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WO2025017033A1 - Édition primaire de la région 115 dans le promoteur hbg1 et/ou hbg2 pour augmenter la teneur en hémoglobine fœtale dans une cellule eucaryote - Google Patents

Édition primaire de la région 115 dans le promoteur hbg1 et/ou hbg2 pour augmenter la teneur en hémoglobine fœtale dans une cellule eucaryote Download PDF

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WO2025017033A1
WO2025017033A1 PCT/EP2024/070167 EP2024070167W WO2025017033A1 WO 2025017033 A1 WO2025017033 A1 WO 2025017033A1 EP 2024070167 W EP2024070167 W EP 2024070167W WO 2025017033 A1 WO2025017033 A1 WO 2025017033A1
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seq
sequence
prime editing
rna
prime
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Annarita MICCIO
Anne CHALUMEAU
Mégane BRUSSON
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Assistance Publique Hopitaux de Paris APHP
Institut National de la Sante et de la Recherche Medicale INSERM
Fondation Imagine
Universite Paris Cite
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Assistance Publique Hopitaux de Paris APHP
Institut National de la Sante et de la Recherche Medicale INSERM
Fondation Imagine
Universite Paris Cite
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    • 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
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    • C12N9/1241—Nucleotidyltransferases (2.7.7)
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    • C12N9/14—Hydrolases (3)
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    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
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    • C12N2800/22—Vectors comprising a coding region that has been codon optimised for expression in a respective host

Definitions

  • the present invention is in the field of medicine, in particular haematology.
  • BACKGROUND OF THE INVENTION ⁇ -hemoglobinopathies, ⁇ -thalassemia and sickle cell disease (SCD), are monogenic diseases caused by mutations in the ⁇ -globin locus, affecting the synthesis or the structure of the adult hemoglobin (Hb).
  • ⁇ -thalassemia is caused by mutations in the ⁇ -globin gene (HBB) locus that reduce ( ⁇ +) or abolish ( ⁇ 0) the production of ⁇ -globin chains included in the adult hemoglobin (HbA) tetramer, leading to the precipitation of uncoupled ⁇ -globin chains, erythroid cell death and severe anemia (Taher, Ali T., David J. Weatherall, and Maria Domenica Cappellini. "Thalassaemia.” The Lancet 391.10116 (2016): 155-167).
  • HBB ⁇ -globin gene
  • HPFH mutations either generate de novo DNA motifs recognized by transcriptional activators (e.g., KLF1) (Wienert, Beeke, et al. "Editing the genome to introduce a beneficial naturally occurring mutation associated with increased fetal globin.” Nature communications 6.1 (2015): 1-8; Wienert, Beeke, et al.
  • KLF1 transcriptional activators
  • KLF1 drives the expression of fetal hemoglobin in British HPFH. Blood, The Journal of the American Society of Hematology 130.6 (2017): 803- 807; Martyn, Gabriella E., et al. "A natural regulatory mutation in the proximal promoter elevates fetal globin expression by creating a de novo GATA1 site.” Blood, The Journal of the American Society of Hematology 133.8 (2019): 852-856.) or disrupt binding sites (BS) for transcriptional repressors (e.g., LRF and BCL11A) (Martyn, Gabriella E., Kate GR Quinlan, and Merlin Crossley.
  • BS disrupt binding sites
  • ⁇ -hemoglobinopathy has its general meaning in the art and refers to any defect in the structure or function of any hemoglobin of an individual, and includes defects in the primary, secondary, tertiary or quaternary structure of hemoglobin caused by any mutation, such as deletion mutations or substitution mutations in the coding regions of the HBB gene, or mutations in, or deletions of, the promoters or enhancers of such gene that cause a reduction in the amount of hemoglobin produced as compared to a normal or standard condition.
  • the term "sickle cell disease” has its general meaning in the art and refers to a group of autosomal recessive genetic blood disorders, which results from mutations in a globin gene and which is characterized by red blood cells that assume an abnormal, rigid, sickle shape. They are defined by the presence of ⁇ S-globin gene coding for a ⁇ -globin chain variant in which glutamic acid is substituted by valine at amino acid position 6 of the peptide: incorporation of the ⁇ S-globin in the Hb tetramers (HbS, sickle Hb) leads to Hb polymerization and to a clinical phenotype.
  • HbSS sickle cell anemia
  • HbSC sickle-hemoglobin C disease
  • HbS/ ⁇ + sickle ⁇ -plus- thalassaemia
  • HbS/ ⁇ 0 sickle ⁇ -zerothalassaemia
  • ⁇ -thalassemia refers to a hemoglobinopathy that results from an altered ratio of ⁇ -globin to ⁇ -like globin polypeptide chains resulting in the underproduction of normal hemoglobin tetrameric proteins and the precipitation of free, unpaired ⁇ -globin chains.
  • alpha globin or “ ⁇ -globin” has its general meaning in the art and refers to protein that is encoded in human by the HBA1 and HBA2 genes.
  • the human alpha globin gene cluster located on chromosome 16 spans about 30 kb and includes seven loci: 5'- zeta - pseudozeta - mu - pseudoalpha-1 - alpha-2 - alpha-1 - theta - 3'.
  • the alpha-2 (HBA2) and alpha-1 (HBA1) coding sequences are identical. These genes differ slightly over the 5' untranslated regions and the introns, but they differ significantly over the 3' untranslated regions.
  • the ENSEMBL IDs i.e.
  • HBA1 and HBA2 are ENSG00000206172 and ENSG00000188536 respectively.
  • ⁇ -globin has its general meaning in the art and refers to a globin protein, which along with alpha globin (HBA), makes up the most common form of haemoglobin (Hb) in adult humans.
  • HBA alpha globin
  • Hb haemoglobin
  • Normal adult human Hb is a heterotetramer consisting of two alpha chains and two beta chains.
  • the ⁇ -globin is encoded by the HBB gene on human chromosome 11. It is 146 amino acids long and has a molecular weight of 15,867 Da.
  • gamma globin or “ ⁇ -globin” has its general meaning in the art and refers to protein that is encoded in human by the HBG1 and HBG2 genes.
  • the HBG1 and HBG2 genes are normally expressed in the fetal liver, spleen and bone marrow.
  • Two ⁇ -globin chains together with two ⁇ -globin chains constitute fetal hemoglobin (HbF) which is normally replaced by adult hemoglobin (HbA) in the year following birth.
  • the ENSEMBL IDs i.e. the gene identifier number from the Ensembl Genome Browser database
  • HBG1 and HBG2 are ENSG00000213934 and ENSG00000196565 respectively.
  • the term “expression” refers to the process by which a polynucleotide is transcribed from a DNA template (such as into and mRNA or other RNA transcript) and/or the process by which a transcribed mRNA is subsequently translated into peptides, polypeptides, or proteins. Transcripts and encoded polypeptides may be collectively referred to as “gene product”. If the polynucleotide is derived from genomic DNA, expression may include splicing of the mRNA in a eukaryotic cell. Any method known in the art can be used to measure the expression of the gene (e. g.
  • the expression "increasing the fetal hemoglobin content” indicates that fetal hemoglobin is at least 5% higher in the eukaryotic cell treated with the prime editing platform, than in a comparable, eukaryotic cell, wherein a prime editing platform targeting an unrelated locus is present or where no prime editing platform is present.
  • the percentage of fetal hemoglobin expression in the eukaryotic cell is at least 10% higher, at least 20% higher, at least 30% higher, at least 40% higher, at least 50% higher, at least 60% higher, at least 70% higher, at least 80% higher, at least 90% higher, at least 1-fold higher, at least 2- fold higher, at least 5-fold higher, at least 10-fold higher, at least 100 fold higher, at least 1000- fold higher, or more than an eukaryotic cell.
  • promoter has its general meaning in the art and refers to a nucleic acid sequence which is required for expression of a gene operably linked to the promoter sequence.
  • HBG1 promoter refers to the promoter of the HBG1 gene.
  • HBG2 promoter refers to the promoter of the HBG2 gene.
  • HBG1 and HBG2 promoters are identical up to –221 bp and comprise the nucleic acid sequence as set forth in SEQ ID NO:1 and depicted in Figure 1.
  • the first nucleotide in SEQ ID NO:1 denotes the nucleotide located at position -147 upstream of the HBG transcription starting site and the last nucleotide in SEQ ID NO:1 denotes the nucleotide located at position -91 upstream of the HBG transcription starting site.
  • the “-115 region” in the HBG1 or HBG2 promoter refers to the region which encompasses the nucleotides at position -123, -124 and -113 and thus relates to the region encompassing the region starting from the nucleotide at position 24 to the nucleotide at position 35 in SEQ ID NO:1.
  • the term “activator” refers to a transcriptional activator that is a protein (transcription factor) that increases gene transcription of a gene or set of genes.
  • activators are DNA-binding proteins that bind to enhancers or promoter-proximal elements.
  • the activator is KLF1 or GATA1.
  • transcriptional activator binding site refers to a site present on DNA whereby the transcriptional activator according to the present disclosure binds.
  • the prime-editing platform of the present invention edits the genome sequence of the eukaryotic cell so that the activator is able to bind to its transcriptional activator binding sites.
  • KLF1 has its general meaning in the art and refers to the Kruppel like factor 1 protein. The term is also known as EKLF; EKLF/KLF1.
  • KLF1 is a hematopoietic-specific transcription factor that induces high-level expression of adult beta-globin and other erythroid genes.
  • the zinc-finger protein binds to a DNA sequence found in the beta globin promoter.
  • GATA1 has its general meaning in the art and refers to the erythroid differentiation factor. The term is also known as ERYF1 or GF1.
  • repressor refers to a transcriptional repressor that is a protein (transcription factor) that decreases gene transcription of a gene or set of genes. Most repressors are DNA-binding proteins that bind to enhancers or promoter-proximal elements.
  • the repressor is BCL11A.
  • transcriptional repressor binding site refers to a site present on DNA whereby the transcription repressor binds.
  • the prime-editing platform of the present invention edits the genome sequence of the eukaryotic cell so that the transcriptional repressor is not able to bind to its transcriptional repressor binding sites.
  • the prime-editing platform of the present invention of the present invention will inhibit the binding of BCL11A to its binding site.
  • BCL11A has its general meaning in the art and refers to the gene encoding for BAF chromatin remodeling complex subunit BCL11A (Gene ID: 53335).
  • the term is also known as EVI9; CTIP1; DILOS; ZNF856; HBFQTL5; BCL11A-L; BCL11A-S; BCL11a-M; or BCL11A-XL.
  • the protein associates with the SWI/SNF complex that regulates gene expression via chromatin remodeling.
  • BCL11A is highly expressed in several hematopoietic lineages, and plays a role in the switch from ⁇ - to ⁇ -globin expression during the fetal to adult erythropoiesis transition (Sankaran VJ et al.
  • polypeptide “peptide”, and “protein” are used interchangeably herein to refer to polymers of amino acids of any length. The terms also encompass an amino acid polymer that has been modified; for example, disulfide bond formation, glycosylation, lipidation, phosphorylation, or conjugation with a labeling component.
  • Polypeptides when discussed in the context of gene therapy refer to the respective intact polypeptide, or any fragment or genetically engineered derivative thereof, which retains the desired biochemical function of the intact protein.
  • polynucleotide refers to a polymeric form of nucleotides of any length, including deoxyribonucleotides or ribonucleotides, or analogs thereof.
  • a polynucleotide may comprise modified nucleotides, such as methylated nucleotides and nucleotide analogs, and may be interrupted by non-nucleotide components. If present, modifications to the nucleotide structure may be imparted before or after assembly of the polymer.
  • the term polynucleotide, as used herein, refers interchangeably to double- and single-stranded molecules.
  • any embodiment of the invention described herein that is a polynucleotide encompasses both the double-stranded form and each of two complementary single-stranded forms known or predicted to make up the double-stranded form.
  • the expression “derived from” refers to a process whereby a first component (e.g., a first polypeptide), or information from that first component, is used to isolate, derive or make a different second component (e.g., a second polypeptide that is different from the first one).
  • fusion protein means a protein created by joining two or more polypeptide sequences together.
  • the fusion polypeptides encompassed in this invention include translation products of a chimeric gene construct that joins the nucleic acid sequences encoding a first polypeptide, e.g., an RNA-binding domain, with the nucleic acid sequence encoding a second polypeptide, e.g., an effector domain, to form a single open-reading frame.
  • a “fusion protein” is a recombinant protein of two or more proteins which are joined by a peptide bond or via several peptides.
  • the fusion protein may also comprise a peptide linker between the two domains.
  • the comparison of sequences and determination of percent identity between two sequences can be accomplished using a mathematical algorithm, as described below.
  • the percent identity between two amino acid sequences can be determined using the Needleman and Wunsch algorithm (Needleman, Saul B. & Wunsch, Christian D. (1970). "A general method applicable to the search for similarities in the amino acid sequence of two proteins". Journal of Molecular Biology.48 (3): 443–53.).
  • the percent identity between two nucleotide or amino acid sequences may also be determined using for example algorithms such as EMBOSS Needle (pair wise alignment; available at www.ebi.ac.uk).
  • EMBOSS Needle may be used with a BLOSUM62 matrix, a “gap open penalty” of 10, a “gap extend penalty” of 0.5, a false “end gap penalty”, an “end gap open penalty” of 10 and an “end gap extend penalty” of 0.5.
  • the “percent identity” is a function of the number of matching positions divided by the number of positions compared and multiplied by 100. For instance, if 6 out of 10 sequence positions are identical between the two compared sequences after alignment, then the identity is 60%.
  • % identity is typically determined over the whole length of the query sequence on which the analysis is performed.
  • Two molecules having the same primary amino acid sequence or nucleic acid sequence are identical irrespective of any chemical and/or biological modification.
  • a first amino acid sequence having at least 90% of identity with a second amino acid sequence means that the first sequence has 90; 91; 92; 93; 94; 95; 96; 97; 98; 99 or 100% of identity with the second amino acid sequence.
  • linker refers to any means, entity or moiety used to join two or more entities.
  • a linker can be a covalent linker or a non-covalent linker.
  • covalent linkers include covalent bonds or a linker moiety covalently attached to one or more of the proteins or domains to be linked.
  • the linker can also be a non-covalent bond, e.g., an organometallic bond through a metal center such as platinum atom.
  • various functionalities can be used, such as amide groups, including carbonic acid derivatives, ethers, esters, including organic and inorganic esters, amino, urethane, urea and the like.
  • the domains can be modified by oxidation, hydroxylation, substitution, reduction etc. to provide a site for coupling. Methods for conjugation are well known by persons skilled in the art and are encompassed for use in the present invention.
  • Linker moieties include, but are not limited to, chemical linker moieties, or for example a peptide linker moiety (a linker sequence). It will be appreciated that modification which do not significantly decrease the function of the RNA- binding domain and effector domain are preferred.
  • the “linked” as used herein refers to the attachment of two or more entities to form one entity.
  • a conjugate encompasses both peptide-small molecule conjugates as well as peptide-protein/peptide conjugates.
  • complementarity refers to the ability of a nucleic acid to form hydrogen bond(s) with another nucleic acid sequence by either traditional Watson-Crick base- pairing or other non-traditional types.
  • a percent complementarity indicates the percentage of residues in a nucleic acid molecule which can form hydrogen bonds (e.g., Watson-Crick base pairing) with a second nucleic acid sequence (e.g., 5, 6, 7, 8, 9, 10 out of 10 being 50%, 60%, 70%, 80%, 90%, and 100% complementary).
  • Perfectly complementary means that all the contiguous residues of a nucleic acid sequence will hydrogen bond with the same number of contiguous residues in a second nucleic acid sequence.
  • “Substantially complementary” as used herein refers to a degree of complementarity that is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% over a region of 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50, or more nucleotides, or refers to two nucleic acids that hybridize under stringent conditions.
  • stringent conditions for hybridization refer to conditions under which a nucleic acid having complementarity to a target sequence predominantly hybridizes with the target sequence, and substantially does not hybridize to non-target sequences. Stringent conditions are generally sequence-dependent, and vary depending on a number of factors.
  • hybridization or “hybridizing” refers to a process where completely or partially complementary nucleic acid strands come together under specified hybridization conditions to form a double-stranded structure or region in which the two constituent strands are joined by hydrogen bonds.
  • the technology can mediate targeted insertions, deletions, and base-to-base conversions without the need for double strand breaks (DSBs) or donor DNA templates.
  • the term “prime editing enzyme” refers to a fusion protein comprising a defective CRISPR/Cas nuclease linked to a reverse transcriptase. The term is also known as “prime editor”.
  • the term “nuclease” includes an enzyme that induces a break in a nucleic acid sequence, e.g., a single or a double strand break in a double-stranded DNA sequence.
  • CRISPR/Cas nuclease has its general meaning in the art and refers to segments of prokaryotic DNA containing clustered regularly interspaced short palindromic repeats (CRISPR) and associated nucleases encoded by Cas genes.
  • CRISPR clustered regularly interspaced short palindromic repeats
  • the CRISPR/Cas loci encode RNA-guided adaptive immune systems against mobile genetic elements (viruses, transposable elements and conjugative plasmids).
  • CRISPR clusters contain spacers, the sequences complementary to antecedent mobile elements.
  • CRISPR clusters are transcribed and processed into mature CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) RNA (crRNA).
  • the CRISPR/Cas nucleases Cas9 and Cpf1 belong to the type II and type V CRISPR/Cas system and have strong endonuclease activity to cut target DNA.
  • Cas9 is guided by a mature crRNA that contains about 20 nucleotides of unique target sequence (called spacer) and a trans-activating small RNA (tracrRNA) that also serves as a guide for ribonuclease III-aided processing of pre-crRNA.
  • spacer a mature crRNA that contains about 20 nucleotides of unique target sequence
  • tracrRNA trans-activating small RNA
  • the crRNA:tracrRNA duplex directs Cas9 to target DNA via complementary base pairing between the spacer on the crRNA and the complementary sequence (called protospacer) on the target DNA.
  • Cas9 recognizes a trinucleotide (NGG for S.
  • Cas9 Pyogenes Cas9 protospacer adjacent motif (PAM) to specify the cut site (the 3rd or the 4th nucleotide upstream from PAM).
  • Cas9 or “Cas9 nuclease” refers to an RNA-guided nuclease comprising a Cas9 protein, or a fragment thereof (e.g., a protein comprising an active or inactive DNA cleavage domain of Cas9, and/or the gRNA binding domain of Cas9).
  • a Cas9 nuclease is also referred to sometimes as a casn1 nuclease or a CRISPR (clustered regularly interspaced short palindromic repeat)-associated nuclease.
  • CRISPR is an adaptive immune system that provides protection against mobile genetic elements (viruses, transposable elements and conjugative plasmids).
  • CRISPR clusters contain spacers, sequences complementary to antecedent mobile elements, and target invading nucleic acids.
  • CRISPR clusters are transcribed and processed into CRISPR RNA (crRNA).
  • crRNA CRISPR RNA
  • type II CRISPR systems correct processing of pre-crRNA requires a trans-encoded small RNA (tracrRNA), endogenous ribonuclease 3 (rnc) and a Cas9 protein.
  • tracrRNA serves as a guide for ribonuclease 3-aided processing of pre- crRNA.
  • Cas9/crRNA/tracrRNA endonucleolytically cleaves linear or circular dsDNA target complementary to the spacer.
  • the target strand not complementary to crRNA is first cut endonucleolytically, then trimmed 3’-5’ exonucleolytically.
  • DNA-binding and cleavage typically requires protein and both RNAs.
  • single guide RNAs (“sgRNA”, or simply “gRNA”) can be engineered so as to incorporate aspects of both the crRNA and tracrRNA into a single RNA species. See, e.g., Jinek M., Chylinski K., Fonfara I., Hauer M., Doudna J. A., Charpentier E.
  • Cas9 recognizes a short motif in the CRISPR repeat sequences (the PAM or protospacer adjacent motif) to help distinguish self versus non-self.
  • Cas9 nuclease sequences and structures are well known to those of skill in the art (see, e.g., “Complete genome sequence of an M1 strain of Streptococcus pyogenes.” Ferretti et al., J. J., McShan W. M., Ajdic D. J., Savic D. J., Savic G., Lyon K., Primeaux C., Sezate S., Suvorov A. N., Kenton S., Lai H.
  • Cas9 nucleases and sequences include Cas9 sequences from the organisms and loci disclosed in Chylinski, Rhun, and Charpentier, “The tracrRNA and Cas9 families of type II CRISPR-Cas immunity systems” (2013) RNA Biology 10:5, 726-737; the entire contents of which are incorporated herein by reference.
  • Cas9 refers to Cas9 from: Corynebacterium ulcerans (NCBI Refs: NC_015683.1, NC_017317.1); Corynebacterium diphtheria (NCBI Refs: NC_016782.1, NC_016786.1); Spiroplasma syrphidicola (NCBI Ref: NC_021284.1); Prevotella intermedia (NCBI Ref: NC_017861.1); Spiroplasma taiwanense (NCBI Ref: NC_021846.1); Streptococcus iniae (NCBI Ref: NC_021314.1); Belliella baltica (NCBI Ref: NC_018010.1); Psychroflexus torquisI (NCBI Ref: NC_018721.1); Streptococcus thermophilus (NCBI Ref: YP_820832.1); Listeria innocua (NCBI Ref: NP_472073.1);
  • Cas9 nuclease comprises the amino acid sequence as set forth in SEQ ID NO: 2.
  • SEQ ID NO:2 Cas9 sequence MDKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGETAEATRLKRTAR RRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHL RKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVD AKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLD NLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQQ
  • nickase has its general meaning in the art and refers to an endonuclease which cleaves only a single strand of a DNA duplex (“nicking”). Accordingly, the term “Cas9 nickase” refers to a nickase derived from a Cas9 protein, typically by inactivating one nuclease domain of Cas9 protein.
  • protospacer adjacent motif sequence or “PAM” refers to an approximately 2-6 base pair DNA sequence that is an important targeting component of a Cas9 nuclease. Typically, the PAM sequence is on either strand, and is downstream in the 5’ to 3’ direction of Cas9 cut site.
  • the canonical PAM sequence i.e., the PAM sequence that is associated with the Cas9 nuclease of Streptococcus pyogenes is 5’-NGG-3’ wherein “N” is any nucleobase followed by two guanine (“G”) nucleobases.
  • Different PAM sequences can be associated with different Cas9 nucleases or equivalent proteins from different organisms.
  • any given Cas9 nuclease e.g., SpCas9, may be modified to alter the PAM specificity of the nuclease such that the nuclease recognizes alternative PAM sequence.
  • the term “protospacer” refers to the sequence ( ⁇ 20 bp) in DNA adjacent to the PAM (protospacer adjacent motif) sequence.
  • the protospacer shares the same sequence as the spacer sequence of the guide RNA.
  • the guide RNA anneals to the complement of the protospacer sequence on the target DNA (specifically, one strand thereof, i.e., the “target strand” versus the “non-target strand” of the target sequence).
  • PAM protospacer adjacent motif
  • guide RNA or “gRNA” has its general meaning in the art and is a particular type of guide nucleic acid which associates with a CRISPR/Cas nuclease (e.g. Cas9), directing the nuclease to a specific sequence in a DNA molecule that includes complementarity to protospacer sequence of the guide RNA.
  • CRISPR/Cas nuclease e.g. Cas9
  • the terms “prime editing guide RNA” or “pegRNA” refers to a specialized form of a guide RNA that has been modified to include one or more additional sequences for implementing the prime editing as described herein.
  • the pegRNAs of the present invention comprise in the 5’ to 3’ direction a spacer, a gRNA core, and an extension arm.
  • the pegRNA of the present invention may also further comprise elements, such as, but not limited to aptamers, stem loops, hairpins, toe loops (e.g., a 3’ toeloop), or an RNA-protein recruitment domain (e.g., MS2 hairpin).
  • the pegRNA may contain one or more structural elements for minimizing its degradation.
  • the pegRNA of the present invention incorporates one or more stable pseudoknots at its 3’ end such as a modified prequeosine1-1 riboswitch aptamer (evopreQ1) or the frameshifting pseudoknot from Moloney murine leukemia virus (MMLV), hereafter referred to as “mpknot” as described in Nelson, James W., et al. "Engineered pegRNAs improve prime editing efficiency.” Nature biotechnology 40.3 (2022): 402-410 for which the teaching is incorporated by reference.
  • the pegRNA may comprise a transcriptional termination sequence at the 3’ of the molecule.
  • the term “spacer” in connection with a guide RNA or a pegRNA refers to the portion of the guide RNA or pegRNA of about 20 nucleotides which contains a nucleotide sequence that is complementary to the protospacer sequence in the target nucleic sequence.
  • the spacer sequence anneals to the protospacer sequence to form a ssRNA/ssDNA hybrid structure at the target site and a corresponding R loop ssDNA structure of the endogenous DNA strand that is complementary to the protospacer sequence.
  • the term “gRNA core” or “gRNA scaffold” refers to the sequence within the gRNA that is responsible for Cas9 binding, it does not include the 20 bp spacer/targeting sequence that is used to guide Cas9 to target DNA.
  • the term “extension arm” refers to a nucleotide sequence component of a pegRNA which provides several functions. Typically, the extension arm is located at the 3’ end of the guide RNA, and comprises the following components in a 5’ to 3’ direction: a reverse transcriptase (RT) template and the primer binding site.
  • RT reverse transcriptase
  • the term “reverse transcriptase template” or “RT template” or “RTT” refers to the portion of the extension arm that spans from the 5’ end of the primer binding site (PBS) to 3’ end of the gRNA core that may operate as a template for the synthesis of a single-strand of DNA by the reverse transcriptase of the prime editing enzyme.
  • the RT template encodes (by the reverse transcriptase of the prime editing enzyme) a single-stranded DNA which, in turn, has been designed to be (a) homologous with the endogenous target DNA to be edited, and (b) which comprises at least one desired nucleotide change (e.g.
  • the term “primer binding site” or “PBS” refers to the nucleotide sequence located on a pegRNA as component of the extension arm (typically at the 3’ end of the extension arm) and serves to bind to the primer sequence that is formed after Cas9 nicking of the target sequence by the prime editing enzyme.
  • PBS primer binding site
  • the Cas9 nickase component of a prime editing enzyme nicks one strand of the target sequence, a 3’-ended ssDNA flap is formed, which serves a primer sequence that anneals to the primer binding site on the pegRNA to prime reverse transcription.
  • RT reverse transcriptase
  • target nucleic acid refers to a nucleic acid containing a target nucleic acid sequence.
  • a target nucleic acid may be single-stranded or double-stranded, and often is double-stranded DNA.
  • a “target nucleic acid sequence,” “target sequence” or “target region”, as used herein, means a specific sequence or the complement thereof that one wishes to bind to using the CRISPR system as disclosed herein.
  • the term “mutation” has its general meaning in the art and refers to a substitution, deletion or insertion.
  • substitution means that a specific nucleotide at a specific position is removed and another nucleotide is inserted into the same position.
  • deletion means that a specific nucleotide is removed.
  • insertion means that one or more nucleotides are inserted before or after a specific nucleotide.
  • variant refers to a first composition (e.g., a first molecule), that is related to a second composition (e.g., a second molecule, also termed a “parent” molecule).
  • the variant molecule can be derived from, isolated from, based on or homologous to the parent molecule.
  • a variant molecule can have entire sequence identity with the original parent molecule, or alternatively, can have less than 100% sequence identity with the parent molecule.
  • a variant of a sequence can be a second sequence that is at least 50; 51; 52; 53; 54; 55; 56; 57; 58; 59; 60; 61; 62; 63; 64; 65; 66; 67; 68; 69; 70; 71; 72; 73; 74; 75; 76; 77; 78; 79; 80; 81; 82; 83; 84; 85; 86; 87; 88; 89; 90; 91; 92; 93; 94; 95; 96; 97; 98; 99; 100% identical in sequence compare to the original sequence.
  • treatment refers to both prophylactic or preventive treatment as well as curative or disease modifying treatment, including treatment of patient at risk of contracting the disease or suspected to have contracted the disease as well as patients who are ill or have been diagnosed as suffering from a disease or medical condition, and includes suppression of clinical relapse.
  • the treatment may be administered to a subject having a medical disorder or who ultimately may acquire the disorder, in order to prevent, cure, delay the onset of, reduce the severity of, or ameliorate one or more symptoms of a disorder or recurring disorder, or in order to prolong the survival of a subject beyond that expected in the absence of such treatment.
  • therapeutic regimen is meant the pattern of treatment of an illness, e.g., the pattern of dosing used during therapy.
  • a therapeutic regimen may include an induction regimen and a maintenance regimen.
  • the phrase “induction regimen” or “induction period” refers to a therapeutic regimen (or the portion of a therapeutic regimen) that is used for the initial treatment of a disease.
  • the general goal of an induction regimen is to provide a high level of drug to a patient during the initial period of a treatment regimen.
  • An induction regimen may employ (in part or in whole) a "loading regimen", which may include administering a greater dose of the drug than a physician would employ during a maintenance regimen, administering a drug more frequently than a physician would administer the drug during a maintenance regimen, or both.
  • maintenance regimen refers to a therapeutic regimen (or the portion of a therapeutic regimen) that is used for the maintenance of a patient during treatment of an illness, e.g., to keep the patient in remission for long periods of time (months or years).
  • a maintenance regimen may employ continuous therapy (e.g., administering a drug at regular intervals, e.g., weekly, monthly, yearly, etc.) or intermittent therapy (e.g., interrupted treatment, intermittent treatment, treatment at relapse, or treatment upon achievement of a particular predetermined criteria [e.g., pain, disease manifestation, etc.]).
  • the term "therapeutically effective amount” is meant a sufficient amount of population of cells to treat the disease at a reasonable benefit/risk ratio applicable to any medical treatment. It will be understood that the total usage compositions of the present invention will be decided by the attending physician within the scope of sound medical judgment.
  • the specific therapeutically effective dose level for any particular patient will depend upon a variety of factors including the age, body weight, general health, sex and diet of the patient, the time of administration, route of administration, the duration of the treatment, drugs used in combination or coincidental with the population of cells, and like factors well known in the medical arts.
  • the first object of the present invention relates to a method of increasing fetal hemoglobin content in a eukaryotic cell comprising the step of contacting the eukaryotic cell with a prime editing platform that consists of (a) one prime editing enzyme and (b) one prime editing guide RNA (pegRNA) for guiding the prime editing enzyme to one target nucleic acid sequence in the -115 region of the HBG1 and/or HBG2 promoter, thereby prime editing said region and subsequently increasing the expression of gamma-globin in said eukaryotic cell.
  • a prime editing platform that consists of (a) one prime editing enzyme and (b) one prime editing guide RNA (pegRNA) for guiding the prime editing enzyme to one target nucleic acid sequence in the -115 region of the HBG1 and/or HBG2 promoter, thereby prime editing said region and subsequently increasing the expression of gamma-globin in said eukaryotic cell.
  • pegRNA prime editing guide RNA
  • the prime editing platform is suitable for introducing a combination of mutations in the -115 region of HBG1 and/or HBG2 promoter so that i) new transcriptional activator binding sites for KLF1 and GATA1 are introduced in said promoter and ii) the binding site for the BCL11A repressor is disrupted.
  • the prime editing platform herein disclosed introduces i) the -123T>C and -124T>C mutations so that the KFL1 activator can bind to the promoter ii) the - 113 A>G mutation so that the GATA1 activator can bind to the promoter and iii) with or without the complete or partial deletion of the BCL11A binding-motif (i.e TGACCA) so that the binding site for the BCL11A repressor is disrupted.
  • the BCL11A binding-motif i.e TGACCA
  • Hematopoietic stem progenitor cells display a number of phenotypes, such as Lin-CD34+CD38 ⁇ CD90+CD45RA ⁇ , Lin- CD34+CD38 ⁇ CD90 ⁇ CD45RA ⁇ , Lin-CD34+CD38+IL-3aloCD45RA ⁇ , and Lin- CD34+CD38+CD10+(Daley et al., Focus 18:62-67, 1996; Pimentel, E., Ed., Handbook of Growth Factors Vol. III: Hematopoietic Growth Factors and Cytokines, pp. 1-2, CRC Press, Boca Raton, Fla., 1994).
  • the stem cells self-renew and maintain continuous production of hematopoietic stem cells that give rise to all mature blood cells throughout life.
  • the hematopoietic progenitor cells or hematopoietic stem cells are isolated form peripheral blood cells.
  • peripheral blood cells refer to the cellular components of blood, including red blood cells, white blood cells, and platelets, which are found within the circulating pool of blood.
  • the eukaryotic cell is a bone marrow derived stem cell.
  • bone marrow-derived stem cells refers to stem cells found in the bone marrow.
  • Stem cells may reside in the bone marrow, either as an adherent stromal cell type that possess pluripotent capabilities, or as cells that express CD34 or CD45 cell-surface protein, which identifies hematopoietic stem cells able to differentiate into blood cells.
  • the eukaryotic cell results from a stem cell mobilization.
  • the term “mobilization” or “stem cell mobilization” refers to a process involving the recruitment of stem cells from their tissue or organ of residence to peripheral blood following treatment with a mobilization agent. This process mimics the enhancement of the physiological release of stem cells from tissues or organs in response to stress signals during injury and inflammation. The mechanism of the mobilization process depends on the type of mobilization agent administered.
  • mobilization agents act as agonists or antagonists that prevent the attachment of stem cells to cells or tissues of their microenvironment. Other mobilization agents induce the release of proteases that cleave the adhesion molecules or support structures between stem cells and their sites of attachment.
  • the term “mobilization agent” refers to a wide range of molecules that act to enhance the mobilization of stem cells from their tissue or organ of residence, e.g., bone marrow (e.g., CD34+ stem cells) and spleen (e.g., Hox11+ stem cells), into peripheral blood.
  • Mobilization agents include chemotherapeutic drugs, e.g., cyclophosphamide and cisplatin; cytokines, and chemokines, e.g., granulocyte colony- stimulating factor (G-CSF), granulocyte-macrophage colony-stimulating factor (GM-CSF), stem cell factor (SCF), Fms-related tyrosine kinase 3 (flt-3) ligand, stromal cell-derived factor 1 (SDF-1); agonists of the chemokine (C—C motif) receptor 1 (CCR1), such as chemokine (C—C motif) ligand 3 (CCL3, also known as macrophage inflammatory protein-1 ⁇ (Mip-1 ⁇ )); agonists of the chemokine (C—X—C motif) receptor 1 (CXCR1) and 2 (CXCR2), such as chemokine (C—X—C motif) ligand 2 (CXCL2) (also known as
  • the prime editing enzyme of the present invention comprises a defective CRISPR/Cas nuclease.
  • the sequence recognition mechanism is the same as for the non- defective CRISPR/Cas nuclease.
  • the defective CRISPR/Cas nuclease of the invention comprises at least one RNA binding domain.
  • the RNA binding domain interacts with a guide RNA molecule as defined hereinafter.
  • the defective CRISPR/Cas nuclease of the invention is a modified version with no nuclease activity.
  • the CRISPR/Cas nuclease consists of a mutant CRISPR/Cas nuclease i.e. a protein having one or more point mutations, insertions, deletions, truncations, a fusion protein, or a combination thereof.
  • the mutant has the RNA-guided DNA binding activity, but lacks one or both of its nuclease active sites.
  • the mutant comprises an amino acid sequence having at least 50% of identity with the wild type amino acid sequence of the CRISPR/Cas nuclease.
  • Various CRISPR/Cas nucleases can be used in this invention.
  • Non-limiting examples of suitable CRISPR/CRISPR/Cas nucleases include Cas3, Cas4, Cas5, Cas5e (or CasD), Cas6, Cas6e, Cas6f, Cas7, Cas8a1, Cas8a2, Cas8b, Cas8c, Cas9, Cas10, Cas10d, CasF, CasG, CasH, Csy1, Csy2, Csy3, Cse1 (or CasA), Cse2 (or CasB), Cse3 (or CasE), Cse4 (or CasC), Csc1, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmr1, Cmr3, Cmr4, Cmr5, Cmr6, Csb1, Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, Cs
  • the CRISPR/Cas nuclease is derived from a type II CRISPR-Cas system. In some embodiments, the CRISPR/Cas nuclease is derived from a Cas9 protein.
  • the Cas9 protein can be from Streptococcus pyogenes, Streptococcus thermophilus, Streptococcus sp., Nocardiopsis rougevillei, Streptomyces pristinaespiralis, Streptomyces viridochromogenes, Streptomyces viridochromogenes, Streptosporangium roseum, Streptosporangium roseum, Alicyclobacillus acidocaldarius, Bacillus pseudomycoides, Bacillus selenitireducens, Exiguobacterium sibiricum, Lactobacillus delbrueckii, Lactobacillus salivarius, Microscilla marina, Burkholderiales bacterium, Polaromonas naphthalenivorans, Polaromonas sp., Crocosphaera watsonii, Cyanothece sp., Microcystis aeruginosa, Synechococcus s
  • the CRISPR/Cas nuclease is a mutant of a wild type CRISPR/Cas nuclease (such as Cas9) or a fragment thereof.
  • the CRISPR/Cas nuclease is a mutant Cas9 protein from S. pyogenes.
  • Methods for generating a Cas9 protein (or a fragment thereof) having an inactive DNA cleavage domain are known (See, e.g., Jinek et al., Science.337:816-821(2012); Qi et al., “Repurposing CRISPR as an RNA-Guided Platform for Sequence-Specific Control of Gene Expression” (2013) Cell.
  • the DNA cleavage domain of Cas9 is known to include two subdomains, the HNH nuclease subdomain and the RuvC1 subdomain.
  • the HNH subdomain cleaves the strand complementary to the gRNA, whereas the RuvC1 subdomain cleaves the non-complementary strand. Mutations within these subdomains can silence the nuclease activity of Cas9.
  • the mutations D10A and H840A completely inactivate the nuclease activity of S.
  • the CRISPR/Cas nuclease of the present invention is a nickase and more particularly a Cas9 nickase i.e. the Cas9 from S. pyogenes having one mutation selected from the group consisting of D10A and H840A.
  • the nickase of the present invention comprises the amino acid sequence as set forth in SEQ ID NO: 3 or SEQ ID NO:4. SEQ ID NO: 3> S.
  • variants of dCas9 are provided which are at least about 70% identical, at least about 80% identical, at least about 90% identical, at least about 95% identical, at least about 98% identical, at least about 99% identical, at least about 99.5% identical, or at least about 99.9% to SEQ ID NO: 3 or 4.
  • the nickase of the present invention comprises the amino acid sequence as set forth in SEQ ID NO: 3 or SEQ ID NO:4 and further comprises the R221K and N394K mutations that was previously shown to improve Cas9 nuclease activity (Spencer, Jeffrey M., and Xiaoliu Zhang. "Deep mutational scanning of S. pyogenes Cas9 reveals important functional domains.” Scientific reports 7.1 (2017): 1-14).
  • variants of dCas9 are provided having amino acid sequences which are shorter, or longer than SEQ ID NO: 3 or 4, by about 5 amino acids, by about 10 amino acids, by about 15 amino acids, by about 20 amino acids, by about 25 amino acids, by about 30 amino acids, by about 40 amino acids, by about 50 amino acids, by about 75 amino acids, by about 100 amino acids or more.
  • Some aspects of the disclosure provide Cas9 proteins that have different PAM specificities. Typically, Cas9 proteins, such as Cas9 from S. pyogenes (spCas9), require a canonical NGG PAM sequence to bind a particular nucleic acid region.
  • any of the Cas proteins provided herein may be capable of binding a nucleotide sequence that does not contain a canonical (e.g., NGG) PAM sequence.
  • a canonical PAM sequence e.g., NGG
  • Cas9 proteins that bind non-canonical PAM sequences have been described in Kleinstiver, B. P., et al., “Engineered CRISPR-Cas9 nucleases with altered PAM specificities” Nature 523, 481- 485 (2015); and Kleinstiver, B.
  • the Cas9 protein of the present invention comprises the following mutations D1135L, S1136W, G1218K, E1219Q, R1335Q and T1337R. This variant is capable of targeting an expanded set of NGN PAMs (i.e.
  • the Ca9 variant SpG can further be optimized to develop a near-PAMless SpCas9 variant named “SpRY” that furthers includes the 5 additional mutations A61R, L1111R, N1317R, A1322R, and R1333P.
  • the second component of the prime editing enzyme herein disclosed comprises a reverse transcriptase.
  • the disclosure contemplates any wild type reverse transcriptase obtained from any naturally- occurring organism or virus, or obtained from a commercial or non-commercial source.
  • the reverse transcriptases can include any naturally-occurring mutant RT, engineered mutant RT, or other variant RT, including truncated variants that retain function.
  • the RTs may also be engineered to contain specific amino acid substitutions, such as those specifically disclosed herein.
  • the reverse transcriptase that is usable in the prime editing enzymes of the present invention has the amino acid sequence as set forth in SEQ ID NO:5 but comprises four mutations: D200N, T306K, W313F, and T330P.
  • the reverse transcriptase is fused to the N-terminus of the defective CRISPR/Cas nuclease.
  • the reverse transcriptase is fused to the C- terminus of the defective CRISPR/Cas nuclease.
  • the defective CRISPR/Cas nuclease and the reverse transcriptase are fused via a linker.
  • the linker comprises a (GGGGS)n (SEQ ID NO:6), a (G)n, an (EAAAK)n (SEQ ID NO: 7), a (GGS)n, an SGSETPGTSESATPES (SEQ ID NO: 8) motif
  • GGGGS GGGGSn
  • EAAAK EAAAK
  • SEQ ID NO: 7 a (GGS)n
  • SGSETPGTSESATPES SEQ ID NO: 8 motif
  • suitable linker motifs and configurations include those described in Chen et al., Fusion protein linkers: property, design and functionality. Adv Drug Deliv Rev.2013; 65(10):1357-69, the entire contents of which are incorporated herein by reference).
  • the prime editing enzyme may comprise additional features.
  • Other exemplary features that may be present are localization sequences, such as nuclear localization sequences (NLS), cytoplasmic localization sequences, export sequences, such as nuclear export sequences, or other localization sequences, as well as sequence tags that are useful for solubilization, purification, or detection of the fusion proteins.
  • the prime editing enzyme incorporates one or more nuclear localization sequence.
  • nuclear localization sequences such as nuclear localization sequences (NLS)
  • cytoplasmic localization sequences such as nuclear export sequences, or other localization sequences
  • sequence tags that are useful for solubilization, purification, or detection of the fusion proteins.
  • the prime editing enzyme incorporates one or more nuclear localization sequence.
  • nuclear localization sequence or “NLS” refers to an amino acid sequence that promotes import of a protein into the cell nucleus, for example, by nuclear transport. Nuclear localization sequences are known in the art and would be apparent to the skilled artisan.
  • NLS sequences are described in Plank et al., international PCT application, PCT/EP2000/011690, filed Nov. 23, 2000, published as WO/2001/038547 on May 31, 2001, the contents of which are incorporated herein by reference for its disclosure of exemplary nuclear localization sequences.
  • a NLS comprises the amino acid sequence PKKKRKV (SEQ ID NO: 9) or MDSLLMNRRKFLYQFKNVRWAKGRRETYLC (SEQ ID NO: 10).
  • Various prime editing enzymes are known in the art and typically include the PE1, PE2, PEmax, PEmax-SpRY prime editors.
  • PE1 protein refers to a prime editing enzyme that consists of a fusion protein having the following structure [NLS]-[Cas9(H840A)]-[linker]- [MMLV_RT(wt)], and having the amino acid sequence as set forth in SEQ ID NO:11.
  • the pegRNA comprises (a) a spacer sequence that comprises a region of complementarity to a first strand of the double-stranded target nucleic sequence located in the HBG1/2 promoters; (b) an extension arm that comprises a RT template and a primer binding site in a 5’ to 3’ orientation, wherein the primer binding site comprises a region of complementarity to a region upstream of a nick site in the second strand of the double- stranded target sequence, and wherein the RT template encodes the desired nucleotide changes (e.g.
  • the pegRNA molecule of the present invention thus comprises a spacer sequence for providing the targeting specificity.
  • this spacer sequence can comprise from about 10 nucleotides to more than about 25 nucleotides.
  • the region of base pairing between the spacer sequence and the corresponding target site sequence can be about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 22, 23, 24, 25, or more than 25 nucleotides in length.
  • the spacer sequence is about 17-20 nucleotides in length, such as 20 nucleotides.
  • a software program is used to identify candidate CRISPR target sequences on both strands of the DNA nucleic acid molecule containing the HBG genes based on desired guide sequence length and a CRISPR motif sequence (PAM) for a specified CRISPR enzyme.
  • PAM CRISPR motif sequence
  • One requirement for selecting a suitable target nucleic acid is that it has a 3′ PAM site/sequence.
  • Each target sequence and its corresponding PAM site/sequence are referred herein as a Cas-targeted site.
  • Type II CRISPR system one of the most well characterized systems, needs only Cas 9 protein and a guide RNA complementary to a target sequence to affect target cleavage. For example, target sites for Cas9 from S.
  • pyogenes with PAM sequences NGG, may be identified by searching for 5′-Nx-NGG- 3′ both on the input sequence and on the reverse-complement of the input. Since multiple occurrences in the genome of the DNA target site may lead to nonspecific genome editing, after identifying all potential sites, the program filters out sequences based on the number of times they appear in the relevant reference genome. For those CRISPR enzymes for which sequence specificity is determined by a “seed” sequence, such as the 11-12 bp 5′ from the PAM sequence, including the PAM sequence itself, the filtering step may be based on the seed sequence. Thus, to avoid editing at additional genomic loci, results are filtered based on the number of occurrences of the seed:PAM sequence in the relevant genome.
  • the user may be allowed to choose the length of the seed sequence.
  • the user may also be allowed to specify the number of occurrences of the seed:PAM sequence in a genome for purposes of passing the filter.
  • the default is to screen for unique sequences. Filtration level is altered by changing both the length of the seed sequence and the number of occurrences of the sequence in the genome.
  • the program may in addition or alternatively provide the sequence of a guide sequence complementary to the reported target sequence(s) by providing the reverse complement of the identified target sequence(s). Further details of methods and algorithms to optimize sequence selection can be found in U.S. application Ser. No.61/836,080; incorporated herein by reference.
  • the spacer sequence is selected from the group consisting of : pegRNA spacer sequence SEQ ID NO : Spacer_10 GTTTGCCTTGTCAAGGCTAT 15 Spacer_11 TGCCTTGTCAAGGCTATTGG 16 Spacer_12 TTGCCTTGTCAAGGCTATTG 17 Spacer_13 TTTGCCTTGTCAAGGCTATT 18 Spacer_14 AGTTTGCCTTGTCAAGGCTA 19
  • the spacer sequence is : Spacer_10 GTTTGCCTTGTCAAGGCTAT SEQ ID NO:15
  • the gRNA core sequence of the pegRNA is selected from the group consisting of : Scaffold sequence SE Q ID NO : Scaffold GTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAG 20 TGGCACCGAGTCGGTGC scaffold GTTTCAGAGCTATGCTGGAAACAGCATAGCAAGTTGAAATAAGGCTAGTCCGTTATCAA 21 _opt CTTGAAAAAGTG
  • the synthesized single-stranded DNA product of the RT template sequence is homologous to the non-target strand and contains desired nucleotide changes (e.g.
  • the single-stranded DNA product of the RT template sequence hybridizes in equilibrium with the complementary target strand sequence, thereby displacing the homologous endogenous target strand sequence.
  • the displaced endogenous strand may be referred to in some embodiments as a 5’ endogenous DNA flap species.
  • This 5’ endogenous DNA flap species can be removed by a 5’ flap endonuclease (e.g., FEN1) and the single-stranded DNA product, now hybridized to the endogenous target strand, may be ligated, thereby creating a mismatch between the endogenous sequence and the newly synthesized strand.
  • the mismatch may be resolved by the cell's innate DNA repair and/or replication processes.
  • the cellular repair of the single-strand DNA flap results in installation of the desired nucleotide changes, thereby forming a desired product.
  • the nucleotide sequence of the RT template sequence corresponds to the nucleotide sequence of the non-target strand which becomes displaced as the 5’ flap species and which overlaps with the site to be edited.
  • the RT template sequence is selected from the group consisting of: RTT sequence SEQ ID NO : RTT_10 GCCccGCCTgATA 22 RTT_10.2 GCCccGCCTTGAgATA 23 RTT_10.3 GCCccGCCTTGACCgATA 24 RTT_11 GCCAGCCTTGCCTG 25 RTT_12 AGCCTTGCCTGA 26 RTT_13 GCCTTGCCTGAT 27 RTT_14 TTGCCTGATAG 28
  • the primer binding site sequence is selected from the group consisting of: PBS sequence SEQ ID NO : PBS_10 GCCTTGACAAGGC 29 PBS_11 ATAGCCTTGACAA 30 PBS_12 TAGCCTTGACAAG 31 PBS_13 AGCCTTGACA
  • Contacting the eukaryotic cell with the prime editing platform of the present invention thus results in i) nicking the second strand of the double-stranded target sequence to form a free 3’ end at the nick site; ii) annealing the primer binding site with the region of the second strand of the double-stranded target sequence upstream of the nick site; iii) synthesizing a single strand of DNA encoded by the RT template from the free 3’ end of the second strand of the double- stranded target sequence; and iv) replacing the region downstream of the nick site in the second strand of the double-stranded target sequence with the single strand of DNA, thereby modifying the sequence of the double-stranded target sequence.
  • the primer binding site of pegRNA binds to the primer sequence that is formed from the endogenous DNA strand of the target sequence when it becomes nicked by the prime editing platform, thereby exposing a 3’ end on the endogenous nicked strand.
  • the binding of the primer sequence to the primer binding site on the extension arm of the pegRNA creates a duplex region with an exposed 3’ end (i.e., the 3’ of the primer sequence), which then provides a substrate for the reverse transcriptase to begin polymerizing a single strand of DNA from the exposed 3’ end along the length of the RT template.
  • the sequence of the single strand DNA product is the complement of the RT template.
  • the RT template represents the portion of the extension arm that is encoded into a single strand DNA product (i.e., the 3’ single strand DNA flap containing the desired genetic edit information) by the reverse transcriptase of the prime editing enzyme complex and which ultimately replaces the corresponding endogenous DNA strand of the target sequence that sits immediate downstream of the PE-induced nick site.
  • the pegRNA of the present invention is selected from the group consisting of : Nam full length 5'-3' S e E Q I D N O : peg GTTTGCCTTGTCAAGGCTATGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTT 3 RNA ATCAACTTGAAAAAGTGGCACCGAGTCGGTGCGCCccGCCTgATAGCCTTGACAAGGC 5 10 peg TGCCTTGTCAAGGCTATTGGGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTT 3 RNA ATCAACTTGAAAAAGTGGCACCGAGTCGGTGCCAGCCTTGCCTGATAGCCTTGACAA 6 11 peg TTGCCTTGTCAAGGCTATTGGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTT 3 RNA ATCAACTTGAAAAAGTGGCACCGAGTCGGTGCCAGCCTTGCCTGATAGCCTTGACAA 6 11 peg TTGCCTTGTCAAGGCTATTGGTTTTAGAGAA
  • ACAAGGC 3 peg GTTTGCCTTGTCAAGGCTATGTTTCAGAGCTATGCTGGAAACAGCATAGCAAGTTGAAATAAGG 4 RNA CTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCTTGGCCAGCCccGCCTTGAgAT 1 10.
  • AGCCTTGACAAGGC 4 epe GTTTGCCTTGTCAAGGCTATGTTTCAGAGCTATGCTGGAAACAGCATAGCAAGTTGAAATAAGG 4 gRN CTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCGCCccGCCTgATAGCCTTGACA 2 A10 AGGCTCCTAATCCGCGGTTCTATCTAGTTACGCGTTAAACCAACTAGAA .1 epe GTTTGCCTTGTCAAGGCTATGTTTCAGAGCTATGCTGGAAACAGCATAGCAAGTTGAAATAAGG 4 gRN CTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCGCCccGCCTTGAgATAGCCTTG 3 A10 ACAAGGCTCCTTATCCGCGGTTCTATCTAGTTACGCGTTAAACCAACTAGAA .2 epe 4 g RN GTTTGCCTTGTCAAGGCTATGTTTCAGAGCTATGCTGGAAACAGCATAGCAAGTTGAAATAAGG 4 A10
  • the ngRNA is designed for temporal control.
  • temporary second-strand nicking refers to a variant of second strand nicking whereby the installation of the second nick in the unedited strand occurs only after the desired edit is installed in the edited strand. This avoids concurrent nicks on both strands that could lead to double-stranded DNA breaks. This is achieved by designing a ngRNA with a spacer sequence that matches only the edited strand, but not the original allele. Using this strategy, mismatches between the protospacer and the unedited allele should disfavor nicking by the ngRNA until after the editing event on the PAM strand takes place.
  • the ngRNA is selected from the group consisting of: ngRNA sequence SEQ ID NO : ngRNA10.1 TAGTCTTAGAGTATCCAGTG 46 ngRNA10.2 TAGAGTATCCAGTGAGGCCA 47 ngRNA10.3 AGAGTATCCAGTGAGGCCAG 48 ngRNA10.4 TATCCAGTGAGGCCAGGGGC 49 ngRNA10.5 GGCTAGGGATGAAGAATAAA 50
  • the ngRNA is : ngRNA10.5 GGCTAGGGATGAAGAATAAA SEQ ID NO :50
  • the guide RNA molecules of the present invention i.e.
  • the pegRNA and optionally the ngRNA can be made by various methods known in the art including cell-based expression, in vitro transcription, and chemical synthesis.
  • cell-based expression in vitro transcription
  • chemical synthesis The ability to chemically synthesize relatively long RNAs (as long as 200 mers or more) using TC-RNA chemistry (see, e.g., U.S. Pat. No. 8,202,983) allows one to produce RNAs with special features that outperform those enabled by the basic four ribonucleotides (A, C, G and U).
  • the RNA molecule of the present invention can be made with recombinant technology using a host cell system or an in vitro translation-transcription system known in the art.
  • the guide RNA molecules may include one or more modifications. Such modifications may include inclusion of at least one non-naturally occurring nucleotide, or a modified nucleotide, or analogs thereof. Modified nucleotides may be modified at the ribose, phosphate, and/or base moiety.
  • Modified nucleotides may include 2’-O-methyl analogs, 2’-deoxy analogs, or 2’-fluoro analogs.
  • the nucleic acid backbone may be modified, for example, a phosphorothioate backbone may be used.
  • LNA locked nucleic acids
  • BNA bridged nucleic acids
  • Further examples of modified bases include, but are not limited to, 2-aminopurine, 5-bromo-uridine, pseudouridine, inosine, 7-methylguanosine.
  • the prime editing platform further involves the transient expression of an engineered DNA mismatch repair (MMR) inhibiting protein (PE5 system) for enhancing the efficiency of the prime editing.
  • MMR engineered DNA mismatch repair
  • PE5 system engineered DNA mismatch repair
  • the MMR inhibiting protein is selected among catalytically impaired mutants of human MSH2, MSH6, PMS2, and MLH1.
  • prime editing platform involves the use of a dominant negative MMR protein (MLH1dn) as described in Chen, Peter J., et al. "Enhanced prime editing systems by manipulating cellular determinants of editing outcomes.” Cell 184.22 (2021): 5635-5652 and having the amino acid sequence as set forth in SEQ ID NO:51.
  • the nucleic acids encoding the pegRNA or the prime editing enzyme can be cloned into one or more vectors for introducing them into the eukaryotic cell.
  • the vectors are typically prokaryotic vectors, e.g., plasmids, or shuttle vectors, or insect vectors, for storage or manipulation of the nucleic acid encoding the pegRNA or the prime editing enzyme herein disclosed.
  • the nucleic acids are isolated and/or purified.
  • the present invention provides recombinant constructs or vectors having sequences encoding one or more of the pegRNA or prime editing enzymes described above.
  • the vector can be capable of autonomous replication or integration into a host DNA.
  • the vector may also include appropriate sequences for amplifying expression.
  • the expression vector preferably contains one or more selectable marker genes to provide a phenotypic trait for selection of transformed host cells such as dihydrofolate reductase or neomycin resistance for eukaryotic cell cultures, or such as tetracycline or ampicillin resistance in E. coli.
  • any of the procedures known in the art for introducing foreign nucleotide sequences into host cells may be used. Examples include the use of calcium phosphate transfection, polybrene, protoplast fusion, electroporation, nucleofection, liposomes, microinjection, naked DNA, plasmid vectors, viral vectors, both episomal and integrative, and any of the other well-known methods for introducing cloned genomic DNA, cDNA, synthetic DNA or other foreign genetic material into a host cell.
  • the different components of the prime editing platform of the present invention are provided to the population of cells through the use of an RNA-encoded system.
  • the editing system may be provided to the population of cells through the use of a chemically modified mRNA-encoded prime editor together with the pegRNA.
  • engineered RNA-encoded prime editing enzymes are prepared by introducing various chemical modifications to both mRNA that encoded the prime editing enzyme and guide RNA.
  • said modifications consist in uridine depleted mRNAs modified with 5- methoxyuridine: synonymous codons may be introduced to deplete uridines as much as possible without altering the coding sequence and replaced all the remaining uridines with 5- methoxyuridine.
  • Said optimized editing system exhibits higher editing efficiency at some genomic sites compared to DNA-encoded system.
  • the different components of the prime editing platform of the present invention are provided to the population of cells through the use of ribonucleoprotein (RNP) complexes.
  • RNP ribonucleoprotein
  • the prime editing enzyme can be pre-complexed with one or more pegRNAs to form a ribonucleoprotein (RNP) complex.
  • RNP ribonucleoprotein
  • the RNP complex can thus be introduced into the eukaryotic cell. Introduction of the RNP complex can be timed.
  • the cell can be synchronized with other cells at G1, S, and/or M phases of the cell cycle.
  • RNP delivery avoids many of the pitfalls associated with mRNA, DNA, or viral delivery.
  • the RNP complex is produced simply by mixing the proteins (i.e. the prime editing enzyme) and one or more pegRNAs in an appropriate buffer. This mixture is incubated for 5-10 min at room temperature before electroporation. Electroporation is a delivery technique in which an electrical field is applied to one or more cells in order to increase the permeability of the cell membrane. In some embodiments, genome editing efficiency can be improved by adding a transfection enhancer oligonucleotide.
  • a further object of the present invention relates to a method for increasing fetal hemoglobin levels in a subject in need thereof, the method comprising transplanting a therapeutically effective amount of the population of eukaryotic cells obtained by the method as above described.
  • the population of cell is autologous to the subject, meaning the population of cells is derived from the same subject.
  • the subject has been diagnosed with a hemoglobinopathy.
  • the method of the present invention is thus particularly suitable for the treatment of hemoglobinopathies.
  • the ⁇ -hemoglobinopathy is a sickle cell disease.
  • the hemoglobinopathy is a ⁇ -thalassemia.
  • the cells are formulated by first harvesting them from their culture medium, and then washing and concentrating the cells in a medium and container system suitable for administration (a "pharmaceutically acceptable" carrier) in a treatment-effective amount.
  • a medium and container system suitable for administration a "pharmaceutically acceptable” carrier
  • Suitable infusion medium can be any isotonic medium formulation, typically normal saline, Normosol R (Abbott) or Plasma-Lyte A (Baxter), but also 5% dextrose in water or Ringer's lactate can be utilized.
  • the infusion medium can be supplemented with human serum albumin.
  • a treatment-effective amount of cells in the composition is dependent on the relative representation of the cells with the desired specificity, on the age and weight of the recipient, and on the severity of the targeted condition.
  • the amount of cells can be as low as approximately 103/kg, preferably 5x103/kg; and as high as 107/kg, preferably 108/kg.
  • the number of cells will depend upon the ultimate use for which the composition is intended, as will the type of cells included therein. Typically, the minimal dose is 2 million of cells per kg. Usually 2 to 20 million of cells are injected in the subject. The desired purity can be achieved by introducing a sorting step.
  • the cells are generally in a volume of a liter or less, can be 500 ml or less, even 250 ml or 100 ml or less.
  • the clinically relevant number of cells can be apportioned into multiple infusions that cumulatively equal or exceed the desired total amount of cells.
  • Kits This invention further provides kits containing reagents for performing the above-described methods, including all component of the prime editing platform as disclosed herein for performing mutagenesis.
  • one or more of the reaction components e.g., guide RNA molecules (i.e. the pegRNA and optionally the ngRNA), and nucleic acid molecules encoding for the prime editing enzymes for the methods disclosed herein can be supplied in the form of a kit for use.
  • the kit comprises one or more prime editing enzymes and one or more guide RNA molecules (i.e. the pegRNA and optionally the ngRNA).
  • the kit consists of one of the following combinations: Prime editor pegRNA ngRNA MLH1 PEmax (SEQ ID pegRNA10.4 (SEQ ngRNA10.5 ( SEQ ID No NO:13) ID NO:41) NO :50) PEmax (SEQ ID pegRNA10.4 (SEQ ngRNA10.5 ( SEQ ID Yes (SEQ ID NO:51) NO:13) ID NO:41) NO :50) PEmax (SEQ ID epegRNA10.2 (SEQ ngRNA10.5 (SEQ ID No NO:13) ID NO:43) NO :50) PEmax (SEQ ID epegRNA10.2 (SEQ ngRNA10.5 (SEQ ID Yes (SEQ ID NO:51) NO:13) ID NO:43) NO :50) PEmax (SEQ ID epegRNA10.2 (SEQ ngRNA10.5 (SEQ ID Yes (SEQ ID NO:51) NO:13) ID NO:43) NO :50) PEmax (SEQ ID epegRNA
  • an appropriate amount of one or more reaction components is provided in one or more containers or held on a substrate.
  • additional components of the kits include, but are not limited to, one or more host cells, one or more reagents for introducing foreign nucleotide sequences into host cells, one or more reagents (e.g., probes or PCR primers) for detecting expression of the guide RNA or prime editing enzymes or verifying the target nucleic acid's status, and buffers or culture media for the reactions.
  • the kit may also include one or more of the following components: supports, terminating, modifying or digestion reagents, osmolytes, and an apparatus for detection.
  • the components used can be provided in a variety of forms.
  • kits and systems include solid matrices (e.g., glass, plastic, paper, foil, micro-particles and the like) that hold the reaction components or detection probes in any of a variety of configurations (e.g., in a vial, microtiter plate well, microarray, and the like).
  • the kits may further include instructions recorded in a tangible form for use of the components.
  • FIGURES Figure 1: Screening of pegRNAs and ngRNAs targeting the HBG1/2 promoters in K562 cells. (A) Upper panel.
  • pegRNAs contain from 5’ to 3’, the spacer sequence (black/dark grey arrows), the scaffold sequence (grey lines) and the 3’ extension composed of the reverse transcription template (RTT, grey light boxes) and the primer binding site (PBS, white boxes).
  • pegRNA10 is compatible with a prime editor harboring the Cas9n that recognizes an NGG PAM (PEmax), while pegRNA11 to pegRNA14 work with a PAM-less (SpRY) prime editor (PEmax-SpRY).
  • pegRNA10 to pegRNA14 were designed to delete the BCL11A-binding motif (TGACC from -114 to -118 upstream of the TSS) (grey light box), insert the -113 A>G (GATA1 BS) and the -123/-124 T>C (KLF1 BS) mutations.
  • C Frequency (%) of InDels generated by the pegRNAs, described in panel B, using a Cas9 (pegRNA10) or a Cas9-SpRY (pegRNA11 to pegRNA14) nuclease in K562 cells. Bars represent the mean ⁇ SEM of 3 biologically independent replicates.
  • C D
  • MOCK samples transfected with TE (Tris-EDTA) buffer were used as controls.
  • PCR products were subjected to Sanger sequencing and InDels were measured using the TIDE software.
  • Figure 2 Prime editing strategies to simultaneously disrupt the BCL11A BS and insert the GATA1 and KLF1 BS in the HBG1/2 promoters using epegRNA10.1 in K562 cells.
  • the epegRNAs contain the tevopreQ1 sequence fused to the 3’ end of the pegRNA via a linker.
  • the epegRNA10.1 deletes the entire BCL11A BS ( ⁇ 5_BCL11A) and inserts the GATA1 (-113 A>G) and the KLF1 (-123/-124 T>C) BSs.
  • B Left panel. Percentage of NGS reads with: (i) desired edits alone, (ii) desired edits with alternative repair events with or without additional mutations, and (iii) Indels in GFPhigh K562 cells.
  • the reads with InDels include InDels located at the nicking site of the epegRNA and of the ngRNA10.3 or ngRNA10.5.
  • Figure 3 Prime editing strategies to simultaneously disrupt the BCL11A BS and insert the GATA1 and KLF1 BS in the HBG1/2 promoters using epegRNA10.2 in K562 cells.
  • A Schematic representation of epegRNA10.2 and ngRNAs (ngRNA10.3 and ngRNA10.5).
  • the epegRNAs contain the tevopreQ1 sequence fused to the 3’ end of the pegRNA via a linker.
  • the epegRNA10.2 deletes two cytosines of the BCL11A BS ( ⁇ 2_BCL11A),and insert the GATA1 and the KLF1 BSs.
  • B Left panel.
  • the reads with InDels include InDels located at the nicking site of the epegRNA and of the ngRNA10.3 or ngRNA10.5.
  • MOCK samples transfected with TE (Tris-EDTA) buffer were used as controls. Data represent the mean ⁇ SEM of 3 biologically independent replicates.
  • FIG. 1 Schematic representation of the resolution of the 3’-5’ flap intermediates using epegRNA10.2.
  • Figure 4 Prime editing strategies to simultaneously disrupt the BCL11A BS and insert the GATA1 and KLF1 BS in the HBG1/2 promoters using epegRNA10.3 in K562 cells.
  • A Schematic representation of epegRNA10.3 and ngRNAs (ngRNA10.3 and ngRNA10.5). The epegRNAs contain the tevopreQ1 sequence fused to the 3’-end of the pegRNA via a linker.
  • the epegRNA10.3 inserts the GATA1 and the KLF1 BSs.
  • B Left panel. Percentage of NGS reads with: (i) desired edits alone, (ii) desired edits with alternative mutations (alt. mut.), (iii) partial edits and (iv) Indels in GFPhigh K562 cells.
  • the reads with InDels include InDels located at the nicking site of the epegRNA and of the ngRNA10.3 or ngRNA10.5.
  • MOCK samples transfected with TE (Tris-EDTA) buffer were used as controls. Data represent the mean ⁇ SEM of 3 biologically independent replicates.
  • Right panel Example of NGS reads.
  • the top line corresponds to the wild-type (WT) sequence of the HBG1/2 promoters aligned to the PBS/RTT of the epegRNA.
  • the different editing profiles are displayed below the WT sequence.
  • the KLF1 BS and the GATA1 BS are respectively highlighted in grey and bold.
  • the desired base conversions are indicated in lower case.
  • Statistical significance was assessed using one-way test ANOVA with multiple comparisons.* p ⁇ 0.05** p ⁇ 0,005, ***p ⁇ 0,0005, **** p ⁇ 0,0001
  • Figure 5 Optimization of the prime editing efficiency in K562 cells.
  • NGS reads containing the desired edits (3 mutations: 1 generating the GATA1 BS, 1 generating the KLF1 BS and the 2-bp deletion in the BCL11A BS), the partial edits (Partial edit 1: 1 mutation generating the GATA1 BS and 2-bp deletion in the BCL11A BS; partial edit 2: only 1 mutation generating the GATA1 BS) or InDels (located at the nicking induced by the epegRNA or the ngRNA) generated using the epegRNA10.2 or the epegRNA10.4 with the ngRNA10.5 in the -115 region of the HBG1/2 promoters.
  • MOCK samples transfected with TE (Tris-EDTA) buffer were used as controls.
  • Prime editing efficiency in HSPCs percentage of NGS reads containing the desired edit (3 mutations: 1 generating the GATA1 BS, 1 generating the KLF1 BS and the 2-bp deletion in the BCL11A BS), the partial edits (1 mutation generating the GATA1 BS and 2-bp deletion in the BCL11A BS) or InDels (located at the nicking induced by the epegRNA and/or the ngRNA) generated using the epegRNA10.4 with or without the ngRNA10.5 (for PE and PEn strategies, respectively) in the -115 region of the HBG1/2 promoters.
  • MOCK samples transfected with TE (Tris-EDTA) buffer were used as controls.
  • HBB ⁇ - globin
  • HbS hemoglobin S
  • Naturally occurring HPFH mutations identified in the promoters of the two ⁇ -globin genes are known to either generate de novo DNA motifs recognized by transcriptional activators (e.g., KLF1, TAL1 and GATA1) or disrupt transcriptional repressor (e.g., LRF and BCL11A) binding sites (BSs).
  • transcriptional activators e.g., KLF1, TAL1 and GATA1
  • disrupt transcriptional repressor e.g., LRF and BCL11A binding sites
  • the disruption of the LRF or BCL11A BS with CRISPR/Cas9- mediated strategy leads to HbF reactivation and correction of the SCD phenotype3.
  • the use of a nuclease can cause cell toxicity and generate several double strand break (DSB) associated with large deletions and high risk of genomic rearrangements4–6.
  • DSB double strand break
  • New DSB-free genome editing tools allowed the development of novel, efficient and safer therapeutic strategies for the development of ⁇ -hemoglobinopathies.
  • the introduction of HPFH mutations generating the KLF1 (-123/-124 T>C)7, TAL1 (-175 T>C)8 or GATA1 (-113 A>G)9 activator BSs in the HBG1/2 promoters results in HbF reactivation using base editing8.
  • the creation of the -113 A>G mutation partially disrupt the BCL11A repressor BS.
  • the co-occurrence of multiple HPFH mutations is associated with higher HbF levels compared to individual mutations10.
  • the prime editing system is a “search and replace” genome editing technology allowing all 12 possible base conversions, insertions, deletions, or the simultaneous combination of these changes11–13 in a specific target region.
  • This system is composed of: (1) the prime editor (PE), a fusion of a Cas9 nickase (Cas9n) and an engineered reverse transcriptase (RT) and (2) a prime editing guide RNA (pegRNA).
  • PE prime editor
  • Cas9n Cas9 nickase
  • RT engineered reverse transcriptase
  • pegRNA prime editing guide RNA
  • the pegRNA contains the spacer sequence that targets a specific region in the genome and the 3’ extension sequence composed of the primer binding site (PBS) that hybridizes to the 3’ end of the nicked DNA strand to start reverse transcription and the RT- template (RTT) that contains the desired edits (DE), which are eventually inserted into the genomic DNA after reverse transcription (PE2 system).
  • PBS primer binding site
  • RTT RT- template
  • DE desired edits
  • PE2 system genomic DNA after reverse transcription
  • the prime editing tool was improved to increase editing efficiency by using a single guide RNA (ngRNA) that nicks the non-edited strand (PE3 system)11 and a dominant negative MLH1 (MLH1dn) that inhibits the mismatch DNA repair pathway (MMR; PE5 system), which reincorporates the original nucleotides in the edited strand14–17.
  • ngRNA single guide RNA
  • MLH1dn dominant negative MLH1
  • MMR mismatch DNA repair pathway
  • the PE by itself was also engineered to further improve its processivity and editing activity by the addition of nuclear localization signal (NLS) sequences, linkers, and two mutations in the Cas9n18. Moreover, its sequence was codon optimized, generating the PEmax15.
  • NLS nuclear localization signal
  • nickase-based PE a Cas9 nuclease to generate the prime editor nuclease (PEn - SEQ ID NO: 86) that facilitates precise insertion of DNA sequences and improves editing efficiency for some pegRNAs that are poorly efficient when used with the nickase-based PE33.
  • This technology allows prime editing by using DSBs and DNA end joining repair pathways33.
  • K562 cell culture Human erythroleukemia K562 cells were maintained at a concentration of 5x105cells/ml in RPMI 1640 containing glutamine (Gibco) and supplemented with 10% fetal bovine serum (Gibco), 2% Hepes (Life Technologies), 1% sodium pyruvate (Life Technologies), and 1% penicillin and streptomycin (Life Technologies) at 37°C and 5%CO2.
  • HSPC purification and culture We obtained human non-mobilized peripheral blood CD34+ HSPCs from patients with SCD from the “Hôpital Necker-Enfantsments” Hospital (Paris, France). Healthy donors were either obtained from the “Hôpital Necker-Enfantsmats” Hospital (Paris, France). All experiments were performed in accordance with the Declaration of Helsinki. The study was approved by the regional investigational review board (reference: DC 2022-5364, CPP Ile-de- France II “Hôpital Necker-Enfants disclosures”). HSPCs were purified by immunomagnetic selection with AutoMACS (Miltenyi Biotec) after immunostaining with the CD34 MicroBead Kit (Miltenyi Biotec).
  • CD34+ cells were thawed and cultured at a concentration of 5 ⁇ 105 cells/ml in the “HSPC medium” containing StemSpan (STEMCELL Technologies) supplemented with penicillin/streptomycin (Gibco), 250 nM StemRegenin1 (STEMCELL Technologies), and the following recombinant human cytokines (PeproTech): human stem-cell factor (SCF) (300 ng/ml), Flt-3L (300 ng/ml), thrombopoietin (TPO) (100 ng/ml), and interleukin-3 (IL-3) (60 ng/ml).
  • SCF human stem-cell factor
  • Flt-3L 300 ng/ml
  • TPO thrombopoietin
  • IL-3 interleukin-3
  • Plasmids used in this study include: pMJ920 Cas9-GFP-expressing plasmid (Addgene #42234) pCMV-T7-SpRY-P2A-EGFP (Addgene #139989) pCMV-PE2 (Addgene #258720) pCMV-PEmax (Addgene #174820) pU6-pegRNA-GG-acceptor (Addgene #132777) pU6-tevopreq1-GG-acceptor (Addgene #174038) pEF1a-hMLH1dn (Addgene #174824) Generation of pegRNA and epegRNA constructs pegRNAs were designed using the web-based tools (PrimeDesign26 https://drugthatgene.pinellolab.partners.org, pegFinder27 http://pegfinder.sidichenlab.org, and EasyPrime28, http://easy-prime.cc).
  • epegRNAs were generated from pegRNAs by adding the tevopreQ1 sequence at the 3’ end of the pegRNA via a linker designed by the web-based tool pegLIT14 (https://peglit.liugroup.us). pegRNAs and epegRNAs were cloned as previously described11,14.
  • oligonucleotide duplexes containing the pegRNAs’ or the epegRNAs’ spacer , scaffold and 3’extension, with or without the tevopreQ1 sequence were cloned using the Golden Gate Assembly method into the pU6-pegRNA-GG-acceptor (Addgene #132777) for pegRNAs and the pU6-tevopreq1-GG-acceptor (Addgene#174038) for epegRNAs. Plasmids were transformed in TOP10 and XL10-gold bacteria for pegRNAs and epegRNAs, respectively, following manufacturer’s instructions. After an overnight culture, transformed bacterial colonies were selected and amplified.
  • ngRNAs were designed using the web-based tool CRISPOR29 (http://crispor.tefor.net/ - version 4.99). We selected ngRNAs with the lowest off-target activity, which was predicted using web- based tools (COSMID, https://crispr.bme.gatech.edu/30; DeepSpCas9, http://deepcrispr.info/DeepSpCas9/31).
  • Oligonucleotide duplexes containing the annealed ngRNA spacer were cloned into the MA128 plasmid (provided by M. Amendola, Genethon, France). Cloning strategies and plasmid selection were similar to those described for peg/epegRNAs. ngRNAs’ sequences are displayed in Table S1.
  • K562 cells 106 cells/condition
  • K562 cells 106 cells/condition
  • Cas9-GFP Additional growth factor
  • Cas9-SpRY-GFP-expressing plasmid Additional growth factor
  • 1.2 ⁇ g of the ngRNA/pegRNAs-containing plasmids using AMAXA Cell Line Nucleofector Kit V (VCA- 1003, Lonza) and U-16 program (Nucleofector 2b, Lonza).
  • TE Tris EDTA
  • RNA transfection 1 x 105 to 2 x 105 HSPCs from SCD donors were transfected per condition 24 h after thawing, with 3.2 or 6.4 ⁇ g of the enzyme-encoding PEmax mRNA (generated using the MEGAscript and Poly-A tailing kits, Ambion and the ARCA, Trilink, as described in Antoniou et al.34) or the PEnmax (provided by AstraZeneca) enzyme-encoding mRNA, and 200 to 400 pmol of synthetic pegRNAs (IDT) and/or ngRNA (Synthego).
  • IDT synthetic pegRNAs
  • Synthego synthetic pegRNAs
  • cells were treated 24h with deoxynucleosides (Sigma-Aldrich; dA, catalog no. D8668; dG, catalog no. D0901; dC, catalog no. D0776; dT, catalog no. T1895) at a final concentration of 100uM, or with small inhibitors at a final concentration of 1 ⁇ M for AZD7648 (DNA-PKi, provided by AstraZeneca), 3 ⁇ M for Pol ⁇ i (ART558, MedChem Express), or a combination of both drug treatments.
  • AZD7648 DNA-PKi, provided by AstraZeneca
  • Pol ⁇ i ART558, MedChem Express
  • Synthetic epegRNAs and ngRNA generation Synthetic epegRNAs were ordered from Integrated DNA Technologies (IDT). Each construct contained 2’-O-methyl modifications at the first and last three nucleotides and phosphorothioate linkages between the three first and last nucleotides. Synthetic nicking gRNAs were obtained from Synthego with 2’-O-methyl modifications at the first and last three nucleotides as well and phosphorothioate linkages between the three first and last two nucleotides. Evaluation of editing efficiency Genomic DNA from K562 cells was extracted using the PureLink Genomic DNA Mini Kit (Invitrogen) following the manufacturer’s instructions.
  • on-target sites (HBG1/2 promoters and ngRNA target sites) were PCR-amplified using the Recombinant Taq DNA Polymerase (Thermo Fisher) according to the manufacturer’s instructions, and subjected to Sanger sequencing. PCR primers are listed in Table S2. Indels were evaluated using the TIDE software32 (http://shinyapps.datacurators.nl/tide/).
  • on-target sites (HBG1/2 promoters) were PCR amplified using the Phusion High-Fidelity polymerase (New England BioLabs), the HF buffer (New England BioLabs) and primers containing specific DNA stretches (MR3 for forward primers and MR4 for reverse primers) 5’ to the sequence recognizing the on-target site. Amplicons were purified using Ampure XP beads (Beckman Coulter).
  • Illumina-compatible barcoded DNA amplicon libraries were prepared by a 2-step PCR using the Phusion High- Fidelity polymerase (New England BioLabs), the HF buffer (New England BioLabs) and primers containing Unique Dual Index (UDI) barcodes and annealing to MR3 and MR4 sequences. Primers used for PCR amplification are listed in Table S2. Libraries were pooled, purified by High Pure PCR Product Purification Kit (Sigma-Aldrich), and sequenced using Illumina NovaSeq 6000 system (paired-end sequencing; 2 ⁇ 100-bp).
  • NGS data were analyzed using a custom python pipeline that allows to align reads to a reference amplicon sequence and to count, (i) reads with sequence modifications (both expected modifications and other ones) in a window including the editing site in the proximity of the PE nicking site, and (ii) reads with Indels occurring between the epegRNA nicking site and the second nicking site, either alone or in combination with sequence modification near the PE nicking site.
  • sequence modifications both expected modifications and other ones
  • Indels occurring between the epegRNA nicking site and the second nicking site, either alone or in combination with sequence modification near the PE nicking site.
  • Amplicons were generated using Phusion Flash High-Fidelity PCR Mastermix (F548, Thermo Scientific) in a 15 ⁇ L reaction, containing 1.5 ⁇ L of genomic DNA extract and 0.5 ⁇ M of target- specific primers (Forward: 5’-AAACGGTCCCTGGCTAAACT-3’ (SEQ ID NO:83) and Reverse: 5’- CCAGAAGCGAGTGTGTGGAA-3’ (SEQ ID NO:84)) with barcodes and NGS adapters.
  • Applied PCR cycling conditions 98 °C for 3 min, 30x (98 °C for 10 s, 60 °C for 20 s, 72 °C for 30 s).
  • PCR products were purified using HighPrep PCR Clean-up System (MagBio Genomics). Size, purity, and concentration of amplicons were determined using a fragment analyzer (Agilent). Amplicons were subjected to the second round of PCR to add unique Illumina indexes. Indexing PCR was performed using KAPA HiFi HotStart Ready Mix (Roche), 1 ng of PCR template and 0.5 ⁇ M of indexed primers in the total reaction volume of 25 ⁇ L. PCR cycling conditions: 72 °C for 3 min, 98 °C for 30 s, 10x (98 °C for 10 s, 63 °C for 30 s, 72 °C for 3 min), 72 °C for 5 min.
  • Indexed amplicons were purified using HighPrep PCR Clean- up System (MagBio Genomics) and analyzed using a fragment analyzer (Agilent). Samples were quantified using Qubit 4 Fluorometer (Life Technologies) and subjected to sequencing using Illumina NextSeq system according to manufacturer’s instructions. Demultiplexing of the NGS sequencing data was performed using bcl2fastq software. The fastq files were analyzed using CRISPResso2 in the prime editing mode with the quantification window of 48 and 18 for pegRNAs targeting the -115 region of HBG1/2 promoters. Prime edited override sequences were used for each site. To generate the representative alignments, the window was extended to 40 to visualize homology arm integrations of different lengths.
  • CFC assay HSPCs were plated at a concentration of 5 ⁇ 102 cells/mL in a methylcellulose-containing medium (GFH4435, STEMCELL Technologies) under conditions supporting erythroid and granulo-monocytic differentiation. After 14 days, BFU-Es were randomly picked and collected as bulk populations (containing at least 25 colonies) to evaluate editing efficiency and globin expression. HPLC Hemoglobin tetramers from BFU-E bulks were separated by CE-HPLC using a 2-cation exchange column (PolyCAT A, PolyLC, Columbia).
  • the pegRNA10 is compatible with the PEmax harboring the Cas9n recognizing an NGG protospacer adjacent motif (PAM) and contains a 13-nucleotide long RTT that generates a de novo BS for GATA1 (-113 A>G), deletes the entire BCL11A BS (TGACC motif from -114 to -118 upstream of the TSS), and creates the KLF1 BS (-123/-124 T>C).
  • the protospacer recognized by pegRNA10 is located close to the first nucleotide to be edited (i.e. the -113 nucleotide located at position +4 from the PE nick) and the PBS has the recommended length of 13 nucleotides.
  • pegRNAs compatible with the PE-SpRY variant which contains a nearly PAM-less Cas9n (pegRNA11 to pegRNA14)21,22 and induce the same mutations as pegRNA10.
  • the -113 base is located in position +1 to +8 (from the PE-induced nick) ( Figure 1B).
  • pegRNAs with a Cas9 nuclease was tested to select the best-performing pegRNAs’ spacers that efficiently bind the HBG1/2 promoters.
  • ngRNA10.1 to ngRNA10.5 we designed five ngRNAs (ngRNA10.1 to ngRNA10.5) inducing a DNA nick in a region spanning from +42 to +81 nucleotides downstream of the nick induced by pegRNA10.
  • ngRNA10.1 to ngRNA10.5 We tested these ngRNAs by plasmid transfection in K562 cells and we selected the most efficient ones based on the efficiency of cleavage induced by the Cas9 nuclease.
  • the ngRNA10.3 and ngRNA10.5 were the most efficient ngRNAs (inducing 39.0% and 45.8% of InDels in K562 cells, respectively) and were selected for further analyses (Figure 1D).
  • the co- transfection with a GFPmax-encoding plasmid enabled us to measure transfection efficiency and evaluate the editing efficiency in FACS-sorted GFPhigh cells.
  • HBG promoters were amplified by PCR and subjected to NGS sequencing. Editing efficiency was calculated using a custom Python pipeline.
  • the deletion of 5- nucleotides of the BCL11A BS and, as a consequence, the reduced complementarity of the 3’ flap to the opposite strand likely determines the annealing of the GCC trinucleotide of the 3’ flap to the closest complementary motif in the DNA target region (Figure 2C).
  • epegRNA10.1 was further modified to generate epegRNA10.2 and epegRNA10.3.
  • the epegRNA10.2 contains the same spacer as epegRNA10.1 and an RTT of 16 nucleotides inserting both GATA1 (-113 A>G) and KLF1 (-123/-124 T>C) BSs, and simultaneously deleting the 2-nucleotide-long core of the BCL11A BS (CC motif located -114 to -115 upstream of the TSS) in the -115 region of the HBG1/2 promoter ( Figure 3A).
  • the epegRNA10.3 contains the same spacer as epegRNA10.1 and an 18-nucleotide-long RTT inserting only the GATA1 (-113 A>G) and the KLF1 (-123/-124 T>C) BS in the -115 region of the HBG1/2 promoter, without deleting the BCL11A BS ( Figure 4A).
  • the introduction of the GATA1 BS partially disrupt the BCL11A BS.
  • Edited promoters contain either both activator BSs or partial edits ( Figure 4B).
  • Partials edits included mainly the introduction of only the GATA1 BS ( Figure 4B), suggesting the low processivity of the RT when the RTT is longer than 13 nucleotides or the degradation of the 3’ end newly synthesized 3’ flap.
  • a low frequency of edited promoters harbored only the KLF1 BS ( Figure 4B), suggesting error-prone and competitive repair pathways mechanisms involved in the resolution of the 3’ flap.
  • Indel frequency tended to be lower when using ngRNA10.5 compared to ngRNA10.3.
  • these results show that our strategy can simultaneously insert multiple HPFH and HPFH-like mutations in the -115 region of the HBG1/2 promoters in K562 cells.
  • epegRNA10.2 that successfully inserted both GATA1 and KLF1 BSs and simultaneously disrupted the BCL11A repressor BS, reaching up to 34% of total editing efficiency in K562 cells.
  • NGS sequencing revealed editing profiles that are indicative of competitive and alternative repair pathways underneath the resolution of the 3’ intermediates.
  • epegRNA10.4 a novel epegRNA (i.e., epegRNA10.4), which contains a 7-nucleotide longer RTT compared to the epegRNA10.2 (Table S1).
  • the elongated RTT should prevent 3’ flap degradation and favor the annealing of the 3’ flap over the 5’ flap to the complementary strand and the desired DNA repair, thus increasing the insertion of the desired edits.
  • the epegRNA10.4 outperformed the epegRNA10.2 in inserting the desired motif reaching up to 50% of precise edits with the PE5max ( Figure 5).
  • InDels remained low and comparable to the frequency observed with the epegRNA10.2 ( Figure 5).
  • G gamma A gamma (beta+) hereditary persistence of fetal hemoglobin the G gamma -158 C-->T mutation in cis to the -175 T-->C mutation of the A gamma-globin gene results in increased G gamma-globin synthesis.

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Abstract

Les beta-hémoglobinopathies, la beta-thalassémie et la drépanocytose (SCD), sont des maladies monogéniques provoquées par des mutations dans le locus de beta-globine, affectant la synthèse ou la structure de l'hémoglobine adulte (Hb). Il a été démontré que la gravité de la SCD et de la bêta-thalassémie est réduite par la persistance héréditaire de l'hémoglobine foetale (HbF) dans l'adulte (HPFH). Or les inventeurs ont exploité la capacité d'édition primaire pour générer simultanément de multiples mutations HPFH et perturber le répresseur BCL11A BS dans la région 115 des promoteurs de gamma-globine HBG1/2 pour amplifier davantage l'expression de HbF et corriger les phénotypes de beta-thalassémie et SCD. En particulier, les inventeurs ont sélectionné deux mutations puissantes de type HPFH/HPFH générant des BS pour les activateurs GATA1 et KLF1, qui sont associées à une forte réactivation de gamma-globine dans des patients et des modèles expérimentaux. Cette stratégie serait très intéressante pour le traitement des β-hémoglobinopathies.
PCT/EP2024/070167 2023-07-17 2024-07-16 Édition primaire de la région 115 dans le promoteur hbg1 et/ou hbg2 pour augmenter la teneur en hémoglobine fœtale dans une cellule eucaryote Pending WO2025017033A1 (fr)

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