and/or 3' terminus region. In some embodiments, a modification pattern is at least 50%, at least 55%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the modification pattern of a sequence over the 5 ' terminus region. In some embodiments, a modification pattern is at least 50%, at least 55%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical over the lower stem. In some embodiments, a modification pattern is at least 50%, at least 55%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%identical over the bulge. In some embodiments, a modification pattern is at least 50%, at least 55%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical over the upper stem. In some embodiments, a modification pattern is at least 50%, at least 55%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical over the nexus. In some embodiments, a modification pattern is at least 50%, at least 55%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%identical over the hairpin 1. In some embodiments, a modification pattern is at least 50%, at least 55%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical over the hairpin 2. In some embodiments, a modification pattern is at least 50%, at least 55%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical over the 3 ' terminus. In some embodiments, a modification pattern differs from the modification pattern of a sequence of Table 4 of W02018107028A1, or a region (e.g. 5' terminus, lower stem, bulge, upper stem, nexus, hairpin 1, hairpin 2, 3' terminus) of such a sequence, e.g., at 0, 1, 2, 3, 4, 5, 6, or more nucleotides. In some embodiments, a gRNA comprises modifications that differ from the modifications of a sequence of Table 4 of W02018107028A1, e.g., at 0, 1, 2, 3, 4, 5, 6, or more nucleotides. In some embodiments, a gRNA comprises modifications that differ from modifications of a region (e.g. 5 ' terminus, lower stem, bulge, upper stem, nexus, hairpin 1, hairpin 2, 3Z terminus) of a sequence of Table 4 of W02018107028A1, e.g., at 0, 1, 2, 3, 4, 5, 6, or more nucleotides.
In some embodiments, a template RNA (e.g., a portion thereof that binds a target site) or a gRNA comprises a 2'-O-methyl (2'-0-Me) modified nucleotide. In some embodiments, a gRNA comprises a 2'-O-(2-methoxy ethyl) (2'-O-moe) modified nucleotide. In some embodiments, a gRNA comprises a 2'-fluoro (2'- F) modified nucleotide. In some embodiments, a gRNA comprises a phosphorothioate (PS) bond between nucleotides. Tn some embodiments, a gRNA comprises a 5' end modification, a 3" end modification, or 5' and 3' end modifications. In some embodiments, a 5 ' end modification comprises a phosphorothioate (PS) bond between nucleotides. In some embodiments, a 5' end modification comprises a 2'-O-methyl (2'-0-Me), 2'-O-(2-methoxy ethyl) (2'-0-M0E), and/or 2'-fluoro (2'-F) modified nucleotide. In some embodiments, a 5' end modification comprises at least one phosphorothioate (PS) bond and one or more of a 2'-O-methyl (2'-O- Me), 2'-O-(2-methoxyethyl) (2'-0-M0E), and/or 2'-fluoro (2'-F) modified nucleotide. An end modification may comprise a phosphorothioate (PS), 2'-O-methyl (2'-0-Me), 2'-O-(2- methoxyethyl) (2'-0-M0E), and/or 2'-fluoro (2'-F) modification. Equivalent end modifications are also encompassed by embodiments described herein. In some embodiments, a template RNA or gRNA comprises an end modification in combination with a modification of one or more regions of the template RNA or the gRNA. Additional exemplary modifications and methods for protecting RNA, e.g., gRNA, and formulae thereof, are described in WO2018126176A1, which is incorporated herein by reference in its entirety.
In some embodiments, a template RNA described herein comprises three phosphorothioate linkages at the 5’ end and three phosphorothioate linkages at the 3’ end. In some embodiments, a template RNA described herein comprises three 2’-O-methyl ribonucleotides at the 5’ end and three 2’-O-methyl ribonucleotides at the 3’ end. In some embodiments, the 5’ most three nucleotides of a template RNA are 2’-O-methyl ribonucleotides, the 5’ most three internucleotide linkages of the template RNA are phosphorothioate linkages, the 3’ most three nucleotides of the template RNA are 2’-O-methyl ribonucleotides, and the 3’ most three intemucleotide linkages of the template RNA are phosphorothioate linkages. In some embodiments, a template RNA comprises alternating blocks of ribonucleotides and 2’-O-methyl ribonucleotides, for instance, blocks of between 12 and 28 nucleotides in length. In some embodiments, the central portion of a template RNA comprises the alternating blocks and the 5’ and 3’ ends each comprise three 2’-O-methyl ribonucleotides and three phosphorothioate linkages.
In some embodiments, structure-guided and systematic approaches are used to introduce modifications (e.g., 2'-0Me-RNA, 2'-F-RNA, and PS modifications) to a template RNA or guide RNA, for example, as described in Mir et al. Nat Commun 9:2641 (2018) (incorporated by reference herein in its entirety). In some embodiments, incorporation of 2'-F-RNAs increases thermal and nuclease stability of RNA:RNA or RNA:DNA duplexes, e.g., while minimally interfering with C3'-cndo sugar puckering. In some embodiments, 2'-F may be better tolerated than 2'-0Me at positions where the 2'-OH is important for RNA:DNA duplex stability. In some embodiments, a crRNA comprises one or more modifications that do not reduce Cas9 activity, e.g., CIO, C20, or C21 (fully modified), e.g., as described in Supplementary Table 1 of Mir et al. Nat Commun 9:2641 (2018), incorporated herein by reference in its entirety. In some embodiments, a tracrRNA comprises one or more modifications that do not reduce Cas9 activity, e.g., T2, T6, T7, or T8 (fully modified) of Suppiemen tary Table 1 of Mir et al. Nat Commun 9:2641 (2018). In some embodiments, a crRNA comprises one or more modifications (e.g., as described herein) may be paired with a tracrRNA comprising one or more modifications, e.g., C20 and T2. In some embodiments, a gRNA comprises a chimera, e.g., of a crRNA and a tracrRNA (e.g., Jinek et al. Science 337(6096):816-821 (2012)). In some embodiments, modifications from a crRNA and tracrRNA are mapped onto a single-guide chimera, e.g., to produce a modified gRNA with enhanced stability.
In some embodiments, a gRNA molecule may be modified by the addition or subtraction of naturally occurring structural components, e.g., hairpins. In some embodiments, a gRNA may comprise a gRNA with one or more 3' hairpin elements deleted, e.g., as described in WO2018106727, incorporated herein by reference in its entirety. In some embodiments, a gRNA may contain an added hairpin structure, e.g., an added hairpin structure in the spacer region, which was shown to increase specificity of a CRISPR-Cas system in the teachings of Kocak et al. Nat Biotechnol 37(6):657-666 (2019). Additional modifications, including examples of shortened gRNA and specific modifications improving in vivo activity, can be found in US20190316121, incorporated herein by reference in its entirety.
In some embodiments, structure-guided and systematic approaches (e.g., as described in Mir et al. Nat Commun 9:2641 (2018); incorporated herein by reference in its entirety) are employed to find modifications for a template RNA. In some embodiments, modifications are identified with the inclusion or exclusion of a guide region of template RNA. In some embodiments, a structure of a polypeptide bound to a template RNA is used to determine nonprotein-contacted nucleotides of the template RNA that may then be selected for modifications, e.g., with lower risk of disrupting the association of the RNA with the polypeptide. Secondary structures in a template RNA can also be predicted in silica by software tools, e.g., the RNAstructure tool available at rna.urmc.rochester.edu/RNAstractureWeb (Bellaousov et al. Nucleic Acids Res 41:W471-W474 (2013); incorporated by reference herein in its entirety), e.g., to determine secondary structures for selecting modifications, e.g., hairpins, stems, and/or bulges.
Production of Compositions and Systems
As will be appreciated by one of skill, methods of designing and constructing nucleic acid constructs and proteins or polypeptides (such as the systems, constructs and polypeptides described herein) are routine in the art. Generally, recombinant methods may be used. See, in general, Smales & James (Eds.), Therapeutic Proteins: Methods and Protocols (Methods in Molecular- Biology), Humana Press (2005); and Crommelin, Sindelar & Meibohm (Eds.), Pharmaceutical Biotechnology: Fundamentals and Applications, Springer (2013). Methods of designing, preparing, evaluating, purifying and manipulating nucleic acid compositions arc described in Green and Sambrook (Eds.), Molecular Cloning: A Laboratory Manual (Fourth Edition), Cold Spring Harbor Laboratory Press (2012).
The present disclosure provides, in part, a nucleic acid, e.g., a vector encoding a gene modifying polypeptide described herein, a template nucleic acid described herein, or both. In some embodiments, a vector comprises a selective marker, e.g., an antibiotic resistance marker. In some embodiments, an antibiotic resistance marker is a kanamycin resistance marker. In some embodiments, an antibiotic resistance marker does not confer resistance to beta-lactam antibiotics. In some embodiments, a vector does not comprise an ampicillin resistance marker. In some embodiments, a vector comprises a kanamycin resistance marker and does not comprise an ampicillin resistance marker. In some embodiments, a vector encoding a gene modifying polypeptide is integrated into a target cell genome (e.g., upon administration to a target cell, tissue, organ, or subject). In some embodiments, a vector encoding a gene modifying polypeptide is not integrated into a target cell genome (e.g., upon administration to a target cell, tissue, organ, or subject). In some embodiments, a vector encoding a template nucleic acid (e.g., a template RNA) is not integrated into a target cell genome (e.g., upon administration to a target cell, tissue, organ, or subject). In some embodiments, if a vector is integrated into a target site in a target cell genome, the selective marker is not integrated into the genome. In some embodiments, if a vector is integrated into a target site in a target cell genome, genes or sequences involved in vector maintenance (e.g., plasmid maintenance genes) are not integrated into the genome. In some embodiments, if a vector is integrated into a target site in a target cell genome, transfer regulating sequences (c.g., inverted terminal repeats, c.g., from an AAV) arc not integrated into the genome. In some embodiments, administration of a vector (e.g., encoding a gene modifying polypeptide described herein, a template nucleic acid described herein, or both) to a target cell, tissue, organ, or subject results in integration of a portion of the vector into one or more target sites in the genome(s) of said target cell, tissue, organ, or subject. In some embodiments, less than 99%, less than 95%, less than 90%, less than 80%, less than 70%, less than 60%, less than 50%, less than 40%, less than 30%, less than 20%, less than 10%, less than 5%, less than 4%, less than 3%, less than 2%, or less than 1% of target sites (e.g., no target sites) comprising integrated material comprise a selective marker (e.g., an antibiotic resistance gene), a transfer regulating sequence (e.g., an inverted terminal repeat, e.g., from an AAV), or both from the vector.
Exemplary methods for producing a therapeutic pharmaceutical protein or polypeptide described herein involve expression in mammalian cells, although recombinant proteins can also be produced using insect cells, yeast, bacteria, or other cells under control of appropriate promoters. Mammalian expression vectors may comprise non-transcribed elements such as an origin of replication, a suitable promoter, and other 5' or 3' flanking non-transcribed sequences, and 5' or 3' non-translated sequences such as necessary ribosome binding sites, a polyadenylation site, splice donor and acceptor sites, and termination sequences. DNA sequences derived from the SV40 viral genome, for example, SV40 origin, early promoter, splice, and polyadenylation sites may be used to provide other genetic elements required for expression of a heterologous DNA sequence. Appropriate cloning and expression vectors for use with bacterial, fungal, yeast, and mammalian cellular hosts are described in Green & Sambrook, Molecular Cloning: A Laboratory Manual (Fourth Edition), Cold Spring Harbor Laboratory Press (2012).
Various mammalian cell culture systems can be employed to express and manufacture recombinant protein. Examples of mammalian expression systems include CHO, COS, HEK293, HeLA, and BHK cell lines. Processes of host cell culture for production of protein therapeutics are described in Zhou and Kantardjieff (Eds.), Mammalian Cell Cultures for Biologies Manufacturing (Advances in Biochemical Engineering/Biotechnology), Springer (2014). Compositions described herein may include a vector, such as a viral vector, e.g., a lentiviral vector, encoding a recombinant protein. In some embodiments, a vector, e.g., a viral vector, may comprise a nucleic acid encoding a recombinant protein.
Purification of protein therapeutics is described in Franks, Protein Biotechnology: Isolation, Characterization, and Stabilization, Humana Press (2013); and in Cutler, Protein Purification Protocols (Methods in Molecular Biology), Humana Press (2010).
The present disclosure also provides compositions and methods for the production of template nucleic acid molecules (e.g., template RNAs) with specificity for a gene modifying polypeptide and/or a genomic target site. In some embodiments, a method comprises production of RNA segments including an upstream homology segment, a heterologous object sequence segment, a gene modifying polypeptide binding motif, and a gRNA segment.
Therapeutic Applications
In some embodiments, a gene modifying system as described herein can be used to modify a cell (e.g., an animal cell, plant cell, or fungal cell). In some embodiments, a gene modifying system as described herein can be used to modify a mammalian cell (e.g., a human cell). In some embodiments, a gene modifying system as described herein can be used to modify a cell from a livestock animal (e.g., a cow, horse, sheep, goat, pig, llama, alpaca, camel, yak, chicken, duck, goose, or ostrich). In some embodiments, a gene modifying system as described herein can be used as a laboratory tool or a research tool, or used in a laboratory method or research method, e.g., to modify an animal cell, e.g., a mammalian cell (e.g., a human cell), a plant cell, or a fungal cell.
By integrating coding genes into a template RNA, a gene modifying system can address therapeutic needs, for example, by providing expression of a therapeutic transgene in individuals with loss-of-function mutations, by replacing gain-of-function mutations with normal transgenes, by providing regulatory sequences to eliminate gain-of-function mutation expression, and/or by controlling the expression of operably linked genes, transgenes and systems thereof. In some embodiments, a template RNA encodes a promotor region specific to the therapeutic needs of the host cell, for example a tissue specific promotor or enhancer. In some embodiments, a promotor can be operably linked to a coding sequence.
Accordingly, provided herein arc methods for treating alpha- 1 antitrypsin deficiency (AATD) in a subject in need thereof. In some embodiments, treatment results in amelioration of one or more symptoms associated with AATD. In some embodiments, a system herein is used to treat a subject having a mutation in E342 (c.g., E342K). In some embodiments, a system of the present disclosure replaces an “A” nucleotide with a “G” nucleotide at the mutation site via gene editing, to reverse an E342K mutation in the corresponding protein. In some embodiments, a system of the present disclosure replaces a “T” nucleotide with a “C” nucleotide at the mutation site via gene editing, to reverse an E342K mutation in the corresponding protein.
In some embodiments, treatment with a system disclosed herein results in correction of the E342K mutation in between about 30-100% (e.g., about 30-40%, about 40-50%, about 50- 60%, about 60-70%, about 70-80%, about 80-90%, about 90-100%, or about 50%) of cells. In some embodiments, treatment with a system disclosed herein results in correction of the E342K mutation in between about 30-60% (e.g., about 30-40%, about 40-50%, about 50-60%, or about 50%) of DNA isolated from the treated cells.
In some embodiments, treatment with a gene modifying system described herein results in one or more of:
(a) an increase in alpha- 1 antitrypsin (AAT) activity and/or function;
(b) an increase in levels of circulating AAT ;
(c) a reduction in protease-induced lung damage and/or inflammation (e.g., a reduction in protease digestion of connective tissue in the lower airway, e.g., alveoli linings));
(d) a reduction in accumulated, polymerized Z-AAT protein within hepatocytes;
(e) a reduction in AAT-induced hepatocyte toxicity;
(f) a reduction of cellular stress, inflammation, fibrosis, cirrhosis, hepatocellular carcinoma (HCC), and/or neonatal liver disease;
(g) an increase in pulmonary function (e.g., an increase in lung elasticity); and/or
(h) a reduction of symptoms associated with emphysema, as compared to a subject having AATD that has not been treated with a gene modifying system described herein.
Administration and Delivery
The compositions and systems described herein may be used in vitro or in vivo. In some embodiments a system or components of a system are delivered to cells (e.g., mammalian cells, e.g., human cells), e.g., in vitro or in vivo. In some embodiments, cells are eukaryotic cells, e.g., cells of a multicellular organism, e.g., an animal, e.g., a mammal (e.g., human, swine, bovine), a bird (e.g., poultry, such as chicken, turkey, or duck), or a fish. In some embodiments, cells arc non-human animal cells (e.g., a laboratory animal, a livestock animal, or a companion animal). In some embodiments, a cell is a primary cell. In some embodiments, a cell is a primary liver cell. In some embodiments, a cell is a primary lung cell. In some embodiments, a cell is a stem cell (e.g., a hematopoietic stem cell), a fibroblast, or a T cell. In some embodiments, a cell is an immune cell, e.g., a T cell (e.g., a Treg, CD4, CD8, y8, or memory T cell), B cell (e.g., memory B cell or plasma cell), or NK cell. In some embodiments, a cell is a non-dividing cell, e.g., a non-dividing fibroblast or non-dividing T cell. In some embodiments, a cell is an HSC and p53 is not upregulated or is upregulated by less than 10%, 5%, 2%, or 1%, e.g., as determined according to the method described in Example 30 of PCT/US2019/048607. The skilled artisan will understand that the components of the gene modifying system may be delivered in the form of polypeptide, nucleic acid (e.g., DNA, RNA), and combinations thereof.
In some embodiments, a system and/or components of the system are delivered as one or more nucleic acids. For example, a gene modifying polypeptide may be delivered in the form of a DNA or RNA encoding the polypeptide, and a template RNA may be delivered in the form of an RNA or its complementary DNA to be transcribed into RNA. In some embodiments, a system or components of the system are delivered on 1, 2, 3, 4, or more distinct nucleic acid molecules. In some embodiments, a system or components of the system are delivered as a combination of DNA and RNA. In some embodiments, a system or components of the system are delivered as a combination of DNA and protein. In some embodiments, a system or components of the system arc delivered as a combination of RNA and protein. In some embodiments, a gene modifying polypeptide is delivered as a protein.
In some embodiments, a system or components of the system are delivered to cells, e.g. mammalian cells or human cells, using a vector, a vector may be, e.g., a plasmid or a virus. In some embodiments, delivery is in vivo, in vitro, ex vivo, or in situ. In some embodiments, a vims is an adeno associated virus (AAV), a lentivirus, or an adenovirus. In some embodiments, a system or components of the system are delivered to cells with a viral-like particle or a virosome. In some embodiments, delivery uses more than one vims, viral-like particle or virosome.
In some embodiments, compositions and systems described herein can be formulated in liposomes or other similar vesicles. Liposomes are spherical vesicle structures composed of a uni- or multilamellar lipid bilayer surrounding internal aqueous compartments and a relatively impermeable outer lipophilic phospholipid bilaycr. Liposomes may be anionic, neutral or cationic. Liposomes are biocompatible, nontoxic, can deliver both hydrophilic and lipophilic drug molecules, protect their cargo from degradation by plasma enzymes, and transport their load across biological membranes and the blood brain barrier (BBB) (see, e.g., Spuch and Navarro, Journal of Drug Delivery, vol. 2011, Article ID 469679, 12 pages, 2011. doi: 10.1155/2011/469679 for review).
Vesicles can be made from several different types of lipids; however, phospholipids are most commonly used to generate liposomes as drug carriers. Methods for preparation of multilamellar vesicle lipids are known in the art (see for example U.S. Pat. No. 6,693,086, the teachings of which relating to multilamellar vesicle lipid preparation are incorporated herein by reference). Although vesicle formation can be spontaneous when a lipid film is mixed with an aqueous solution, it can also be expedited by applying force in the form of shaking by using a homogenizer, sonicator, or an extrusion apparatus (see, e.g., Spuch and Navarro, Journal of Drug Delivery, vol. 2011, Article ID 469679, 12 pages, 2011. doi:10.1155/2011/469679 for review). Extruded lipids can be prepared by extruding through filters of decreasing size, as described in Templeton et al., Nature Biotech, 15:647-652, 1997, the teachings of which relating to extruded lipid preparation are incorporated herein by reference.
A variety of nanoparticles can be used for delivery, such as a liposome, a lipid nanoparticle, a cationic lipid nanoparticle, an ionizable lipid nanoparticle, a polymeric nanoparticle, a gold nanoparticle, a dendrimer, a cyclodextrin nanoparticle, a micelle, or a combination of the foregoing.
Lipid nanoparticles are an example of a carrier that provides a biocompatible and biodegradable delivery system for the pharmaceutical compositions described herein. Nano structured lipid carriers (NLCs) are modified solid lipid nanoparticles (SLNs) that retain the characteristics of the SLN, improve drug stability and loading capacity, and prevent drug leakage. Polymer nanoparticles (PNPs) are an important component of drug delivery. These nanoparticles can effectively direct drug delivery to specific targets and improve drug stability and controlled drug release. Lipid-polymer nanoparticles (PLNs), a type of carrier that combines liposomes and polymers, may also be employed. These nanoparticles possess the complementary advantages of PNPs and liposomes. A PLN is composed of a core-shell structure; the polymer core provides a stable structure, and the phospholipid shell offers good biocompatibility. As such, the two components increase the drug encapsulation efficiency rate, facilitate surface modification, and prevent leakage of water-soluble drugs. For a review, see, e.g., Li et al. 2017, Nanomaterials 7, 122; doi:10.3390/nano7060122.
Exosomes can also be used as drug delivery vehicles for the compositions and systems described herein. For a review, see Ha et al. July 2016. Acta Pharmaceutica Sinica B. Volume 6, Issue 4, Pages 287-296; https://doi.Org/10.1016/j.apsb.2016.02.001.
Fusosomes interact and fuse with target cells, and thus can be used as delivery vehicles for a variety of molecules. They generally consist of a bilayer of amphipathic lipids enclosing a lumen or cavity and a fusogen that interacts with the amphipathic lipid bilayer. The fusogen component has been shown to be engineerable in order to confer target cell specificity for the fusion and payload delivery, allowing the creation of delivery vehicles with programmable cell specificity (see for example Patent Application W02020014209, the teachings of which relating to fusosome design, preparation, and usage are incorporated herein by reference).
In some embodiments, protein component(s) of a gene modifying system may be preassociated with a template nucleic acid (e.g., template RNA). For example, in some embodiments, a gene modifying polypeptide may be first combined with a template nucleic acid (e.g., template RNA) to form a ribonucleoprotein (RNP) complex. In some embodiments, an RNP may be delivered to cells via, e.g., transfection, nucleofection, virus, vesicle, LNP, exosome, fusosome.
A gene modifying system can be introduced into cells, tissues and multicellular organisms. In some embodiments, a system or components of the system are delivered to cells via mechanical means or physical means.
Formulation of protein therapeutics is described in Meyer (Ed.), Therapeutic Protein Drug Products: Practical Approaches to formulation in the Laboratory, Manufacturing, and the Clinic, Woodhead Publishing Series (2012).
Tissue Specific Activity/Administration
In some embodiments, a system described herein can make use of one or more feature (e.g., a promoter or microRNA binding site) to limit activity in off-target cells or tissues.
In some embodiments, a nucleic acid described herein (e.g., a template RNA or a DNA encoding a template RNA) comprises a promoter sequence, e.g., a tissue specific promoter sequence. In some embodiments, a tissue-specific promoter is used to increase target-cell specificity of a gene modifying system. For instance, a promoter can be chosen on the basis that it is active in a target cell type but not active in (or active at a lower level in) a non-target cell type. Thus, even if a promoter integrated into the genome of a non-target cell, it would not drive expression (or only drive low level expression) of an integrated gene. A system having a tissuespecific promoter sequence in the template RNA may also be used in combination with a microRNA binding site, e.g., in a template RNA or a nucleic acid encoding a gene modifying protein, e.g., as described herein. A system having a tissue- specific promoter sequence in a template RNA may also be used in combination with a DNA encoding a gene modifying polypeptide, driven by a tissue-specific promoter, e.g., to achieve higher levels of gene modifying protein in target cells than in non-target cells. In some embodiments, e.g., for liver indications, a tissue-specific promoter is selected from Table 3 of W02020014209, incorporated herein by reference.
In some embodiments, a nucleic acid described herein (e.g., a template RNA or a DNA encoding a template RNA) comprises a microRNA binding site. In some embodiments, a microRNA binding site is used to increase target-cell specificity of a gene modifying system. For instance, a microRNA binding site can be chosen on the basis that is recognized by a miRNA that is present in a non-target cell type, but that is not present (or is present at a reduced level relative to the non-target cell) in a target cell type. Thus, when a template RNA is present in a non-target cell, it would be bound by the miRNA, and when the template RNA is present in a target cell, it would not be bound by the miRNA (or bound but at reduced levels relative to the non-target cell). While not wishing to be bound by any particular theory, binding of a miRNA to a template RNA may interfere with its activity, e.g., may interfere with insertion of a heterologous object sequence into the genome. Accordingly, a system would edit the genome of target cells more efficiently than it edits the genome of non-target cells, e.g., a heterologous object sequence would be inserted into the genome of target cells more efficiently than into the genome of non-target cells, or an insertion or deletion is produced more efficiently in target cells than in non-target cells. A system having a microRNA binding site in a template RNA (or DNA encoding it) may also be used in combination with a nucleic acid encoding a gene modifying polypeptide, wherein expression of the gene modifying polypeptide is regulated by a second microRNA binding site, e.g., as described herein. In some embodiments, e.g., for liver indications, a miRNA is selected from Table 4 of W02020014209, incorporated herein by reference.
In some embodiments, a template RNA comprises a microRNA sequence, an siRNA sequence, a guide RNA sequence, or a piwi RNA sequence.
Promoters
In some embodiments, one or more promoter or enhancer elements are operably linked to a nucleic acid encoding a gene modifying protein or a template nucleic acid, e.g., that controls expression of a heterologous object sequence. In some embodiments, one or more promoter or enhancer elements comprise cell-type or tissue specific elements. In some embodiments, a promoter or enhancer is the same or derived from the promoter or enhancer that naturally controls expression of a heterologous object sequence. For example, the ornithine transcarbomylasc promoter and enhancer may be used to control expression of the ornithine transcarbomylase gene in a system or method provided by the invention for correcting ornithine transcarbomylase deficiencies. In some embodiments, a promoter is a promoter of Table 35 or Table 36 or a functional fragment or variant thereof.
Exemplary tissue specific promoters that are commercially available can be found, for example, at a uniform resource locator (e.g., www.invivogen.com/tissue-specific-promoters). In some embodiments, a promoter is a native promoter or a minimal promoter, e.g.. which consists of a single fragment from the 5' region of a given gene. In some embodiments, a native promoter comprises a core promoter and its natural 5' UTR. In some embodiments, a 5 " UTR comprises an intron. In some embodiments, a promoter may include composite promoters, which combine promoter elements of different origins or were generated by assembling a distal enhancer with a minimal promoter of the same origin.
Exemplary cell or tissue specific promoters are provided in the tables below, and exemplary nucleic acid sequences encoding them are known in the art and can be readily- accessed using a variety of resources, such as the NCBI database, including RefSeq, as well as the Eukaryotic Promoter Database (//epd.epfl.ch//index.php).
Table 35. Exemplary cell or tissue-specific promoters
Table 36. Additional exemplary cell or tissue-specific promoters
Depending on the host/vector system utilized, any of a number of suitable transcription and translation control elements, including constitutive and inducible promoters, transcription enhancer elements, transcription terminators, etc., may be used in the expression vector (see e.g., Bitter el al. (1987) Methods in Enzymology, 153:516-544: incorporated herein by reference in its entirely).
In some embodiments, a nucleic acid encoding a gene modifying protein or template nucleic acid is operably linked to a control element, e.g., a transcriptional control element, such as a promoter. The transcriptional control element may, in some embodiments, be functional in either a eukaryotic cell, e.g., a mammalian cell; or a prokaryotic cell (e.g., bacterial or archaeal cell). In some embodiments, a nucleotide sequence encoding a polypeptide is operably linked to multiple control elements, e.g., that allow expression of the nucleotide sequence encoding the polypeptide in both prokaryotic and eukaryotic cells.
For illustration purposes, examples of spatially restricted promoters include, but are not limited to. hepatic- specific promoters, lung-specific promoters, neuron-specific promoters, adipocyte-specific promoters, cardiomyocyte-speciflc promoters, smooth muscle-specific promoters, photoreceptor- specific promoters, etc. Neuron -specific spatially restricted promoters include, but are not limited to, a neuron -specific enolase (NSE) promoter (see, e.g., EMBL HSEN02, X51956); an aromatic amino acid decarboxylase (AADC) promoter, a neurofilament promoter (see, e.g., GenBank HUMNFL. L04147): a synapsin promoter (see, e.g., GenBank HUMSYN1B, M55301); a thy-1 promoter (see, e.g., Chen et al. (1987) Cell 51 :7-19; and Llewellyn, et al. (2010) Nat. Med. 16(10): 1161-1166): a serotonin receptor promoter (see. e.g., GenBank S62283); a tyrosine hydroxylase promoter (TH) (see, e.g., Oh et al. (2009) Gene Ther 16:437; Sasaoka et al. (1992) Mol. Brain Res. 16:274; Boundy et al. (1998) J. Neurosci. 18:9989; and Kaneda et al. (1991 ) Neuron 6:583-594); a GnR.FI promoter (see, e.g.. Radovick et al. (1991) Proc. Natl. Acad. Sc). USA 88:3402-3406); an L7 promoter (see, e.g., Oberdick et al. (1990) Science 248:223-226); a DNMT promoter (see, e.g,, Badge el al. (1988) Proc. Nail. Acad. Sei. USA 85:3648-3652): an enkephalin promoter (see, e.g., Comb ei al. (1988) EM BO J. 17:3793- 3805); a myelin basic protein (MBP) promoter; a Ca2+-calmodulin-dependent protein kinase II- alpha (CamKIIa) promoter (see, e.g., Mayford et al. (1996) Proc. Natl. Acad. Sci. USA 93:13250; and Casanova et al. (2001) Genesis 31:37); a CMV enhancer/platelet-derived growth factor-p promoter (see, e.g., Liu et al. (2004) Gene Therapy 11:52-60); and the like,
Adipocyte-specific spatially restricted promoters include, but are not limited to, the aP2 gene promoter/enhancer, e.g,, a region from -5.4 kb to +21 bp of a human aP2 gene (see, e.g., Tozzo et al. (1997) Endocrinol. 138: 1604; Ross et al. (1990) Proc. Natl. Acad. Sci. US.A 87:9590: and Pavjani et al. (2005) Nat. Med. 11:797); a glucose transporter-4 (GLUT4) promoter (see, e.g., Knight et ah (2003) Proc. Natl. Acad. Sci. USA 100:14725); a fatty acid translocase (FAT/CD36) promoter (see, e.g., Kuriki et al. (2002) Biol. Pharm. Bull. 25:1476; and Sato el al. (2002) J. Biol. Chem. 277: 15703); a stearoyl-CoA desaturase- 1 (SCD1 ) promoter (Tabor et al. (1999) J . Biol. Chem. 274:20603): a leptin promoter (see, e.g.. Mason et al. (1998) Endocrinol. 139: 1013: and Chen et al, (1999) Biochem, Biophys, Res. Comm, 262: 187); an adiponectin promoter (see, e.g., Kita et al. (2005) Biochem. Biophys. Res. Comm. 331:484; and Chakrabarti (2010) Endocrinol. 151 :24()8); an adipsin promoter (see, e.g., Plait el al. (1989) Proc. Natl. Acad. Sei. USA 86:7490); a resistin promoter (see, e.g., Seo ct al. (2003) Molcc. Endocrinol. 17:1522); and the like.
Cardiomyocyte-specific spatially restricted promoters include, but are not limited to, control sequences derived from the following genes: myosin light chain-2, a-myosin heavy chain, AE3, cardiac troponin C, cardiac actin, and the like. Franz et al. (1997) Cardiovasc. Res. 35:560-566; Robbins et al. (1995) Arm. N.Y. Acad. Sci. 752:492-505; Linn et al, (1995) Circ. Res. 76:584-591 ; Parmacek et al. ( 1994) Mol. Cell. Biol. 14:1870- 1885; Hunter et al. ( 1993) Hypertension 22:608-617; and Sartorelli et al. (1992) Proc. Natl. Acad. Sci. USA 89:4047-4051.
Smooth muscle-specific spatially restricted promoters include, but are not limited to, an SM22a promoter (see. e.g., Akyiirek et al. (2000) Mol. Med. 6:983: and U.S. Pat. No. 7,169,874): a smoothelin promoter (see, e.g., WO 2001/018048 ); an a-smooth muscle actin promoter; and the like. For example, a 0.4 kb region of the SM22a promoter, within which lie two CArG elements, has been shown to mediate vascular smooth muscle cell-specific expression (see, e.g., Kim, et al. (1997) Mol. Cell. Biol. 17, 2266-2278; Li, et al., (1996) J. Cell Biol. 132, 849-859; and Moessler, et al. ( 1996) Development 122, 2415-2425).
Photoreceptor-specific spatially restricted promoters include, but are not limited to, a rhodopsin promoter; a rhodopsin kinase promoter ( Young et al. (2003) Ophthalmol. Vis. Sci, 44:4076): a beta phosphodiesterase gene promoter (Nicoud et al. (2007) J. Gene Med. 9:1015); a retinitis pigmentosa gene promoter (Nicoud et al. (2007) supra); an interphotoreceptor retinoid- binding protein (IRBP) gene enhancer (Nicoud et al, (2007) supra): an IRBP gene promoter (Yokoyama et al. (1992) Exp Eye Res. 55:225); and the like.
In some embodiments, a gene modifying system, e.g., DNA encoding a gene modifying polypeptide, DNA encoding a template RNA, or DNA or RNA encoding a heterologous object sequence, is designed such that one or more elements is operably linked to a tissue-specific promoter, e.g., a promoter that is active in T-cells. In some embodiments, a T-cell active promoter is inactive in other cell types, e.g., B-cells, NK cells. In some embodiments, a T-cell active promoter is derived from a promoter for a gene encoding a component of the T-cell receptor, e.g., TRAC, TRBC, TRGC, TRDC. In some embodiments, a T-cell active promoter is derived from a promoter for a gene encoding a component of a T-cell-specific cluster of differentiation protein, e.g., CD3, e.g., CD3D, CD3E, CD3G, CD3Z. In some embodiments, T- cell-specific promoters in gene modifying systems are discovered by comparing publicly available gene expression data across cell types and selecting promoters from the genes with enhanced expression in T-cells. In some embodiments, promoters may be selecting depending on the desired expression breadth, e.g., promoters that are active in T-cells only, promoters that are active in NK cells only, promoters that are active in both T-cells and NK cells.
Cell-specific promoters known in the art may be used to direct expression of a gene modifying protein, e.g., as described herein. Non-limiting exemplary mammalian cell- specific promoters have been characterized and used in mice expressing Cre recombinase in a cellspecific manner. Certain nonlimiting exemplary mammalian cell-specific promoters are listed in Table 1 of US9845481, incorporated herein by reference.
In some embodiments, a vector as described herein comprises an expression cassette. Typically, an expression cassette comprises the nucleic acid molecule of the instant invention operatively linked to a promoter sequence. For example, a promoter is operatively linked with a coding sequence when it is capable of affecting the expression of that coding sequence (e.g.. the coding sequence is under the transcriptional control of the promoter). Encoding sequences can be operatively linked to regulatory sequences in sense or antisense orientation. In some embodiments, a promoter is a heterologous promoter. In some embodiments, an expression cassette may comprise additional elements, for example, an intron, an enhancer, a polyadenylation site, a woodchuck response element (WRE), and/or other elements known to affect expression levels of the encoding sequence. A promoter typically controls the expression of a coding sequence or functional RNA. In some embodiments, a promoter sequence comprises proximal and more distal upstream elements and can further comprise an enhancer element. An enhancer can typically stimulate promoter activity and may be an innate element of a promoter or a heterologous element inserted to enhance the level or tissue- specificity of a promoter. In some embodiments, a promoter is derived in its entirety from a native gene. In some embodiments, the promoter is composed of different elements derived from different naturally occurring promoters. In some embodiments, a promoter comprises a synthetic nucleotide sequence. It will be understood by those skilled in the art that different promoters will direct the expression of a gene in different tissues or cell types, or at different stages of development, or in response to different environmental conditions or to the presence or the absence of a drug or transcriptional co-factor. Ubiquitous, cell -type-specific, tissue-specific, developmental stage- specific, and conditional promoters, for example, drug-responsive promoters (e.g., tetracycline- rcsponsivc promoters) arc well known to those of skill in the art. Exemplary promoters include, but are not limited io, the phosphoglycerate kinase (PKG) promoter, CAG (composite of the CMV enhancer the chicken beta actin promoter (CBA) and the rabbit beta globin intron), NSE (neuronal specific enolase), synapsin or NeuN promoters, the SV40 early promoter, mouse mammary tumor virus LTR promoter; adenovirus major late promoter (Ad MLP), a herpes simplex virus (MSV) promoter, a cytomegalovirus (CMV) promoter such as the CMV immediate early promoter region (CM VIE), SFFV promoter, rous sarcoma virus (KSV) promoter, synthetic promoters, hybrid promoters, and the like. Other promoters can be of human origin or from other species, including from mice. Common promoters include, e.g., the human cytomegalovirus (CMV) immediate early gene promoter, the SV40 early promoter, the Rous sarcoma virus long terminal repeat, [beta]- actin, rat insulin promoter, the phosphoglycerate kinase promoter, the human alpha- 1 antitrypsin (hAAT) promoter, the transthyretin promoter, the TBG promoter and other liver- specific promoters, the desmin promoter and similar musclespecific promoters, the EFl -alpha promoter, hybrid promoters with multi-tissue specificity, promoters specific for neurons like synapsin and glyceraldehyde-3 -phosphate dehydrogenase promoter, all of which are promoters well known and readily available to those of skill in the art, can be used to obtain high-level expression of the coding sequence of interest. In addition, sequences derived from non-viral genes, such as the murine metallothionein gene, will also find use herein. Such promoter sequences are commercially available from, e.g., Stratagene (San Diego, CA). Additional exemplary promoter sequences are described, for example, in WO2018213786AI (incorporated by reference herein in its entirety).
In some embodiments, the apolipoprotein E enhancer (ApoE) or a functional fragment thereof is used, e.g., to drive expression in the liver. In some embodiments, two copies of the ApoE enhancer or a functional fragment thereof are used. In some embodiments, the ApoE enhancer or functional fragment thereof is used in combination with a promoter, e.g., the human alpha- 1 antitrypsin (hAAT) promoter.
In some embodiments, regulatory sequences impart tissue-specific gene expression capabilities. In some cases, tissue-specific regulatory sequences bind tissue-specific transcription factors that induce transcription in a tissue specific manner. Various tissue-specific regulatory sequences (e.g., promoters, enhancers, etc.) are known in the art. Exemplary? tissue- specific regulatory sequences include, but are not limited to, the following tissue-specific promoters: a liver-specific thyroxin binding globulin (TBG) promoter, an insulin promoter, a glucagon promoter, a somatostatin promoter, a pancreatic polypeptide ( PPY ) promoter, a synapsin-1 (Syn) promoter, a creatine kinase (MCK) promoter, a mammalian desmin (DES) promoter, a a-myosin heavy chain (a-MHC) promoter, or a cardiac Troponin T (cTnT) promoter, Other exemplary promoters include Beta-actin promoter, hepatitis B virus core promoter. Sandig et al., Gene Ther., 3:1002-9 ( 1996); alpha-fetoprotein (AFP) promoter, Arbuthnot et al.. Hum, Gene Ther., 7:1503-14 (19961), bone osteocalcin promoter (Stein el al., Mol. Biol. Rep., 24:185- 96 ( 1997)); bone sialoprotein promoter (Chen et al., J. Bone Miner. Res., 11:654-64 (1996)), CD2 promoter (Hansa! et al., J. Immunol., 161 :1063-8 (1998); immunoglobulin heavy chain promoter; T cell receptor a-chain promoter, neuronal such as neuron-specific enolase (NSE) promoter (Andersen et al., Cell. Mol. Neurobiol,, 13:503-15 ( 1993)), neurofilament light-chain gene promoter (Piccioli et al.. Proc. Natl. Acad. Sci. USA, 88:5611-5 (1991 )), and the neuronspecific vgf gene promoter (Piccioli et al., Neuron, 15:373-84 ( 1995)), and others. Additional exemplary promoter sequences are described, for example, in U.S. Patent No. 10300146 (incorporated herein by reference in its entirety). In some embodiments, a tissue-specific regulatory element, e.g., a tissue-specific promoter, is selected from one known to be operably linked to a gene that is highly expressed in a given tissue, e.g., as measured by RNA-seq or protein expression data, or a combination thereof. Methods for analyzing tissue specificity by expression are taught in Fagerberg et al. Moi Cell Proteomics 13(2):397-406 (2014). which is incorporated herein by reference in its entirety.
In some embodiments, a vector described herein is a multicistronic expression construct. Multicistronic expression constructs include, for example, constructs harboring a first expression cassette, e.g., comprising a first promoter and a first encoding nucleic acid sequence, and a second expression cassette, e.g.. comprising a second promoter and a second encoding nucleic acid sequence. Such multicistronic expression constructs may. in some instances, be particularly useful in the delivery of non-translated gene products, such as hairpin RNAs. together with a polypeptide, for example, a gene modifying polypeptide and gene modifying template. In some embodiments, multicistronic expression constructs may exhibit reduced expression levels of one or more of the included transgenes, for example, because of promoter interference or the presence of incompatible nucleic acid elements in close proximity. If a multicistronic expression construct is part of a viral vector, the presence of a seif-complementary nucleic acid sequence may, in some instances, interfere with the formation of structures necessary for viral reproduction or packaging.
In some embodiments, a sequence encodes an RNA with a hairpin. In some embodiments, a hairpin RNA is a guide RNA, a template RNA, a shRNA, or a microRN A. In some embodiments, a first promoter is an RNA polymerase I promoter. In some embodiments, a first promoter is an RNA polymerase II promoter. Tn some embodiments, a second promoter is an RNA polymerase III promoter. In some embodiments, a second promoter is a U6 or Hl promoter,
Without wishing to be bound by theory , multicistronic expression constructs may not achieve optimal expression levels as compared to expression systems containing only one cistron. One of the suggested causes of lower expression levels achieved with multicistronic expression constructs comprising two or more promoter elements is the phenomenon of promoter interference (see. e.g., Curtin J A, Dane A P, Swanson A, Alexander I E, Ginn S L. Bidirectional promoter interference between two widely used internal heterologous promoters in a late- generation lenllviral construct. Gene Ther. 2008 March; 15(5):384-90; and Martin-Duque P, Jezzard S, Kaftan si s L, Vassaux G. Direct comparison of the insulating properties of two genetic elements in an adenoviral vector containing two different expression cassettes. Hum Gene Ther. 2004 October; 15(10):995-1002; both references incorporated herein by reference for disclosure of promoter interference phenomenon). In some embodiments, the problem of promoter interference may be overcome, e,g., by producing multicistronic expression constructs comprising only one promoter driving transcription of multiple encoding nucleic acid sequences separated by internal ribosomal entry sites, or by separating cistrons comprising their own promoter with transcriptional insulator elements. In some embodiments, single-promoter driven expression of multiple cistrons may result in uneven expression levels of the cistrons. In some embodiments, a promoter cannot efficiently be isolated and isolation dements may not be compatible with some gene transfer vectors, for example, some retroviral vectors.
MicroRNAs
MicroRNAs (miRNAs) and other small interfering nucleic acids generally regulate gene expression via target RNA transcript cleavage/degradation or translational repression of the target messenger RN/k (mRNA). miRNAs may, in some instances, be natively expressed, typically as final 19-25 non -Iran slated RNA products. miRNAs generally exhibit their activity through sequence-specific interactions with the 3' untranslated regions (UTR) of target mRNAs. These endogenously expressed miRNAs may form hairpin precursors that are subsequently processed into an miRNA duplex, and further into a mature single stranded miRNA molecule. This mature miRNA generally guides a multi protein complex, miRISC, which identifies target 3' UTR regions of target mRNzAs based upon their complementarity to the mature miRNzA. Useful transgene products may include, for example, miRNAs or miRNA binding sites that regulate the expression of a linked polypeptide. A non- limiting list of miRNA genes; the products of these genes and their homologues are useful as transgenes or as targets for small interfering nucleic acids (e.g., miR NA sponges, antisense oligonucleotides), e.g., in methods such as those listed in US10300146, 22:25-25:48, are herein incorporated by reference. In some embodiments, one or more binding sites for one or more of the foregoing miRNAs are incorporated in a transgene, e.g., a transgene delivered by a r.AAV vector, e.g., to inhibit the expression of the transgene in one or more tissues of an animal harboring the transgene. In some embodiments, a binding site may be selected to control the expression of a transgene in a tissue specific manner. For example, binding sites for the liver-specific miR-122 may be incorporated into a transgene to inhibit expression of that transgene in the liver. Additional exemplary miRNA sequences are described, for example, in U.S. Patent No. 10,300,146 (incorporated herein by reference in its entirety).
An miR inhibitor or miRNA inhibitor is generally an agent that blocks miRNA expression and/or processing. Examples of such agents include, but are not. limited to. microRNA antagonists, microRNA specific antisense, niicroRN A sponges, and microRNA oligonucleotides (double-stranded, hairpin, short oligonucleotides) that inhibit miRNA interaction with a Drosha complex. MicroRNA inhibitors, e.g., miRNA sponges, can be expressed in cells from transgenes (e.g., as described in Ebert. M. S. Nature Methods. Epub Aug. 12, 2007; incorporated by reference herein in its entirety). In some embodiments, microRNA sponges, or other miR inhibitors, are used with the AAVs. microRNzA sponges generally specifically inhibit miRNAs through a complementary heptameric seed sequence. In some embodiments, an entire family of miRNAs can be silenced using a single sponge sequence. Other methods for silencing miRNA function (derepression of miRN A targets) in cells will be apparent to one of ordinary skill in the art. In some embodiments, a gene modifying system, template RNA, or polypeptide described herein is administered to or is active in (c.g., is more active in) a target tissue, c.g., a first tissue. In some embodiments, a gene modifying system, template RNA, or polypeptide is not administered to or is less active in (e.g., not active in) a non-target tissue. In some embodiments, a gene modifying system, template RNA, or polypeptide described herein is useful for modifying DNA in a target tissue, e.g., a first tissue, (e.g., and not modifying DNA in a nontarget tissue).
In some embodiments, a gene modifying system comprises (a) a polypeptide described herein or a nucleic acid encoding the same, (b) a template nucleic acid (e.g., template RNA) described herein, and (c) one or more first tissue- specific expression-control sequences specific to the target tissue, wherein the one or more first tissue-specific expression-control sequences specific to the target tissue are in operative association with (a), (b), or (a) and (b), wherein, when associated with (a), (a) comprises a nucleic acid encoding the polypeptide.
In some embodiments, the nucleic acid in (b) comprises RNA.
In some embodiments, the nucleic acid in (b) comprises DNA.
In some embodiments, the nucleic acid in (b): (i) is single-stranded or comprises a singlestranded segment, e.g., is single-stranded DNA or comprises a single- stranded segment and one or more double stranded segments; (ii) has inverted terminal repeats; or (iii) both (i) and (ii).
In some embodiments, the nucleic acid in (b) is double-stranded or comprises a doublestranded segment.
In some embodiments, (a) comprises a nucleic acid encoding the polypeptide.
In some embodiments, the nucleic acid in (a) comprises RNA.
In some embodiments, the nucleic acid in (a) comprises DNA.
In some embodiments, the nucleic acid in (a): (i) is single-stranded or comprises a singlestranded segment, e.g., is single-stranded DNA or comprises a single- stranded segment and one or more double stranded segments; (ii) has inverted terminal repeats; or (iii) both (i) and (ii).
In some embodiments, the nucleic acid in (a) is double-stranded or comprises a doublestranded segment.
In some embodiments, the nucleic acid in (a), (b), or (a) and (b) is linear.
In some embodiments, the nucleic acid in (a), (b), or (a) and (b) is circular, e.g., a plasmid or minicircle. In some embodiments, the heterologous object sequence is in operative association with a first promoter.
In some embodiments, the one or more first tissue-specific expression-control sequences comprises a tissue specific promoter.
In some embodiments, the tissue-specific promoter comprises a first promoter in operative association with: (i) the heterologous object sequence, (ii) a nucleic acid encoding the retroviral RT, or (iii) (i) and (ii).
In some embodiments, the one or more first tissue- specific expression-control sequences comprises a tissue- specific microRNA recognition sequence in operative association with: (i) the heterologous object sequence, (ii) a nucleic acid encoding the retroviral RT domain, or (iii) (i) and (ii).
In some embodiments, a system comprises a tissue- specific promoter, and the system further comprises one or more tissue-specific microRNA recognition sequences, wherein: (i) the tissue specific promoter is in operative association with: (I) the heterologous object sequence, (II) a nucleic acid encoding the retroviral RT domain, or (III) (I) and (II); and/or (ii) the one or more tissue-specific microRNA recognition sequences are in operative association with: (I) the heterologous object sequence, (II) a nucleic acid encoding the retroviral RT, or (III) (I) and (II).
In some embodiments, wherein (a) comprises a nucleic acid encoding a gene modifying polypeptide, the nucleic acid comprises a promoter in operative association with the nucleic acid encoding the gene modifying polypeptide.
In some embodiments, a nucleic acid encoding a gene modifying polypeptide comprises one or more second tissue-specific expression-control sequences specific to the target tissue in operative association with the gene modifying polypeptide coding sequence.
In some embodiments, one or more second tissue-specific expression-control sequences comprises a tissue specific promoter.
In some embodiments, a tissue- specific promoter is the promoter in operative association with a nucleic acid encoding a gene modifying polypeptide.
In some embodiments, one or more second tissue-specific expression-control sequences comprises a tissue- specific microRNA recognition sequence. In some embodiments, a promoter in operative association with a nucleic acid encoding a gene modifying polypeptide is a tissue-specific promoter, the system further comprising one or more tissue-specific microRNA recognition sequences.
In some embodiments, a nucleic acid component of a system of the present disclosure is a sequence (e.g., encoding the polypeptide or comprising a heterologous object sequence) flanked by untranslated regions (UTRs) that modify protein expression levels. Various 5' and 3' UTRs can affect protein expression. For example, in some embodiments, the coding sequence may be preceded by a 5' UTR that modifies RNA stability or protein translation. In some embodiments, a sequence may be followed by a 3' UTR that modifies RNA stability or translation. In some embodiments, a sequence may be preceded by a 5' UTR and followed by a 3' UTR that modify RNA stability or translation. In some embodiments, the 5' and/or 3' UTR may be selected from the 5' and 3' UTRs of complement factor 3 (C3) (CACTCCTCCCCATCCTCTCCCTCTGTCCCTCTGTCCCTCTGACCCTGCACTGTCCCAG CACC; SEQ ID NO: 11,004) or orosomucoid 1 (0RM1) (CAGGACACAGCCTTGGATCAGGACAGAGACTTGGGGGCCATCCTGCCCCTCCAACC CGACATGTGTACCTCAGCTTTTTCCCTCACTTGCATCAATAAAGCTTCTGTGTTTGGA ACAGCTAA; SEQ ID NO: 11,005) (Asrani et al. RNA Biology 2018). In certain embodiments, the 5' UTR is the 5' UTR from C3 and the 3' UTR is the 3' UTR from ORMl. In some embodiments, a 5 ' UTR and 3' UTR for protein expression, e.g., mRNA (or DNA encoding the RNA) for a gene modifying polypeptide or heterologous object sequence, comprise optimized expression sequences. In some embodiments, the 5' UTR comprises GGGAAAUAAGAGAGAAAAGAAGAGUAAGAAGAAAUAUAAGAGCCACC (SEQ ID NO: 11,006) and/or the 3' UTR comprising UGAUAAUAGGCUGGAGCCUCGGUGGCCAUGCUUCUUGCCCCUUGGGCCUCCCCCC AGCCCCUCCUCCCCUUCCUGCACCCGUACCCCCGUGGUCUUUGAAUAAAGUCUGA (SEQ ID NO: 11,007), e.g., as described in Richner et al. Cell 168(6): Pl 114-1125 (2017), the sequences of which are incorporated herein by reference.
In some embodiments, a 5' and/or 3' UTR may be selected to enhance protein expression. In some embodiments, a 5' and/or 3' UTR may be selected to modify protein expression such that overproduction inhibition is minimized. In some embodiments, UTRs are around a coding sequence, e.g., outside the coding sequence and in other embodiments proximal to the coding sequence. In some embodiments, additional regulatory elements (e.g., miRNA binding sites, cis-rcgulatory sites) arc included in the UTRs.
In some embodiments, an open reading frame of a gene modifying system, e.g., an ORF of an mRNA (or DNA encoding an mRNA) encoding a gene modifying polypeptide or one or more ORFs of an mRNA (or DNA encoding an mRNA) of a heterologous object sequence, is flanked by a 5' and/or 3' untranslated region (UTR) that enhances the expression thereof. In some embodiments, the 5' UTR of an mRNA component (or transcript produced from a DNA component) of the system comprises the sequence 5'- GGGAAAUAAGAGAGAAAAGAAGAGUAAGAAGAAAUAUAAGAGCCACC-3'; SEQ ID NO: 11,008). In some embodiments, the 3' UTR of an mRNA component (or transcript produced from a DNA component) of the system comprises the sequence 5'- UGAUAAUAGGCUGGAGCCUCGGUGGCCAUGCUUCUUGCCCCUUGGGCCUCCCCCC AGCCCCUCCUCCCCUUCCUGCACCCGUACCCCCGUGGUCUUUGAAUAAAGUCUGA- 3' (SEQ ID NO: 11,009). This combination of 5' UTR and 3' UTR has been shown to result in desirable expression of an operably linked ORF by Richner et al. Cell 168(6): Pl 114-1125 (2017), the teachings and sequences of which are incorporated herein by reference. In some embodiments, a system described herein comprises a DNA encoding a transcript, wherein the DNA comprises the corresponding 5' UTR and 3' UTR sequences, with T substituting for U in the above- listed sequence). In some embodiments, a DNA vector used to produce an RNA component of the system further comprises a promoter upstream of the 5' UTR for initiating in vitro transcription, e.g, a T7, T3, or SP6 promoter. The 5' UTR above begins with GGG, which is a suitable start for optimizing transcription using T7 RNA polymerase. For tuning transcription levels and altering the transcription start site nucleotides to fit alternative 5' UTRs, the teachings of Davidson et al. Pac Symp Biocomput 433-443 (2010) describe T7 promoter variants, and the methods of discovery thereof, that fulfill both of these traits.
Viral vectors and components thereof
Viruses arc a useful source of delivery vehicles for the systems described herein, in addition to a source of relevant enzymes or domains as described herein, e.g., as sources of polymerases and polymerase functions used herein, e.g., DNA-dependent DNA polymerase, RNA-dependent RNA polymerase, RNA-dependent DNA polymerase, DNA-dependent RNA polymerase, reverse transcriptase. Some enzymes, e.g., reverse transcriptases, may have multiple activities, e.g., be capable of both RNA-dependent DNA polymerization and DNA- dcpcndcnt DNA polymerization, c.g., first and second strand synthesis. In some embodiments, the virus used as a gene modifying delivery system or a source of components thereof may be selected from a group as described by Baltimore Bacterial Rev 35(3):235-241 (1971).
In some embodiments, a virus is selected from a Group I virus, e.g., is a DNA virus and packages dsDNA into virions. In some embodiments, the Group I virus is selected from, e.g., Adenoviruses, Herpesviruses, Poxviruses.
In some embodiments, a virus is selected from a Group II virus, e.g., is a DNA virus and packages ssDNA into virions. In some embodiments, the Group II virus is selected from, e.g., Parvoviruses. In some embodiments, the parvovirus is a dependoparvovirus, e.g., an adeno- associated virus (AAV).
In some embodiments, a virus is selected from a Group III virus, e.g., is an RNA virus and packages dsRNA into virions. In some embodiments, a Group III virus is selected from, e.g., Reoviruses. In some embodiments, one or both strands of the dsRNA contained in such virions is a coding molecule able to serve directly as mRNA upon transduction into a host cell, e.g., can be directly translated into protein upon transduction into a host cell without requiring any intervening nucleic acid replication or polymerization steps.
In some embodiments, a virus is selected from a Group IV virus, e.g., is an RNA virus and packages ssRNA(+) into virions. In some embodiments, a Group IV virus is selected from, e.g., Coronaviruses, Picomaviruses, Togaviruses. In some embodiments, a ssRNA(+) contained in such virions is a coding molecule able to serve directly as mRNA upon transduction into a host cell, e.g., can be directly translated into protein upon transduction into a host cell without requiring any intervening nucleic acid replication or polymerization steps.
In some embodiments, a virus is selected from a Group V virus, e.g., is an RNA virus and packages ssRNA(-) into virions. In some embodiments, a Group V virus is selected from, e.g., Orthomyxoviruses, Rhabdoviruses. In some embodiments, an RNA virus with an ssRNA(-) genome also carries an enzyme inside the virion that is transduced to host cells with the viral genome, e.g., an RNA-dependent RNA polymerase, capable of copying the ssRNA(-) into ssRNA(+) that can be translated directly by the host.
In some embodiments, a virus is selected from a Group VI virus, e.g., is a retrovirus and packages ssRNA(+) into virions. In some embodiments, a Group VI virus is selected from, e.g., retroviruses. In some embodiments, a retrovirus is a lentivirus, e.g., HIV-1 , HIV-2, SIV, BIV. In some embodiments, a retrovirus is a spumavirus, e.g., a foamy virus, e.g., HFV, SFV, BFV. In some embodiments, the ssRNA(+) contained in such virions is a coding molecule able to serve directly as mRNA upon transduction into a host cell, e.g., can be directly translated into protein upon transduction into a host cell without requiring any intervening nucleic acid replication or polymerization steps. In some embodiments, an ssRNA(+) is first reverse transcribed and copied to generate a dsDNA genome intermediate from which mRNA can be transcribed in the host cell. In some embodiments, an RNA virus with an ssRNA(+) genome also carries an enzyme inside the virion that is transduced to host cells with the viral genome, e.g., an RNA-dependent DNA polymerase, capable of copying the ssRNA(+) into dsDNA that can be transcribed into mRNA and translated by the host. In some embodiments, a reverse transcriptase from a Group VI retrovirus is incorporated as the reverse transcriptase domain of a gene modifying polypeptide.
In some embodiments, a virus is selected from a Group VII virus, e.g., is a retrovirus and packages dsRNA into virions. In some embodiments, a Group VII virus is selected from, e.g., Hepadnaviruses. In some embodiments, one or both strands of a dsRNA contained in such virions is a coding molecule able to serve directly as mRNA upon transduction into a host cell, e.g., can be directly translated into protein upon transduction into a host cell without requiring any intervening nucleic acid replication or polymerization steps. In some embodiments, one or both strands of a dsRNA contained in such virions is first reverse transcribed and copied to generate a dsDNA genome intermediate from which mRNA can be transcribed in the host cell. In some embodiments, an RNA virus with a dsRNA genome also carries an enzyme inside the virion that is transduced to host cells with the viral genome, e.g., an RNA-dependent DNA polymerase, capable of copying the dsRNA into dsDNA that can be transcribed into mRNA and translated by the host. In some embodiments, a reverse transcriptase from a Group VII retrovirus is incorporated as the reverse transcriptase domain of a gene modifying polypeptide.
In some embodiments, virions used to deliver nucleic acid in this invention may also carry enzymes involved in the process of gene modification. For example, a retroviral virion may contain a reverse transcriptase domain that is delivered into a host cell along with the nucleic acid. In some embodiments, an RNA template may be associated with a gene modifying polypeptide within a virion, such that both arc co-delivered to a target cell upon transduction of the nucleic acid from the viral particle. In some embodiments, a nucleic acid in a virion may comprise DNA, e.g., linear ssDNA, linear dsDNA, circular ssDNA, circular dsDNA, minicircle DNA, dbDNA, ccDNA. In some embodiments, the nucleic acid in a virion may comprise RNA, e.g., linear ssRNA, linear dsRNA, circular ssRNA, circular dsRNA. In some embodiments, a viral genome may circularize upon transduction into a host cell, e.g., a linear ssRNA molecule may undergo a covalent linkage to form a circular ssRNA, a linear dsRNA molecule may undergo a covalent linkage to form a circular dsRNA or one or more circular ssRNA. In some embodiments, a viral genome may replicate by rolling circle replication in a host cell. In some embodiments, a viral genome may comprise a single nucleic acid molecule, e.g., comprise a nonsegmented genome. In some embodiments, a viral genome may comprise two or more nucleic acid molecules, e.g., comprise a segmented genome. In some embodiments, a nucleic acid in a virion may be associated with one or proteins. In some embodiments, one or more proteins in a virion may be delivered to a host cell upon transduction. In some embodiments, a natural virus may be adapted for nucleic acid delivery by the addition of virion packaging signals to the target nucleic acid, wherein a host cell is used to package the target nucleic acid containing the packaging signals.
In some embodiments, a virion used as a delivery vehicle may comprise a commensal human virus. In some embodiments, a virion used as a delivery vehicle may comprise an anello virus, the use of which is described in WO2018232017A1, which is incorporated herein by reference in its entirety. AAV Administration
In some embodiments, an adeno-associated virus (AAV) is used in conjunction with a system, a template nucleic acid, and/or a polypeptide (e.g., a gene modifying polypeptide) described herein. In some embodiments, an AAV is used to deliver, administer, or package a system, template nucleic acid, and/or polypeptide (e.g., gene modifying polypeptide) described herein. In some embodiments, an AAV is a recombinant AAV (rAAV).
In some embodiments, a system comprises (a) a gene modifying polypeptide described herein or a nucleic acid encoding the same, (b) a template nucleic acid (e.g., template RNA) described herein, and (c) one or more first tissue- specific expression-control sequences specific to the target tissue, wherein the one or more first tissue-specific expression-control sequences specific to the target tissue are in operative association with (a), (b), or (a) and (b), wherein, when associated with (a), (a) comprises a nucleic acid encoding the polypeptide. In some embodiments, a system described herein further comprises a first recombinant adcno-associatcd virus (rAAV) capsid protein; wherein the at least one of (a) or (b) is associated with the first rAAV capsid protein, wherein at least one of (a) or (b) is flanked by AAV inverted terminal repeats (ITRs).
In some embodiments, (a) and (b) are associated with the first rAAV capsid protein.
In some embodiments, (a) and (b) are on a single nucleic acid.
In some embodiments, the system further comprises a second rAAV capsid protein, wherein at least one of (a) or (b) is associated with the second rAAV capsid protein, and wherein the at least one of (a) or (b) associated with the second rAAV capsid protein is different from the at least one of (a) or (b) is associated with the first rAAV capsid protein.
In some embodiments, the at least one of (a) or (b) is associated with the first or second rAAV capsid protein is dispersed in the interior of the first or second rAAV capsid protein, which first or second rAAV capsid protein is in the form of an AAV capsid particle.
In some embodiments, a system further comprises a nanoparticle, wherein the nanoparticle is associated with at least one of (a) or (b).
In some embodiments, (a) and (b), respectively are associated with: a) a first rAAV capsid protein and a second rAAV capsid protein; b) a nanoparticle and a first rAAV capsid protein; c) a first rAAV capsid protein; d) a first adenovirus capsid protein; e) a first nanoparticle and a second nanoparticle; or f) a first nanoparticle.
Viral vectors are useful for delivering all or part of a system provided by the invention, e.g., for use in methods provided by the invention. Systems derived from different viruses have been employed for the delivery of polypeptides or nucleic acids; for example: integrase-deficient lentivirus, adenovirus, adeno-associated virus (AAV), herpes simplex virus, and baculovirus (reviewed in Hodge et al. Hum Gene Ther 2017; Narayanavari et al. Crit Rev Biochem Mol Biol 2017; Boehme et al. Curr Gene Ther 2015).
Adenoviruses are common viruses that have been used as gene delivery vehicles given well-defined biology, genetic stability, high transduction efficiency, and ease of large-scale production (see, for example, review by Lee et al. Genes & Diseases 2017). They possess linear dsDNA genomes and come in a variety of serotypes that differ in tissue and cell tropisms. In order to prevent replication of infectious virus in recipient cells, adenovirus genomes used for packaging are deleted of some or all endogenous viral proteins, which are provided in trans in viral production cells. This renders the genomes helper-dependent, meaning they can only be replicated and packaged into viral particles in the presence of the missing components provided by so-called helper functions. A helper-dependent adenovirus system with all viral ORFs removed may be compatible with packaging foreign DNA of up to ~37 kb (Parks et al. J Virol 1997). In some embodiments, an adenoviral vector is used to deliver DNA corresponding to the polypeptide or template component of the gene modifying system, or both are contained on separate or the same adenoviral vector. In some embodiments, the adenovirus is a helperdependent adenovirus (HD-AdV) that is incapable of self-packaging. In some embodiments, the adenovirus is a high-capacity adenovirus (HC-AdV) that has had all or a substantial portion of endogenous viral ORFs deleted, while retaining the necessary sequence components for packaging into adenoviral particles. For this type of vector, the only adenoviral sequences required for genome packaging are noncoding sequences: the inverted terminal repeats (ITRs) at both ends and the packaging signal at the 5'-end (Jager et al. Nat Protoc 2009). In some embodiments, the adenoviral genome also comprises stuffer DNA to meet a minimal genome size for optimal production and stability (see, for example, Hausl et al. Mol Ther 2010). hi some embodiments, an adenovirus is used to deliver a gene modifying system to the liver.
In some embodiments, an adenovirus is used to deliver a gene modifying system to HSCs, c.g., HDAd5/35++. HDAd5/35++ is an adenovirus with modified serotype 35 fibers that de-target the vector from the liver (Wang et al. Blood Adv 2019). In some embodiments, the adenovirus that delivers a gene modifying system to HSCs utilizes a receptor that is expressed specifically on primitive HSCs, e.g., CD46.
Adeno-associated viruses (AAV) belong to the parvoviridae family and more specifically constitute the dependoparvovirus genus. The AAV genome is composed of a linear singlestranded DNA molecule which contains approximately 4.7 kilobases (kb) and consists of two major open reading frames (ORFs) encoding the non- structural Rep (replication) and structural Cap (capsid) proteins. A second ORF within the cap gene was identified that encodes the assembly-activating protein (AAP). The DNAs flanking the AAV coding regions are two cisacting inverted terminal repeat (ITR) sequences, approximately 145 nucleotides in length, with interrupted palindromic sequences that can be folded into energetically stable hairpin structures that function as primers of DNA replication. In addition to their role in DNA replication, the ITR sequences have been shown to be involved in viral DNA integration into the cellular genome, rescue from the host genome or plasmid, and encapsidation of viral nucleic acid into mature virions (Muzyczka, (1992) Curr. Top. Micro. Immunol. 158:97-129). In some embodiments, one or more gene modifying nucleic acid components is flanked by ITRs derived from AAV for viral packaging. See, e.g., WO2019113310.
In some embodiments, one or more components of the gene modifying system are carried via at least one AAV vector. In some embodiments, the at least one AAV vector is selected for tropism to a particular cell, tissue, organism. In some embodiments, the AAV vector is pseudotyped, e.g., AAV2/8, wherein AAV2 describes the design of the construct but the capsid protein is replaced by that from AAV8. It is understood that any of the described vectors could be pseudotype derivatives, wherein the capsid protein used to package the AAV genome is derived from that of a different AAV serotype. Without wishing to be limited in vector choice, a list of exemplary AAV serotypes can be found in Table 18. In some embodiments, an AAV to be employed for gene modifying may be evolved for novel cell or tissue tropism as has been demonstrated in the literature (e.g., Davidsson et al. Proc Natl Acad Sci U S A 2019).
In some embodiments, an AAV delivery vector is a vector which has two AAV inverted terminal repeats (ITRs) and a nucleotide sequence of interest (for example, a sequence coding for a gene modifying polypeptideor a DNA template, or both), each of said ITRs having an interrupted (or noncontiguous) palindromic sequence, i.e., a sequence composed of three segments: a first segment and a last segment that are identical when read 5'— > 3' but hybridize when placed against each other, and a segment that is different that separates the identical segments. See, for example, WO2012123430.
Conventionally, AAV virions with capsids are produced by introducing a plasmid or plasmids encoding the rAAV or scAAV genome, Rep proteins, and Cap proteins (Grimm et al, 1998). Upon introduction of these helper plasmids in trans, the AAV genome is “rescued” (i.e., released and subsequently recovered) from the host genome, and is further encapsidated to produce infectious AAV. In some embodiments, one or more gene modifying nucleic acids are packaged into AAV particles by introducing the ITR-flanked nucleic acids into a packaging cell in conjunction with the helper functions.
In some embodiments, an AAV genome is a so called self-complementary genome (referred to as scAAV), such that the sequence located between the ITRs contains both the desired nucleic acid sequence (e.g., DNA encoding the gene modifying polypeptide or template, or both) in addition to the reverse complement of the desired nucleic acid sequence, such that these two components can fold over and sclf-hybridizc. In some embodiments, self- complementary modules are separated by an intervening sequence that permits the DNA to fold back on itself, e.g., forms a stem-loop. An scAAV has the advantage of being poised for transcription upon entering the nucleus, rather than being first dependent on ITR priming and second-strand synthesis to form dsDNA. In some embodiments, one or more gene modifying components is designed as an scAAV, wherein the sequence between the AAV ITRs contains two reverse complementing modules that can self-hybridize to create dsDNA.
In some embodiments, nucleic acid (e.g., encoding a polypeptide, or a template, or both) delivered to cells is closed-ended, linear duplex DNA (CELiD DNA or ceDNA). In some embodiments, ceDNA is derived from the replicative form of the AAV genome (Li et al. PLoS One 2013). In some embodiments, the nucleic acid (e.g., encoding a polypeptide, or a template DNA, or both) is flanked by ITRs, e.g., AAV ITRs, wherein at least one of the ITRs comprises a terminal resolution site and a replication protein binding site (sometimes referred to as a replicative protein binding site). In some embodiments, ITRs are derived from an adeno- associated virus, e.g., AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, or a combination thereof. In some embodiments, ITRs are symmetric. In some embodiments, ITRs are asymmetric. In some embodiments, at least one Rep protein is provided to enable replication of the construct. In some embodiments, at least one Rep protein is derived from an adeno-associated virus, e.g., AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, or a combination thereof. In some embodiments, ceDNA is generated by providing a production cell with (i) DNA flanked by ITRs, e.g., AAV ITRs, and (ii) components required for ITR-dependent replication, e.g., AAV proteins Rep78 and Rep52 (or nucleic acid encoding the proteins). In some embodiments, ceDNA is free of any capsid protein, e.g., is not packaged into an infectious AAV particle. In some embodiments, ceDNA is formulated into LNPs (see, for example, WO2019051289A1).
In some embodiments, a ceDNA vector comprises two self-complementary sequences, e.g., asymmetrical or symmetrical or substantially symmetrical ITRs as defined herein, flanking said expression cassette, wherein the ceDNA vector is not associated with a capsid protein. In some embodiments, a ceDNA vector comprises two self-complementary sequences found in an AAV genome, where at least one ITR comprises an operative Rep-binding element (RBE) (also sometimes referred to herein as “RBS”) and a terminal resolution site (trs) of AAV or a functional variant of the RBE. See, for example, WO2019113310.
In some embodiments, an AAV genome comprises two genes that encode four replication proteins and three capsid proteins, respectively. In some embodiments, the genes are flanked on either side by 145-bp inverted terminal repeats (ITRs). In some embodiments, a virion comprises up to three capsid proteins (Vpl, Vp2, and/or Vp3), e.g., produced in a 1:1:10 ratio. In some embodiments, capsid proteins are produced from the same open reading frame and/or from differential splicing (Vpl) and alternative translational start sites (Vp2 and Vp3, respectively). Generally, Vp3 is the most abundant subunit in the virion and participates in receptor recognition at the cell surface defining the tropism of the virus. In some embodiments, Vpl comprises a phospholipase domain, e.g., which functions in viral infectivity, in the N-terminus of Vpl.
In some embodiments, packaging capacity of viral vectors limits the size of a gene modifying system that can be packaged into the vector. For example, the packaging capacity of AAVs can be about 4.5 kb (e.g., about 3.0, about 3.5, about 4.0, about 4.5, about 5.0, about 5.5, or about 6.0 kb), e.g., including one or two inverted terminal repeats (ITRs), e.g., 145 base ITRs.
In some embodiments, recombinant AAV (rAAV) comprises cis-acting 145-bp ITRs flanking vector transgene cassettes, e.g., providing up to 4.5 kb for packaging of foreign DNA. Subsequent to infection, rAAV can, in some instances, express a fusion protein of the invention and persist without integration into the host genome by existing episomally in circular head-to- tail concatemers. rAAV can be used, for example, in vitro and in vivo. In some embodiments, AAV-mediated gene delivery requires that the length of the coding sequence of the gene is equal or greater in size than the wild-type AAV genome.
AAV delivery of genes that exceed this size and/or the use of large physiological regulatory elements can be accomplished, for example, by dividing the protein(s) to be delivered into two or more fragments. In some embodiments, the N-terminal fragment is fused to an intein- N sequence. In some embodiments, the C- terminal fragment is fused to an intein-C sequence. In embodiments, the fragments are packaged into two or more AAV vectors.
In some embodiments, dual AAV vectors are generated by splitting a large transgene expression cassette in two separate halves (5' and 3' ends, or head and tail), e.g., wherein each half of the cassette is packaged in a single AAV vector (of <5 kb). The re-assembly of the full- length transgene expression cassette can, in some embodiments, then be achieved upon co- infection of the same cell by both dual AAV vectors. In some embodiments, co-infection is followed by one or more of: (1) homologous recombination (HR) between 5' and 3' genomes (dual AAV overlapping vectors); (2) ITR-mediated tail-to-head concatemerization of 5' and 3' genomes (dual AAV trans-splicing vectors); and/or (3) a combination of these two mechanisms (dual AAV hybrid vectors). In some embodiments, the use of dual AAV vectors in vivo results in the expression of full-length proteins. In some embodiments, the use of the dual AAV vector platform represents an efficient and viable gene transfer strategy for transgenes of greater than about 4.0, greater than about 4.1, greater than about 4.2, greater than about 4.3, greater than about 4.4, greater than about 4.5, greater than about 4.6, greater than about 4.7, greater than about 4.8, greater than about 4.9, or greater than about 5.0 kb in size. In some embodiments, AAV vectors can also be used to transduce cells with target nucleic acids, e.g., in the in vitro production of nucleic acids and peptides. In some embodiments, AAV vectors can be used for in vivo and ex vivo gene therapy procedures (see, e.g., West et al., Virology 160:38-47 (1987); U.S. Patent No. 4,797,368; WO 93/24641; Kotin, Human Gene Therapy 5:793-801 (1994);
Muzyczka, J. Clin. Invest.94: 1351 (1994); each of which is incorporated herein by reference in their entirety). The construction of recombinant AAV vectors is described in a number of publications, including U.S. Patent No.5, 173,414; Tratschin et al., Mol. Cell. Biol.5:3251- 3260 (1985); Tratschin, et al., Mol. Cell. Biol.4:2072-2081 (1984); Hermonat & Muzyczka, PNAS 81:6466-6470 (1984); and Samulski et al., J. Virol.63:03822-3828 (1989) (incorporated by reference herein in their entirety).
In some embodiments, a gene modifying polypeptide described herein (e.g., with or without one or more guide nucleic acids) can be delivered using AAV, lentivirus, adenovirus or other plasmid or viral vector types, in particular, using formulations and doses from, for example, U.S. Patent No. 8,454,972 (formulations, doses for adenovirus), U.S. Patent No.8, 404, 658 (formulations, doses for AAV) and U.S. Patent No.5, 846, 946 (formulations, doses for DNA plasmids) and from clinical trials and publications regarding the clinical trials involving lentivirus, AAV and adenovirus. For example, for AAV, the route of administration, formulation and dose can be as described in U.S. Patent No.8, 454, 972 and as in clinical trials involving AAV. For adenovirus, the route of administration, formulation and dose can be as described in U.S. Patent No.8, 404, 658 and as in clinical trials involving adenovirus. For plasmid delivery, the route of administration, formulation and dose can be as described in U.S. Patent No.5, 846, 946 and as in clinical studies involving plasmids. Doses can be based on or extrapolated to an average 70 kg individual (c.g. a male adult human), and can be adjusted for patients, subjects, mammals of different weight and species. Frequency of administration is within the ambit of the medical or veterinary practitioner (e.g., physician, veterinarian), depending on usual factors including the age, sex, general health, other conditions of the patient or subject and the particular condition or symptoms being addressed. In some embodiments, the viral vectors can be injected into the tissue of interest. For cell-type specific gene modifying, the expression of the gene modifying polypeptide and optional guide nucleic acid can, in some embodiments, be driven by a cell-type specific promoter.
In some embodiments, AAV allows for low toxicity, for example, due to the purification method not requiring ultracentrifugation of cell particles that can activate the immune response. In some embodiments, AAV allows low probability of causing insertional mutagenesis, for example, because it does not substantially integrate into the host genome.
In some embodiments, AAV has a packaging limit of about 4.4, about 4.5, about 4.6, about 4.7, or about 4.75 kb. In some embodiments, a gene modifying polypeptide-encoding sequence, promoter, and transcription terminator can fit into a single viral vector. SpCas9 (4.1 kb) may, in some instances, be difficult to package into AAV. Therefore, in some embodiments, a gene modifying polypeptide coding sequence is used that is shorter in length than other gene modifying polypeptide coding sequences or base editors. In some embodiments, the gene modifying polypeptide encoding sequences are less than about 4.5 kb, about 4.4 kb, about 4.3 kb, about 4.2 kb, about 4.1 kb, about 4 kb, about 3.9 kb, about 3.8 kb, about 3.7 kb, about 3.6 kb, about 3.5 kb, about 3.4 kb, about 3.3 kb, about 3.2 kb, about 3.1 kb, about 3 kb, about 2.9 kb, about 2.8 kb, about 2.7 kb, about 2.6 kb, about 2.5 kb, about 2 kb, or about 1.5 kb.
An AAV can be AAV1, AAV2, AAV5 or any combination thereof. In some embodiments, the type of AAV is selected with respect to the cells to be targeted; e.g., AAV serotypes 1, 2, 5 or a hybrid capsid AAV1, AAV2, AAV5 or any combination thereof can be selected for targeting brain or neuronal cells; or AAV4 can be selected for targeting cardiac tissue. In some embodiments, AAV8 is selected for delivery to the liver. Exemplary AAV serotypes as to these cells are described, for example, in Grimm, D. et al, J. Virol.82: 5887-5911 (2008) (incorporated herein by reference in its entirety). In some embodiments, AAV refers all serotypes, subtypes, and naturally-occurring AAV as well as recombinant AAV. AAV may be used to refer to the virus itself or a derivative thereof. Tn some embodiments, AAV includes AAV1, AAV2, AAV3, AAV3B, AAV4, AAV5, AAV6, AAV6.2, AAV7, AAVrh.64Rl, AAVhu.37, AAVrh.8, AAVrh.32.33, AAV8, AAV9, AAV-DJ, AAV2/8, AAVrhlO, AAVLK03, AV10, AAV11, AAV 12, rhlO, and hybrids thereof, avian AAV, bovine AAV, canine AAV, equine AAV, primate AAV, non-primate AAV, and ovine AAV. The genomic sequences of various serotypes of AAV, as well as the sequences of the native terminal repeats (TRs), Rep proteins, and capsid subunits are known in the art. Such sequences may be found in the literature or in public databases such as GenBank. Additional exemplary AAV serotypes are listed in Table 37. Table 37. Exemplary AAV serotypes.
In some embodiments, a pharmaceutical composition (e.g., comprising an AAV as described herein) has less than 10% empty capsids, less than 8% empty capsids, less than 7% empty capsids, less than 5% empty capsids, less than 3% empty capsids, or less than 1 % empty capsids. In some embodiments, a pharmaceutical composition has less than about 5% empty capsids. In some embodiments, the number of empty capsids is below the limit of detection. In some embodiments, it is advantageous for the pharmaceutical composition to have low amounts of empty capsids, e.g., because empty capsids may generate an adverse response (e.g., immune response, inflammatory response, liver response, and/or cardiac response), e.g., with little or no substantial therapeutic benefit.
In some embodiments, the residual host cell protein (rHCP) in a pharmaceutical composition is less than or equal to 100 ng/ml rHCP per 1 x 1013 vg/ml, e.g., less than or equal to 40 ng/ml rHCP per 1 x 1013 vg/ml or 1-50 ng/ml rHCP per 1 x 1013 vg/ml. In some embodiments, a pharmaceutical composition comprises less than 10 ng rHCP per 1.0 x 1013 vg, or less than 5 ng rHCP per 1.0 x 1013 vg, less than 4 ng rHCP per 1.0 x 1013 vg, or less than 3 ng rHCP per 1.0 x 1013 vg, or any concentration in between. In some embodiments, the residual host cell DNA (hcDNA) in a pharmaceutical composition is less than or equal to 5 x 106 pg/ml hcDNA per 1 x 1013 vg/ml, less than or equal to 1.2 x 106 pg/ml hcDNA per 1 x 1013 vg/ml, or 1 x 105 pg/ml hcDNA per 1 x 1013 vg/ml. In some embodiments, the residual host cell DNA in a pharmaceutical composition is less than 5.0 x 105 pg per 1 x 1013 vg, less than 2.0 x 105 pg per 1.0 x 1013 vg, less than 1.1 x 105 pg per 1.0 x 1013 vg, less than 1.0 x 105 pg hcDNA per 1.0 x 1013 vg, less than 0.9 x 105 pg hcDNA per 1.0 x 1013 vg, less than 0.8 x 105 pg hcDNA per 1.0 x 1013 vg, or any concentration in between.
In some embodiments, the residual plasmid DNA in a pharmaceutical composition is less than or equal to 1.7 x 105 pg/ml per 1.0 x 1013 vg/ml, or 1 x 105 pg/ml per 1 x 1.0 x 1013 vg/ml, or 1.7 x 106 pg/ml per 1.0 x 1013 vg/ml. In some embodiments, the residual DNA plasmid in a pharmaceutical composition is less than 10.0 x 10 5 pg by 1.0 x 10 13 vg, less than 8.0 x 10 5 pg by 1.0 x 10 13 vg or less than 6.8 x 10 5 pg by 1.0 x 10 13 vg. In some embodiments, a pharmaceutical composition comprises less than 0.5 ng per 1.0 x 1013 vg, less than 0.3 ng per 1.0 x 1013 vg, less than 0.22 ng per 1.0 x 1013 vg or less than 0.2 ng per 1.0 x 1013 vg or any intermediate concentration of bovine serum albumin (BSA). Insome embodiments, benzonase in a pharmaceutical composition is less than 0.2 ng by 1.0 x 1013 vg, less than 0.1 ng by 1.0 x 1013 vg, less than 0.09 ng by 1.0 x 1013 vg, less than 0.08 ng by 1.0 x 1013 vg or any intermediate concentration. In some embodiments, Poloxamer 188 in a pharmaceutical composition is about 10 to 150 ppm, about 15 to 100 ppm or about 20 to 80 ppm. In embodiments, cesium in a pharmaceutical composition is less than 50 pg I g (ppm), less than 30 pg / g (ppm) or less than 20 pg / g (ppm) or any intermediate concentration.
In some embodiments, a pharmaceutical composition comprises total impurities, e.g., as determined by SDS-PAGE, of less than 10%, less than 8%, less than 7%, less than 6%, less than 5%, less than 4%, less than 3%, less than 2%, or any percentage in between. In some embodiments, the total purity, e.g., as determined by SDS-PAGE, is greater than 90%, greater than 92%, greater than 93%, greater than 94%, greater than 95%, greater than 96%, greater than 97%, greater than 98%, or any percentage in between. In some embodiments, no single unnamed related impurity, e.g., as measured by SDS-PAGE, is greater than 5%, greater than 4%, greater than 3% or greater than 2%, or any percentage in between. In embodiments, the pharmaceutical composition comprises a percentage of filled capsids relative to total capsids (e.g., peak 1 + peak 2 as measured by analytical ultracentrifugation) of greater than 85%, greater than 86%, greater than 87%, greater than 88%, greater than 89%, greater than 90%, greater than 91%, greater than 91.9%, greater than 92%, greater than 93%, or any percentage in between. In some embodiments of the pharmaceutical composition, the percentage of filled capsids measured in peak 1 by analytical ultracentrifugation is 20-80%, 25-75%, 30-75%, 35-75%, or 37.4-70.3%. In embodiments of the pharmaceutical composition, the percentage of filled capsids measured in peak 2 by analytical ultracentrifugation is 20-80%, 20-70%, 22-65%, 24-62%, or 24.9-60.1%.
In some embodiments, a pharmaceutical composition comprises a genomic titer of 1.0 to 5.0 x 1013 vg / mL, 1.2 to 3.0 x 1013 vg I mL or 1.7 to 2.3 x 1013 vg I ml. In some embodiments, a pharmaceutical composition exhibits a biological load of less than 5 CFU / mL, less than 4 CFU I mL, less than 3 CFU / mL, less than 2 CFU I mL or less than 1 CFU / mL or any intermediate contraction. In some embodiments, the amount of endotoxin according to USP, for example, USP <85> (incorporated by reference in its entirety) is less than 1.0 EU I mL, less than 0.8 EU I mL or less than 0.75 EU / mL. In embodiments, the osmolarity of a pharmaceutical composition according to USP, for example, USP <785 > (incorporated by reference in its entirety) is 350 to 450 mOsm / kg, 370 to 440 mOsm / kg or 390 to 430 mOsm / kg. In some embodiments, a pharmaceutical composition contains less than 1200 particles that are greater than 25 pm per container, less than 1000 particles that are greater than 25 pm per container, less than 500 particles that are greater than 25 pm per container or any intermediate value. In embodiments, the pharmaceutical composition contains less than 10,000 particles that are greater than 10 pm per container, less than 8000 particles that are greater than 10 pm per container or less than 600 particles that arc greater than 10 pm per container.
In some embodiments, a pharmaceutical composition has a genomic titer of 0.5 to 5.0 x 10 13 vg I mL, 1.0 to 4.0 x 10 13 vg I mL, 1.5 to 3.0 x 10 13 vg / mL or 1.7 to 2.3 x 10 13 vg / mL. In some embodiments, a pharmaceutical composition described herein comprises one or more of the following: less than about 0.09 ng benzonase per 1.0 x 10 13 vg, less than about 30 pg / g (ppm ) of cesium, about 20 to 80 ppm Poloxamer 188, less than about 0.22 ng BSA per 1.0 x 10 13 vg, less than about 6.8 x 105 pg of residual DNA plasmid per 1.0 x 10 13 vg, less than about 1.1 x 10 5 pg of residual hcDNA per 1.0 x 10 13 vg, less than about 4 ng of rHCP per 1.0 x 10 13 vg, pH 7.7 to 8.3, about 390 to 430 mOsm / kg, less than about 600 particles that are > 25 pm in size per container, less than about 6000 particles that are > 10 pm in size per container, about 1.7 x 10 13 - 2.3 x 10 13 vg I mL genomic titer, infectious titer of about 3.9 x 108 to 8.4 x 1010 IU per 1.0 x 10 13 vg, total protein of about 100-300 pg per 1.0 x 10 13 vg, mean survival of >24 days in A7SMA mice with about 7.5 x 10 13 vg / kg dose of viral vector, about 70 to 130% relative potency based on an in vitro cell based assay and / or less than about 5% empty capsid. In some embodiments, a pharmaceutical composition described herein comprises any of the viral particles discussed here, retain a potency of between ± 20%, between ± 15%, between ± 10% or within ± 5% of a reference standard. In some embodiments, potency is measured using a suitable in vitro cell assay or in vivo animal model.
Additional methods of preparation, characterization, and dosing AAV particles are taught in WO2019094253, which is incorporated herein by reference in its entirety.
Additional rAAV constructs that can be employed consonant with the invention include those described in Wang et al 2019, available at: //doi.org/10.1038/s41573-019-0012-9, including Table 1 thereof, which is incorporated by reference in its entirety.
Lipid Nanoparticles
Methods and systems provided herein may employ any suitable carrier or delivery modality, including, in certain embodiments, lipid nanoparticles (LNPs). Lipid nanoparticles, in some embodiments, comprise one or more ionic lipids, such as non-cationic lipids (e.g., neutral or anionic, or zwitterionic lipids); one or more conjugated lipids (such as PEG-conjugated lipids or lipids conjugated to polymers described in Table 5 of WO2019217941; incorporated herein by reference in its entirety); one or more sterols (e.g., cholesterol); and, optionally, one or more targeting molecules (e.g., conjugated receptors, receptor ligands, antibodies); or combinations of the foregoing.
Lipids that can be used in nanoparticle formations (e.g., lipid nanoparticles) include, for example those described in Table 4 of WO2019217941, which is incorporated by reference — e.g., a lipid-containing nanoparticle can comprise one or more of the lipids in Table 4 of WO2019217941. Lipid nanoparticles can include additional elements, such as polymers, such as the polymers described in Table 5 of WO2019217941, incorporated by reference.
In some embodiments, conjugated lipids, when present, can include one or more of PEG- diacylglycerol (DAG) (such as l-(monomethoxy-polyethyleneglycol)-2,3- dimyristoylglycerol (PEG-DMG)), PEG-dialkyloxypropyl (DAA), PEG-phospholipid, PEG- ceramide (Cer), a pegylated phosphatidylethanoloamine (PEG-PE), PEG succinate diacylglycerol (PEGS-DAG) (such as 4-0-(2',3'-di(tetradecanoyloxy)propyl-l-0-(w- methoxy(polyethoxy)ethyl) butanedioate (PEG-S-DMG)), PEG dialkoxypropylcarbam, N- (carbonyl-methoxypoly ethylene glycol 2000)- 1 ,2-distearoyl-sn-glycero-3-phosphoethanolamine sodium salt, and those described in Table 2 of WO2019051289 (incorporated by reference), and combinations of the foregoing.
In some embodiments, sterols that can be incorporated into lipid nanoparticles include one or more of cholesterol or cholesterol derivatives, such as those in W02009/127060 or US2010/0130588, which are incorporated by reference. Additional exemplary sterols include phytosterols, including those described in Eygeris et al (2020), dx.doi.org/10.1021/acs.nanolett.0c01386, incorporated herein by reference.
In some embodiments, a lipid particle comprises an ionizable lipid, a non-cationic lipid, a conjugated lipid that inhibits aggregation of particles, and a sterol. The amounts of these components can be varied independently and to achieve desired properties. For example, in some embodiments, a lipid nanoparticle comprises an ionizable lipid is in an amount from about 20 mol % to about 90 mol % of the total lipids (in other embodiments it may be 20-70% (mol), 30-60% (mol) or 40-50% (mol); about 50 mol % to about 90 mol % of the total lipid present in the lipid nanoparticle), a non-cationic lipid in an amount from about 5 mol % to about 30 mol % of the total lipids, a conjugated lipid in an amount from about 0.5 mol % to about 20 mol % of the total lipids, and a sterol in an amount from about 20 mol % to about 50 mol % of the total lipids. The ratio of total lipid to nucleic acid (e.g., encoding the gene modifying polypeptide or template nucleic acid) can be varied as desired. For example, the total lipid to nucleic acid (mass or weight) ratio can be from about 10: 1 to about 30: 1.
In some embodiments, an ionizable lipid may be a cationic lipid, an ionizable cationic lipid, e.g., a cationic lipid that can exist in a positively charged or neutral form depending on pH, or an amine-containing lipid that can be readily protonated. In some embodiments, a cationic lipid is a lipid capable of being positively charged, e.g., under physiological conditions. Exemplary cationic lipids include one or more amine group(s) which bear the positive charge. In some embodiments, a lipid particle comprises a cationic lipid in formulation with one or more of neutral lipids, ionizable amine-containing lipids, biodegradable alkyn lipids, steroids, phospholipids including polyunsaturated lipids, structural lipids (e.g., sterols), PEG, cholesterol and polymer conjugated lipids. In some embodiments, a cationic lipid may be an ionizable cationic lipid. An exemplary cationic lipid as disclosed herein may have an effective pKa over 6.0. In some embodiments, a lipid nanoparticle may comprise a second cationic lipid having a different effective pKa (e.g., greater than the first effective pKa), than the first cationic lipid. A lipid nanoparticle may comprise between 40 and 60 mol percent of a cationic lipid, a neutral lipid, a steroid, a polymer conjugated lipid, and a therapeutic agent, e.g., a nucleic acid (e.g., RNA) described herein (e.g., a template nucleic acid or a nucleic acid encoding a gene modifying polypeptide), encapsulated within or associated with the lipid nanoparticle. In some embodiments, the nucleic acid is co-formulated with the cationic lipid. The nucleic acid may be adsorbed to the surface of an LNP, e.g., an LNP comprising a cationic lipid. In some embodiments, a nucleic acid may be encapsulated in an LNP, e.g., an LNP comprising a cationic lipid. In some embodiments, a lipid nanoparticle may comprise a targeting moiety, e.g., coated with a targeting agent. In some embodiments, a LNP formulation is biodegradable. In some embodiments, a lipid nanoparticle comprising one or more lipid described herein, e.g., Formula (i), (ii), (ii), (vii) and/or (ix) encapsulates at least 1%, at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 92%, at least 95%, at least 97%, at least 98% or 100% of an RNA molecule, e.g., template RNA and/or a mRNA encoding a gene modifying polypeptide.
In some embodiments, a lipid to nucleic acid ratio (mass/mass ratio; w/w ratio) can be in the range of from about 1 : 1 to about 25: 1, from about 10: 1 to about 14: 1, from about 3 : 1 to about 15: 1, from about 4: 1 to about 10: 1, from about 5: 1 to about 9: 1, or about 6: 1 to about 9: 1 . The amounts of lipids and nucleic acid can be adjusted to provide a desired N/P ratio, for example, N/P ratio of 3, 4, 5, 6, 7, 8, 9, 10 or higher. Generally, a lipid nanoparticlc formulation’s overall lipid content can range from about 5 mg/ml to about 30 mg/mL.
Exemplary ionizable lipids that can be used in lipid nanoparticle formulations include, without limitation, those listed in Table 1 of WO2019051289, incorporated herein by reference. Additional exemplary lipids include, without limitation, one or more of the following formulae: X of US2016/0311759; I of US20150376115 or in US2016/0376224; I, II or III of US20160151284; I, IA, II, or IIA of US20170210967; I-c of US20150140070; A of US2013/0178541; I of US2013/0303 87 or US2013/0123338; I of US2015/0141678; II, III, IV, or V of US2015/0239926; I of US2017/0119904; I or II of WO2017/117528; A of US2012/0149894; A of US2015/0057373; A of WO2013/116126; A of US2013/0090372; A of US2013/0274523; A of US2013/0274504; A of US2013/0053572; A of W02013/016058; A of W02012/162210; I of US2008/042973; I, II, III, or IV of US2012/01287670; I or II of US2014/0200257; I, II, or III of US2015/0203446; I or III of US2015/0005363; I, IA, IB, IC, ID, II, IIA, IIB, IIC, IID, or III-XXIV of US2014/0308304; of US2013/0338210; I, II, III, or IV of W02009/132131; A of US2012/01011478; I or XXXV of US2012/0027796; XIV or XVII of US2012/0058144; of US2013/0323269; I of US2011/0117125; I, II, or III of US2011/0256175; I, II, III, IV, V, VI, VII, VIII, IX, X, XI, XII of US2012/0202871; I, II, III, IV, V, VI, VII, VIII, X, XII, XIII, XIV, XV, or XVI of US2011/0076335; I or II of US2006/008378; I of US2013/0123338; I or X-A-Y-Z of US2015/0064242; XVI, XVII, or XVIII of US2013/0022649; I, II, or III of US2013/0116307; I, II, or III of US2013/0116307; I or II of US2010/0062967; I-X of US2013/0189351; I of US2014/0039032; V of US2018/0028664; I of US2016/0317458; I of US2013/0195920; 5, 6, or 10 of US10,221,127; III-3 of W02018/081480; 1-5 or 1-8 of W02020/081938; 18 or 25 of US9,867,888; A of US2019/0136231; II of WO2020/219876; 1 of US2012/0027803; OF-02 of US2019/0240349; 23 of US1O,O86,O13; CKK-E12/A6 of Miao et al (2020); C12-200 of W02010/053572; 7C1 of Dahlman et al (2017); 304-013 or 503-013 of Whitehead et al; TS-P4C2 of US9,708,628; I of W02020/ 106946; I of W02020/106946.
In some embodiments, an ionizable lipid is MC3 (6Z,9Z,28Z,3 lZ)-heptatriaconta- 6,9,28,3 l-tetraen-19-yl-4-(dimethylamino) butanoate (DLin-MC3-DMA or MC3), e.g., as described in Example 9 of WO2019051289A9 (incorporated by reference herein in its entirety). In some embodiments, an ionizable lipid is the lipid ATX-002, e.g., as described in Example 10 of WO2019051289A9 (incorporated by reference herein in its entirety). In some embodiments, an ionizable lipid is (13Z,16Z)-A,A-dimethyl-3- nonyldocosa-13, 16-dien-l-amine (Compound 32), e.g., as described in Example 11 of WO2019051289A9 (incorporated by reference herein in its entirety). In some embodiments, an ionizable lipid is Compound 6 or Compound 22, e.g., as described in Example 12 of WO2019051289A9 (incorporated by reference herein in its entirety). In some embodiments, an ionizable lipid is heptadecan-9-yl 8-((2-hydroxyethyl)(6-oxo-6- ( undecyloxy )hexyl)amino)octanoate (SM-102); e.g., as described in Example 1 of US9,867,888(incorporated by reference herein in its entirety). In some embodiments, an ionizable lipid is 9Z,12Z)-3-((4,4-bis(octyloxy)butanoyl)oxy)-2-((((3- (diethylamino)propoxy)carbonyl)oxy)methyl)propyl octadeca-9,12-dienoate (LP01) e.g., as synthesized in Example 13 of W02015/095340(incorporated by reference herein in its entirety). In some embodiments, an ionizable lipid is Di((Z)-non-2-en-l-yl) 9-((4- dimethylamino)butanoyl)oxy)heptadecanedioate (L319), e.g. as synthesized in Example 7, 8, or 9 of US2012/0027803(incorporated by reference herein in its entirety). In some embodiments, an ionizable lipid is l,l'-((2-(4-(2-((2-(Bis(2-hydroxydodecyl)amino)ethyl)(2-hydroxydodecyl) amino)ethyl)piperazin-l-yl)ethyl)azanediyl)bis(dodecan-2-ol) (C12-200), e.g., as synthesized in Examples 14 and 16 of W02010/053572(incorporated by reference herein in its entirety). In some embodiments, an ionizable lipid is; Imidazole cholesterol ester (ICE) lipid (3S, 10R, 13R, 17R)-10, 13-dimethyl-17- ((R)-6-methylheptan-2-yl)-2, 3, 4, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17-tetradecahydro-lH- cyclopenta[a]phenanthren-3-yl 3-(lH-imidazol-4-yl)propanoate, e.g., Structure (I) from W02020/ 106946 (incorporated by reference herein in its entirety).
Some non-limiting examples of lipid compounds that may be used (e.g., in combination with other lipid components) to form lipid nanoparticles for the delivery of compositions described herein, e.g., nucleic acid (e.g., RNA) described herein (e.g., a template nucleic acid or a nucleic acid encoding a gene modifying polypeptide) includes, In some embodiments an LNP comprising Formula (i) is used to deliver a gene modifying composition described herein to the liver and/or hepatocyte cells.
In some embodiments an LNP comprising Formula (ii) is used to deliver a gene modifying composition described herein to the liver and/or hepatocyte cells.
In some embodiments an LNP comprising Formula (iii) is used to deliver a gene modifying composition described herein to the liver and/or hepatocyte cells.
In some embodiments an LNP comprising Formula (v) is used to deliver a gene modifying composition described herein to the liver and/or hepatocyte cells.
In some embodiments an LNP comprising Formula (vi) is used to deliver a gene modifying composition described herein to the liver and/or hepatocyte cells.
In some embodiments an LNP comprising Formula (viii) is used to deliver a gene modifying composition described herein to the liver and/or hepatocyte cells.
In some embodiments an LNP comprising Formula (ix) is used to deliver a gene modifying composition described herein to the liver and/or hepatocyte cells.
wherein
In some embodiments an LNP comprising Formula (xii) is used to deliver a gene modifying composition described herein to the liver and/or hepatocyte cells.
In some embodiments an LNP comprising Formula (xi) is used to deliver a gene modifying composition described herein to the liver and/or hepatocyte cells.
(xiv)
In some embodiments an LNP comprises a compound of Formula (xiii) and a compound of Formula (xiv). In some embodiments an LNP comprising Formula (xv) is used to deliver a gene modifying composition described herein to the liver and/or hepatocyte cells. In some embodiments an LNP comprising a formulation of Formula (xvi) is used to deliver a gene modifying composition described herein to the lung endothelial cells.
(xviii) (a) (xviii)(b)
In some embodiments, a lipid compound used to form lipid nanoparticles for the delivery of compositions described herein, e.g., nucleic acid (e.g., RNA) described herein (e.g., a template nucleic acid or a nucleic acid encoding a gene modifying polypeptide) is made by one of the following reactions:
5
Exemplary non-cationic lipids include, but are not limited to, distearoyl-sn-glycero- phosphoethanolamine, distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG), dioleoyl-phosphatidylethanolamine (DOPE), 1,2- dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), palmitoyloleoylphosphatidylcholine (POPC), palmitoyloleoylphosphatidylethanolamine (POPE), dioleoyl-phosphatidylethanolamine 4-(N-maleimidomethyl)-cyclohexane- 1 - carboxylate (DOPE-mal), dipalmitoyl phosphatidyl ethanolamine (DPPE), dimyristoylphosphoethanolamine (DMPE), distearoyl-phosphatidyl- ethanolamine (DSPE), monomethyl-phosphatidylethanolamine (such as 16-0-monomethyl PE), dimethyl- phosphatidylethanolamine (such as 16-O-dimethyl PE), 18-1-trans PE, l-stearoyl-2- oleoyl- phosphatidyethanolamine (SOPE), hydrogenated soy phosphatidylcholine (HSPC), egg phosphatidylcholine (EPC), dioleoylphosphatidylserine (DOPS), sphingomyelin (SM), dimyristoyl phosphatidylcholine (DMPC), dimyristoyl phosphatidylglycerol (DMPG), di stearoylphosphatidylglycerol (DSPG), dierucoylphosphatidylcholine (DEPC), palmitoylolcyolphosphatidylglyccrol (POPG), diclaidoyl-phosphatidylcthanolaminc (DEPE), lecithin, phosphatidylethanolamine, lysolecithin, lysophosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, sphingomyelin, egg sphingomyelin (ESM), cephalin, cardiolipin, phosphatidicacid, cerebrosides, dicetylphosphate, lysophosphatidylcholine, dilinoleoylphosphatidylcholine, or mixtures thereof. It is understood that other diacylphosphatidylcholine and diacylphosphatidylethanolamine phospholipids can also be used. The acyl groups in these lipids are preferably acyl groups derived from fatty acids having C10- C24 carbon chains, e.g., lauroyl, myristoyl, paimitoyl, stearoyl, or oleoyl. Additional exemplary lipids, in certain embodiments, include, without limitation, those described in Kim et al. (2020) dx.doi.org/10.1021/acs.nanolett.0c01386, incorporated herein by reference. Such lipids include, in some embodiments, plant lipids found to improve liver transfection with mRNA (e.g., DGTS). In some embodiments, the non-cationic lipid may have the following structure, (xxi )
Other examples of non-cationic lipids suitable for use in the lipid nanopartieles include, without limitation, nonphosphorous lipids such as, e.g., stearylamine, dodeeylamine, hexadecylamine, acetyl palmitate, glycerol ricinoleate, hexadecyl stereate, isopropyl myristate, amphoteric acrylic polymers, triethanolamine-lauryl sulfate, alkyl-aryl sulfate polyethyloxylated fatty acid amides, dioctadecyl dimethyl ammonium bromide, ceramide, sphingomyelin, and the like. Other non-cationic lipids are described in WO2017/099823 or US patent publication US2018/0028664, the contents of which is incorporated herein by reference in their entirety.
In some embodiments, a non-cationic lipid is oleic acid or a compound of Formula I, II, or IV of US2018/0028664, incorporated herein by reference in its entirety. A non-cationic lipid can comprise, for example, 0-30% (mol) of the total lipid present in the lipid nanoparticle. In some embodiments, non-cationic lipid content is 5-20% (mol) or 10-15% (mol) of the total lipid present in a lipid nanoparticle. In some embodiments, the molar ratio of ionizable lipid to neutral lipid ranges from about 2:1 to about 8:1 (e.g., about 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, or 8:1).
In some embodiments, lipid nanoparticles do not comprise any phospholipids. In some embodiments, a lipid nanoparticle can further comprise a component, such as a sterol, to provide membrane integrity. One exemplary sterol that can be used in the lipid nanoparticle is cholesterol and derivatives thereof. Non-limiting examples of cholesterol derivatives include polar analogues such as 5a-choiestanol, 53-coprostanol, choiesteryl-(2’- hydroxy)-ethyl ether, choiesteryl-(4'- hydroxy)-butyl ether, and 6-ketocholestanol; non-polar analogues such as 5a-cholestane, cholestenone, 5a-cholestanone, 5p-cholestanone, and cholesteryl decanoate; and mixtures thereof. In some embodiments, a cholesterol derivative is a polar analogue, e.g., choiesteryl-(4'-hydroxy)-butyl ether. Exemplary cholesterol derivatives are described in PCT publication W02009/127060 and US patent publication US2010/0130588, each of which is incorporated herein by reference in its entirety.
In some embodiments, a component providing membrane integrity, such as a sterol, can comprise 0-50% (mol) (e.g., 0-10%, 10-20%, 20-30%, 30-40%, or 40-50%) of the total lipid present in a lipid nanoparticle. In some embodiments, such a component is 20-50% (mol) 30- 40% (mol) of the total lipid content of the lipid nanoparticle.
In some embodiments, a lipid nanoparticle can comprise a polyethylene glycol (PEG) or a conjugated lipid molecule. Generally, these are used to inhibit aggregation of lipid nanoparticles and/or provide steric stabilization. Exemplary conjugated lipids include, but are not limited to, PEG-lipid conjugates, polyoxazoline (POZ)-lipid conjugates, polyamide-lipid conjugates (such as ATTA-lipid conjugates), cationic -polymer lipid (CPL) conjugates, and mixtures thereof. In some embodiments, the conjugated lipid molecule is a PEG-lipid conjugate, for example, a (methoxy polyethylene glycol)-conjugated lipid.
Exemplary PEG-lipid conjugates include, but are not limited to, PEG-diacylglycerol (DAG) (such as l-(monomethoxy-polyethyleneglycol)-2,3-dimyristoylglycerol (PEG-DMG)), PEG-dialkyloxypropyl (DAA), PEG-phospholipid, PEG-ceramide (Cer), a pegylated phosphatidylethanoloamine (PEG-PE), 1,2-dimyristoyl-sn-glycerol, methoxypoly ethylene glycol (DMG-PEG-2K), PEG succinate diacylglycerol (PEGS -DAG) (such as 4-0-(2’,3’- di(tetradecanoyloxy)propyl-l-0-(w-methoxy (polyethoxy )ethyl) butanedioate (PEG-S-DMG)), PEG dialkoxypropylcarbam, N-(carbonyl-methoxypolyethylene glycol 2000)-l,2-distearoyl-sn- glycero-3-phosphoethanolamine sodium salt, or a mixture thereof. Additional exemplary PEG- lipid conjugates are described, for example, in US5,885,613, US6,287,591, US2003/0077829, US2003/0077829, US2005/0175682, US2008/0020058, US201 1/0117125, US2010/0130588, US2016/0376224, US2017/0119904, and US/099823, the contents of all of which are incorporated herein by reference in their entirety. In some embodiments, a PEG-lipid is a compound of Formula III, III-a-I, III-a-2, III-b-1, III-b-2, or V of US2018/0028664, the content of which is incorporated herein by reference in its entirety. In some embodiments, a PEG-lipid is of Formula II of US20150376115 or US2016/0376224, the content of both of which is incorporated herein by reference in its entirety. In some embodiments, the PEG-DAA conjugate can be, for example, PEG-dilauryloxypropyl, PEG- dimyristyloxypropyl, PEG- dipalmityloxypropyl, or PEG-distearyloxypropyl. The PEG-lipid can be one or more of PEG- DMG, PEG-dilaurylglycerol, PEG-dipalmitoylglycerol, PEG- disterylglycerol, PEG- dilaurylglycamide, PEG-dimyristylglycamide, PEG- dipalmitoylglycamide, PEG- disterylglycamide, PEG-cholesterol (l-[8’-(Cholest-5-en-3[beta]- oxy)carboxamido-3’,6’- dioxaoctanyl] carbamoyl- [omega] -methyl-poly (ethylene glycol), PEG- DMB (3,4- Ditetradecoxylbenzyl- [omega]-methyl-poly(ethylene glycol) ether), and 1,2- dimyristoyl- sn- glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000]. In some embodiments, the PEG-lipid comprises PEG-DMG, 1,2- dimyristoyl-sn-glycero-3-phosphoethanolamine-N- [methoxy(polyethylene glycol)-2000]. In some embodiments, the PEG-lipid comprises a structure selected from:
(xxiii),
In some embodiments, lipids conjugated with a molecule other than a PEG can also be used in place of PEG-lipid. For example, polyoxazoline (POZ)-lipid conjugates, polyamide-lipid conjugates (such as ATTA-lipid conjugates), and cationic-polymer lipid (GPL) conjugates can be used in place of or in addition to the PEG-lipid.
Exemplary conjugated lipids, i.e., PEG-lipids, (POZ)-lipid conjugates, ATTA-lipid conjugates and cationic polymer-lipids are described in the PCT and LIS patent applications listed in Table 2 of WO2019051289 A9 and in W02020106946A1, the contents of all of which are incorporated herein by reference in their entirety.
In some embodiments, an LNP comprises a compound of Formula (xix), a compound of Formula (xxi) and a compound of Formula (xxv). In some embodiments, an LNP comprising a formulation of Formula (xix), Formula (xxi) and Formula (xxv)is used to deliver a gene modifying composition described herein to the lung or pulmonary cells.
In some embodiments, a lipid nanoparticle may comprise one or more cationic lipids selected from Formula (i), Formula (ii), Formula (iii), Formula (vii), and Formula (ix). In some embodiments, the LNP may further comprise one or more neutral lipid, e.g., DSPC, DPPC, DMPC, DOPC, POPC, DOPE, SM, a steroid, e.g., cholesterol, and/or one or more polymer conjugated lipid, e.g., a pegylated lipid, e.g., PEG-DAG, PEG-PE, PEG-S-DAG, PEG-cer or a PEG dialkyoxypropylcarbamatc.
In some embodiments, PEG or conjugated lipid can comprise 0-20% (mol) of the total lipid present in a lipid nanoparticle. In some embodiments, PEG or conjugated lipid content is 0.5- 10% or 2-5% (mol) of the total lipid present in a lipid nanoparticle. Molar ratios of the ionizable lipid, non-cationic-lipid, sterol, and PEG/conjugated lipid can be varied as needed. For example, a lipid particle can comprise 30-70% ionizable lipid by mole or by total weight of the composition, 0-60% cholesterol by mole or by total weight of the composition, 0-30% non- cationic-lipid by mole or by total weight of the composition and 1-10% conjugated lipid by mole or by total weight of the composition. Preferably, a composition comprises 30-40% ionizable lipid by mole or by total weight of the composition, 40-50% cholesterol by mole or by total weight of the composition, and 10- 20% non-cationic-lipid by mole or by total weight of the composition. In some embodiments, a composition is 50-75% ionizable lipid by mole or by total weight of the composition, 20-40% cholesterol by mole or by total weight of the composition, and 5 to 10% non-cationic-lipid, by mole or by total weight of the composition and 1-10% conjugated lipid by mole or by total weight of the composition. The composition may contain 60-70% ionizable lipid by mole or by total weight of the composition, 25-35% cholesterol by mole or by total weight of the composition, and 5-10% non-cationic-lipid by mole or by total weight of the composition. The composition may also contain up to 90% ionizable lipid by mole or by total weight of the composition and 2 to 15% non-cationic lipid by mole or by total weight of the composition. The formulation may also be a lipid nanoparticle formulation, for example comprising 8-30% ionizable lipid by mole or by total weight of the composition, 5-30% noncationic lipid by mole or by total weight of the composition, and 0-20% cholesterol by mole or by total weight of the composition; 4-25% ionizable lipid by mole or by total weight of the composition, 4-25% non-cationic lipid by mole or by total weight of the composition, 2 to 25% cholesterol by mole or by total weight of the composition, 10 to 35% conjugate lipid by mole or by total weight of the composition, and 5% cholesterol by mole or by total weight of the composition; or 2-30% ionizable lipid by mole or by total weight of the composition, 2-30% non-cationic lipid by mole or by total weight of the composition, 1 to 15% cholesterol by mole or by total weight of the composition, 2 to 35% conjugate lipid by mole or by total weight of the composition, and 1-20% cholesterol by mole or by total weight of the composition; or even up to 90% ionizable lipid by mole or by total weight of the composition and 2-10% non-cationic lipids by mole or by total weight of the composition, or even 100% cationic lipid by mole or by total weight of the composition. In some embodiments, a lipid particle formulation comprises ionizable lipid, phospholipid, cholesterol and a PEG-ylated lipid in a molar ratio of 50: 10:38.5: 1 .5. In some other embodiments, the lipid particle formulation comprises ionizable lipid, cholesterol and a PEG-ylatcd lipid in a molar ratio of 60:38.5: 1.5.
In some embodiments, a lipid particle comprises ionizable lipid, non-cationic lipid (e.g. phospholipid), a sterol (e.g., cholesterol) and a PEG-ylated lipid, where the molar ratio of lipids ranges from 20 to 70 mole percent for the ionizable lipid, with a target of 40-60, the mole percent of non-cationic lipid ranges from 0 to 30, with a target of 0 to 15, the mole percent of sterol ranges from 20 to 70, with a target of 30 to 50, and the mole percent of PEG-ylated lipid ranges from 1 to 6, with a target of 2 to 5.
In some embodiments, a lipid particle comprises ionizable lipid / non-cationic- lipid I sterol / conjugated lipid at a molar ratio of 50: 10:38.5: 1.5.
In some embodiments, the disclosure provides a lipid nanoparticle formulation comprising phospholipids, lecithin, phosphatidylcholine and phosphatidylethanolamine.
In some embodiments, one or more additional compounds can also be included. Those compounds can be administered separately or the additional compounds can be included in lipid nanoparticles of the present disclosure. In other words, lipid nanoparticles can contain other compounds in addition to the nucleic acid or at least a second nucleic acid, different than the first. Without limitations, other additional compounds can be selected from the group consisting of small or large organic or inorganic molecules, monosaccharides, disaccharides, trisaccharides, oligosaccharides, polysaccharides, peptides, proteins, peptide analogs and derivatives thereof, peptidomimetics, nucleic acids, nucleic acid analogs and derivatives, an extract made from biological materials, or any combinations thereof.
In some embodiments, a lipid nanoparticle (or a formulation comprising lipid nanoparticles) lacks reactive impurities (e.g., aldehydes or ketones), or comprises less than a preselected level of reactive impurities (e.g., aldehydes or ketones). While not wishing to be bound by theory, in some embodiments, a lipid reagent is used to make a lipid nanoparticle formulation, and the lipid reagent may comprise a contaminating reactive impurity (e.g., an aldehyde or ketone). A lipid regent may be selected for manufacturing based on having less than a preselected level of reactive impurities (e.g., aldehydes or ketones). Without wishing to be bound by theory, in some embodiments, aldehydes can cause modification and damage of RNA, e.g., cross-linking between bases and/or covalently conjugating lipid to RNA (e.g., forming lipid- RNA adducts). This may, in some instances, lead to failure of a reverse transcriptase reaction and/or incorporation of inappropriate bases, e.g., at the site(s) of lesion(s), e.g., a mutation in a newly synthesized target DNA.
In some embodiments, a lipid nanoparticle formulation is produced using a lipid reagent comprising less than 5%, less than 4%, less than 3%, less than 2%, less than 1%, less than 0.9%, less than 0.8%, less than 0.7%, less than 0.6%, less than 0.5%, less than 0.4%, less than 0.3%, less than 0.2%, or less than 0.1% total reactive impurity (e.g., aldehyde) content. In some embodiments, a lipid nanoparticle formulation is produced using a lipid reagent comprising less than 5%, less than 4%, less than 3%, less than 2%, less than 1%, less than 0.9%, less than 0.8%, less than 0.7%, less than 0.6%, less than 0.5%, less than 0.4%, less than 0.3%, less than 0.2%, or less than 0.1% of any single reactive impurity (e.g., aldehyde) species. In some embodiments, a lipid nanoparticle formulation is produced using a lipid reagent comprising: (i) less than 5%, less than 4%, less than 3%, less than 2%, less than 1%, less than 0.9%, less than 0.8%, less than 0.7%, less than 0.6%, less than 0.5%, less than 0.4%, less than 0.3%, less than 0.2%, or less than 0.1% total reactive impurity (e.g., aldehyde) content; and (ii) less than 5%, less than 4%, less than 3%, less than 2%, less than 1%, less than 0.9%, less than 0.8%, less than 0.7%, less than 0.6%, less than 0.5%, less than 0.4%, less than 0.3%, less than 0.2%, or less than 0.1% of any single reactive impurity (e.g., aldehyde) species. In some embodiments, the lipid nanoparticle formulation is produced using a plurality of lipid reagents, and each lipid reagent of the plurality independently meets one or more criterion described in this paragraph. In some embodiments, each lipid reagent of the plurality meets the same criterion, e.g., a criterion of this paragraph.
In some embodiments, a lipid nanoparticle formulation comprises less than 5%, less than 4%, less than 3%, less than 2%, less than 1%, less than 0.9%, less than 0.8%, less than 0.7%, less than 0.6%, less than 0.5%, less than 0.4%, less than 0.3%, less than 0.2%, or less than 0.1% total reactive impurity (e.g., aldehyde) content. In some embodiments, a lipid nanoparticle formulation comprises less than 5%, less than 4%, less than 3%, less than 2%, less than 1%, less than 0.9%, less than 0.8%, less than 0.7%, less than 0.6%, less than 0.5%, less than 0.4%, less than 0.3%, less than 0.2%, or less than 0.1% of any single reactive impurity (e.g., aldehyde) species. In some embodiments, a lipid nanoparticle formulation comprises: (i) less than 5%, less than 4%, less than 3%, less than 2%, less than 1%, less than 0.9%, less than 0.8%, less than 0.7%, less than 0.6%, less than 0.5%, less than 0.4%, less than 0.3%, less than 0.2%, or less than 0.1% total reactive impurity (e.g., aldehyde) content; and (ii) less than 5%, less than 4%, less than 3%, less than 2%, less than 1 %, less than 0.9%, less than 0.8%, less than 0.7%, less than 0.6%, less than 0.5%, less than 0.4%, less than 0.3%, less than 0.2%, or less than 0.1% of any single reactive impurity (e.g., aldehyde) species.
In some embodiments, one or more, or optionally all, of the lipid reagents used for a lipid nanoparticle as described herein or a formulation thereof comprise less than 5%, less than 4%, less than 3%, less than 2%, less than 1%, less than 0.9%, less than 0.8%, less than 0.7%, less than 0.6%, less than 0.5%, less than 0.4%, less than 0.3%, less than 0.2%, or less than 0.1% total reactive impurity (e.g., aldehyde) content. In some embodiments, one or more, or optionally all, of the lipid reagents used for a lipid nanoparticle as described herein or a formulation thereof comprise less than 5%, less than 4%, less than 3%, less than 2%, less than 1%, less than 0.9%, less than 0.8%, less than 0.7%, less than 0.6%, less than 0.5%, less than 0.4%, less than 0.3%, less than 0.2%, or less than 0.1% of any single reactive impurity (e.g., aldehyde) species. In some embodiments, one or more, or optionally all, of the lipid reagents used for a lipid nanoparticle as described herein or a formulation thereof comprise: (i) less than 5%, less than 4%, less than 3%, less than 2%, less than 1%, less than 0.9%, less than 0.8%, less than 0.7%, less than 0.6%, less than 0.5%, less than 0.4%, less than 0.3%, less than 0.2%, or less than 0.1% total reactive impurity (e.g., aldehyde) content; and (ii) less than 5%, less than 4%, less than 3%, less than 2%, less than 1%, less than 0.9%, less than 0.8%, less than 0.7%, less than 0.6%, less than 0.5%, less than 0.4%, less than 0.3%, less than 0.2%, or less than 0.1% of any single reactive impurity (e.g., aldehyde) species.
In some embodiments, total aldehyde content and/or quantity of any single reactive impurity (e.g., aldehyde) species is determined by liquid chromatography (LC), e.g., coupled with tandem mass spectrometry (MS/MS), e.g., according to the method described in Example 40 of PCT/US21/20948. In some embodiments, reactive impurity (e.g., aldehyde) content and/or quantity of reactive impurity (e.g., aldehyde) species is determined by detecting one or more chemical modifications of a nucleic acid molecule (e.g., an RNA molecule, e.g., as described herein) associated with the presence of reactive impurities (e.g., aldehydes), e.g., in the lipid reagents. In some embodiments, reactive impurity (e.g., aldehyde) content and/or quantity of reactive impurity (e.g., aldehyde) species is determined by detecting one or more chemical modifications of a nucleotide or nucleoside (e.g., a ribonucleotide or ribonucleoside, e.g., comprised in or isolated from a template nucleic acid, e.g., as described herein) associated with the presence of reactive impurities (e.g., aldehydes), e.g., in the lipid reagents, e.g., according to the method described in Example 41 of PCT/US21/20948. In embodiments, chemical modifications of a nucleic acid molecule, nucleotide, or nucleoside are detected by determining the presence of one or more modified nucleotides or nucleosides, e.g., using LC-MS/MS analysis, e.g., according to the method described in Example 41 of PCT/US21/20948.
In some embodiments, a nucleic acid (e.g., RNA) described herein (e.g., a template nucleic acid or a nucleic acid encoding a gene modifying polypeptide) does not comprise an aldehyde modification, or comprises less than a preselected amount of aldehyde modifications. In some embodiments, on average, a nucleic acid has less than 50, less than 20, less than 10, less than 5, less than 2, or less than 1 aldehyde modifications per 1000 nucleotides, e.g., wherein a single cross-linking of two nucleotides is a single aldehyde modification. In some embodiments, the aldehyde modification is an RNA adduct (e.g., a lipid-RNA adduct). In some embodiments, the aldehyde-modified nucleotide is cross-linking between bases. In some embodiments, a nucleic acid (e.g., RNA) described herein comprises less than 50, less than 20, less than 10, less than 5, less than 2, or less than 1 nucleotide cross-links.
In some embodiments, LNPs are directed to specific tissues by the addition of targeting domains. For example, biological ligands may be displayed on the surface of LNPs to enhance interaction with cells displaying cognate receptors, thus driving association with and cargo delivery to tissues wherein cells express the receptor. In some embodiments, a biological ligand may be a ligand that drives delivery to the liver, e.g., LNPs that display GalNAc result in delivery of nucleic acid cargo to hepatocytes that display asialoglycoprotein receptor (ASGPR). The work of Akinc et al. Mol Ther 18(7): 1357-1364 (2010) teaches the conjugation of a trivalent GalNAc ligand to a PEG-lipid (GalNAc-PEG-DSG) to yield LNPs dependent on ASGPR for observable LNP cargo effect (see, e.g., Figure 6 therein). Other ligand-displaying LNP formulations, e.g., incorporating folate, transferrin, or antibodies, are discussed in WO2017223135, which is incorporated herein by reference in its entirety, in addition to the references used therein, namely Kolhatkar et al., Curr Drug Discov Technol. 2011 8:197-206; Musacchio and Torchilin, Front Biosci. 2011 16:1388-1412; Yu et al., Mol Membr Biol. 2010 27:286-298; Patil et al., Crit Rev Ther Drug Carrier Syst. 2008 25:1-61; Benoit et al., Biomacromolecules. 2011 12:2708-2714; Zhao et al., Expert Opin Drug Deliv. 2008 5:309-319; Akinc et al., Mol Ther. 2010 18:1357-1364; Srinivasan et al., Methods Mol Biol. 2012 820:105- 116; Ben-Arie et al., Methods Mol Biol. 2012757:497-507; Peer 2010 J Control Release. 20:63- 68; Peer ct al., Proc Natl Acad Sci U S A. 2007 104:4095-4100; Kim ct al., Methods Mol Biol. 2011 721:339-353; Subramanya et al., Mol Ther. 2010 18:2028-2037; Song et al., Nat Biotechnol. 2005 23:709-717; Peer et al., Science. 2008 319:627-630; and Peer and Lieberman, Gene Ther. 2011 18:1127-1133.
In some embodiments, LNPs are selected for tissue- specific activity by the addition of a Selective ORgan Targeting (SORT) molecule to a formulation comprising traditional components, such as ionizable cationic lipids, amphipathic phospholipids, cholesterol and poly(ethylene glycol) (PEG) lipids. The teachings of Cheng et al. Nat Nanotechnol 15(4):313- 320 (2020) demonstrate that the addition of a supplemental “SORT” component precisely alters the in vivo RNA delivery profile and mediates tissue-specific (e.g., lungs, liver, spleen) gene delivery and editing as a function of the percentage and biophysical property of the SORT molecule.
In some embodiments, LNPs comprise biodegradable, ionizable lipids. In some embodiments, LNPs comprise (9Z,12Z)-3-((4,4-bis(octyloxy)butanoyl)oxy)-2-((((3- (diethylamino)propoxy)carbonyl)oxy)methyl)propyl octadeca-9,12-dienoate, also called 3- ((4,4- bis(octyloxy)butanoyl)oxy)-2-((((3-(diethylamino)propoxy)carbonyl)oxy)methyl)propyl (9Z,12Z)-octadeca-9,12-dienoate) or another ionizable lipid. See, e.g, lipids of WO2019/067992, WO/2017/173054, WO2015/095340, and WO2014/136086, as well as references provided therein. In some embodiments, the term cationic and ionizable in the context of LNP lipids is interchangeable, e.g., wherein ionizable lipids are cationic depending on the pH.
In some embodiments, an LNP described herein comprises a lipid described in Table 39.
Table 38: Exemplary Lipids
In some embodiments, multiple components of a gene modifying system may be prepared as a single LNP formulation, e.g., an LNP formulation comprises mRNA encoding for the gene modifying polypeptide and an RNA template. Ratios of nucleic acid components may be varied in order to maximize the properties of a therapeutic. In some embodiments, the ratio of RNA template to mRNA encoding a gene modifying polypeptide is about 1:1 to 100:1, e.g., about 1 : 1 to 20: 1 , about 20: 1 to 40: 1 , about 40: 1 to 60: 1 , about 60: 1 to 80: 1 , or about 80:1 to 100:1, by molar ratio. In other embodiments, a system of multiple nucleic acids may be prepared by separate formulations, e.g., one LNP formulation comprising a template RNA and a second LNP formulation comprising an mRNA encoding a gene modifying polypeptide. In some embodiments, a system may comprise more than two nucleic acid components formulated into LNPs. In some embodiments, a system may comprise a protein, e.g., a gene modifying polypeptide, and a template RNA formulated into at least one LNP formulation.
In some embodiments, the average LNP diameter of an LNP formulation may be between 10s of nm and 100s of nm, e.g., measured by dynamic light scattering (DLS). In some embodiments, the average LNP diameter of the LNP formulation may be from about 40 nm to about 150 nm, such as about 40 nm, about 45 nm, about 50 nm, about 55 nm, about 60 nm, about 65 nm, about 70 nm, about 75 nm, about 80 nm, about 85 nm, about 90 nm, about 95 nm, about 100 nm, about 105 nm, about 110 nm, about 115 nm, about 120 nm, about 125 nm, about 130 nm, about 135 nm, about 140 nm, about 145 nm, or about 150 nm. In some embodiments, the average LNP diameter of an LNP formulation may be from about 50 nm to about 100 nm, from about 50 nm to about 90 nm, from about 50 nm to about 80 nm, from about 50 nm to about 70 nm, from about 50 nm to about 60 nm, from about 60 nm to about 100 nm, from about 60 nm to about 90 nm, from about 60 nm to about 80 nm, from about 60 nm to about 70 nm, from about 70 nm to about 100 nm, from about 70 nm to about 90 nm, from about 70 nm to about 80 nm, from about 80 nm to about 100 nm, from about 80 nm to about 90 nm, or from about 90 nm to about 100 nm. In some embodiments, the average LNP diameter of an LNP formulation may be from about 70 nm to about 100 nm. In a particular embodiment, the average LNP diameter of an LNP formulation may be about 80 nm. In some embodiments, the average LNP diameter of an LNP formulation may be about 100 nm. Tn some embodiments, the average LNP diameter of an LNP formulation ranges from about 1 mm to about 500 mm, from about 5 mm to about 200 mm, from about 10 mm to about 100 mm, from about 20 mm to about 80 mm, from about 25 mm to about 60 mm, from about 30 mm to about 55 mm, from about 35 mm to about 50 mm, or from about 38 mm to about 42 mm.
An LNP may, in some embodiments, be relatively homogenous. A polydispersity index may be used to indicate the homogeneity of an LNP, e.g., the particle size distribution of the lipid nanoparticles. A small (e.g., less than 0.3) polydispersity index generally indicates a narrow particle size distribution. An LNP may have a polydispersity index from about 0 to about 0.25, such as 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.10, 0.11, 0.12, 0.13, 0.14, 0.15, 0.16, 0.17, 0.18, 0.19, 0.20, 0.21, 0.22, 0.23, 0.24, or 0.25. In some embodiments, the poly dispersity index of an LNP may be from about 0.10 to about 0.20.
The zeta potential of an LNP may be used to indicate the electrokinetic potential of the composition. In some embodiments, the zeta potential may describe the surface charge of an LNP. Lipid nanoparticles with relatively low charges, positive or negative, are generally desirable, as more highly charged species may interact undesirably with cells, tissues, and other elements in the body. In some embodiments, the zeta potential of an LNP may be from about -10 mV to about +20 mV, from about -10 mV to about +15 mV, from about -10 mV to about +10 mV, from about -10 mV to about +5 mV, from about -10 mV to about 0 mV, from about -10 mV to about -5 mV, from about -5 mV to about +20 mV, from about -5 mV to about +15 mV, from about -5 mV to about +10 mV, from about -5 mV to about +5 mV, from about -5 mV to about 0 mV, from about 0 mV to about +20 mV, from about 0 mV to about +15 mV, from about 0 mV to about +10 mV, from about 0 mV to about +5 mV, from about +5 mV to about +20 mV, from about +5 mV to about +15 mV, or from about +5 mV to about +10 mV.
The efficiency of encapsulation of a protein and/or nucleic acid, e.g., gene modifying polypeptide or mRNA encoding the polypeptide, describes the amount of protein and/or nucleic acid that is encapsulated or otherwise associated with an LNP after preparation, relative to the initial amount provided. The encapsulation efficiency is desirably high (e.g., close to 100%). The encapsulation efficiency may be measured, for example, by comparing the amount of protein or nucleic acid in a solution containing the lipid nanoparticle before and after breaking up the lipid nanoparticle with one or more organic solvents or detergents. An anion exchange resin may be used to measure the amount of free protein or nucleic acid (e.g., RNA) in a solution. Fluorescence may be used to measure the amount of free protein and/or nucleic acid (e.g., RNA) in a solution. For the lipid nanoparticles described herein, the encapsulation efficiency of a protein and/or nucleic acid may be at least 50%, for example 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%. In some embodiments, the encapsulation efficiency may be at least 80%. In some embodiments, the encapsulation efficiency may be at least 90%. In some embodiments, the encapsulation efficiency may be at least 95%.
An LNP may optionally comprise one or more coatings. In some embodiments, an LNP may be formulated in a capsule, film, or table having a coating. A capsule, film, or tablet including a composition described herein may have any useful size, tensile strength, hardness or density.
Additional exemplary lipids, formulations, methods, and characterization of LNPs are taught by W02020061457, which is incorporated herein by reference in its entirety.
In some embodiments, in vitro or ex vivo cell lipofections are performed using Lipofectamine MessengerMax (Thermo Fisher) or TransIT-mRNA Transfection Reagent (Mirus Bio). In some embodiments, LNPs are formulated using the GenVoy_ILM ionizable lipid mix (Precision NanoSystems). In some embodiments, LNPs are formulated using 2,2-dilinoleyl-4- dimethylaminoethyl-[l,3]-dioxolane (DLin-KC2-DMA) or dilinoleylmethyl-4- dimethylaminobutyrate (DLin-MC3-DMA or MC3), the formulation and in vivo use of which are taught in layaraman et al. Angew Chem Int Ed Engl 51(34):8529-8533 (2012), incorporated herein by reference in its entirety.
LNP formulations optimized for the delivery of CRISPR-Cas systems, e.g., Cas9-gRNA RNP, gRNA, Cas9 mRNA, are described in WO2019067992 and W02019067910, both incorporated by reference.
Additional specific LNP formulations useful for delivery of nucleic acids are described in US8158601 and US8168775, both incorporated by reference, which include formulations used in patisiran, sold under the name ONPATTRO.
Exemplary dosing of gene modifying LNP may include about 0.1, about 0.25, about 0.3, about 0.5, about 1, about 2, about 3, about 4, about 5, about 6, about 8, about 10, or about 100 mg/kg (RNA). Exemplary dosing of AAV comprising a nucleic acid encoding one or more components of the system may include an MOT of about 1011, about 1012, about 1013, and about 1014 vg/kg.
An mRNA encoding a gene modifying polypeptide may have a cap, 5' UTR containing a Kozak, 3' UTR, and polyA tail containing at least 60 As. In some embodiments, the polyA tail does not comprise any nucleotides other than As. In some embodiments, the polyA tail comprises primarily As, and also comprises one or more Us. An mRNA encoding a gene modifying polypeptide may have a reduced Uridine content through codon selection/optimization. An mRNA encoding a gene modifying polypeptide may have uridines that are about 1%, about 2%, about 5%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or about 100% substituted with 5-methoxy uridine. An mRNA encoding a gene modifying polypeptide may have uridines that are about 1%, about 2%, about 5%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or about 100% substituted with Nl-methyl- pseudouridine. An mRNA encoding a gene modifying polypeptide may have cytosines in the mRNA are about 1%, about 2%, about 5%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or about 100% substituted with 5- methylcytosine. An mRNA encoding a gene modifying polypeptide may have a combination of about 1%, about 2%, about 5%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or about 100% substitution of cytosine with 5- methylcytosine and about 1%, about 2%, about 5%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or about 100% substitution of uridine with 5-methoxy uridine. An mRNA encoding a gene modifying polypeptide may have a combination of about 1%, about 2%, about 5%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or about 100% substitution of cytosine with 5-methylcytosine and about 1%, about 2%, about 5%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or about 100% substitution of uridine with Nl-methyl-pseudouridine.
A guide RNA may be synthesized by T7 RNA polymerase. A guide RNA may be chemically synthesized and contain modifications such as, e.g., 2'-O-methyl, 2'-Fluoro, and/or phosphorothioate. The 3 most terminal nucleotides of a guide RNA may contain 2'-O-methyl modifications with 3 phosphorothioate linkages between the nucleotides. A guide RNA may contain 2'-(9-methyl modified nucleotides where there are cytosines and uridines, except at nucleotides found in the “seed” of the guide RNA where cytosines and uridines contain 2'-fluoro modifications.
A gene modifying mRNA and a guide RNA may be co-formulated in an LNP as described herein. They may be separately formulated. They may be combined prior to injection. They may be combined at a molar ratio in the range of about 1:10 to 1:250 mRNA:gRNA. They may be formulated in a molar ratio of about 1:20, about 1:30, about 1:40, about 1:50, about 1:60, about 1:70, about 1:80, about 1:90, about 1:100, about 1:110, about 1:120, about 1:130, about 1:140, about 1:150, about 1:160, about 1:170, about 1:180, about 1:190, about 1:200, about 1:210, about 1:220, about 1:230, about 1:240, or about 1:250 mRNA:gRNA. A gene modifying mRNA and guide RNA may be injected 30-180 minutes apart where the mRNA LNPs are delivered first followed by the guide RNA LNPs. They may be delivered about 30, about 40, about 50, about 60, about 70, about 80, about 90, about 100, about 110, about 120, about 130, about 140, about 150, about 160, about 170, or about 180 minutes apart. A gene modfiyng mRNA and/or gRNA may be dosed at 0.01 - 6 mg/kg either separately or together as a total amount of RNA-LNP. RNA-LNPs may be injected as an intravenous (IV) bolus. RNA-LNPs may be infused over a period of 30-360 minutes. RNA-LNPs may be infused over a period of about 30, about 60, about 90, about 120, about 150, about 180, about 210, about 240, about 270, about 300, about 330 or about 360 minutes.
Exemplary Gene Modifying Systems for Correcting an E342K Mutation
In some embodiments, compositions of a gene modifying system used to correct the E342K mutation in the PiZ model, as described herein, are modified as follows to optimize efficiency and precision of editing.
Gene modifying olypeptide-encoding mRNA.
In some embodiments, a gene modifying polypeptide comprises the bipartite SV40 NLS sequences (doi: 10.1074/jbc.M601718200) at its N-terminus and C-terminus. In some embodiments, a gene modifying system construct contains modified c-myc NLS and bipartite SV40 NLS at its N-terminus and at the C-terminus a modified bipartite SV40 NLS followed by a SV40 NLS is linked to the reverse transcriptase through a SGGS linker. In some embodiments, a linker between each NLS and the NLS and a fusion protein is a SGGS linker. In some embodiments, a 32 amino acid linker of a fusion protein encoded by an mRNA is: SGGSSGGSSGSETPGTSESATPESSGGSSGGSS (SEQ ID NO: 19525).
In some embodiments, a catalytic mutation of the Cas9 domain to generate Cas9 nickase activity is H840A or N863A. In some embodiments, an mRNA has a cap, 5' UTR containing a Kozak sequence, 3' UTR, and a polyA tail containing at least 60 As. In some embodiments, an mRNA has reduced uridine content through codon selection/optimization. In some embodiments, uridines in an mRNA are 100% substituted with 5-methoxy uridine. In some embodiments, uridines in an mRNA are 100% substituted with Nl-methyl-pseudouridine. In some embodiments, cytosines in an mRNA are 100% substituted with 5-methylcytosine. In some embodiments, mRNA contains a combination of 100% substitution of cytosine with 5- methylcytosine and 100% substitution of uridine with 5-methoxy uridine. In some embodiments, mRNA contains a combination of 100% substitution of cytosine with 5-methylcytosine and 100% substitution of uridine with Nl-methyl-pseudouridine. In some embodiments, combinations of modifications described above include 0-100% substitution of unmodified nucleotides, e.g., 0-10%, 10-20%, 20-30%, 30-40%, 40-50%, 50-60%, 60-70%, 70-80%, 80- 90%, or less than 90-100% substitution. In some embodiments, a gene modifying polypeptide encoded by an mRNA of the system comprises the sequence: c-Myc NLS-BPSV40 NLS-SpCas9H840A-linker-M-MLV reverse transcriptase-
SGGS linker-BPSV40 NLS-SV40
Template RNA and optional second-nick guide RNA.
In some embodiments, a gene modifying system employs only a template RNA in addition to an mRNA encoding a gene modifying polypeptide. In some embodiments, a gene modifying system additionally employs a second-nick guide RNA that targets the Cas9 nickase of the system to the non-edited strand of the target DNA. In some embodiments, a gene modifying template RNA for targeting SERPINA1 is:
In some embodiments, a template RNA and optional second-nick guide RNA are synthesized by T7 RNA polymerase. In some embodiments, a template RNA and optional second- nick guide RNA are chemically synthesized and contain a combination of one or multiple modifications of the following: 2'-O-methyl, 2'-Fluoro, and/or Phosphorothioate. In some embodiments, the 3 most terminal nucleotides contain 2'-O-methyl modifications with 3 phosphorothioatc linkages between the nucleotides. In some embodiments, a template RNA and optional second-nick guide RNA contain 2'-(9-methyl modified nucleotides, where there are cytosines and uridines, except at nucleotides found in the seed sequence of the gRNA spacers, e.g., the seed sequences in the 3’ end of the spacer regions, where cytosines and uridines contain 2'-fluoro modifications and/or combination of 2'-fluoro and 2' hydroxyl. In some embodiments, combinations of modifications described above include 0-100% substitution of unmodified nucleotides, e.g., 0-10%, 10-20%, 20-30%, 30-40%, 40-50%, 50-60%, 60-70%, 70-80%, 80- 90%, or less than 90-100% substitution.
Formulations.
In some embodiments, a gene modifying polypeptide mRNA and template RNA (and optional second-nick guide RNA) are separately formulated as described above, combined prior to injection at a 1:20 RNA molar ratio, mRNA:template RNA (and optionally mRNA:second- nick guide RNA), respectively. In some embodiments, a gene modifying polypeptide mRNA and template RNA (and optional second-nick guide RNA) are separately formulated as described above, combined prior to injection at a 1:50 RNA molar ratio, mRNA:guide RNAs (and optionally mRNA:second-nick guide RNA), respectively. In some embodiments, a gene modifying polypeptide mRNA and template RNA (and optional second-nick guide RNA) are separately formulated, combined prior to injection at ratio ranges from 1:10-1:250, mRNA:template RNA (and optionally mRNA: second-nick guide RNA), respectively. In some embodiments, a gene modifying polypeptide mRNA and template RNA (and optional second- nick guide NRA) are mixed together at a 1:10-1:250, gene modifying polypeptide mRNA:template RNA (and optionally mRNA: second-nick guide RNA), and then formulated as described above, where the RNA concentration going into formulation is 0.1 mg/mL. In some embodiments, a gene modifying polypeptide mRNA and template RNA (and optional second- nick guide RNA) are formulated separately and are injected 30-180 minutes apart, where the gene modifying polypeptide mRNA LNPs are delivered first followed by the template RNA (and optional second-nick guide RNA) LNPs. In some embodiments, an ionizable lipid is LIPIDV005 from Table 38. Dosing.
In some embodiments, a gene modifying polypeptide mRNA and/or template RNA (and optional second-nick guide RNA) are dosed at 0.01 - 6 mg/kg, either separately or together as a total amount of RNA-LNP. In some embodiments, RNA-LNPs are injected as an IV bolus. In some embodiments, RNA-LNPs are infused over a period of 30-360 minutes.
Kits, Articles of Manufacture, and Pharmaceutical Compositions
In some embodiments, the present disclosure provides a kit comprising a gene modifying polypeptide or a gene modifying system, e.g., as described herein. In some embodiments, a kit comprises a gene modifying polypeptide (or a nucleic acid encoding the polypeptide) and a template RNA (or DNA encoding the template RNA). In some embodiments, a kit further comprises a reagent for introducing the system into a cell, e.g., transfection reagent, LNP, and the like. In some embodiments, a kit is suitable for any of the methods described herein. In some embodiments, a kit comprises one or more elements, compositions (e.g., pharmaceutical compositions), gene modifying polypeptides, and/or gene modifying systems, or a functional fragment or component thereof, e.g., disposed in an article of manufacture. In some embodiments, a kit comprises instructions for use thereof.
In some embodiments, the present disclosure provides an article of manufacture, e.g., in which a kit as described herein, or a component thereof, is disposed.
In some embodiments, the present disclosure provides a pharmaceutical composition comprising a gene modifying polypeptide or a gene modifying system, e.g., as described herein. In some embodiments, a pharmaceutical composition further comprises a pharmaceutically acceptable carrier or excipient. In some embodiments, a pharmaceutical composition comprises a template RNA and/or an RNA encoding a gene modifying polypeptide. In some embodiments, a pharmaceutical composition has one or more (e.g., 1, 2, 3, or 4) of the following characteristics:
(a) less than 1% (e.g., less than 0.5%, less than 0.4%, less than 0.3%, less than 0.2%, or less than 0.1%) DNA template relative to template RNA and/or RNA encoding a gene modifying polypeptide, e.g., on a molar basis;
(b) less than 1% (e.g., less than 0.5%, less than 0.4%, less than 0.3%, less than 0.2%, or less than 0.1 %) uncapped RNA relative to template RNA and/or RNA encoding a gene modifying polypeptide, e.g., on a molar basis; (c) less than 1 % (e.g., less than 0.5%, less than 0.4%, less than 0.3%, less than 0.2%, or less than 0.1%) partial length RNAs relative to template RNA and/or RNA encoding a gene modifying polypeptide, e.g., on a molar basis; and
(d) substantially lacks unreacted cap dinucleotides.
Chemistry, Manufacturing, and Controls (CMC)
Purification of protein therapeutics is described, for example, in Franks, Protein Biotechnology: Isolation, Characterization, and Stabilization, Humana Press (2013); and in Cutler, Protein Purification Protocols (Methods in Molecular Biology), Humana Press (2010).
In some embodiments, a gene modifying system, polypeptide, and/or template nucleic acid (e.g., template RNA) conforms to certain quality standards. In some embodiments, a gene modifying system, polypeptide, and/or template nucleic acid (e.g., template RNA) produced by a method described herein conforms to certain quality standards. Accordingly, the disclosure is directed, in some emboidments, to methods of manufacturing a gene modifying system, polypeptide, and/or template nucleic acid (e.g., template RNA) that conforms to certain quality standards, e.g., in which said quality standards are assayed. The present disclosure is also directed, in some embodiments, to methods of assaying said quality standards in a gene modifying system, polypeptide, and/or template nucleic acid (e.g., template RNA). In some embodiments, quality standards include, but are not limited to, one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12) of the following:
(i) the length of template RNA, e.g., whether the template RNA has a length that is above a reference length or within a reference length range, e.g., whether at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% of the template RNA present is greater than 100, greater than 125, greater than 150, greater than 175, or greater than 200 nucleotides long;
(ii) the presence, absence, and/or length of a polyA tail on a template RNA, e.g., whether at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% of a template RNA present contains a polyA tail (e.g., a polyA tail that is at least 5, 10, 20, 30, 50, 70, 100 nucleotides in length);
(iii) the presence, absence, and/or type of a 5 ' cap on a template RNA, e.g., whether at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% of the template RNA present contains a 5' cap, e.g., whether that cap is a 7-mcthylguanosinc cap, e.g., a 0-Mc-m7G cap;
(iv) the presence, absence, and/or type of one or more modified nucleotides (e.g., selected from pseudouridine, dihydrouridine, inosine, 7-methylguanosine, 1-N- methylpseudouridine ( l -Mc- ), 5-methoxy uridine (5-MO-U), 5-methylcytidine (5mC), or a locked nucleotide) in the template RNA, e.g., whether at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% of a template RNA present contains one or more modified nucleotides;
(v) the stability of a template RNA (e.g., over time and/or under a pre-selected condition), e.g., whether at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% of a template RNA remains intact (e.g., greater than 100, greater than 125, greater than 150, greater than 175, or greater than 200 nucleotides long) after a stability test;
(vi) the potency of a template RNA in a system for modifying DNA, e.g., whether at least 1% of target sites are modified after a system comprising the template RNA is assayed for potency;
(vii) the length of a polypeptide, first polypeptide, or second polypeptide, e.g., whether the polypeptide, first polypeptide, or second polypeptide has a length that is above a reference length or within a reference length range, e.g., whether at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% of the polypeptide, first polypeptide, or second polypeptide present is greater than 600, greater than 650, greater than 700, greater than 750, greater than 800, greater than 850, greater than 900, greater than 950, greater than 1000, greater than 1050, greater than
1100, greater than 1150, greater than 1200, greater than 1250, greater than 1300, greater than 1350, greater than 1400, greater than 1450, greater than 1500, greater than 1600, greater than 1700, greater than 1800, greater than 1900, or greater than 2000 amino acids long (and optionally, no larger than 2500, 2000, 1500, 1400, 1300, 1200, 1100, 1000, 900, 800, 700, or 600 amino acids long);
(viii) the presence, absence, and/or type of post-translational modification on a polypeptide, first polypeptide, or second polypeptide, e.g., whether at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% of the polypeptide, first polypeptide, or second polypeptide contains phosphorylation, methylation, acetylation, myristoylation, palmitoylation, isoprcnylation, glipyatyon, or lipoylation, or any combination thereof;
(ix) the presence, absence, and/or type of one or more artificial, synthetic, or non- canonical amino acids (e.g., selected from ornithine, 0-alanine, GABA, 8-Aminolevulinic acid, PABA, a D-amino acid (e.g., D-alanine or D-glutamate), aminoisobutyric acid, dehydroalanine, cystathionine, lanthionine, Djenkolic acid, Diaminopimelic acid, Homoalanine, Norvaline, Norleucine, Homonorleucine, homoserine, O-methyl- homoserine and O-ethyl-homoserine, ethionine, selenocysteine, selenohomocysteine, selenomethionine, selenoethionine, tellurocysteine, or telluromethionine) in apolypeptide, first polypeptide, or second polypeptide, e.g., whether at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% of the polypeptide, first polypeptide, or second polypeptide present contains one or more artificial, synthetic, or non-canonical amino acids;
(x) the stability of a polypeptide, first polypeptide, or second polypeptide (e.g., over time and/or under a pre-selected condition), e.g., whether at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% of the polypeptide, first polypeptide, or second polypeptide remains intact (e.g., greater than 600, greater than 650, greater than 700, greater than 750, greater than 800, greater than 850, greater than 900, greater than 950, greater than 1000, greater than 1050, greater than 1100, greater than 1150, greater than 1200, greater than 1250, greater than 1300, greater than 1350, greater than 1400, greater than 1450, greater than 1500, greater than 1600, greater than 1700, greater than 1800, greater than 1900, or greater than 2000 amino acids long (and optionally, no larger than 2500, 2000, 1500, 1400, 1300, 1200, 1100, 1000, 900, 800, 700, or 600 amino acids long)) after a stability test;
(xi) the potency of a polypeptide, first polypeptide, or second polypeptide in a system for modifying DNA, e.g., whether at least 1% of target sites are modified after a system comprising the polypeptide, first polypeptide, or second polypeptide is assayed for potency; or (xii) the presence, absence, and/or level of one or more of a pyrogen, virus, fungus, bacterial pathogen, or host cell protein, c.g., whether a system is free or substantially free of pyrogen, virus, fungus, bacterial pathogen, or host cell protein contamination.
In some embodiments, a system or pharmaceutical composition described herein is endotoxin free.
In some embodiments, the presence, absence, and/or level of one or more of a pyrogen, virus, fungus, bacterial pathogen, and/or host cell protein is determined. In some embodiments, whether a system is free or substantially free of pyrogen, virus, fungus, bacterial pathogen, and/or host cell protein contamination is determined.
In some embodiments, a pharmaceutical composition or system as described herein has one or more (e.g., 1, 2, 3, or 4) of the following characteristics:
(a) less than 1% (e.g., less than 0.5%, less than 0.4%, less than 0.3%, less than 0.2%, or less than 0.1%) DNA template relative to the template RNA and/or the RNA encoding the polypeptide, e.g., on a molar basis;
(b) less than 1% (e.g., less than 0.5%, less than 0.4%, less than 0.3%, less than 0.2%, or less than 0.1%) uncapped RNA relative to the template RNA and/or the RNA encoding the polypeptide, e.g., on a molar basis;
(c) less than 1% (e.g., less than 0.5%, less than 0.4%, less than 0.3%, less than 0.2%, or less than 0.1%) partial length RNAs relative to the template RNA and/or the RNA encoding the polypeptide, e.g., on a molar basis;
(d) substantially lacks unreacted cap dinucleotides.
EXAMPLES
Example 1: Design and generation of template RNAs for SERPINA1 PiZ correction.
This example describes the generation and design of exemplary SERPINA1 template RNAs comprising varied spacers, scaffolds, lengths and compositions of heterologous object sequences, and PBS sequences to quantify the activity of template RNAs for correction of the PiZ mutation at the human SERPINA1 locus.
An StlCas9 compatible spacer sequence in the human genome was identified for the use of gene writing in PiZ correction. The spacer is proximal to the PiZ site and has previously demonstrated good indel activity (data not shown), both factors considered potentially beneficial to robust gene writing. To improve the editing efficiency of gene modifying systems comprising StlCas9 gene modifying polypeptides (StlCas9-bascd gene modifying systems), the guide RNA scaffold sequence was engineered (see, e.g., Table 26) to create a variant gRNA scaffold. Specifically, (1) the stem loop 2 motif at the 3’ end of the RNA was truncated, and (2) the tetraloop (TL) upper stem was thermodynamically strengthened by stem elongation and substitution of more stable loop bases. Without wishing to be bound by theory, (1) was hypothesized to mitigate the catalytically negative effects of scaffold 3’ extension and to improve target engagement by the PBS, and (2) was hypothesized to overcome RNA misfolding and facilitate Cas9 loading. Both changes, when adopted in template guide RNA designs, resulted in significant improvement in editing efficiencies over the wild-type scaffold (e.g., as seen in Example 2).
The length of the spacer sequence was also varied in engineered StlCas9-based gene modifying system guide RNA sequences. Without wishing to be bound by theory, it is hypothesized that spacer length modulates the thermodynamic stability of the R-loop that is formed by the annealing of spacer and target DNA, which then can impact the editing efficiencies.
In addition, the lengths of PBS sequence and the heterologous object sequence were also varied, and editing efficiencies of said engineered StlCas9-based gene modifying systems (see, e.g., Example 2 and Example 3). The sequences of these template RNAs are provided in Table 24. Second nick gRNAs are provided in Table 29.
Example 2: Evaluating the impact of scaffold engineering on editing efficiency of StlCas9- based gene modifying systems
This example describes the use of exemplary gene modifying systems containing a gene modifying polypeptide (containing an StlCas9 domain) and template RNAs comprising StlCas9 variant gRNA scaffolds, a spacer, and varied lengths and compositions of heterologous object sequences and PBS sequences to quantify the activity of template RNAs for correction of the PiZ (E342K, G>A) mutation at the human SERPINA1 locus.
In this example, a template RNA contained:
• a gRNA spacer;
• a variant gRNA scaffold;
• a heterologous object sequence; and
• a primer binding site (PBS) sequence. In this example, a gene modifying polypeptide contained:
• an endonuclease and/or DNA binding domain;
• a peptide linker; and
• a reverse transcriptase (RT) domain.
Exemplary template RNAs evaluated are given in Table 39, column 3 (with chemical modifications). Nucleotide modifications are noted as follows: phosphorothioate linkages denoted by an asterisk, 2’-O-methyl groups denoted by an ‘m’ preceding a nucleotide. The exemplary gene modifying polypeptide has the amino acid sequence of SEQ ID NO: 26,002 (RNAIVT1790). In SEQ ID NO: 26,002 below, the StlCas9 domain is underlined once, the N622A mutation is shown in bold, and the RT domain is double-underlined.
Table 39 describes template RNAs having the following nomenclature: the first set of characters indicates the compatible Cas (e.g., Stl indicates StlCas9), the second set of characters indicates the name of the variant gRNA scaffold (e.g., dSL2), the third set of characters indicates the target gene or protein encoded by the target gene (e.g., A1AT), the fourth set of characters indicates the name of the spacer (e.g., ED4), the fifth set of characters indicates the length of the PBS and heterologous object sequence (e.g., P12R7 indicates a PBS of length 12 and a heterologous object sequence of length 7), and the sixth set of characters indicates the edit in a strand of the DNA template (e.g., TtoC). Column 4 shows the unmodified sequence corresponding to the chemically modified sequence of column 3.
Table 39. Tested template RNA sequences.
20,000 HEK293T cells carrying the PiZ mutation (CELLengl716) were transfected using MessengerMax. The gene modifying system included RNAIVT1790 gene modifying polypeptide and a template RNA described above. Specifically, 75 ng of RNAIVT1790 mRNA and 1 pmol tgRNA were diluted to 10 pl and mixed with 35 pl Opti-MEM containing 0.5 pl MessengerMax. The lipoplexes were mixed with the cells in suspension and plated into 96-well plates. After transfection, cells were grown at 37°C, 5% CO2 in DMEM media supplemented with 10% serum for 3 days prior to cell lysis and genomic DNA extraction. Editing of the SERPINA1 target nucleic acid sequence was assessed using amplicon sequencing (Amp-SEQ) using primers flanking the SERPINA1 gene.
FIG. 3A shows a graph of the rewriting performance of StlCas9-based gene modifying systems comprising exemplary template RNAs comprising various variant scaffolds truncated in the SL2 region. The results showed significantly higher gene rewriting percent with templates containing variant scaffolds (RNACS6751 and RNACS6752), compared to templates carrying the wild-type scaffold (RNACS6724). The results suggest that truncation of the SL2 region may improve rewriting performance.
FIG. 3B and FIG. 3C show graphs of rewriting by StlCas9-based gene modifying systems comprising exemplary template RNAs comprising variant scaffolds further engineered in the TL and RAR region by the use of various modified tetraloops. Without wishing to be bound by theory, it is hypothesized that engineering the TL and/or RAR regions may confer higher thermodynamic structural stability. The results showed that various modified tetraloopcontaining templates facilitated rewriting. The results further showed that relative to a template that contains WT tetraloop (RNACS9208), several modified tetraloops boosted rewriting efficiency. For example, the highest rewriting efficiency was achieved with RNACS 10529 (GGGA tetraloop), RNACS 10524 (GAAA tetraloop) and RNACS 10539 (UUCG tetraloop), which performed significantly better at the lowest dose evaluated relative to the WT tetraloop (RNACS9208). The results showed that various modified RAR-containing templates facilitated rewriting. The results also showed that various compositions of RAR significantly improved rewriting relative to the wt RAR (RNACS9209); without wishing to be bound by theory, this may be due to the modified RAR further strengthening thermodynamic stability. Examples include RNACS 10547, RNACS 10548, RNACS 10550 and RNACS 10551 which showed substantially higher rewriting efficiencies at the lowest dose of 0.01 pmol.
FIG. 3D shows a graph of rewriting by StlCas9-based gene modifying systems comprising exemplary template RNAs comprising various lengths of spacers. The results showed that RNACS 10553 (having a spacer length of 23 nt) facilitated a higher rewriting efficiency relative to RNACS9208 (having a WT spacer length of 20 nt).
Taken together, the results show that modified StlCas9-compatible template RNAs comprising various variant scaffolds and spacers enable high levels of PiZ correction with StlCas9-based gene modifying polypeptides.
Example 3: Evaluating the rewriting efficiency of StlCas9-based gene modifying systems comprising various template RNAs at the SERPINA locus
This example describes the use of exemplary gene modifying systems containing a gene modifying polypeptide comprising an StlCas9 domain, and template RNAs comprising StlCas9- compatible variant scaffolds and spacer and varied lengths and compositions of heterologous object sequences and PBS sequences to quantify the activity of template RNAs for correction of the PiZ (E342K, G>A mutation) mutation at human SERPINA 1 locus.
In this example, a template RNA contained:
• a gRNA spacer;
• a variant gRNA scaffold;
• a heterologous object sequence; and
• a primer binding site (PBS) sequence. In this example, a gene modifying polypeptide contained:
• an endonuclease and/or DNA binding domain;
• a peptide linker; and
• a reverse transcriptase (RT) domain.
Exemplary template RNAs evaluated are given in Table 39, column 3 (with chemical modifications). Nucleotide modifications are noted as follows: phosphorothioate linkages denoted by an asterisk, 2’-O-methyl groups denoted by an ‘m’ preceding a nucleotide. The exemplary gene modifying polypeptide is RNAIVT1790, comprising the amino acid sequence of SEQ ID NO: 26002. 20,000 HEK293T cells carrying the PiZ mutation (CELLengl716) were transfected using McsscngcrMax. The gene modifying system includes RNAIVT1790 gene modifying polypeptide and template RNAs described above. Specifically, 75 ng of RNAIVT1790 mRNA and 1 pmol tgRNA were diluted to 10 pl and mixed with 35 pl Opti-MEM containing 0.5 pl MessengerMax. The lipoplexes were mixed with the cells in suspension and plated into 96- well plates. After transfection, cells were grown at 37°C, 5% CO2 in DMEM media supplemented with 10% serum for 3 days prior to cell lysis and genomic DNA extraction. Editing of the SERPINA1 target nucleic acid sequence was assessed using amplicon sequencing (Amp-SEQ) using primers flanking the SERPINA1 gene.
FIG. 4A shows a graph of the rewriting efficiency of gene modifying systems comprising different StlCas9-compatible template RNAs comprising variant scaffold sequences. The results showed that a number of different template RNAs facilitate rewriting of the PiZ locus of the SERPINA1 gene. The results further showed several template RNAs achieved high rewriting efficiencies of up to 40% with <1% indels. RNACS9201 (containing a heterologous object sequence length of 10 nucleotides and a primer binding sequence length of 8 nucleotides) achieved the highest rewriting efficiency. The results showed that priming lengths of < 10 nt and > 6nt appear advantageous for rewriting, whereas heterologous object sequence lengths of less than 14 nt appear advantageous for rewriting.
FIG. 4B shows a graph of the % indel levels of the same gene modifying systems evaluated in FIG. 4A. The results showed that the % indel levels are low for all evaluated template RNAs.
Several template RNAs were retested in the same assay as shown in FIG. 4A. FIG. 4C shows the results of the retest. The results showed that some template RNAs facilitated >60% rewriting with <5% indels. RNACS9201 was the top performer with >75% rewriting efficiency and less than 5% indels.
Taken together, these results show that gene modifying systems comprising StlCas9-based gene modifying polypeptides and template RNAs comprising variant scaffold sequences can correct the E342K mutation in SERPINA1 with very high efficiency and low %indel in 293T cells. It is expected that template RNAs comprising variant scaffold and spacer variants, e.g., template RNAs described herein, e.g., described in Example 1, combined with template and PBS lengths described in this Example may further increase the efficiency of rewriting. Example 4: Evaluating the rewriting efficiency of StlCas9-based gene modifying systems comprising 2nd nick guide RNAs
This example describes the use of exemplary gene modifying systems containing a gene modifying polypeptide comprising an StlCas9 domain, template RNAs comprising StlCas9 variant gRNA scaffold, spacer, and varied lengths and compositions of heterologous object sequences and PBS sequences, and a second strand-targeting gRNA (ngRNA) to quantify the activity of said systems for installation of a G to A nucleotide mutation in wildtype cells or correction (A to G) of the PiZ (E342K, G>A) mutation at human SERPINA1 locus.
In this example, a template RNA contained:
• a gRNA spacer;
• a variant gRNA scaffold;
• a heterologous object sequence; and
• a primer binding site (PBS) sequence.
In this example, a ngRNA contained:
• a gRNA spacer;
• a gRNA scaffold;
In this example, a gene modifying polypeptide contained:
• an endonuclease and/or DNA binding domain;
• a peptide linker; and
• a reverse transcriptase (RT) domain.
Exemplary correction template RNAs used are given in Table 39, column 3 (with chemical modifications). Exemplary C to T mutation template RNAs are given in Table 40 below. Exemplary ngRNAs used are given in Table 29 (column 2, with chemical modifications). Nucleotide modifications are noted as follows: phosphorothioate linkages denoted by an asterisk, 2’-O-methyl groups denoted by an ‘m’ preceding a nucleotide. The exemplary gene modifying polypeptide is RNAIVT1790, comprising the amino acid sequence of SEQ ID NO: 26002.
Table 40: Exemplary C to T mutation template RNAs
The gene modifying system comprising mRNA encoding the gene modifying polypeptide, a template RNA, and a ngRNA were transfected into primary human hepatocytes. The gene modifying polypeptide, template RNA, and second strand-targeting gRNA were delivered by nucleofection in RNA format. Specifically, 4 pg of gene modifying polypeptide mRNA were combined with 10 pg of chemically synthesized template RNA in 5 pL of water. The transfection mix was added to 100,000 primary hepatocytes in Buffer P3 [Lonza], and cells were nucleofected using program DG-138. After nucleofection, cells were grown at 37 °C, 5% CO2 for 3 days prior to cell lysis and genomic DNA extraction. Editing of the SERPINA1 target nucleic acid sequence was assessed using amplicon sequencing (Amp-SEQ) using primers flanking the SERPINA1 gene.
FIG. 5 shows a graph of rewriting efficiency of gene modifying systems comprising StlCas9-based gene modifying polypeptide, one of three G to A mutation install template RNAs (one with a WT scaffold and two comprising variant scaffolds), and one of three different ngRNAs. The results showed that RNACS7555 (with a nick site 97 bp away from the tgRNA nick site) improved the editing efficiency by 2.5-fold compared to editing in the absence of a second nick guide. The results further showed that this enhancement of rewriting with ngRNA (RNACS7555) was seen with all three different mutation install templates: RNACS7410, RNACS9285, and RNACS9286.
FIG. 6 and FIG. 6B shows a graph of rewriting efficiency of gene modifying systems comprising StlCas9-based gene modifying polypeptide, an exemplary template RNA correcting the PiZ mutation and containing a variant scaffold, and with (FIG. 6A, closed bars) or without (FIG. 6A, open bars) exemplary ngRNA RNACS7555. The results showed that rewriting efficiency of gene modifying systems correcting the PiZ mutation is increased by the presence of the exemplary ngRNA in combination with all evaluated templates (left panel). The results further showed that % indel was low relative to rewriting across all evaluated gene modifying systems, with or without ngRNA (FIG. 6B).
Example 5: Validation of Stl Cas9 template RNAs in primary mouse hepatocytes This example describes the use of exemplary gene modifying systems containing a gene modifying polypeptide comprising an StlCas9 domain, exemplary template RNAs comprising a StlCas9 variant gRNA scaffold, spacer, and varied lengths and compositions of heterologous object sequences and PBS sequences, and optionally exemplary ngRNA to quantify the activity of said systems for correction of the PiZ (E342K, G>A) mutation at human SERPINA1 locus in primary mouse hepatocytes derived from PiZ mouse.
In this example, a template RNA contains:
• a gRNA spacer;
• a variant gRNA scaffold;
• a heterologous object sequence; and
• a primer binding site (PBS) sequence.
In this example, a ngRNA contains:
• a gRNA spacer;
• a gRNA scaffold;
In this example, a gene modifying polypeptide contains:
• an endonuclease and/or DNA binding domain;
• a peptide linker; and
• a reverse transcriptase (RT) domain.
Exemplary template RNAs and ngRNAs are given in Table 39 and Table 29, respectively. Nucleotide modifications are noted as follows: phosphorothioate linkages denoted by an asterisk, 2’-O-methyl groups denoted by an ‘m’ preceding a nucleotide. The exemplary gene modifying polypeptide is RNAIVT1790, comprising the amino acid sequence of SEQ ID NO: 26002.
The gene modifying system comprising mRNA encoding the gene modifying polypeptide, a template RNA, and a second strand-targeting gRNA (ngRNA) are transfected into primary mouse hepatocytes. The gene modifying polypeptide, template RNA, and second strandtargeting gRNA are delivered by nucleofection in the RNA format. Specifically, 4 pg of gene modifying polypeptide mRNA are combined with 10 pg of chemically synthesized template RNA in 5 pL of water. The transfection mix is added to 100,000 primary hepatocytes in Buffer P3 [Lonza], and cells are nucleofected using program DG-138. After nucleofection, cells are grown at 37 °C, 5% CO2 for 3 days prior to cell lysis and genomic DNA extraction. Editing of the SERPINA1 target nucleic acid sequence is assessed using amplicon sequencing (Amp-SEQ) using primers flanking the SERPINA1 gene. tgRNA candidates that confer high editing efficiency in HEK293T cells are also tested in primary mouse hepatocytes carrying the human SERPINA1 with the PiZ mutation, both in the presence and absence of an ngRNA (e.g., RNACS7555). The PiZ correction editing efficiency is expected to validate the efficacy of the template RNAs using the engineered variant scaffold and demonstrate a significant improvement over the editing by systems using StlCas9 tgRNAs with the wild-type scaffold.
Example 6: Optimization of lipid nanoparticle compositions for delivery of gene modifying systems to correct the pathogenic E342K mutation associated with alpha-1 antitrypsin deficiency.
In this example, lipid nanoparticle (LNP) components are formulated as described in Example 44 of WO2021/ 178720. Specifically, the lipid nanoparticle (LNP) components (ionizable lipid, helper lipid, sterol, PEG) are dissolved in 100% ethanol with the lipid component molar ratios of 50:10:38.5:1.5, respectively. An mRNA encoding a gene modifying polypeptide as described herein is produced by in vitro transcription and purified mRNA is dissolved in 25 mM sodium citrate, pH 4, to a final concentration of RNA cargo of 0.1 mg/mL. Similarly, a template RNA designed to correct the E342K mutation in SERPINA1 and optionally optimized for use with the specific gene modifying polypeptide (as described herein) is dissolved in 25 mM sodium citrate, pH 4. Optionally, a second-nick gRNA as described herein is dissolved in 25 mM sodium citrate, pH 4.
Each RNA is separately formulated into distinct LNPs with a lipid amine to RNA phosphate (N:P) molar ratio of 6. The LNPs are formed by microfluidic mixing of the lipid and RNA solutions using a Precision Nanosystems NanoAssemblr™ Benchtop Instrument, using the manufacturer’s recommended settings. A 3:1 ratio of aqueous to organic solvent is maintained during mixing using differential flow rates. After mixing, the LNPs are collected and dialyzed in 15 mM Tris, 5% sucrose buffer at 4°C overnight. Formulations are concentrated by centrifugation with Amicon 10 kDa centrifugal filters (Millipore). The resulting mixture is then filtered using a 0.2 pm sterile filter. The final LNP composition is stored at -80°C until further use. Additional LNP formulations are generated to optimize the formulation composition and process for delivery and function of a gene modifying system. The lipid nanoparticlc components are varied according to the following parameters: 30-60% ionizable lipid, e.g., an ionizable lipid in Table 38 or described elsewhere in this application, 5-15% helper phospholipid Distearoylphosphatidylcholine (DSPC), 30-50% cholesterol, and 0.5-5% Polyethylene glycol (PEG). Beyond the lipid composition, additional formulations comprising combinations of gene modifying components are generated, e.g., an mRNA encoding the gene modifying polypeptide is co-formulated with a template RNA for correcting the disease-causing mutation, and optionally a second-nick gRNA is either co-formulated with the mRNA and template RNA, or formulated separately. In some embodiments, the mRNA and template RNA, and optionally a second-nick gRNA, are co-formulated with the lipid nanoparticle components to make the total RNA cargo at a concentration approximately 0.1 mg/mL. The RNA composition for coformulation is a mix of the mRNA and template RNA at a 1-4:1-10 ratio by weight, respectively, or is a mix of mRNA, template RNA, and second-nick gRNA at a ratio of 1-4:1-10:1-10, respectively.
Alternate formulations described in this example include RNAs of the system, e.g., mRNA encoding a gene modifying polypeptide, template RNA, and optional second-nick gRNA, being separately formulated using identical or different ionizable lipids, or identical ionizable lipids formulated with different lipid component ratios as described herein. An exemplary formulation has a gene modifying polypeptide mRNA formulated using the ionizable lipid LIPIDV004, where the formulation is a ratio of 50:10:38.5:1.5 of ionizable lipid, helper lipid, sterol, and PEG, respectively. The RNA is mixed with the lipid at a lipid amine to RNA phosphate (N:P) ratio of 6. An exemplary template RNA for use with the exemplary mRNA is formulated using the ionizable lipid LIPIDV004, where the formulation is a ratio of 50:10:38.5:1.5 of ionizable lipid, helper lipid, sterol, and PEG, respectively. The template RNA is mixed with the lipid at an N:P ratio of 4. An exemplary optional second- nick RNA for further use in this system is formulated using the ionizable lipid LIPIDV004, where the formulation is a ratio of 50:10:38.5:1.5 of ionizable lipid, helper lipid, sterol, and PEG, respectively, with the optional second-nick gRNA being mixed with lipid at an N:P ratio of 4.
As described herein, a single-nucleotide polymorphism in the SERPINA1 gene causes the pathogenic E342K mutation that leads alpha- 1 anti-trypsin deficiency (AATD). This particular amino acid change, known as the Pi*Z allele in humans, has been modeled in the transgenic mouse line B6.Cg-Tg (SERPINA1*E342K) Z11.03Slcw/ChmuJ (stock# 035411, The Jackson Laboratory), which expresses the Pi*Z allele of human SERPINA1 in the liver and kidney at levels similar to human patients with AATD. To correct the amino acid substitution and ameliorate the effects caused by the non-functional AAT protein an optimized gene modifying system described herein, e.g., a gene modifying system composition described in Table 18, or a composition from Table 18 further modified to utilize an RT template region introducing a PAM disruption at the target site as in Table 19, is delivered to a transgenic mouse model of AATD by an LNP formulation described in Example 46 of WO2021/178720 or Example 4, below. To determine any efficacy-modifying effects of a second-nick gRNA, formulations including or lacking the second-nick gRNA are prepared along with the gene modifying polypeptide mRNA and disease-modifying template RNA, and additionally prepared as separate LNPs or coformulations. LNPs of this example are prepared as described in an example of this application and delivered intravenously to disease model mice at a total RNA amount of 1 mg/kg. Mice are monitored for correction in the liver and kidneys through various immunological, physiological, and molecular assays, including detection of wild-type human AAT, e.g., hAAT-specific ELISA, histology for detection of changes in liver and/or kidney fibrosis, immunohistochemistry to stain for intracellular hAAT, and amplicon sequencing for the genomic edit. As described herein, amplicon sequencing comprises using locus- specific primers to amplify across the target site containing the mutation, next-generation sequencing of purified amplicons, e.g., Illumina MiSeq, and computational analysis of amplicon sequencing data, e.g., analysis of editing outcome using the CRISPResso2 pipeline (Clement et al. Nat Biotechnol. 37(3):224-226 (2019)).
Example 7: Validation of Stl Cas9 template gRNAs in primary mouse hepatocytes
This example describes the use of exemplary gene modifying systems containing a gene modifying polypeptide and template RNAs comprising StlCas9 spacer, varied lengths and compositions of heterologous object sequences, and primer binding site (PBS) sequences to quantify the activity of template RNAs for correction of the PiZ (E342K, G>A) mutation at human SERPINA1 locus in primary mouse hepatocytes derived from hSERPINAl E342K +/- (exogenous human SERPINA1 inserted in mouse genome) that is hemizygous for PiZ mutation.
In this example, a template RNA contained:
• a gRNA spacer; • a gRNA scaffold;
• a heterologous object sequence; and
• a primer binding site (PBS) sequence.
In this example, a gene modifying polypeptide contained:
• an endonuclease and/or DNA binding domain;
• a peptide linker; and
• a reverse transcriptase (RT) domain.
Exemplary template RNAs generated are given in Table 39 and Table 41. Nucleotide modifications are noted as follows: phosphorothioate linkages denoted by an asterisk, 2’-O- methyl groups denoted by an ‘m’ preceding a nucleotide. The exemplary gene modifying polypeptide is RNAIVT1798, comprising the amino acid sequence of
The gene modifying polypeptide of RNAIVT 1798 comprises, in an N-terminal to C- terminal direction, a first NLS, StlCas9 nickase domain, linker, MMLV RT domain, and second NLS.
Table 41: Exemplary template RNAs
The gene modifying system comprising mRNA encoding the gene modifying polypeptide and a template RNA were nucleofected into primary mouse hepatocytes. The gene modifying polypeptide and a template RNA were delivered by nucleofection in the RNA format. Specifically, 4 pg of gene modifying polypeptide mRNA were combined with 3 pg of chemically synthesized template RNA in 5 pL of water. The transfection mix was added to 100,000 primary hepatocytes in Buffer P3 (Lonza), and cells were nucleofected using program DG-138. After nucleofection, cells were grown at 37°C, 5% CO2 for 3 days prior to cell lysis and genomic DNA extraction. Editing of the SERPINA1 target nucleic acid sequence was assessed using amplicon sequencing (Amp-SEQ) using primers flanking the SERPINA1 gene.
FIGs. 7A and 7B show graphs of the rewriting activity of StlCas9-based gene modifying systems comprising of exemplary template RNAs containing various lengths of PBS sequences and heterologous object sequences. The template RNA sequences corresponding to FIGs. 7A and 7B are provided in the Column 3 of Table 41 herein. Each exemplary template RNA comprised an exemplary variant gRNA scaffold (e.g., dSL2). The results showed that many exemplary template RNAs facilitated rewriting at the PiZ locus of SERPINA1 (FIG. 7A). The average rewriting activity was about 35%, with the highest rewriting activity observed being about 73% with samples containing template RNA, RNACS9201, and mRNA encoding RNAIVT1798. The results also showed that the percentage of unwanted indels was less than 1% for all examined template RNAs (FIG. 7B). The result suggests that the exemplary StlCas9- based gene modifying systems are highly active and efficient in correcting the PiZ mutation at human SERPINA1 locus in primary mouse hepatocytes derived from PiZ mouse.
Example 8: Tetraloop structure engineering of template RNAs for StlCas9-based gene modifying systems
This example describes the use of exemplary gene modifying systems containing a gene modifying polypeptide and template RNAs comprising a StlCas9 spacer, heterologous object sequences, PBS sequences, and variant scaffolds containing various exemplary variant tetraloop structures aimed to enhance the potency of the RNA molecule to quantify the activity of template RNAs bearing distinct tetraloop structures for correction of the PiZ (E342K, G>A) mutation at human SERPINA1 locus in HEK293T cells and primary mouse hepatocytes derived from PiZ mouse.
In this example, a template RNA contained:
• a gRNA spacer; • a variant gRNA scaffold bearing a dSL2 truncation and variant tetraloops and/or altered lengths of the stem-loop encompassing the tetraloop structure;
• a heterologous object sequence; and
• a primer binding site (PBS) sequence. In this example, a gene modifying polypeptide contained:
• an endonuclease and/or DNA binding domain;
• a peptide linker; and
• a reverse transcriptase (RT) domain.
Exemplary template RNAs generated are given in Table E8 and also in Table 20. Nucleotide modifications are noted as follows: phosphorothioate linkages denoted by an asterisk, 2’-O-methyl groups denoted by an ‘m’ preceding a nucleotide. The exemplary gene modifying polypeptide is RNAIVT1790 (or RNAIVT1798), comprising the amino acid sequence of SEQ ID NO: 26002 (or SEQ ID NO: 26004)
Table 42: Exemplary template RNA The gene modifying system comprising mRNA encoding the gene modifying polypeptide and a template RNA were transfected into HEK293T cells (containing an exogenous chromosomal copy of a PiZ mutated human SERPINA1 locus) and primary mouse hepatocytes derived from a PiZ mouse. The PiZ mouse model is described, e.g., in Guo et al. J Clin Invest. 124(1):251-61 (2014). For HEK293T cells, the gene modifying polypeptide and template RNA were delivered by lipofection using Lipofectamine MessengerMAX. Specifically, 1 pmol (high dose) or 0.01 pmol (low dose) of chemically synthesized template RNA were mixed with 75 ng of gene modifying polypeptide mRNA in 10 pL of water, before mixing with 34.5 L Opti- MEM and 0.5 pL Lipofectamine MessengerMAX reagent. The lipoplexes were subsequently mixed with 20,000 suspended cells and plated in 96- well plates and incubated at 37 °C, 5% CO2 for 3 days prior to cell lysis and genomic DNA extraction. For primary hepatocytes, the gene modifying polypeptide and template RNA were delivered by nucleofection in the RNA format. Specifically, 4 pg of gene modifying polypeptide mRNA were combined with 10 pg of chemically synthesized template RNA in 5 pL of water. The transfection mix was added to 100,000 primary hepatocytes in Buffer P3 [Lonza], and cells were nucleofected using program DG-138. After nucleofection, cells were grown at 37 °C, 5% CO2 for 3 days prior to cell lysis and genomic DNA extraction. Editing of the SERPINA1 target nucleic acid sequence was assessed using amplicon sequencing (Amp-SEQ) using primers flanking the SERPINA1 gene.
FIGs. 8A to 8C show % rewriting achieved in HEK293T cells or primary hepatocytes treated as described. The results showed that many variant tetraloop sequences, either containing changes in tetraloop base identities or from elongations of the stem structure by 2, 3, 4, or 5 bp, resulted in higher editing efficiencies at both low and high dose compared to an otherwise similar template RNAs with the unmodified tetraloop sequence. The results in primary hepatocytes recapitulated the improved rewriting efficiencies seen in HEK293T cells, except for template RNAs with tetraloop substitutions GGAG and UGAA. Among the improved template RNA designs, those bearing UUCG, CUUG, CUCG, GAAA, GUAA, and GAGA tetraloops, and those with stem elongations (especially t-lock-1) to distinct extents resulted in the highest gains in editing efficiencies over the control template RNA not comprising a modified tetraloop (StldSL2_R10P9-WT TL, also called RNACS9208). FIG. 8D illustrates the hypothesized secondary structure of the dSL2 truncated StlCas9 gRNA scaffold, and is overlaid with description of variants described herein. Taken together, RNA scaffold enhancement through tetraloop structure reinforcement improves editing efficiency in HEK293T cells and in primary hepatocytes. The results show that template RNAs for targeting the PiZ mutation in the human SERPINA1 locus can be improved using variant tetraloop sequences.
Example 9: Evaluating Chemical modification patterns for wild-type StlCas9-based gRNA scaffolds in gRNAs of gene modifying systems
This example describes the use of systems containing a StlCas9 domain-containing fusion polypeptide and gRNAs comprising a gRNA scaffold, a spacer, and different patterns of
2’-O-methyl chemical modifications in the gRNA scaffold region, to quantify the activity of gRNAs to direct StlCas9 activity at the SERPINA1 locus carrying the PiZ mutation (E342K, G>A).
In this example, a gRNA contained:
• a gRNA spacer; and
• a StlCas9-compatible gRNA scaffold having a wild-type sequence.
In this example, the fusion polypeptide contained:
• an StlCas9 domain having wildtype endonuclease activity;
• a peptide linker; and
• a reverse transcriptase (RT) domain.
Exemplary gRNAs evaluated are given in Table 43. Nucleotide modifications are noted as follows: phosphoro thioate linkages denoted by an asterisk, 2’-O-methyl groups denoted by an
‘m’ preceding a nucleotide The fusion polypeptide has the amino acid sequence of:
Table 43: Exemplary template RNAs
20,000 HEK293T cells containing an exogenous chromosomal copy of a PiZ mutated human SERPINA1 locus were transfected using MessengerMax. The system included RNAV253 fusion polypeptide and a gRNA described above. Specifically, 75 ng of RNAV253 mRNA and 1 pmol gRNA were diluted to 10 pl and mixed with 35 pl Opti-MEM containing 0.5 pl MessengerMax. The lipoplexes were mixed with the cells in suspension and plated into 96- well plates. After transfection, cells were grown at 37 °C, 5% CO2 in DMEM media supplemented with 10% serum for 3 days prior to cell lysis and genomic DNA extraction. Insertions or deletions (indels) at the hSERPINAl target nucleic acid sequence was assessed using amplicon sequencing (Amp-SEQ) using primers flanking the loci. FIG. 9A contains the graph showing indel activity of the systems comprising WT
StlCas9 fusion polypeptides and the indicated exemplary gRNAs containing wildtype scaffolds with various 2’-O-methyl chemical modifications in the gRNA scaffold region, and FIGs. 9B to 9K show schematic diagrams of each tested chemical modification pattern. All the exemplary template RNAs used in this Example contain a full, non-truncated stem loop scaffold structure. The results showed that a number of chemical modification patterns of the wildtype template RNAs resulted in significant indel levels, and suggested regions of the scaffold that tolerate chemical modification (FIG. 9A). The highest indel activity of 80.3% was observed with samples containing gRNA wt_design8_7 compared to 50.7%, the indel activity of samples containing template RNAs containing unmodified scaffold (wt_control). The results suggest that chemical modifications are well tolerated in specific positions, in particular in the repeat:anti- repeat region and stem loop 2 of the scaffold. The results demonstrate that a system comprising an StlCas9 domain with a gRNA comprising a chemically modified gRNA scaffold is highly active and efficient in producing indels in the SERPINA1 locus in 293T landing pad cells. Accordingly, a similar system comprising a template RNA comprising a similar chemically modified gRNA scaffold were tested for editing at the SERPINA1 locus.
Example 10: Evaluating Chemical modification patterns for exemplary variant StlCas9-based scaffold (dSL2) for Rewriting
This example describes the use of exemplary gene modifying systems containing a gene modifying polypeptide and template RNAs comprising a gRNA scaffold, a spacer, a heterologous object sequence, a PBS sequence, and different patterns of 2’-O-methyl chemical modifications in the gRNA scaffold region, to quantify the gene correction activity of template RNAs with StlCas9 at the SERPINA1 locus carrying the PiZ mutation (E342K, G>A).
In this example, a template RNA contained:
• a gRNA spacer;
• a dSL2 gRNA scaffold;
• a heterologous object sequence; and
• a primer binding site (PBS) sequence.
In this example, a gene modifying polypeptide contained:
• an endonuclease and/or DNA binding domain;
• a peptide linker; and
• a reverse transcriptase (RT) domain. Exemplary gRNAs evaluated are given in Table 44. Nucleotide modifications are noted as follows: phosphorothioatc linkages denoted by an asterisk, 2’-0-mcthyl groups denoted by an ‘m’ preceding a nucleotide. The exemplary gene modifying polypeptide has the amino acid sequence of SEQ ID NO: 26004. Table 44: Exemplary template RNAs
20,000 HEK293T cells containing an exogenous chromosomal copy of a PiZ mutated human SERPINA1 locus were transfected using MessengerMax. The gene modifying system included RNAIVT1790 gene modifying polypeptide and a gRNA described above. Specifically, 75 ng of RNAIVT1790 mRNA and 1 pmol gRNA were diluted to 10 pl and mixed with 35 pl Opti-MEM containing 0.5 pl MessengerMax. The lipoplexes were mixed with the cells in suspension and plated into 96-well plates. After transfection, cells were grown at 37 °C, 5% CO2 in DMEM media supplemented with 10% serum for 3 days prior to cell lysis and genomic DNA extraction. Editing of the hSERPINAl target nucleic acid sequence was assessed using amplicon sequencing (Amp-SEQ) using primers flanking the loci.
FIG. 10A contains a graph showing the rewriting activity of exemplary StlCas9-based gene modifying systems comprising variant template RNAs having the nucleotide sequence of exemplary template RNA, RNACS9201, (see Example 3 above) with various designed patterns of 2-O'-methyl chemical modifications in the gRNA scaffold region. All the exemplary template RNAs used in this Example contain an exemplary variant scaffold sequence containing a truncated stem loop structure. The results showed that a number of variant template RNAs containing truncated stem loops and differently positioned chemical modifications facilitated rewriting at the PiZ mutation of the human SERPINA1 locus and that some variant template RNAs facilitated rewriting at levels comparable to control (not chemically modified). The results further suggested that some positions tolerate chemical modifications better than other positions. The positions of chemical modifications in exemplary template RNAs tested are also shown in FIGs. 10D-10L.
FIG. 10B shows the results of modifying three nucleotides of the scaffold at a time (a modification “walk” approach) with 2’-O-methyl chemical modifications (see Table 44). The results showed that modifications in positions 1 -3 and positions 43 - 54 decreased % rewriting. The results further showed that modifications in positions 7 - 12 and 25 - 33 are somewhat tolerated and modifications in positions 13 - 24 and 34 - 42 are well tolerated, (summarized in FIG. IOC). The highest rewriting activity of 67.95% was observed in samples containing template RNA dSL2_design8 compared to 73.05%, the rewriting activity observed for samples containing template RNA dSL2_control (the unmodified scaffold, RNACS9201). The results suggest that chemical modifications are well tolerated in specific positions in the template RNA scaffold and that the StlCas9 based gene modifying system with a modified template RNA scaffold is highly active and efficient in correcting the PiZ mutation at human SERPINA1 locus in landing pad 293T cells.
Example 11: Evaluating Chemical modification patterns for Exemplary template RNAs containing dSL2 StlCas9 scaffold for Rewriting PiZ in human SERPINA1 in primary mouse hepatocytes
This example describes the use of exemplary gene modifying systems containing a gene modifying polypeptide and template RNAs comprising spacer, heterologous object sequences, PBS sequences, and a St1Cas9 gRNA scaffold region containing different patterns of 2’-O- mcthyl chemical modifications (designed to enhance the activity of the template RNA) to quantify the activity of template RNAs for correction of the PiZ (E342K, G>A) mutation at human SERPINA1 locus in primary mouse hepatocytes derived from PiZ mouse.
In this example, a template RNA contained:
• a gRNA spacer;
• a dSL2 gRNA scaffold;
• a heterologous object sequence; and
• a primer binding site (PBS) sequence.
In this example, a gene modifying polypeptide contained:
• an endonuclease and/or DNA binding domain;
• a peptide linker; and
• a reverse transcriptase (RT) domain.
Exemplary template RNAs generated are given in Table 43 and Table 44. Nucleotide modifications are noted as follows: phosphorothioate linkages denoted by an asterisk, 2’-O- methyl groups denoted by an ‘m’ preceding a nucleotide. The exemplary gene modifying polypeptide is RNAIVT1790 (or RNAIVT1798), comprising the amino acid sequence of SEQ ID NO: 26002 (or SEQ ID NO: 26004).
The gene modifying system, comprising mRNA encoding the gene modifying polypeptide and a template RNA, was nucleofected into primary mouse hepatocytes. Specifically, 4 pg of gene modifying polypeptide mRNA were combined with 3 pg of chemically synthesized template RNA in 5 pL of water. The transfection mix was added to 100,000 primary hepatocytes in Buffer P3 (Lonza), and cells were nucleofected using program DG-138. After nucleofection, cells were grown at 37 °C, 5% CO2 for 3 days prior to cell lysis and genomic DNA extraction. Editing of the SERPINA1 target nucleic acid sequence was assessed using amplicon sequencing (Amp-SEQ) using primers flanking the SERP1NA1 gene.
FIG. 11 contains a graph showing the rewriting activity of the gene modifying systems. Each template RNA comprised of the nucleotide sequence of exemplary template RNA, RNACS9201, with various 2-O'-methyl chemical modifications in the gRNA scaffold region. Each template RNA comprised the exemplary variant scaffold sequence dSL2, containing truncated stem loop scaffold structure. The highest rewriting activity of 71.89% was observed with samples containing template RNA dSL2_design8, compared to 74.56%, the rewriting activity observed with template RNA dSL2_control (the template RNA with the unmodified scaffold). The results further suggest that chemical modifications are well tolerated in specific positions in the template RNA scaffold and that the StlCas9 based gene modifying system with a modified template RNA scaffold is highly active and efficient in correcting the PiZ mutation at human SERPINA1 locus in primary hepatocytes.
Example 12: Evaluating Rewriting Activity of Exemplary Human Template RNAs in Correcting the SERPINA1 PiZ Mutation in hSERPINAl Transgenic Mice
This example describes the use of exemplary gene modifying systems containing a gene modifying polypeptide and template RNA to quantify the activity of template RNAs for correction of the PiZ mutation (corresponding to a A>G base change) in a human SERPINA1 (hSERPINAl) gene in vivo in mice modified to carry hSERPINAl *E342K (PiZ) (NSG-PiZ (NOD.Cg-Prkdcscid I12rgtmlWjl Tg(SERPINAl*E342K)#Slcw/SzJ)) encoding alpha- 1 -antitrypsin (A1AT) protein.
In this example, a template RNA contained:
• a gRNA spacer;
• a gRNA scaffold;
• a heterologous object sequence; and
• a primer binding site (PBS) sequence.
In this example, a gene modifying polypeptide contained:
• an endonuclease and/or DNA binding domain;
• a peptide linker; and
• a reverse transcriptase (RT) domain.
Exemplary template RNAs generated are given in Table 45 and Table 46. Nucleotide modifications are noted as follows: phosphoro thioate linkages denoted by an asterisk, 2’-O-methyl groups denoted by an ‘m’ preceding a nucleotide. The exemplary gene modifying polypeptide is RNA1VT1798, comprising the amino acid sequence of SEQ ID NO: 26004
Table 45 - Exemplary template RNAs and sequences (with chemical modifications)
Table 46 shows the sequences of Table 45 without chemical modifications. In some embodiments, the sequences of Table 46 may be used without chemical modifications, or with one or more chemical modifications.
Table 46: Exemplary template RNAs and sequences (without chemical modifications) The gene modifying system comprising RNAIVT1798 gene modifying polypeptide and a template RNA described above were formulated in LNP and delivered to mice. Specifically, 2 mg/kg of total RNA equivalent formulated in LNPs (4:1 N:P ratio for mRNA, and for tgRNA), combined at 2: 1 (w/w) of mRNA and template RNA were dosed intravenously in about 8-week- old, female NSG-PiZ mice (0.66 mg/kg each of template RNA and 1.33 mg/kg each of mRNA) in a 10 ml/kg bolus. Mice were administered a dose at time 0 (t = 0). 7 days post-dosing (as used herein post-dosing refers to time since the first dose), animals were sacrificed, and their liver and serum were collected for analyses.
7-day liver samples were analyzed by using Amp-Seq to determine % rewriting and % indels in target liver cells. To analyze gene editing activity, primers flanking the target mutation site locus were used to amplify across the locus in the genomic DNA of liver samples. Amplicons were analyzed via short read sequencing using an Illumina MiSeq. Conversion of an A nucleotide to a G nucleotide at position c.1096 in exon 5 in the human SERPINA1 gene indicated successful editing.
FIG. 12A shows a graph of % rewriting in liver samples from treated mice as assessed using Amp-Seq. The results showed detectable rewriting for each of the tested template RNAs. RNACS9201 showed the highest rewriting activity with an average 22.5% +SEM. FIG. 12B shows a graph of % indels in the same liver samples from treated mice. The results showed low levels (about 2.6% ±SEM) of indels for each of the tested template RNAs in NSG-PiZ mouse liver. The results show that exemplary gene modifying systems can be used to specifically correct a clinically relevant mutation in human SERPINA1 gene in vivo in mice.
7-day serum samples were analyzed using Human alpha 1 Antitrypsin ELISA Kit (abl08799) following the manufacturer's instructions to determine the circulating hAlAT level. NSG-PiZ transgenic mice harbor the E342K mutation in the human SERPINA1 gene that causes misfolding and aggregation of the protein inside the hepatocytes and results in low circulating Al AT levels in serum. Successful rewriting of the hSERPINAl transgene would be expected to result in a substantial increase of Al AT level by rescued secretion from liver into blood.
FIG. 12C shows a graph of Al AT levels in serum from treated mice. The results showed that Al AT levels were significantly higher than baseline level in NSG-PiZ mice treated with exemplary gene modifying systems. The results further showed that the highest A1AT levels observed were in mice treated with RNACS9208 and RNACS9201 template RNAs, each about 4-fold higher than in untreated mice (baseline, marked by a dotted line). These results showed that exemplary gene modifying systems targeting mutant hSERPINA! in vivo achieved editing that resulted in measurable phenotype changes relevant to improving therapeutic outcomes in AATD patients.
Example 13: Evaluating Efficacy of Rewriting Activity of Exemplary Human Template RNAs with Different Scaffold Chemical Modifications in Correcting the human SERPINA1 PiZ Mutation in Transgenic Mice
This example describes the use of exemplary gene modifying systems containing a gene modifying polypeptide and template RNA comprising two distinct scaffold sequences and varied chemical modifications in combinations to quantify the activity of template RNAs for correction of the PiZ mutation (corresponding to a A>G base change) in a human SERPINA1 (hSERPINA I ) gene in vivo in mice modified to carry hSERPINAl*E342K (PiZ) encoding alpha- 1 -antitrypsin (A1AT) protein.
In this example, a template RNA contained:
• a gRNA spacer;
• a gRNA scaffold;
• a heterologous object sequence; and
• a primer binding site (PBS) sequence.
In this example, a gene modifying polypeptide contained:
• an endonuclease and/or DNA binding domain;
• a peptide linker; and
• a reverse transcriptase (RT) domain.
Exemplary template RNAs generated are given in Table 47. Nucleotide modifications are noted as follows: phosphorothioate linkages denoted by an asterisk, 2’-O-methyl groups denoted by an ‘m’ preceding a nucleotide. The exemplary gene modifying polypeptide is RNAIVT1798, comprising the amino acid sequence of SEQ ID NO: 26004. In TABLE 47, the names of the template RNAs can include: the compatible Cas (e.g., Stl indicates StlCas9); the name of the variant gRNA scaffold (e.g., dSL2); the target gene or protein encoded by the target gene (e.g., A1AT); the name of the spacer (e.g., ED4); the length of the heterologous object sequence and PBS (e.g., R10P8 indicates a heterologous object sequence of length 10 and a PBS of length 8); and an indication of the chemical modification (e.g., 8 or Mod8 refers to the chemical modification pattern called design 8 illustrated in FIGs. 10E to 10L, and end_mod refers to three 2-O-methyls and three phosphorothioates at each end).
Table 47 - Exemplary template RNAs and sequences (with chemical modifications)
Table 48 shows the sequences of Table 47 without chemical modifications. In some embodiments, the sequences of Table 48 may be used without chemical modifications, or with one or more chemical modifications. Table 48 - Exemplary template RNAs and sequences (without chemical modifications)
The gene modifying system comprising RNAIVT1798 gene modifying polypeptide and template RNAs described above were formulated in LNP and delivered to mice. Specifically, 1 mg/kg of total RNA equivalent formulated in LNPs (4:1 N:P ratio for mRNA, and for tgRNA), combined at 2: 1 (w/w) of mRNA and template RNA were dosed intravenously in about 8-week- old, female NSG-PiZ mice (0.25 mg/kg of template RNA and 0.75 mg/kg of mRNA) in a 10 ml/kg bolus. Mice were administered a dose at time 0 (t = 0). 7 days post-dosing (as used herein postdosing refers to time since the first dose), animals were sacrificed, and their liver and serum were collected for analyses.
Liver samples were analyzed by using Amp-Seq to determine % rewriting and % indels in target liver cells. To analyze gene editing activity, primers flanking the target mutation site locus were used to amplify across the locus in the genomic DNA of liver samples. Amplicons were analyzed via short read sequencing using an Illumina MiSeq. Conversion of a A nucleotide to a G nucleotide at position c.1096 in exon 5 in the human SERPINA1 gene indicated successful editing.
FIG. 13A shows a graph of Amp-Seq results of % editing in liver. The results show that template RNAs RNACS 12268 and RNACS 13597, comprising two distinct scaffold sequences and identical chemical modifications on those nucleotides present in both scaffold sequences, show improvement in rewriting activity compared to control template RNA RNACS9201, which does not contain any chemical modification in the scaffold region. The results further show that certain other chemical modification patterns tested in this assay did not improve or sometimes decreased the efficacy of the rewriting activity of the template RNAs. The highest, rewriting activity was observed for RNACS 13597, showing approximately a 50% increase over RNACS9201. FIG. 13B shows a graph of Amp-Seq results of indel levels in liver. The results show less than 0.2% ±SEM indel activity for each condition in NSG-PiZ mouse liver. The results show that exemplary gene modifying systems with certain chemical modifications in the scaffold region of the template RNA enable efficient rewriting in vivo without a 2nd nick, and that said systems can be used to specifically correct a clinically relevant mutation in human SERPINA1 gene in vivo in mice.
Serum samples were analyzed using Human alpha 1 Antitrypsin ELISA Kit (ab 108799) following the manufacturer's instructions to determine the circulating hAlAT level. Transgenic mice harboring the E342K mutation in the human SERPINA1 gene that causes misfolding and aggregation of the protein inside the hepatocytes and results in low circulating A1AT levels in serum. Successful rewriting of the hSERPINAl transgene would be expected to result in a substantial increase of Al AT level by rescued secretion from liver into blood.
FIG. 13C shows a graph of hAlAT levels in serum from treated mice. The results show that hAlAT levels correlate with the rewriting activity. The highest serum hAlAT level was observed in mice treated with template RNAs RNACS 12268 and RNACS 13597, and these levels were higher than levels in mice treated with control template RNA RNACS9201. These results show that exemplary gene modifying systems containing chemically modified template RNAs targeting mutant hSERPINAl in vivo can achieve more efficient editing that results in measurable phenotype differences.
Example 14: Evaluating Efficacy of Rewriting Activity of Exemplary Human Template RNAs with Scaffold Chemical Modifications in Combination with Fluoro Modification at RT-PBS sequence in Correcting the human SERPINA1 PiZ Mutation in Transgenic Mice
This example describes the use of exemplary gene modifying systems containing a gene modifying polypeptide and template RNA comprising scaffold chemical modifications and chemical modifications of the RT-PBS sequence to quantify the activity of template RNAs for correction of the PiZ mutation (corresponding to a A>G base change) in a human SERPINA1 (hSERPINAl) gene in vivo in mice modified to carry hSERPINAl *E342K (PiZ) encoding alpha- 1-antitrypsin (A1AT) protein.
In this example, a template RNA contains:
• a gRNA spacer;
• a gRNA scaffold;
• a heterologous object sequence; and
• a primer binding site (PBS) sequence. In this example, a gene modifying polypeptide contains:
• an endonuclease and/or DNA binding domain;
• a peptide linker; and
• a reverse transcriptase (RT) domain. Exemplary template RNAs are given in Table 49. Nucleotide modifications arc noted as follows: phosphorothioate linkages denoted by an asterisk, 2’-O-methyl groups denoted by an ‘m’ preceding a nucleotide, 2’ -Fluororibose denoted by an ‘i2F’ preceding a nucleotide.
Table 49 - Exemplary template RNAs and sequences (with chemical modifications)
Table 50 shows the sequences of Table 49 without chemical modifications. In some embodiments, the sequences of Table 50 may be used without chemical modifications, or with one or more chemical modifications.
Table 50 - Exemplary template RNAs and sequences (without chemical modifications)
The gene modifying system comprising RNAIVT4479 gene modifying polypeptide and template RNAs described above are formulated in LNP and delivered to mice. Specifically, 0.8 mg/kg of total RNA equivalent formulated in LNPs (4:1 N:P ratio for mRNA, and for tgRNA), combined at 1 : 1 (w/w) of mRNA and template RNA are dosed intravenously in about 8-week-old, gender balanced hSERPINAlE342K mice (0.4 mg/kg of template RNA and 0.4 mg/kg of mRNA) in a 10 ml/kg bolus. Mice are administered a dose at time 0 (t = 0). 7 days post-dosing (as used herein post-dosing refers to time since the first dose), animals are sacrificed, and their liver and serum are collected for analyses.
Liver samples are analyzed by using Amp-Seq to determine % rewriting and % indels in target liver cells. To analyze gene editing activity, primers flanking the target mutation site locus are used to amplify across the locus in the genomic DNA of liver samples. Amplicons are analyzed via short read sequencing using an Illumina MiSeq. Conversion of a A nucleotide to a G nucleotide at position c.1096 in exon 5 in the human SERPINA1 gene will indicate successful editing.
Example 15: Evaluating Efficacy of Rewriting Activity of Exemplary Human Template RNAs with Scaffold Chemical Modifications in Correcting the human SERPINA1 PiZ Mutation in Transgenic Mice
This example describes the use of exemplary gene modifying systems containing a gene modifying polypeptide and template RNA comprising scaffold chemical modifications to quantify the activity of template RNAs for correction of the PiZ mutation (corresponding to a A>G base change) in a human SERPINA1 (hSERPINA I) gene in vivo in mice modified to carry hSERPlNAl *E342K (PiZ) encoding alpha- 1 -antitrypsin (A1AT) protein.
In this example, a template RNA contained:
• a gRNA spacer;
• a gRNA scaffold;
• a heterologous object sequence; and • a primer binding site (PBS) sequence.
In this example, a gene modifying polypeptide contained:
• an endonuclease and/or DNA binding domain;
• a peptide linker; and
• a reverse transcriptase (RT) domain.
Exemplary template RNAs generated are given in Table 51. Nucleotide modifications are noted as follows: phosphorothioate linkages denoted by an asterisk, 2’-O-methyl groups denoted by an ‘m’ preceding a nucleotide, 2-Fluororibose denoted by an ‘i2F’ preceding a nucleotide. The exemplary gene modifying polypeptide is RNAIVT4479, comprising the amino acid sequence of SEQ ID NO: 10,001.
RNAIVT4479
PAAKRVKLDGGSDLVLGLDIGIGSVGVGILNKVTGEIIHKNSRIFPAAQAENN LVRRTNRQGRRLARRKKHRRVRLNRLFEESGLITDFTKISINLNPYQLRVKGLTD ELSNEELFIALKNMVKHRGISYLDDASDDGNSSVGDYAQIVKENSKQLETKTPGQ IQLERYQTYGQLRGDFTVEKDGKKHRLINVFPTSAYRSEALRILQTQQEFNPQITD EFINRYLEILTGKRKYYHGPGNEKSRTDYGRYRTSGETLDNIFGILIGKCTFYPDEF RAAKASYTAQEFNLLNDLNNLTVPTETKKLSKEQKNQIINYVKNEKAMGPAKLF KYIAKLLSCDVADIKGYRIDKSGKAEIHTFEAYRKMKTLETLDIEQMDRETLDKL AYVLTLNTEREGIQEALEHEFADGSFSQKQVDELVQFRKANSSIFGKGWHNFSV KLMMELIPELYETSEEQMTILTRLGKQKTTSSSNKTKYIDEKLLTEEIYNPVVAKS VRQAIKIVNAAIKEYGDFDNIVIEMARETNEDDEKKAIQKIQKANKDEKDAAML KAANQYNGKAELPHSVFHGHKQLATKIRLWHQQGERCLYTGKTISIHDLINNSN QFEVDHILPLSITFDDSLANKVLVYATAAQEKGQRTPYQALDSMDDAWSFRELK AFVRESKTLSNKKKEYLLTEEDISKFDVRKKFIERNLVDTRYASRVVLNALQEHF RAHKIDTKVSVVRGQFTSQLRRHWGIEKTRDTYHHHAVDALIIAASSQLNLWKK QKNTLVSYSEDQLLDIETGELISDDEYKESVFKAPYQHFVDTLKSKEFEDSILFSY QVDSKFNRKISDATIYATRQAKVGKDKADETYVLGKIKDIYTQDGYDAFMKIYK KDKSKFLMYRHDPQTFEKVIEPILENYPNKQINEKGKEVPCNPFLKYKEEHGYIR KYSKKGNGPEIKSLKYYDSKLGNHIDITPKDSNNKVVLQSVSPWRADVYFNKTT GKYEILGLKYADLQFEKGTGTYKISQEKYNDIKKKEGVDSDSEFKFTLYKNDLLL VKDTETKEQQLFRFLSRTMPKQKHYVELKPYDKQKFEGGEALIKVLGNVANSG QCKKGLGKSNISIYKVRTDVLGNQHIIKNEGDKPKLDFGGAEAAAKEAAAKEAA AKEAAAKALEAEAAAKEAAAKEAAAKEAAAKAGGTAPLEEEYRLFLEAPIQNV TLLEQWKREIPKVWAEINPPGLASTQAPIHVQLLSTALPVRVRQYPITLEAKRSLR ETIRKFRAAGILRPVHSPWNTPLLPVRKSGTSEYRMVQDLREVNKRVETIHPTVP NPYTLLSLLPPDRIWYSVLDLKDAFFCIPLAPESQLIFAFEWADAEEGESGQLTWT RLPQGFKNSPTLFNEALNRDLQGFRLDHPSVSLLQYVDDLLIAADTQAACLSATR DLLMTLAELGYRVSGKKAQLCQEEVTYLGFKIHKGSRSLSNSRTQAILQIPVPKT KRQVREFLGKIGYCRLFIPGFAELAQPLYAATRPGNDPLVWGEKEEEAFQSLKLA LTQPPALALPSLDKPFQLFVEETSGAAKGVLTQALGPWKRPVAYLSKRLDPVAA GWPRCLRAIAAAALLTREASKLTFGQDIEITSSHNLESLLRSPPDKWLTNARITQY
Table 51: Exemplary template RNAs and sequences (with chemical modifications)
Table 52 shows the sequences of Table 51 without chemical modifications. In some embodiments, the sequences of Table 52 may be used without chemical modifications, or with one or more chemical modifications. Table 52 - Exemplary template RNAs and sequences (without chemical modifications)
The gene modifying systems comprising RNAIVT4479 gene modifying polypeptide anc template RNAs described above were formulated in LNP and delivered to mice. Specifically, 0.5 mg/kg of total RNA equivalent formulated in LNPs (4:1 N:P ratio for mRNA, and for tgRNA), combined at 1 : 1 (w/w) of mRNA and template RNA were dosed intravenously in about 8-week- old, gender balanced hSERPlNAlE342K mice (0.25 mg/kg of template RNA and 0.25 mg/kg of mRNA) in a 10 ml/kg bolus. Mice were administered a dose at time 0 (t = 0). 7 days post-dosing (as used herein post-dosing refers to time since the first dose), animals were sacrificed, and their liver and scrum were collected for analyses.
Liver samples were analyzed by using Amp-Seq to determine % rewriting and % indels in target liver cells. To analyze gene editing activity, primers flanking the target mutation site locus were used to amplify across the locus in the genomic DNA of liver samples. Amplicons were analyzed via short read sequencing using an Illumina MiSeq. Conversion of an A nucleotide to a G nucleotide at position c.1096 in exon 5 in the human SERPINA1 gene indicated successful editing.
FIGs. 16A and 16B show graphs of % editing (FIG. 16A) and % indels (FIG. 16B) in liver as measured by Amp-Seq. RNACS 13597 was used as “benchmark” and each exemplary tgRNA has distinct scaffold chemical modifications compared to the benchmark. The results show that many of the evaluated exemplary tgRNAs containing distinct chemical modifications facilitated editing. In particular, the results show that a number of 2’-0-methyl modification patterns in the scaffold show improvement in rewriting activities. 2-Fluororibose modification patterns also improved editing over the benchmark, with the exception of RNACS 19674. The combination of 2’-O-methyl and 2-Fluororibose modification patterns resulted in the highest observed rewriting activities. FIG. 16B shows less than 0.18% +SEM indel activity for each condition in hSERPINAl E342K mouse liver. The results show that exemplary gene modifying systems with tgRNAs containing certain chemical modifications in the scaffold enable efficient rewriting in vivo and can be used to specifically correct a clinically relevant mutation in human SERPINA1 gene in vivo in mice.
Example 16: Evaluating Efficacy of Rewriting Activity of Exemplary Human Template RNAs with Scaffold Chemical Modifications in Combination with Fluoro Modification at heterologous object sequence in Correcting the human SERPINA1 PiZ Mutation in Transgenic Mice
This example describes the use of exemplary gene modifying systems containing a gene modifying polypeptide and template RNA comprising scaffold chemical modifications and heterologous object sequence chemical modifications to quantify the activity of template RNAs for correction of the PiZ mutation (corresponding to a A>G base change) in a human SERPINA1 (hSERPINAl) gene in vivo in mice modified to carry hSERPINAl *E342K (PiZ) encoding alpha- 1-antitrypsin (A1AT) protein.
In this example, a template RNA contained: • a gRNA spacer;
• a gRNA scaffold;
• a heterologous object sequence; and
• a primer binding site (PBS) sequence.
In this example, a gene modifying polypeptide contained:
• an endonuclease and/or DNA binding domain;
• a peptide linker; and
• a reverse transcriptase (RT) domain.
Exemplary template RNAs generated are given in Table 53. Nucleotide modifications are noted as follows: phosphorothioate linkages denoted by an asterisk, 2’-O-methyl groups denoted by an ‘m’ preceding a nucleotide, 2-Fluororibose denoted by an ‘i2F’ preceding a nucleotide. The exemplary gene modifying polypeptide is RNAIVT6289 (comprising a PERV RT domain and
PAPEAAAKGGS linker), comprising the amino acid sequence of SEQ ID NO: 10,011.
Table 53: Exemplary template RNAs and sequences (with chemical modifications)
Table 54 shows the sequences of Table 53 without chemical modifications. In some embodiments, the sequences of Table 54 may be used without chemical modifications, or with one or more chemical modifications.
Table 54 - Exemplary template RNAs and sequences (without chemical modifications)
The gene modifying system comprises RNAIVT6289 gene modifying polypeptide and template RNAs described above were formulated in LNP and delivered to mice. Specifically, 0.5 mg/kg of total RNA equivalent formulated in LNPs (4:1 N:P ratio for mRNA, and for tgRNA), combined at 1:1 (w/w) of mRNA and template RNA were dosed intravenously in about 8-week- old, gender balanced NSG-P Z mice (0.25 mg/kg of template RNA and 0.25 mg/kg of mRNA) in a 10 ml/kg bolus. Mice were administered a dose at time 0 (t = 0). 7 days post-dosing (as used herein post-dosing refers to time since the first dose), animals were sacrificed, and their liver and scrum arc collected for analyses.
Liver samples were analyzed by using Amp-Seq to determine % rewriting and % indels in target liver cells. To analyze gene editing activity, primers flanking the target mutation site locus were used to amplify across the locus in the genomic DNA of liver samples. Amplicons were analyzed via short read sequencing using an Illumina MiSeq. Conversion of a A nucleotide to a G nucleotide at position c.1096 in exon 5 in the human SERPINA1 gene indicated successful editing.
FIGs. 17A and 17B show graphs of % editing (FIG. 17A) and % indels (FIG. 17B) in liver as measured by Amp-Seq. RNACS 13597 was used as a “benchmark” and each exemplary tgRNA evaluated contains distinct scaffold chemical modifications and/or a distinct fluoro modification pattern of the heterologous object sequence compared to the benchmark. The results show that the examined chemical modification of the scaffold shows improvement in rewriting activities. The results further show that 2-Fluororibose modification of the heterologous object sequence, e.g., in the patterns evaluated, also improves rewriting. Combination of scaffold and heterologous object sequence modification patterns resulted in the highest rewriting observed, except for RNACS20687. FIG. 17B shows less than 2% ±SEM indel activity for each condition inNSG-PIZ mouse liver. The results show that exemplary gene modifying system with certain chemical modifications in the scaffold enable efficient rewriting in vivo and can be used to specifically correct a clinically relevant mutation in human SERPINA1 gene in vivo in mice.
Example 17: Evaluating Efficacy of Rewriting Activity of Exemplary Human Template RNAs with Scaffold Chemical Modifications in Combination with Fluoro Modification at heterologous object sequence Using Different Optimized mRNAJPolypeptide Constructs in Correcting the Human SERPINA1 PiZ Mutation in Transgenic Mice
This example describes the use of exemplary gene modifying systems containing optimized gene modifying polypeptide variants and template RNAs comprising scaffold chemical modifications and heterologous object sequence chemical modifications to quantify the activity of template RNAs for correction of the PiZ mutation (corresponding to a A>G base change) in a human SERPINA1 hSERPINAl) gene in vivo in mice modified to carry hSERPINAl 'E342K (PiZ) encoding alpha- 1 -antitrypsin (Al AT) protein.
In this example, a template RNA contained:
• a gRNA spacer; • a gRNA scaffold;
• a heterologous object sequence; and
• a primer binding site (PBS) sequence.
In this example, a gene modifying polypeptide contained:
• an endonuclease and/or DNA binding domain;
• a peptide linker; and
• a reverse transcriptase (RT) domain.
Exemplary template RNAs generated are given in Table 55. Nucleotide modifications are noted as follows: phosphorothioate linkages denoted by an asterisk, 2’-O-methyl groups denoted by an ‘m’ preceding a nucleotide, 2-Fluororibose denoted by an ‘i2F’ preceding a nucleotide.
Exemplary gene modifying polypeptides included RNAIVT7200 (comprising a PERV RT domain and AEIKYDGV linker), RNAIVT6241 (comprising a BAEVM RT domain and
EAAAKEAAAKEAAAKEAAAKALEAEAAAKEAAAKEAAAKEAAAK linker (SEQ ID
N0:30004)), and RNAIVT6898 (comprising a AVIRE RT domain and WQAAESYEV linker
(SEQ ID NO: 30005)), comprising the amino acid sequences of SEQ ID NOs: 10,018-10,020 below. Table 55: - Exemplary template RNAs and sequences (with chemical modifications)
Table 56 shows the sequences of Table 55 without chemical modifications. In some embodiments, the sequences of Table 56 may be used without chemical modifications, or with one or more chemical modifications.
Table 56: - Exemplary template RNAs and sequences (without chemical modifications)
Gene modifying systems comprising RNAIVT7200, RNAIVT6241 or RNAIVT6898 gene modifying polypeptides and template RNAs described above were formulated in LNP and delivered to mice. Specifically, two dose levels, 0.15 mg/kg and 0.45 mg/kg of total RNA equivalent formulated in LNPs (4:1 N:P ratio for mRNA, and for tgRNA), combined at 1:1 (w/w) of mRNA and template RNA were dosed intravenously in about 8-week-old, gender balanced hSERPINAl E342K mice (0.075 mg/kg or 0.225 mg/kg of template RNA and 0.075 mg/kg or 0.225 mg/kg of mRNA) in a 10 ml/kg bolus. Mice were administered a dose at time 0 (t = 0). 7 days post-dosing (as used herein post-dosing refers to time since the first dose), animals were sacrificed, and their liver and serum are collected for analyses.
Liver samples were analyzed by using Amp-Seq to determine % rewriting and % indels in target liver cells. To analyze gene editing activity, primers flanking the target mutation site locus were used to amplify across the locus in the genomic DNA of liver samples. Amplicons were analyzed via short read sequencing using an Illumina MiSeq. Conversion of a A nucleotide to a G nucleotide at position c.1096 in exon 5 in the human SERPINA1 gene indicated successful editing.
FIGs. 18A and 18B show graphs of % editing (FIG. 18A) and % indels (FIG. 18B) in liver as measured by Amp-Seq. The results showed that each evaluated gene modifying system facilitated editing. RNACS20686 tgRNA (containing 2’-O-methyl and 2-Fluororibose modification patterns of the scaffold and the heterologous object sequence) exhibited the highest rewriting activity at 0.15 mg/kg dose among the examined tgRNAs when combined with the exemplary gene modifying polypeptide RNAIVT6898. RNACS20684 and RNACS 19669 (containing 2’-O- methyl and 2-Fluororibose modification patterns of the scaffold only or 2’-O-methyl modification of the scaffold and 2-Fluororibose modification on the heterologous object sequence, respectively) performed similarly in combination with RNAIVT6898. The examined tgRNAs showed comparable rewriting activity in combination with RNAIVT7200 andRNAIVT6241 at 0.15 mg/kg dose. The results show that the examined systems perform similarly at 0.45 mg/kg dose; without wishing to be bound by theory, said 583imilarity suggests such a dose may be saturating, producing rewriting around 45% for all evaluated combinations of tgRNA and gene modifying polypeptide. FIG. 18B shows less than 0.2% ±SEM indel activity for each condition in hSERPINAl E342K mouse liver. The results show that exemplary gene modifying systems containing gene modifying polypeptides having AVIRE, BAEVM, and PERV RT domains (e.g., and paired with these exemplary linkers) facilitate a high level of editing. The results further show that these gene modifying polypeptides combined with tgRNAs with certain chemical modifications in the scaffold and heterologous object sequence enable efficient rewriting in vivo and can be used to specifically correct a clinically relevant mutation in human SERPINA1 gene in vivo in mice.
Example 18: Evaluating Efficacy of Rewriting Activity of Different Optimized mRNA/Polypeptide Constructs Using a Human Template RNA in Correcting the Human SERPINA1 PiZ Mutation in Different A A ll) Mouse Models.
This example describes the use of exemplary gene modifying systems containing optimized gene modifying polypeptide variants and a partially optimized template RNA to quantify the activity of gene modifying polypeptides combined with template RNA for correction of the PiZ mutation (corresponding to a A>G base change) in a human SERP1NA1 (hSERPINAl) gene in vivo in human transgenic mice. Two different transgenic mouse stains (hSERPINAl E342K mice and NSG-PiZ mice) were used in this example, both were modified to carry hSERPINAl *E342K (PiZ) encoding alpha- 1 -antitrypsin (Al AT) protein.
In this example, a template RNA contained:
• a gRNA spacer;
• a gRNA scaffold;
• a heterologous object sequence; and
• a primer binding site (PBS) sequence.
In this example, a gene modifying polypeptide contained:
• an endonuclease and/or DNA binding domain;
• a peptide linker; and
• a reverse transcriptase (RT) domain. The exemplary template RNA used is given in Table 57. Nucleotide modifications are noted as follows: phosphorothioatc linkages denoted by an asterisk, 2’-0-mcthyl groups denoted by an ‘m’ preceding a nucleotide, 2-Fluororibose denoted by an ‘i2F’ preceding a nucleotide. Exemplary gene modifying polypeptides used included RNAIVT6241 (comprising a BAEVM RT domain,
EAAAKEAAAKEAAAKEAAAKEAAAK linker (SEQ ID NO: 5129), and a nickase StlCas9 domain), and RNAIVT6898 (comprising a AVIRE RT domain, an WQAAESYEV linker (SEQ
ID NO: 30005), and a nickase StlCas9 domain), comprising the amino acid sequences of SEQ ID
NOs: 31453 and 31454 below. AVGNRQADRVARQAAMAEVLTLATEPDNTSHITAGKRTADGSEFEKRTADGSE
FESPKKKAKVE*** (SEQ ID NO: 31453)
Table 57: Exemplary template RNA sequence (with chemical modifications)
Table 58 shows the sequence of Table 57 without chemical modifications. In some embodiments, the sequence of Table 58 may be used without chemical modifications, or with one or more chemical modifications.
Table 58: Exemplary template RNA sequence (without chemical modifications)
Gene modifying systems comprising RNAIVT6241 or RNAIVT6898 gene modifying polypeptides and template RNA described above were formulated in LNP and delivered to either hSERPINAlE342K or NSG-PiZ mice. Specifically, three dose levels, 0.15 mg/kg, 0.5 mg/kg, and 1.5 mg/kg of total RNA equivalent formulated in LNPs (4: 1 N:P ratio for mRNA, and for tgRNA), combined at 1:1 (w/w) of mRNA and template RNA (0.075 mg/kg, 0.25 mg/kg or 0.75 mg/kg of template RNA and 0.075 mg/kg, 0.25 mg/kg or 0.75 mg/kg of mRNA) were dosed intravenously in about 8-week-old, gender balanced hSERPINAl E342K or NSG-PiZ mice in a 10 ml/kg bolus. Mice were administered a dose at time 0 (t = 0). 7 days or 28 days post-dosing (as used herein post-dosing refers to time since the first dose), animals were sacrificed, and their liver and serum are collected for analyses.
Liver samples were analyzed by using Amp-Seq to determine % rewriting and % indels in target liver cells. To analyze gene editing activity, primers flanking the target mutation site locus were used to amplify across the locus in the genomic DNA and in the mRNA transcript of liver samples. Amplicons were analyzed via short read sequencing using an Illumina NovaSeq sequencing system. Conversion of an A nucleotide to a G nucleotide at position c.1096 in exon 5 in the human SERPINA1 gene indicated successful editing.
FIGs. 19A-19C show graphs of % corrected gDNA for both exemplary gene modifying polypeptides over evaluated dosages in hSERPINAl E342K mice (FIG. 19A), % corrected gDNA for RNAIVT6898 systems over evaluated dosages in NSG-PiZ mice at 7 day (FIG. 19B) and 28 day time points (FIG. 19C), % edited mRNA for RNAIVT6898 systems over evaluated dosages in both mouse models (FIG. 19D), and % indels (FIG. 19E) in the liver of hSERPINAl E342K for both exemplary gene modifying polypeptides over evaluated dosages, and % indels in the liver of NSG-PiZ mouse models at 7 day (FIG. 19F) and 28 day (FIG. 19G) time points; all readouts as measured by Amp-Seq. The results indicate that both evaluated gene modifying polypeptides facilitated editing at comparable levels in the hSERPINAl E342K mouse strain; systems containing RNAIVT6241 and RNAIVT6898 demonstrated comparable rewriting activity at each dose levels when combined with exemplary tgRNA, RNACS20686. Rewriting values reach around 25% at 0.15 mg/kg dosage and are similar at 0.5 mg/kg and 1.5 mg/kg doses in both cohorts of the hSERPINAl E342K mouse strain; that similarity suggests such doses may be saturating, producing rewriting around 50% for both evaluated gene modifying polypeptides. Results further show comparable editing of the PiZ mutation of the hSERPINAl gene in both transgenic mouse models (NSG-PiZ mice and hSERPINAl E342K mice) when using RNAIVT6898 gene modifying polypeptide; about 50% rewriting in the hSERPINAl E342K model and about 40% rewriting in the NSG-PiZ model at 0.5 mg/kg and reaches saturation at 1.5 mg/kg dosage (see FIG. Al right and A2 left). Results further demonstrate durable correction of PiZ mutation when rewriting levels were analyzed from samples collected either 7 days or 28 days post dose (FIG. 1A2). Fig. IB shows the level of corrected mRNA transcripts after treatment with RNAIVT6898 containing gene modifying systems. The results show up to 74% in hSERPINAl E342K mice and 95% in NSG- PiZ mice, respectively; this suggests that despite 40-50% levels of detected DNA editing, such editing results in the substantial majority of mRNA transcripts contain the desired mutation correction. The results in Fig. 1C1 indicates less than 0.5% +SEM indel activity for each condition in hSERPINAl E342K mouse liver. The results in FIG. 1C2 show that at either 7 or 28 days post dosing in NSG-PiZ mouse liver indel levels were low, reaching 0.5% at the highest dose of 1.5 mpk at 28 days post dosing.
The results demonstrate that exemplary gene modifying systems containing gene modifying polypeptides having AVIRE or BAEVM RT domains facilitate comparably high levels of editing. The results further show that systems utilizing these gene modifying polypeptides enable efficient and durable rewriting in vivo and can be used to specifically correct a clinically relevant mutation in human SERPINA1 gene in vivo in mice. Serum samples were analyzed using Human alpha 1 Antitrypsin ELISA Kit (ab 108799) following the manufacturer's instructions to determine the circulating hAlAT level. Transgenic mice harboring the E342K mutation in the human SERPINA1 gene that causes misfolding and aggregation of the protein inside the hepatocytes and results in low circulating Al AT levels in serum. Successful rewriting of the hSERPINAl transgene would be expected to result in a substantial increase of Al AT level by rescued secretion from liver into blood.
FIG. 20A shows a graph of human Al AT levels in serum from hSERPINAl E342K mice treated with gene modifying systems containing either RNAIVT6241 or RNAIVT6898 polypeptides at varying doses. The results show that serum hAlAT levels increase in a dosedependent manner. Changes in the serum hAlAT levels at each dose levels are comparable when RNAIVT6241 and RNAIVT6898 treatment groups are compared side by side. FIGs. 20B and 20C shows graphs of human A1AT levels in serum from NSG-piZ mice treated with RNAIVT6898- containing gene modifying system at varying dose at either 7 (FIG. 20B) or 28 days (FIG. 20C) post dosing. The results show a durable increase of A1AT levels in treated mice out through 28 days post dosing. High rewriting activity leads to increased (>3.5-fold) serum hAlAT over baseline in hSERPINAl E342K mouse cohorts (FIG. 2A) and about 6-fold increase over baseline in NSG-PiZ mice (FIG. 2B). These results show that exemplary gene modifying systems targeting mutant hSERPINAl in vivo can achieve more efficient editing that results in measurable and durable phenotype differences.
Liver sections of NSG-PiZ mouse after treatment with RNAIVT6898 gene modifying system correction were stained with Periodic Acid-Schiff-Diastase (PAS-D) to visualize hepatocytes with intracellular Al AT mutant protein aggregates. Histology slides were scanned with the Ay oka Vectra Polaris scanner and digital images were analyzed by QuPath morphometric analysis software to measure the percent liver area occupied by PAS positive globules.
FIGs. 21A and 21B show % liver area occupied by globules as described above in liver sections of NSG-PiZ mice after treatment. Histology evaluation shows that the percentage area of the liver occupied by A1AT polymer globules are significantly reduced 7 days post dose (FIG. 21A) in a dose-dependent manner and the aggregate reduction is more prominent in liver samples collected 28 days post dose (FIG. 21B). These results show that exemplary gene modifying systems targeting mutant hSERPINAl in vivo can achieve efficient editing that results in measurable and durable liver phenotype correction. Example 19: Evaluating Efficacy of Rewriting Activity of an Exemplary Human Template RNA Combined with an Exemplary mRNA encoding a Gene Modifying Polypeptide in Correcting the E342K Mutation in NSG-PiZ Transgenic Mice
This example describes the use of exemplary gene modifying system containing an exemplary gene modifying polypeptide and exemplary template RNA to evaluate editing efficacy at multiple dose levels for correction of the PiZ mutation (corresponding to a A>G base change) in the human SERPINA1 (hSERPINA 1 ) gene in vivo in mice modified to carry hSERPINAl *E342K (PiZ) encoding alpha- 1 -antitrypsin (Al AT) protein.
In this example, a template RNA contained:
• a gRNA spacer;
• a gRNA scaffold;
• a heterologous object sequence; and
• a primer binding site (PBS) sequence.
In this example, a gene modifying polypeptide contained:
• an endonuclease and/or DNA binding domain;
• a peptide linker; and
• a reverse transcriptase (RT) domain.
Exemplary template RNAs generated are given in Table 59. Nucleotide modifications are noted as follows: phosphorothioate linkages denoted by an asterisk, 2’-0-methyl groups denoted by an ‘m’ preceding a nucleotide, 2-Fluororibose denoted by an ‘i2F’ preceding a nucleotide. The exemplary gene modifying polypeptide used was RNAIVT9315 (comprising a AVIRE RT domain and WQAAESYEV linker (SEQ ID NO: 30005)), comprising the amino acid sequences of SEQ ID NOs: 31455 below.
Table 59: Exemplary template RNA sequence (with chemical modifications)
Table 60 shows the sequence of Table 59 without chemical modifications. In some embodiments, the sequence of Table 60 may be used without chemical modifications, or with one or more chemical modifications.
Table 60: Exemplary template RNA sequence (without chemical modifications)
Gene modifying systems comprising RNAIVT9315 gene modifying polypeptide and RNACS22230 template RNA described above were formulated in LNP and delivered to mice. Specifically, single administrations of varying dose levels (0.03, 0.06, 0.1, 0.25 and 0.5 mg/kg) of total RNA equivalent formulated in LNPs (4: 1 N:P ratio for mRNA, and for tgRNA), combined at 1 : 1 (w/w) were dosed intravenously in about 8-week-old male NSG-PiZ mice in a 10 ml/kg bolus. Mice were administered a dose at time 0 (t = 0). 7 days post-dosing (as used herein post-dosing refers to time since the first dose), animals were sacrificed, and their liver and serum are collected for analyses.
Liver samples were analyzed by using Amp-Seq to determine % rewriting and % indels in target liver cells. To analyze gene editing activity, primers flanking the target mutation site locus were used to amplify across the locus in the genomic DNA and in the mRNA transcript of liver samples. Amplicons were analyzed via short read sequencing using an Illumina NovaSeq sequencing system. Conversion of an A nucleotide to a G nucleotide at position c. 1096 in exon 5 in the human SERPINA1 gene indicated successful editing.
FIGs. 22A-22C show graphs of % corrected gDNA editing (Al), % corrected mRNA (IB) and % indels (1C) in the liver of NSG-PiZ mice as measured by Amp-Seq. The results showed (FIG. 22A) that the evaluated gene modifying system facilitated editing in a dose dependent manner. A rewriting level around 50% was observed at 0.5 mg/kg dose. FIG. 22B shows the corrected mRNA transcript level in NSG-PiZ mice livers reaches 93% rewriting. The results suggests that despite an approximately 50% level of detected DNA editing, such editing results in the substantial majority of mRNA transcripts contain the desired mutation correction. FIG. 22C shows less than 0.77% indel activity for each condition evaluated in NSG-PiZ mouse liver. The results demonstrate that exemplary gene modifying systems containing exemplary gene modifying polypeptides and exemplary tgRNAs enable efficient rewriting in vivo and can be used to specifically correct a clinically relevant mutation in human SERPINA1 gene in vivo in mice.
Serum samples were analyzed using Human alpha 1 Antitrypsin ELISA Kit (ab 108799) following the manufacturer's instructions to determine the circulating hAlAT level. Transgenic mice harboring the E342K mutation in the human SERPINA1 gene that causes misfolding and aggregation of the protein inside the hepatocytes and results in low circulating Al AT levels in scrum. Successful rewriting of the hSERPINAl transgcnc would be expected to result in a substantial increase of Al AT level by rescued secretion from liver into blood.
FIGs. 23A and 23B show graphs of human Al AT levels in serum from mice treated with exemplary gene modifying system as described above, with FIG. 23A showing serum Al AT over dosage administered and FIG. 23B showing serum A1AT as a function of % rewriting. The results demonstrate that the serum hAlAT levels increased in a dose dependent manner. Serum hAlAT concentration reached protective threshold ( I I pmol/L over baseline) at 0.06 mg/kg dosage and increased up to 54 pmol/L at 0.5 mg/kg dosage resulting in a 6-fold increase in NSG-PiZ mice. FIG. 23B indicates a strong correlation between Al AT levels and rewriting activity. These results show that exemplary gene modifying systems targeting mutant hSERPINAl in vivo can achieve highly efficient editing that results in measurable phenotype differences.
Example 20: Evaluating Efficacy of Rewriting Activity of Optimized mRNA Variants combined with an Optimized Human Template RNA in Correcting the E342K Mutation in hSERPINAl E342K Transgenic Mice
This example describes the use of exemplary gene modifying system containing optimized gene modifying polypeptide encoded by different mRNA variants and optimized template RNA to evaluate editing efficacy at multiple dose levels for correction of the PiZ mutation (corresponding to a A>G base change) in the human SERPINA1 (hSERPINAl) gene in vivo in mice modified to carry hSERPINAl *E342K (PiZ) encoding alpha- 1 -antitrypsin (A1AT) protein.
In this example, a template RNA contained:
• a gRNA spacer;
• a gRNA scaffold;
• a heterologous object sequence; and
• a primer binding site (PBS) sequence
In this example, a gene modifying polypeptide contained:
• an endonuclease and/or DNA binding domain;
• a peptide linker; and
• a reverse transcriptase (RT) domain.
Exemplary template RNA generated are given in Table 24. Nucleotide modifications are noted as follows: phosphorothioate linkages denoted by an asterisk, 2’-O-methyl groups denoted by an ‘m’ preceding a nucleotide, 2-Fluororibose denoted by an ‘i2F’ preceding a nucleotide. Exemplary gene modifying polypeptides included RNAIVT9315 (SEQ ID NO:31455) and RNAIVT9318 (comprising a AVIRE RT domain and WQAAESYEV linker (SEQ ID NO: 30005)), comprising the amino acid sequence of SEQ ID NO: 31458 below. Gene modifying systems comprising either RNAIVT931 or RNAIVT9318 gene modifying polypeptides and RNACS22230 template RNA described above were formulated in LNP and delivered to mice. Specifically, single administration of different dose levels (0.05 and 0.1 mg/kg) of total RNA equivalent formulated in LNPs (4: 1 N:P ratio for mRNA, and for tgRNA), combined at 1:1 (w/w) of mRNA and template RNA were dosed intravenously in about 8-week-old gender balanced hSERPINAl E342K mice in a 10 ml/kg bolus. Mice were administered a dose at time 0 (t = 0). 7 days post-dosing (as used herein post-dosing refers to time since the first dose), animals were sacrificed, and their liver and serum are collected for analyses.
Liver samples were analyzed by using Amp-Seq to determine % rewriting and % indels in target liver cells. To analyze gene editing activity, primers flanking the target mutation site locus were used to amplify across the locus in the genomic DNA of liver samples. Amplicons were analyzed via short read sequencing using an Illumina NovaSeq sequencing system. Conversion of an A nucleotide to a G nucleotide at position c.1096 in exon 5 in the human SERPINA1 gene indicated successful editing.
FIG. 24 shows graph of % corrected gDNA editing (FIG. 24A) and % indels (FIG. 24B) in the liver of hSERPINA!E342K mice as measured by Amp-Seq. The results showed (FIG. 24A) that the evaluated gene modifying system facilitated editing in a dose dependent manner. The rewriting activity of the examined polypeptides are comparable at both tested dose levels. FIG. 24B shows less than 0.3% indel activity for each condition in hSERPINAl E342K mouse liver. The results demonstrate that exemplary gene modifying systems containing each optimized gene modifying polypeptide and optimized tgRNA enable efficient and comparable rewriting in vivo and can be used to specifically correct a clinically relevant mutation in human SERPINA1 gene in vivo in mice.
Example 21: Evaluating Efficacy of Rewriting Activity of Exemplary Human Template RNAs with Engineered StlCas9-based Scaffold (dSL2) Variants Combined with Optimized Heterologous Object Sequences Using Optimized mRNA/Polypeptide Constructs in Correcting the Human SERPINA1 PiZ Mutation in Transgenic Mice
This example describes the use of exemplary gene modifying systems containing an optimized gene modifying polypeptide and template RNAs comprising variants of engineered gRNA scaffold, varying heterologous object sequences, and an identical spacer and PBS site to quantify the activity of template RNAs for correction of the PiZ mutation (corresponding to a A>G base change) in a human SERPINA 1 (hSERPINA I) gene in vivo in mice modified to carry hSERPENAl *E342K (PiZ) encoding alpha- 1 -antitrypsin (A1AT) protein.
In this example, a template RNA contained:
• a gRNA spacer; • a gRNA scaffold;
• a heterologous object sequence; and
• a primer binding site (PBS) sequence.
In this example, a gene modifying polypeptide contained:
• an endonuclease and/or DNA binding domain; • a peptide linker; and
• a reverse transcriptase (RT) domain.
Exemplary template RNAs generated are given in Table 61. Nucleotide modifications are noted as follows: phosphorothioate linkages denoted by an asterisk, 2’-O-methyl groups denoted by an ‘m’ preceding a nucleotide, 2-Fluororibose denoted by an ‘i2F’ preceding a nucleotide. The exemplary gene modifying polypeptide is RNAIVT9315, comprising the amino acid sequence of SEQ ID NO:31455).
Table 61: Exemplary template RNA sequence (with chemical modifications) Table 62 shows the sequence of Table 61 without chemical modifications. In some embodiments, the sequence of Table 62 may be used without chemical modifications, or with one or more chemical modifications.
Table 62: Exemplary template RNA sequence (without chemical modifications)
The gene modifying system comprising RNAIVT9315 gene modifying polypeptide and template RNAs described above were formulated in LNP and delivered to mice. Specifically, four dose levels, 0.012 mg/kg, 0.025 mg/kg, 0.05 mg/kg, and 0.1 mg/kg of total RNA equivalent formulated in LNPs (4:1 N:P ratio for mRNA and tgRNA, combined at 1:1 (w/w) of mRNA and template RNA) were dosed intravenously in about 8-week-old, gender balanced hSERPINA!E342K mice (0.006, 0.0125, 0.025, or 0.05 mg/kg of template RNA and 0.006, 0.0125, 0.025, or 0.05 mg/kg of mRNA) in a 10 ml/kg bolus. Mice were administered a dose at time 0 (t = 0). 7 days post-dosing (as used herein post-dosing refers to time since the first dose), animals were sacrificed, and their liver and serum are collected for analyses.
Liver samples were analyzed by using Amp-Seq to determine % rewriting and % indels in target liver cells. To analyze gene editing activity, primers flanking the target mutation site locus were used to amplify across the locus in the genomic DNA of liver samples. Amplicons were analyzed via short read sequencing using an Illumina MiSeq. Conversion of a A nucleotide to a G nucleotide at position c.1096 in exon 5 in the human SERPINA1 gene indicated successful editing.
FIGs. 25A-25D show graphs of % editing and FIGs. 26A-26D show graphs of % indels in liver as measured by Amp-Seq. RNACS24757 exemplary tgRNA exhibits the highest rewriting activity at 0.012 and 0.025 mg/kg doses compared to RNACS24756. The examined tgRNAs show comparable rewriting activity at higher doses, reaches the saturation level around 50% at 0.1 mg/kg dose. FIGs. 26A-26D show less than 0.3% ±SEM indel activity for each condition in hSERPINA l E342K mouse liver. The results show that exemplary gene modifying systems (e.g., containing certain chemical modifications in the scaffold and PBS regions of their tgRNAs) enable efficient rewriting in vivo and can be used to efficiently and specifically correct a clinically relevant mutation in human SERPINA1 gene in vivo in mice.
Example 22: Evaluating Efficacy of Rewriting Activity of Optimized Human Template RNA Combined with Optimized mRNA/Polypeptide Construct in Correcting the E342K Mutation in hSERPINAl E342K Transgenic Mice
This example describes the use of an exemplary gene modifying system containing an optimized gene modifying polypeptide and optimized template RNA to evaluate editing efficacy at multiple dose levels for correction of the PiZ mutation (corresponding to a A>G base change) in the human SERPINA1 (hSERPINAl) gene in vivo in mice modified to carry hSERPTNAl *E342K (PiZ) encoding alpha- 1 -antitrypsin (A1AT) protein.
In this example, a template RNA contained:
• a gRNA spacer;
• a gRNA scaffold;
• a heterologous object sequence; and
• a primer binding site (PBS) sequence.
In this example, a gene modifying polypeptide contained:
• an endonuclease and/or DNA binding domain;
• a peptide linker; and
• a reverse transcriptase (RT) domain.
Exemplary template RNAs generated are given in Table 61. Nucleotide modifications are noted as follows: phosphorothioate linkages denoted by an asterisk, 2’-O-methyl groups denoted by an ‘m’ preceding a nucleotide, 2-Fluororibose denoted by an ‘i2F’ preceding a nucleotide. Exemplary gene modifying polypeptide was RNA1VT9315 (comprising a AV1RE RT domain and WQAAESYEV linker (SEQ ID NO: 30005)), comprising the amino acid sequence of SEQ ID NO: 31455.
Gene modifying systems comprising mRNA encoding RNAIVT9315 gene modifying polypeptide and RNACS24757 template RNA described above were formulated in LNP and delivered to mice. Specifically, single administration of increasing dose levels (0.01, 0.025, 0.05, 0.1 , 0.25, 0.5 and 1 mg/kg) of total RNA equivalent formulated in LNPs (6:1 N:P ratio for mRNA and tgRNA), combined at 2:1 (w/w) of mRNA and template RNA were dosed intravenously in about 8-week-old hSERPINAl E342K mice in a 10 ml/kg bolus. Mice were administered a dose at time 0 (t = 0). 7 days post-dosing (as used herein post-dosing refers to time since the first dose), animals were sacrificed, and their liver and serum are collected for analyses.
Liver samples were analyzed by using Amp-Seq to determine % rewriting and % indels in target liver cells. To analyze gene editing activity, primers flanking the target mutation site locus were used to amplify across the locus in the genomic DNA and in the mRNA transcript of liver samples. Amplicons were analyzed via short read sequencing using an Illumina NovaSeq sequencing system. Conversion of an A nucleotide to a G nucleotide at position c.1096 in exon 5 in the human SERPINA1 gene indicated successful editing.
FIGs. 27A-27C show graphs of % corrected gDNA editing (FIG. 27A), % corrected mRNA (FIG. 27B) and % indels ( FIG. 27C) in the liver of hSERPINAl E342K mice as measured by Amp-Seq. The results showed (FIG. 27A) that the evaluated gene modifying system facilitated high levels of editing in a dose dependent manner. The maximum rewriting activity around 55% was observed at 0.25 mg/kg dose. FIG. 27B shows the level of corrected mRNA transcripts in hSERPINAl E342K mice reaches about 88% rewriting. FIG. 27C shows less than 0.75% indel activity for each condition in hSERPINAl E342K mouse liver. The results demonstrate that exemplary gene modifying systems containing optimized gene modifying polypeptide and optimized tgRNA enable efficient rewriting in vivo and can be used to specifically correct a clinically relevant mutation in human SERPINA1 gene in vivo in mice.
Serum samples were analyzed using Human alpha 1 Antitrypsin ELISA Kit (ab 108799) following the manufacturer's instructions to determine the circulating hAlAT level. Transgenic mice harboring the E342K mutation in the human SERPINA1 gene that causes misfolding and aggregation of the protein inside the hepatocytes exhibit low circulating Al AT levels in serum. Successful rewriting of the hSERPINAl transgene would be expected to result in a substantial increase of Al AT level by rescued secretion from liver into blood.
FIGs. 28A-28B show human A1AT levels in serum from treated mice (FIG. 28A) and a correlation plot of A1AT levels and rewriting levels from FIG. 27A (FIG. 28B). The results demonstrate that the serum hAlAT levels increased in a dose dependent manner. Serum hAlAT concentration reached maximal levels at 0.25 mg/kg dosage resulting in a 3-fold increase in hSERPINAl E342K mice. FIG. 28B indicates a strong correlation between A1AT levels and rewriting activity. These results show that exemplary gene modifying systems targeting mutant hSERPINAl in vivo can achieve highly efficient editing that results in measurable phenotype differences.
EQUIVALENTS
It should be understood that for all numerical bounds describing some parameter in this application, such as “about,” “at least,” “less than,” and “more than,” the description also necessarily encompasses any range bounded by the recited values. Accordingly, for example, the description “at least 1, 2, 3, 4, or 5” also describes, inter alia, the ranges 1-2, 1-3, 1-4, 1-5, 2- 3, 2-4, 2-5, 3-4, 3-5, and 4-5, et cetera.
For all patents, applications, or other reference cited herein, such as non-patent literature and reference sequence information, it should be understood that they are incorporated by reference in their entirety for all purposes as well as for the proposition that is recited. Where any conflict exists between a document incorporated by reference and the present application, this application will control. All information associated with reference gene sequences disclosed in this application, such as GenelDs or accession numbers (typically referencing NCBI accession numbers), including, for example, genomic loci, genomic sequences, functional annotations, allelic variants, and reference mRNA (including, e.g., exon boundaries or response elements) and protein sequences (such as conserved domain structures), as well as chemical references (e.g., PubChem compound, PubChem substance, or PubChem Bioassay entries, including the annotations therein, such as structures and assays, et cetera), are hereby incorporated by reference in their entirety.
Headings used in this application are for convenience only and do not affect the interpretation of this application.
Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. The scope of the present invention is not intended to be limited to the above Description, but rather is as set forth in the following claims.

Claims

1. A nucleic acid molecule comprising: an StlCas9 scaffold; wherein the StlCas9 scaffold comprises a chemically modified nucleotide at one or more of (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38 or all of) positions 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, and 42 relative to SEQ ID NO: 25999.
2. A nucleic acid molecule comprising: an StlCas9 scaffold comprising: a) a Repeat: anti-repeat (RAR) region, wherein optionally the RAR region comprises a RAR lower stem, a RAR upper stem, and an RAR loop (e.g., a tetraloop); b) a stem-loop 1 (SL1) region that is optionally 3’ of the RAR region, and c) optionally, a stem loop 2 (SL2) region that is optionally 3’ of the SL1 region; wherein the StlCas9 scaffold comprises a chemically modified nucleotide in one or both of the RAR region or the SL1 region.
3. The nucleic acid of claim 1 or 2, wherein at least 15-20%, 20-30%, 30-40%, 40-50%, 50- 60%, 60-70, or 70-75% of nucleotides in the StlCas9 scaffold are chemically modified.
4. The nucleic acid of any of the preceding claims, wherein the StlCas9 scaffold comprises a RAR region, wherein the RAR region comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or 31 chemically modified nucleotides (e.g., wherein the chemically modified nucleotides have the same chemical modification).
5. The nucleic acid of any of the preceding claims, wherein the StlCas9 scaffold comprises a SL1 region, wherein the SL1 region comprises 1, 2, 3, 4, 5, 6, 7, or 8 chemically modified nucleotides (e.g., wherein the chemically modified nucleotides have the same chemical modification).
6. The nucleic acid of any of the preceding claims, wherein positions 1, 2, and 3 (if present) do not comprise a 2’-O-methyl chemically modified nucleotide.
7. The nucleic acid of any of the preceding claims, wherein positions 43 through 54 (if present) do not comprise a 2’-O-methyl chemically modified nucleotide.
8. The nucleic acid of any of the preceding claims, wherein the StlCas9 scaffold comprises a chemically modified nucleotide at each of positions 4 through 6 (if present).
9. The nucleic acid of any of the preceding claims, wherein the StlCas9 scaffold comprises a chemically modified nucleotide at each of positions 13 through 15 (if present).
10. The nucleic acid of any of the preceding claims, wherein the StlCas9 scaffold comprises a chemically modified nucleotide at each of positions 16 through 18 (if present).
11. The nucleic acid of any of the preceding claims, wherein the StlCas9 scaffold comprises a chemically modified nucleotide at each of positions 19 through 21 (if present).
12. The nucleic acid of any of the preceding claims, wherein the StlCas9 scaffold comprises a chemically modified nucleotide at each of positions 22 through 24 (if present).
13. The nucleic acid of any of the preceding claims, wherein the StlCas9 scaffold comprises a chemically modified nucleotide at each of positions 34 through 36 (if present).
14. The nucleic acid of any of the preceding claims, wherein the StlCas9 scaffold comprises a chemically modified nucleotide at each of positions 37 through 39 (if present).
15. The nucleic acid of any of the preceding claims, wherein the StlCas9 scaffold comprises a chemically modified nucleotide at each of positions 40 through 42 (if present).
16. The nucleic acid of any of the preceding claims, wherein the StlCas9 scaffold comprises a chemically modified nucleotide at each of positions 13 through 24 (if present).
17. The nucleic acid of any of the preceding claims, wherein the StlCas9 scaffold comprises a chemically modified nucleotide at each of positions 34 through 42 (if present).
18. The nucleic acid of any of the preceding claims, wherein the chemically modified nucleotide is a modification to a sugar group, e.g., a modification to the 2’-0 of ribose, e.g., a 2’-O-Methyl chemically modified nucleotide.
19. The nucleic acid of any of the preceding claims, which further comprises a second chemically modified nucleotide.
20. The nucleic acid of any of the preceding claims, which comprises a sequence according to SEQ ID NO: 26000, or a sequence having at least 70%, 80%, 90%, 95%, 97%, 98%, or 99% identity thereto.
21. The nucleic acid of any of the preceding claims, wherein the StlCas9 scaffold binds to an StlCas9 protein having a sequence of SEQ ID NO: 23818.
22. A nucleic acid comprising a Repeat: anti-repeat (RAR) region which comprises a chemically modified nucleotide.
23. A nucleic acid comprising a stem-loop 1 (SL1) region which comprises a chemically modified nucleotide.
24. A nucleic acid molecule comprising: an StlCas9 scaffold; wherein the StlCas9 scaffold comprises a chemically modified nucleotide.
25. A template RNA comprising (tgRNA) comprising, from 5’ to 3’:
(1) a gRNA spacer;
(2) a chemically modified StlCas9 scaffold comprising a nucleic acid of any of claims 1-
24;
(3) a heterologous object sequence; and
(4) a primer binding site (PBS) sequence.
26. A system comprising: a nucleic acid of any of claims 1-24 or a template RNA of claim 25; and a polypeptide comprising a StlCas9 domain, or a nucleic acid encoding the polypeptide.
27. A gene modifying system comprising: a template RNA of claim 25 ; and a gene modifying polypeptide, or a nucleic acid encoding the gene modifying polypeptide, the gene modifying polypeptide comprising:
(1) a StlCas9 domain;
(2) a linker; and
(3) a reverse transcriptase (RT) domain.
28. A pharmaceutical composition, comprising the nucleic acid or template RNA of any one of claims 1-25 or the system of claim 27, and a pharmaceutically acceptable excipient or carrier.
29. A host cell (e.g., a mammalian cell, e.g., a human cell) comprising the gene modifying system, template RNA, or nucleic acid of any one of the preceding claims.
30. A method of making the nucleic acid or template RNA of any one of claims 1-25, the method comprising synthesizing the template RNA in vitro (e.g., by in vitro transcription or solid state synthesis).
31. A method for modifying a target site (c.g., a target site in the human SERPINA1 gene) in a cell, the method comprising contacting the cell with the gene modifying system of claim 27, or DNA encoding the same, or the pharmaceutical composition of claim 28, thereby modifying the target site.
32. A method for treating a subject having a disease or condition associated with a mutation in a gene (e.g., the human SERPINA1 gene), the method comprising administering to the subject the gene modifying system of claim 27, or DNA encoding the same, or the pharmaceutical composition of claim 28, thereby treating the subject having a disease or condition.
33. A method for treating a subject having AATD, the method comprising administering to the subject the gene modifying system of claim 27, or DNA encoding the same, or the pharmaceutical composition of claim 28, thereby treating the subject having AATD.
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Citations (53)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US789538A (en) 1904-11-11 1905-05-09 Colin E Ham Dumb-bell.
WO1995019431A1 (en) 1994-01-18 1995-07-20 The Scripps Research Institute Zinc finger protein derivatives and methods therefor
WO1996006166A1 (en) 1994-08-20 1996-02-29 Medical Research Council Improvements in or relating to binding proteins for recognition of dna
US5789538A (en) 1995-02-03 1998-08-04 Massachusetts Institute Of Technology Zinc finger proteins with high affinity new DNA binding specificities
WO1998037186A1 (en) 1997-02-18 1998-08-27 Actinova Limited In vitro peptide or protein expression library
WO1998053058A1 (en) 1997-05-23 1998-11-26 Gendaq Limited Nucleic acid binding proteins
WO1998053059A1 (en) 1997-05-23 1998-11-26 Medical Research Council Nucleic acid binding proteins
WO1998054311A1 (en) 1997-05-27 1998-12-03 The Scripps Research Institute Zinc finger protein derivatives and methods therefor
US5925523A (en) 1996-08-23 1999-07-20 President & Fellows Of Harvard College Intraction trap assay, reagents and uses thereof
GB2338237A (en) 1997-02-18 1999-12-15 Actinova Ltd In vitro peptide or protein expression library
WO2000027878A1 (en) 1998-11-09 2000-05-18 Gendaq Limited Screening system for zinc finger polypeptides for a desired binding ability
US6242568B1 (en) 1994-01-18 2001-06-05 The Scripps Research Institute Zinc finger protein derivatives and methods therefor
WO2001060970A2 (en) 2000-02-18 2001-08-23 Toolgen, Inc. Zinc finger domains and methods of identifying same
WO2001068895A1 (en) 2000-03-15 2001-09-20 Invitrogen Corporation High fidelity reverse transcriptases and uses thereof
WO2001088197A2 (en) 2000-05-16 2001-11-22 Massachusetts Institute Of Technology Methods and compositions for interaction trap assays
WO2002016536A1 (en) 2000-08-23 2002-02-28 Kao Corporation Bactericidal antifouling detergent for hard surface
US6410248B1 (en) 1998-01-30 2002-06-25 Massachusetts Institute Of Technology General strategy for selecting high-affinity zinc finger proteins for diverse DNA target sites
US6453242B1 (en) 1999-01-12 2002-09-17 Sangamo Biosciences, Inc. Selection of sites for targeting by zinc finger proteins and methods of designing zinc finger proteins to bind to preselected sites
WO2002077227A2 (en) 2000-11-20 2002-10-03 Sangamo Biosciences, Inc. Iterative optimization in the design of binding proteins
US6479626B1 (en) 1998-03-02 2002-11-12 Massachusetts Institute Of Technology Poly zinc finger proteins with improved linkers
WO2002099084A2 (en) 2001-04-04 2002-12-12 Gendaq Limited Composite binding polypeptides
US6503717B2 (en) 1999-12-06 2003-01-07 Sangamo Biosciences, Inc. Methods of using randomized libraries of zinc finger proteins for the identification of gene function
WO2003016496A2 (en) 2001-08-20 2003-02-27 The Scripps Research Institute Zinc finger binding domains for cnn
US6534261B1 (en) 1999-01-12 2003-03-18 Sangamo Biosciences, Inc. Regulation of endogenous gene expression in cells using zinc finger proteins
US6599692B1 (en) 1999-09-14 2003-07-29 Sangamo Bioscience, Inc. Functional genomics using zinc finger proteins
US6689558B2 (en) 2000-02-08 2004-02-10 Sangamo Biosciences, Inc. Cells for drug discovery
US20050064474A1 (en) 2003-08-08 2005-03-24 Sangamo Biosciences, Inc. Methods and compositions for targeted cleavage and recombination
US20050267061A1 (en) 2004-04-08 2005-12-01 Sangamo Biosciences, Inc. Methods and compositions for treating neuropathic and neurodegenerative conditions
US7030215B2 (en) 1999-03-24 2006-04-18 Sangamo Biosciences, Inc. Position dependent recognition of GNN nucleotide triplets by zinc fingers
US7067317B2 (en) 2000-12-07 2006-06-27 Sangamo Biosciences, Inc. Regulation of angiogenesis with zinc finger proteins
US7070934B2 (en) 1999-01-12 2006-07-04 Sangamo Biosciences, Inc. Ligand-controlled regulation of endogenous gene expression
US7253273B2 (en) 2004-04-08 2007-08-07 Sangamo Biosciences, Inc. Treatment of neuropathic pain with zinc finger proteins
US7262054B2 (en) 2002-01-22 2007-08-28 Sangamo Biosciences, Inc. Zinc finger proteins for DNA binding and gene regulation in plants
US20070218528A1 (en) 2004-02-05 2007-09-20 Sangamo Biosciences, Inc. Methods and compositions for targeted cleavage and recombination
US7361635B2 (en) 2002-08-29 2008-04-22 Sangamo Biosciences, Inc. Simultaneous modulation of multiple genes
WO2010028347A2 (en) 2008-09-05 2010-03-11 President & Fellows Of Harvard College Continuous directed evolution of proteins and nucleic acids
WO2012088381A2 (en) 2010-12-22 2012-06-28 President And Fellows Of Harvard College Continuous directed evolution
US8394604B2 (en) 2008-04-30 2013-03-12 Paul Xiang-Qin Liu Protein splicing using short terminal split inteins
WO2014004336A2 (en) 2012-06-27 2014-01-03 The Trustees Of Princeton University Split inteins, conjugates and uses thereof
WO2015134121A2 (en) 2014-01-20 2015-09-11 President And Fellows Of Harvard College Negative selection and stringency modulation in continuous evolution systems
WO2016044416A1 (en) 2014-09-16 2016-03-24 Sangamo Biosciences, Inc. Methods and compositions for nuclease-mediated genome engineering and correction in hematopoietic stem cells
WO2016168631A1 (en) 2015-04-17 2016-10-20 President And Fellows Of Harvard College Vector-based mutagenesis system
WO2017004279A2 (en) * 2015-06-29 2017-01-05 Massachusetts Institute Of Technology Compositions comprising nucleic acids and methods of using the same
WO2017132580A2 (en) 2016-01-29 2017-08-03 The Trustees Of Princeton University Split inteins with exceptional splicing activity
US20180127780A1 (en) 2016-10-14 2018-05-10 President And Fellows Of Harvard College Aav delivery of nucleobase editors
WO2018107028A1 (en) * 2016-12-08 2018-06-14 Intellia Therapeutics, Inc. Modified guide rnas
WO2019023680A1 (en) 2017-07-28 2019-01-31 President And Fellows Of Harvard College Methods and compositions for evolving base editors using phage-assisted continuous evolution (pace)
WO2019213776A1 (en) * 2018-05-11 2019-11-14 UNIVERSITé LAVAL Crispr/cas9 system and uses thereof
WO2020051561A1 (en) 2018-09-07 2020-03-12 Beam Therapeutics Inc. Compositions and methods for delivering a nucleobase editing system
WO2022081890A1 (en) * 2020-10-14 2022-04-21 Beam Therapeutics Inc. Compositions and methods for treating glycogen storage disease type 1a
WO2023039447A2 (en) * 2021-09-08 2023-03-16 Flagship Pioneering Innovations Vi, Llc Serpina-modulating compositions and methods
WO2023108153A2 (en) * 2021-12-10 2023-06-15 Flagship Pioneering Innovations Vi, Llc Cftr-modulating compositions and methods
WO2024192299A2 (en) * 2023-03-15 2024-09-19 Flagship Pioneering Innovations Vi, Llc Serpina-modulating compositions and methods

Patent Citations (67)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US789538A (en) 1904-11-11 1905-05-09 Colin E Ham Dumb-bell.
WO1995019431A1 (en) 1994-01-18 1995-07-20 The Scripps Research Institute Zinc finger protein derivatives and methods therefor
US6242568B1 (en) 1994-01-18 2001-06-05 The Scripps Research Institute Zinc finger protein derivatives and methods therefor
US6140466A (en) 1994-01-18 2000-10-31 The Scripps Research Institute Zinc finger protein derivatives and methods therefor
US6007988A (en) 1994-08-20 1999-12-28 Medical Research Council Binding proteins for recognition of DNA
WO1996006166A1 (en) 1994-08-20 1996-02-29 Medical Research Council Improvements in or relating to binding proteins for recognition of dna
US6013453A (en) 1994-08-20 2000-01-11 Medical Research Council Binding proteins for recognition of DNA
US5789538A (en) 1995-02-03 1998-08-04 Massachusetts Institute Of Technology Zinc finger proteins with high affinity new DNA binding specificities
US6200759B1 (en) 1996-08-23 2001-03-13 President And Fellows Of Harvard College Interaction trap assay, reagents and uses thereof
US5925523A (en) 1996-08-23 1999-07-20 President & Fellows Of Harvard College Intraction trap assay, reagents and uses thereof
WO1998037186A1 (en) 1997-02-18 1998-08-27 Actinova Limited In vitro peptide or protein expression library
GB2338237A (en) 1997-02-18 1999-12-15 Actinova Ltd In vitro peptide or protein expression library
WO1998053057A1 (en) 1997-05-23 1998-11-26 Gendaq Limited Nucleic acid binding polypeptide library
WO1998053060A1 (en) 1997-05-23 1998-11-26 Gendaq Limited Nucleic acid binding proteins
WO1998053059A1 (en) 1997-05-23 1998-11-26 Medical Research Council Nucleic acid binding proteins
WO1998053058A1 (en) 1997-05-23 1998-11-26 Gendaq Limited Nucleic acid binding proteins
WO1998054311A1 (en) 1997-05-27 1998-12-03 The Scripps Research Institute Zinc finger protein derivatives and methods therefor
US6410248B1 (en) 1998-01-30 2002-06-25 Massachusetts Institute Of Technology General strategy for selecting high-affinity zinc finger proteins for diverse DNA target sites
US6479626B1 (en) 1998-03-02 2002-11-12 Massachusetts Institute Of Technology Poly zinc finger proteins with improved linkers
US6903185B2 (en) 1998-03-02 2005-06-07 Massachusetts Institute Of Technology Poly zinc finger proteins with improved linkers
US7153949B2 (en) 1998-03-02 2006-12-26 Massachusetts Institute Of Technology Nucleic acid encoding poly-zinc finger proteins with improved linkers
WO2000027878A1 (en) 1998-11-09 2000-05-18 Gendaq Limited Screening system for zinc finger polypeptides for a desired binding ability
US6534261B1 (en) 1999-01-12 2003-03-18 Sangamo Biosciences, Inc. Regulation of endogenous gene expression in cells using zinc finger proteins
US7070934B2 (en) 1999-01-12 2006-07-04 Sangamo Biosciences, Inc. Ligand-controlled regulation of endogenous gene expression
US6453242B1 (en) 1999-01-12 2002-09-17 Sangamo Biosciences, Inc. Selection of sites for targeting by zinc finger proteins and methods of designing zinc finger proteins to bind to preselected sites
US7030215B2 (en) 1999-03-24 2006-04-18 Sangamo Biosciences, Inc. Position dependent recognition of GNN nucleotide triplets by zinc fingers
US6599692B1 (en) 1999-09-14 2003-07-29 Sangamo Bioscience, Inc. Functional genomics using zinc finger proteins
US6503717B2 (en) 1999-12-06 2003-01-07 Sangamo Biosciences, Inc. Methods of using randomized libraries of zinc finger proteins for the identification of gene function
US6689558B2 (en) 2000-02-08 2004-02-10 Sangamo Biosciences, Inc. Cells for drug discovery
WO2001060970A2 (en) 2000-02-18 2001-08-23 Toolgen, Inc. Zinc finger domains and methods of identifying same
WO2001068895A1 (en) 2000-03-15 2001-09-20 Invitrogen Corporation High fidelity reverse transcriptases and uses thereof
WO2001088197A2 (en) 2000-05-16 2001-11-22 Massachusetts Institute Of Technology Methods and compositions for interaction trap assays
WO2002016536A1 (en) 2000-08-23 2002-02-28 Kao Corporation Bactericidal antifouling detergent for hard surface
US6794136B1 (en) 2000-11-20 2004-09-21 Sangamo Biosciences, Inc. Iterative optimization in the design of binding proteins
WO2002077227A2 (en) 2000-11-20 2002-10-03 Sangamo Biosciences, Inc. Iterative optimization in the design of binding proteins
US7067317B2 (en) 2000-12-07 2006-06-27 Sangamo Biosciences, Inc. Regulation of angiogenesis with zinc finger proteins
WO2002099084A2 (en) 2001-04-04 2002-12-12 Gendaq Limited Composite binding polypeptides
WO2003016496A2 (en) 2001-08-20 2003-02-27 The Scripps Research Institute Zinc finger binding domains for cnn
US7262054B2 (en) 2002-01-22 2007-08-28 Sangamo Biosciences, Inc. Zinc finger proteins for DNA binding and gene regulation in plants
US7361635B2 (en) 2002-08-29 2008-04-22 Sangamo Biosciences, Inc. Simultaneous modulation of multiple genes
US20050064474A1 (en) 2003-08-08 2005-03-24 Sangamo Biosciences, Inc. Methods and compositions for targeted cleavage and recombination
US20070218528A1 (en) 2004-02-05 2007-09-20 Sangamo Biosciences, Inc. Methods and compositions for targeted cleavage and recombination
US7253273B2 (en) 2004-04-08 2007-08-07 Sangamo Biosciences, Inc. Treatment of neuropathic pain with zinc finger proteins
US20050267061A1 (en) 2004-04-08 2005-12-01 Sangamo Biosciences, Inc. Methods and compositions for treating neuropathic and neurodegenerative conditions
US8394604B2 (en) 2008-04-30 2013-03-12 Paul Xiang-Qin Liu Protein splicing using short terminal split inteins
WO2010028347A2 (en) 2008-09-05 2010-03-11 President & Fellows Of Harvard College Continuous directed evolution of proteins and nucleic acids
US9771574B2 (en) 2008-09-05 2017-09-26 President And Fellows Of Harvard College Apparatus for continuous directed evolution of proteins and nucleic acids
US9023594B2 (en) 2008-09-05 2015-05-05 President And Fellows Of Harvard College Continuous directed evolution of proteins and nucleic acids
WO2012088381A2 (en) 2010-12-22 2012-06-28 President And Fellows Of Harvard College Continuous directed evolution
US9394537B2 (en) 2010-12-22 2016-07-19 President And Fellows Of Harvard College Continuous directed evolution
US20150344549A1 (en) 2012-06-27 2015-12-03 The Trustees Of Princeton University Split inteins, conjugates and uses thereof
WO2014004336A2 (en) 2012-06-27 2014-01-03 The Trustees Of Princeton University Split inteins, conjugates and uses thereof
US10179911B2 (en) 2014-01-20 2019-01-15 President And Fellows Of Harvard College Negative selection and stringency modulation in continuous evolution systems
WO2015134121A2 (en) 2014-01-20 2015-09-11 President And Fellows Of Harvard College Negative selection and stringency modulation in continuous evolution systems
WO2016044416A1 (en) 2014-09-16 2016-03-24 Sangamo Biosciences, Inc. Methods and compositions for nuclease-mediated genome engineering and correction in hematopoietic stem cells
WO2016168631A1 (en) 2015-04-17 2016-10-20 President And Fellows Of Harvard College Vector-based mutagenesis system
WO2017004279A2 (en) * 2015-06-29 2017-01-05 Massachusetts Institute Of Technology Compositions comprising nucleic acids and methods of using the same
WO2017132580A2 (en) 2016-01-29 2017-08-03 The Trustees Of Princeton University Split inteins with exceptional splicing activity
US20180127780A1 (en) 2016-10-14 2018-05-10 President And Fellows Of Harvard College Aav delivery of nucleobase editors
WO2018107028A1 (en) * 2016-12-08 2018-06-14 Intellia Therapeutics, Inc. Modified guide rnas
WO2019023680A1 (en) 2017-07-28 2019-01-31 President And Fellows Of Harvard College Methods and compositions for evolving base editors using phage-assisted continuous evolution (pace)
WO2019213776A1 (en) * 2018-05-11 2019-11-14 UNIVERSITé LAVAL Crispr/cas9 system and uses thereof
WO2020051561A1 (en) 2018-09-07 2020-03-12 Beam Therapeutics Inc. Compositions and methods for delivering a nucleobase editing system
WO2022081890A1 (en) * 2020-10-14 2022-04-21 Beam Therapeutics Inc. Compositions and methods for treating glycogen storage disease type 1a
WO2023039447A2 (en) * 2021-09-08 2023-03-16 Flagship Pioneering Innovations Vi, Llc Serpina-modulating compositions and methods
WO2023108153A2 (en) * 2021-12-10 2023-06-15 Flagship Pioneering Innovations Vi, Llc Cftr-modulating compositions and methods
WO2024192299A2 (en) * 2023-03-15 2024-09-19 Flagship Pioneering Innovations Vi, Llc Serpina-modulating compositions and methods

Non-Patent Citations (61)

* Cited by examiner, † Cited by third party
Title
"American thoracic society/European respiratory society", AM J RESPIR CRIT CARE MED, vol. 168, no. 7, 2003, pages 818 - 900
"UniProt", Database accession no. AOA142BKH1
"UniProtKB", Database accession no. P03360
AAMIR MIR ET AL: "Heavily and fully modified RNAs guide efficient SpyCas9-mediated genome editing", NATURE COMMUNICATIONS, vol. 9, no. 1, 6 July 2018 (2018-07-06), pages 1 - 9, XP055580482, DOI: 10.1038/s41467-018-05073-z *
ANZALONE ET AL., NATURE, vol. 576, 2019, pages 149 - 157
ASMARI ET AL., METHODS, vol. 146, 2018, pages 107 - 119
BEERLI ET AL., NATURE BIOTECHNOL., vol. 20, 2002, pages 135 - 141
BIBILLOEICKBUSH, J BIOL CHEM, vol. 277, no. 38, 2002, pages 34836 - 34845
BIRBACH ET AL., JOURNAL OF CELL SCIENCE, vol. 117, no. 3615-3624, 2004
BOTHMER ET AL., NAT COMMUN, vol. 8, 2017, pages 13905
CHAUDHRYWEINFELD, NUCLEIC ACIDS RES, vol. 23, no. 19, 1995, pages 3805 - 3809
CHEN ET AL., SCIENCE, vol. 360, no. 6387, 2018, pages 436 - 439
CHOO ET AL., CURR. OPIN. STRUCT. BIOL., vol. 10, 2000, pages 411 - 416
CHRISTENSEN ET AL., PNAS, 2006
COLLIAS ET AL., NATURE COMMUNICATIONS, vol. 12, 2021, pages 555
ELACQUA ET AL., BIORXIV, 2019
FONFARA ET AL., NUCLEIC ACIDS RES, vol. 40, no. 2, 2012, pages 847 - 860
FREGONESESTOLK, ORPHANET J RARE DIS., vol. 33, 2008, pages 16
GOVINDARAJU ET AL., NUCLEIC ACIDS RESEARCH, vol. 44, 2016, pages 7
GUHAEDGELL, INT J MOL SCI, vol. 18, no. 22, 2017, pages 2565
HAO YIN ET AL: "Structure-guided chemical modification of guide RNA enables potent non-viral in vivo genome editing", NATURE BIOTECHNOLOGY, vol. 35, no. 12, 13 November 2017 (2017-11-13), New York, pages 1179 - 1187, XP055484407, ISSN: 1087-0156, DOI: 10.1038/nbt.4005 *
HENDEL ET AL., NATURE BIOTECHNOL., 2015, pages 985 - 991
HERSCHHORNHIZI, CELL MOL LIFE SCI, vol. 67, no. 16, 2010, pages 2717 - 2747
ISALAN ET AL., NATURE BIOTECHNOL., vol. 19, 2001, pages 656 - 660
IVICS ET AL., CELL, 1997, pages 501 - 510
JAMBURUTHUGODAEICKBUSH, J MOL BIOL, vol. 407, no. 5, 2011, pages 661 - 672
JIANG, SCIENCE, vol. 351, 2016, pages 867 - 871
KARST ET AL., BIORXIV, 2020
KLEINSTIVER ET AL., PNAS, 2012
KOTEWICZ ET AL., NUCLEIC ACIDS RES, vol. 16, no. 1, 1988, pages 265 - 277
LIN ET AL., BMC BIOINFORMAT, vol. 13, 2012, pages 157
LINMILES, NUCLEIC ACIDS RES, vol. 47, no. 11, 2019, pages 5490 - 5501
LUBELSKYULITSKY, NATURE, vol. 555, no. 107-111, 2018
MAHBUB ET AL., MOBILE DNA, vol. 8, 2017, pages 16
MCCONNELL SMITH ET AL., PNAS, vol. 106, no. 48, 2009, pages 20294 - 20299
MINCZUK ET AL., NUCLEIC ACIDS RES, vol. 36, no. 12, 2008, pages 3926 - 3938
MIYAGAWA ET AL., RNA, vol. 18, no. 738-751, 2012
MOLINA ET AL., J BIOL CHEM, 2015
MULEPATI ET AL., SCIENCE, vol. 345, 19 September 2014 (2014-09-19), pages 1479 - 1484
NISHIMASU ET AL., CELL, vol. 156, 2014, pages 935 - 949
NIU ET AL., J MOL BIOL, 2008
OGUSHI ET AL., J CLIN INVEST., vol. 80, no. 5, 1987, pages 1366 - 74
PABO ET AL., ANN. REV. BIOCHEM., vol. 70, 2001, pages 313 - 340
PIITULAINENTANASH, COPD, vol. 12, no. 1, 2015, pages 36 - 41
REMADE ET AL., EMBO JOURNAL, vol. 18, no. 18, 1999, pages 5073 - 5084
RODRIGUEZ-FORNES ET AL., GENE THERAPY, 2020
SEGAL ET AL., CURR. OPIN. BIOTECHNOL., vol. 12, 2001, pages 632 - 637
SHAH ET AL., CHEM SCI., vol. 5, no. 1, 2014, pages 446 - 461
SHUKLA ET AL., THE EMBO JOURNAL, 2018, pages 98452
STEVENS ET AL., J AM CHEM SOC., vol. 138, no. 7, 24 February 2016 (2016-02-24), pages 2162 - 5
SUZUKI T. ET AL.: "Crystal structures reveal an elusive functional domain of pyrrolysyl-tRNA synthetase", NAT CHEM BIOL., vol. 13, no. 12, 2017, pages 1261 - 1266, XP055915912, DOI: 10.1038/nchembio.2497
TANASH ET AL., INT J CL-IRON OBSTRUCT PULM DIS., vol. 11, 2016, pages 1663 - 9
WAGSTAFF ET AL., MOLECULAR BIOLOGY AND EVOLUTION, 2013, pages 88 - 99
WOLFS ET AL., NUCLEIC ACIDS RES, 2014
WOLFS ET AL., PNAS, 2016
WOOD ET AL., J. BIOL. CHEM., vol. 289, no. 21, 2014, pages 14512 - 9
YANG ET AL., JOURNAL OF BIOMEDICAL SCIENCE, vol. 22, no. 33, 2015
YASUKAWA ET AL., BIOCHEM BIOPHYS RES COMMUN, vol. 492, no. 2, 2017, pages 147 - 153
YOURIK, RNA, vol. 25, no. 1, 2019, pages 35 - 44
ZETSCHE ET AL., CELL, vol. 163, 2015, pages 759 - 771
ZHANG ET AL., MOLECULAR AND CELLULAR BIOLOGY, vol. 34, 2014, pages 2318 - 2329

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