HK40033100A

HK40033100A - In vitro method of mrna delivery using lipid nanoparticles

Info

Publication number: HK40033100A
Application number: HK62021023222.4A
Authority: HK
Inventors: M·H·斯图尔特; M·P·瓦拉斯特; A·蒙蒂; P·K·纳伦德拉; Q·侯
Original assignee: 英特利亚治疗股份有限公司
Priority date: 2017-09-29
Filing date: 2018-09-28
Publication date: 2021-04-01

Description

In vitro MRNA delivery methods using lipid nanoparticles

This application claims priority from U.S. provisional patent application No. 62/566,232, filed 2017, 9, 29, the contents of which are incorporated herein by reference in their entirety.

Introduction of genetic changes into stem cells, including Hematopoietic Stem Cells (HSCs), and their progeny, is of great significance for gene editing and gene therapy approaches. Stem cells (such as HSCs) lose proliferative capacity in mature cells and committed progenitors, making them particularly useful for gene editing techniques. For example, the ability to modify HSCs and stem cells in vitro is important, and methods of delivering biological agents to HSCs and other stem cells in culture are needed. There is a particular need for techniques for delivering human HSCs in culture.

HSCs are essential for lifelong hematopoiesis. HSCs are able to maintain long-term functional hematopoiesis by being able to differentiate to give rise to mature progeny of all myeloid and lymphoid blood lineages or to self-renew to replace cells that are progressively committed to differentiation. HSCs can be used to restore blood and immune cells in transplant recipients, immunocompromised patients, or other patients. In particular, autologous or allogeneic transplantation of HSCs can be used to treat patients with inherited immunodeficiency and autoimmune diseases, as well as various hematopoietic disorders, to reconstitute hematopoietic cell lineage and immune system defense.

Methods of delivering components of the CRISPR/Cas gene editing system to HSCs in culture are of particular interest. Provided herein are methods of delivering RNA (including CRISPR/Cas system components) to hematopoietic cell cultures including HSCs. The methods deliver active proteins to stem cells (including HSCs) cultured in vitro and comprise contacting the cells with a Lipid Nanoparticle (LNP) composition that provides mRNA encoding the protein. In addition, methods of in vitro gene editing in stem cells, such as HSCs, and methods of producing engineered cells are provided.

In some embodiments, methods of in vitro gene editing in HSCs are provided, as well as methods of generating engineered HSC cells. In additional embodiments, provided herein is a method of delivering mRNA to a hematopoietic stem and/or progenitor cell (HSPC) or HSPC population. In some embodiments, the method comprises pre-incubating serum factors with an LNP composition comprising mRNA, amine lipids, helper lipids, neutral lipids, and PEG lipids. In some embodiments, the method further comprises contacting the HSPC or HSPC population with a pre-incubated LNP composition in vitro. In some embodiments, the method further comprises culturing the HSPC or HSPC population in vitro. In some embodiments, the method results in delivery of mRNA to the HSPC or HSPC population.

In some embodiments, provided herein is a method of introducing Cas nuclease mRNA and gRNA into a stem cell, e.g., HSPC. In some embodiments, the methods comprise preincubating the serum factors with an LNP composition comprising Cas nuclease mRNA, grnas, amine lipids, helper lipids, neutral lipids, and PEG lipids. In some embodiments, the method further comprises contacting the HSPCs with a pre-incubated LNP composition in vitro. In some embodiments, the method further comprises culturing HSPCs. In some embodiments, the method results in introducing Cas nuclease mRNA and gRNA into HSPCs.

In some embodiments, provided herein is a method of producing genetically engineered stem cells, such as HSPCs, in vitro. In some embodiments, the methods comprise preincubating the serum factors with an LNP composition comprising Cas nuclease mRNA, grnas, amine lipids, helper lipids, neutral lipids, and PEG lipids. In some embodiments, the method further comprises contacting the HSPCs with a pre-incubated LNP composition in vitro. In some embodiments, the method further comprises culturing HSPCs in vitro. In some embodiments, the method results in the production of genetically engineered HSPCs.

In some embodiments, there is provided a method of delivering mRNA to a HSPC or HSPC population, the method comprising pre-incubating a LNP composition with serum factors, contacting a cell or population with the pre-incubated LNP composition in vitro; and culturing the cell or population in vitro; thereby delivering mRNA to HSPCs. In some embodiments, the HSPCs are HSCs. In some embodiments, the methods deliver mRNA, such as Cas nuclease mRNA, to a HSPC population (e.g., a CD34+ cell population). In certain embodiments, a guide rna (grna) is delivered to the cell, optionally in combination with Cas nuclease mRNA.

Drawings

Figure 1 shows Green Fluorescent Protein (GFP) mRNA delivery in CD34+ bone marrow cells using LNP.

Figure 2 shows that mRNA delivery in CD34+ bone marrow cells depends on preincubation with serum.

Fig. 3A and 3B show B2M editing in CD34+ bone marrow cells under serum pre-incubation, where fig. 3A depicts the percentage of B2M-cells (protein expression knockouts) and fig. 3B depicts the percentage of editing achieved in the experiment.

Fig. 4A and 4B show the effective delivery under serum pre-incubation and ApoE3 pre-incubation. Fig. 4A depicts the percentage of B2M-cells, and fig. 4B provides the edit percentage achieved in the experiment.

Figure 5 shows the effect of pre-incubation of LNP with various serum factor preparations on LNP delivery to CD34+ cells.

Fig. 6A and 6B show viability and compiled data for CD34+ cells exposed to LNP treatment at different intervals. Fig. 6A shows the survival of CD34+ cells at 2 hours, 6 hours, and 24 hours after exposure to LNP. Fig. 6B provides edit percentage data for the 2 hour, 6 hour, and 24 hour treatment groups.

Detailed Description

The present disclosure provides methods of delivering RNA, including CRISPR/Cas component RNA ("cargo"), in vitro to CD34+ cells, e.g., HSC-containing cell populations, using LNP compositions. The methods may exhibit improved properties compared to previous delivery techniques, for example, the methods provide for efficient RNA delivery while reducing cell death caused by transfection.

In some embodiments, provided herein is a method of delivering mRNA to a stem cell, e.g., a HSPC or HSPC population. In some embodiments, the method comprises pre-incubating serum factors with an LNP composition comprising mRNA, amine lipids, helper lipids, neutral lipids, and PEG lipids. In some embodiments, the method further comprises contacting the HSPC or HSPC population with a pre-incubated LNP composition in vitro. In some embodiments, the method further comprises culturing the HSPC or HSPC population in vitro. In some embodiments, the method results in delivery of mRNA to the HSPC or HSPC population. In some embodiments, the mRNA encodes a Cas nuclease.

In some embodiments, provided herein is a method of introducing Cas nuclease mRNA and gRNA into a stem cell, e.g., a HSPC or a population of HSPCs. In some embodiments, the methods comprise preincubating the serum factors with an LNP composition comprising Cas nuclease mRNA, grnas, amine lipids, helper lipids, neutral lipids, and PEG lipids. In some embodiments, the method further comprises contacting the HSPCs with a pre-incubated LNP composition in vitro. In some embodiments, the method further comprises culturing HSPCs. In some embodiments, the method results in introducing Cas nuclease mRNA and gRNA into HSPCs.

In some embodiments, the LNP composition further comprises a gRNA. In some embodiments, the mRNA encodes a class 2 Cas nuclease. In certain embodiments, the cargo or RNA component comprises a Cas nuclease mRNA, such as a class 2 Cas nuclease mRNA. In certain embodiments, the cargo or RNA component comprises a CRISPR/Cas system gRNA or a nucleic acid encoding a gRNA. Methods of gene editing and methods of making engineered cells are also provided.

In vitro methods

The method delivers RNA to CD34+ cells in vitro. "CD 34+ cells" refers to cells expressing a CD34 marker on their surface. CD34+ cells can be detected and counted using, for example, flow cytometry and fluorescently labeled anti-human CD34 antibodies.

In some embodiments, a method of delivering mRNA to a stem cell, e.g., a HSPC or HSPC population, is provided, the method comprising (a) preincubating a serum factor with a LNP composition comprising mRNA, amine lipids, helper lipids, neutral lipids, and PEG lipids; (b) contacting in vitro a HSPC or population of HSPCs with a pre-incubated LNP composition; and (c) culturing the HSPC or HSPC population in vitro; thereby delivering mRNA to HSPCs. In some embodiments, the mRNA encodes a Cas nuclease, such as a class 2 Cas nuclease. In some aspects, the class 2 Cas nuclease mRNA is Cas9 mRNA or Cpf1 mRNA. In certain embodiments, the class 2 Cas nuclease is streptococcus pyogenes Cas 9. In some embodiments, the LNP composition further comprises a gRNA. In additional embodiments, the method introduces Cas nuclease mRNA and gRNA into HSPCs, the method comprising (a) preincubating serum factors with an LNP composition comprising Cas nuclease mRNA, gRNA, amine lipids, helper lipids, neutral lipids, and PEG lipids; (b) contacting HSPCs with a pre-incubated LNP composition in vitro; and (c) culturing HSPCs; thereby introducing the Cas nuclease and the gRNA into HSPC.

In various embodiments, the gRNA of the methods described herein can be a double-guide rna (dgrna) or a single-guide rna (sgrna).

In some embodiments of the in vitro methods, LNP transfection can reduce HSPC or CD34+ cell death compared to known techniques such as electroporation. In some embodiments, LNP transfection may result in less than 5%, less than 10%, less than 20%, less than 30%, or less than 40% cell death. In certain embodiments, the cell viability after transfection is at least 60%, 70%, 80%, 90% or 95%.

Stem cells are characterized by the ability to self-renew and differentiate into multiple cell types. Two broad types of mammalian stem cells are Embryonic Stem (ES) cells and adult stem cells. Adult stem or progenitor cells can complement specialized cells. Most adult stem cells are lineage restricted and may be referred to as being of their tissue origin. The ES cell line is derived from the epiblast tissue of the inner cell mass of blastocysts or early morula stage embryos. ES cells are pluripotent, producing three germinal layers, derivatives of ectoderm, endoderm and mesoderm. Induced pluripotent stem cells (ipscs) are adult cells that have been genetically reprogrammed into an embryonic stem cell-like state by being forced to express genes and factors that are critical to maintaining certain characteristics of embryonic stem cells. For example, a "stem cell" can be an ESC, iPSC, progenitor cell, or HSPC.

The terms "hematopoietic stem and/or progenitor cells" and "HSPCs" are used interchangeably and refer to a population of cells that have both HSCs and hematopoietic progenitor cells ("HPCs"). Such cells can be characterized, for example, as CD34 +. In an exemplary embodiment, the HSPCs are isolated from bone marrow. In other exemplary embodiments, the HSPCs are isolated from peripheral blood. In other exemplary embodiments, HSPCs are isolated from umbilical cord blood.

HSPCs may be derived from bone marrow, peripheral blood or umbilical cord blood, and may be autologous (patient's own stem cells) or allogeneic (stem cells from a donor).

As used herein, the term "hematopoietic progenitor cell" or "HPC" refers to the following primitive hematopoietic cells: it has limited self-renewal capacity and the potential for multipotent (e.g., myeloid, lymphoid), unipotent (e.g., myeloid or lymphoid) or Cell type-restricted (e.g., erythroid progenitor) differentiation, depending on its location in the hematopoietic hierarchy (dolatov et al, CellStem Cell 2012).

As used herein, the term "hematopoietic stem cell" or "HSC" also refers to immature blood cells that are capable of self-renewal and differentiation into more mature blood cells, including granulocytes (e.g., promyelocytes, neutrophils, eosinophils, basophils), erythrocytes (e.g., reticulocytes, erythrocytes), thrombocytes (e.g., promegakaryocytes, thrombopoietic megakaryocytes, platelets), and monocytes (e.g., monocytes, macrophages). It is known in the art that such cells may or may not include CD34+ cells. CD34+ cells are immature cells expressing CD34 cell surface markers. It is believed that CD34+ cells comprise a subpopulation of cells having the characteristics of stem cells described above. Transplantation of cell populations, such as HSPCs comprising pluripotent HSCs, can be used to treat leukemia, lymphoma, and other disorders.

HSCs are pluripotent cells that give rise to primitive progenitors (e.g., pluripotent progenitors) and/or progenitors that are committed to a particular hematopoietic lineage (e.g., lymphoid progenitors). Stem cells targeted to a particular hematopoietic lineage can be T cell lineage, B cell lineage, dendritic cell lineage, langerhans cell lineage, and/or lymphoid tissue-specific macrophage lineage. In addition, HSC are also referred to as long-term HSC (LT-HSC) and short-term HSC (ST-HSC). ST-HSCs are more active and proliferative than LT-HSCs. However, LT-HSCs have unlimited self-renewal capacity (i.e., they can survive throughout adulthood), while ST-HSCs have limited self-renewal capacity (i.e., they can only survive for a limited period of time). Any of these HSCs can be used in any of the methods described herein. Optionally, ST-HSCs are useful because they are highly proliferative, thus rapidly increasing the number of HSCs and their progeny.

HSCs, HPCs, and HSPCs are optionally obtained from blood products. Blood products include products obtained from the body or body organ containing cells of hematopoietic origin. These sources include bone marrow, umbilical cord, peripheral blood (e.g., mobilized peripheral blood, e.g., with a mobilizing agent such as G-CSF or(AMD3100) mobilized peripheral blood), liver, thymus, lymph, and spleen. All of the aforementioned blood products can be enriched (e.g., in crude, unfractionated, or fractionated form) in a manner known to those skilled in the art to obtain cells having HSC characteristics. Likewise, these blood products can be enriched for HPC and/or HSPC population characteristics. In one embodiment, the HSC are characterized as CD34+/CD38-/CD90+/CD45 RA-. In various embodiments, the HSC are characterized as CD34+/CD90+/CD49f + cells. In a further embodiment, the HSC are characterized as CD34+/CD38-/CD90+/CD45 RA-. In various embodiments, the HSC are characterized as lineage-CD 34+/CD90+/CD49f + cellsWherein "lineage" means a marker omitting terminally differentiated cells such as T cells, B cells, etc. These markers can be removed by staining the cells with antibodies against surface markers expressed by cells that have been targeted to the hematopoietic lineage. These markers may include, but are not limited to: CD3(T cells), CD19(B cells), CD33 (myeloid cells), CD56(NK cells), CD235a (erythroid cells), CD71 (erythroid cells).

When used in the context of a cell population, "enriched" refers to a cell population that is selected based on the presence of one or more markers (e.g., CD34 +). A cell population, such as a stem cell population or a HSPC population, refers to eukaryotic mammalian (preferably human) cells isolated from a biological source (e.g., a blood product or tissue) and derived from more than one cell.

During the pre-incubation, serum factors can be contacted with the LNP composition and then delivered to the HSPC cells in vitro.

Some embodiments of the in vitro methods comprise preincubating the serum factor and LNP composition together for about 30 seconds to overnight. In some embodiments, the pre-incubation step comprises pre-incubating the serum factors and LNP composition together for about 1 minute to 1 hour. In some embodiments, the pre-incubation step comprises pre-incubation for about 1-30 minutes. In other embodiments, the pre-incubation step comprises pre-incubation for about 1-10 minutes. A further embodiment comprises a pre-incubation of about 5 minutes. In certain embodiments, the endpoints and values of the ranges provided above can be ± 0.5, 1,2, 3, or 4 minutes.

In certain embodiments, the pre-incubation step occurs at about 4 ℃. In certain embodiments, the pre-incubation step occurs at about 25 ℃. In certain embodiments, the pre-incubation step occurs at about 37 ℃. The pre-incubation step may include a buffer, such as sodium bicarbonate or HEPES. In certain embodiments, the buffer can comprise HSPC medium. In further embodiments, the buffer may consist of HSPC medium.

Pre-incubation of the LNP composition with serum factors may include pre-incubation with serum, serum fractions, or isolated serum factors. In some embodiments, the LNP composition is preincubated with serum. The serum can be mammalian, mouse, primate or human serum. In some embodiments, the LNP composition is preincubated with the isolated serum factors. In certain embodiments, the serum factor is ApoE. In certain embodiments, the serum factor is selected from ApoE2, ApoE3, and ApoE 4. In further embodiments, the ApoE is a recombinant protein, such as a recombinant human protein. ApoE may be recombinant human ApoE 3. It may be recombinant human ApoE 4.

In some embodiments, the methods comprise contacting stem cells (e.g., HSPCs) or stem cell populations (e.g., HSPC populations) after the pre-incubation step, e.g., contacting the cells with a pre-incubated LNP composition. In some embodiments, the methods comprise contacting a population of stem cells, such as a population of ES or ipscs, after the pre-incubation step, for example contacting these cells with a pre-incubated LNP composition. In some embodiments, the method comprises contacting the cells with the pre-incubated LNP composition for about 1 minute to about 72 hours. In some embodiments, the method comprises contacting the cells with the pre-incubated LNP composition for about 1 hour to about 24 hours. In some embodiments, the method comprises contacting the cells with the pre-incubated LNP composition for about 4 hours to about 24 hours. In some embodiments, the method comprises contacting the cells with the pre-incubated LNP composition for about 4 hours to about 12 hours. In some embodiments, the method comprises contacting the cells with the pre-incubated LNP composition for about 2 hours to about 12 hours. In some embodiments, the method comprises contacting the cells with the pre-incubated LNP composition for about 6 hours to about 8 hours. In some embodiments, the method comprises contacting the cells with the pre-incubated LNP composition for about 6 hours to about 24 hours. In some embodiments, the method comprises contacting the cells with the pre-incubated LNP composition for about 6 hours to about 24 hours. In some embodiments, the method comprises contacting the cells with the pre-incubated LNP composition for about 4 hours to about 12 hours. In some embodiments, the method comprises contacting the cell with the pre-incubated LNP composition for at least about 0.5, 1,2, 4, 6, 8, 10, or 12 hours. In some embodiments, the method comprises a washing step after the contacting step. The washing step may comprise a medium.

In some embodiments, the method comprises Cas nuclease mRNA. In some embodiments, the method comprises a class 2 Cas nuclease mRNA. In some embodiments, the methods include gRNA nucleic acids, such as grnas. In certain embodiments, the method includes at least two gRNA nucleic acids. In additional embodiments, the method includes 3 or more gRNA nucleic acids. In some embodiments, mRNA (such as Cas nuclease mRNA) and gRNA are formulated in a single LNP composition. In some embodiments, the methods include mRNA (such as Cas nuclease mRNA) and gRNA nucleic acid co-encapsulated in an LNP composition. In additional embodiments, the methods include mRNA and gRNA nucleic acids encapsulated separately in LNPs. In certain embodiments, the mRNA is formulated in a first LNP composition and the gRNA nucleic acid is formulated in a second LNP composition. In some embodiments, the first LNP composition and the second LNP composition are administered simultaneously. In other embodiments, the first LNP composition and the second LNP composition are administered sequentially. In some embodiments of the in vitro methods, the first LNP composition and the second LNP composition are combined prior to the pre-incubation step. In some embodiments, the first LNP composition and the second LNP composition are pre-incubated separately.

In one embodiment, an LNP composition comprising mRNA encoding a Cas nuclease (such as a class 2 Cas nuclease) can be administered to a cell or population of cells, such as, for example, a HSPC or HSPC population, separately from the administration of the composition comprising the gRNA. In one embodiment, an LNP composition comprising mRNA encoding a Cas nuclease (such as a class 2 Cas nuclease) and a gRNA can be administered to, for example, a HSPC or a population of HSPCs, separately from the administration of the template nucleic acid to the cell. In one embodiment, an LNP composition comprising mRNA encoding a Cas nuclease (such as a class 2 Cas nuclease) can be administered to a population such as HSPC or HSPC, followed by sequential administration of the LNP composition comprising a gRNA and a template to a cell or population. In embodiments where an LNP composition comprising Cas nuclease-encoding mRNA is administered before an LNP composition comprising grnas, the administration may be about 4, 6, 8, 12, 24, 36, 48, or 72 hours apart; or about 1,2, or 3 days.

In some embodiments of the in vitro methods described herein, the stem cell, HSPC, or HSPC population can be cultured in vitro following transfection via LNPs.

In some embodiments, the transfected stem cell, HSPC or HSPC population is expanded in a stem cell culture medium, such as a HSPC culture medium. In the context of cells, "expansion" refers to an increase in the number of one or more cell types characteristic of an initial population of cells from which the cells (which may be the same or different) are derived. The initial cells used for expansion may be different from the cells produced from expansion. Some embodiments of the in vitro method comprise culturing the HSPC or HSPC population in a HSPC medium. Some embodiments further comprise expanding HSPCs in HSPC media comprising stem cell expanding agents. See, e.g., WO2010/059401 (e.g., the compound of example 1), WO2013/110198, and WO2017115268, the contents of which are incorporated herein by reference for suitable compounds for stem cell expansion. "Stem cell expansion agent" refers to a compound that: it allows cells such as HSPCs, HSCs, and/or HPCs to proliferate at a faster rate (e.g., increased number) relative to the same cell type without the agent. In one exemplary aspect, the stem cell expansion agent is an inhibitor of the arene receptor pathway.

In additional embodiments, the in vitro method further comprises changing the culture medium between the contacting step and the culturing step. In additional embodiments, the culturing step comprises a cell culture medium comprising thrombopoietin (Tpo), Flt3 ligand (Flt-3L), and human Stem Cell Factor (SCF). In various embodiments, the cell culture medium further comprises human interleukin-6 (IL-6). In various embodiments, the cell culture medium comprises thrombopoietin (Tpo), Flt3 ligand (Flt-3L), and human Stem Cell Factor (SCF).

CRISPR/Cas cargo

The CRISPR/Cas cargo delivered via the LNP formulation comprises an mRNA molecule encoding a protein of interest. For example, mRNA for expression of proteins such as Green Fluorescent Protein (GFP), and RNA-guided DNA binding agents, or Cas nucleases are included. LNP compositions are provided that include Cas nuclease mrnas, e.g., class 2 Cas nuclease mrnas that allow for expression of Cas9 protein in cells. In addition, the cargo can contain one or more guide RNAs or nucleic acids encoding guide RNAs. For example, a template nucleic acid for repair or recombination may also be included in the composition or may be used in the methods described herein.

"mRNA" refers to a polynucleotide that is not DNA and comprises an open reading frame that can be translated into a polypeptide (i.e., can serve as a substrate for translation by ribosomes and aminoacylated tRNA's). The mRNA may comprise a phosphate-sugar backbone comprising ribose residues or analogs thereof, such as 2' -methoxy ribose residues. In some embodiments, the sugar of the mRNA phosphate-sugar backbone consists essentially of ribose residues, 2' -methoxy ribose residues, or a combination thereof. Generally, the mRNA does not contain significant thymidine residues (e.g., 0 residues or less than 30, 20, 10, 5, 4, 3, or 2 thymidine residues; or less than 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.2%, or 0.1% thymidine content). The mRNA may contain modified uridines at some or all of its uridine positions.

CRISPR/Cas nuclease system

One component of the disclosed formulations is mRNA encoding an RNA-guided DNA binding agent (such as a Cas nuclease).

As used herein, "RNA-guided DNA binding agent" means a polypeptide or polypeptide complex having RNA and DNA binding activity, or a DNA-binding subunit of such a complex, wherein the DNA binding activity is sequence-specific and depends on the sequence of the RNA. Exemplary RNA-guided DNA binding agents include Cas lyase/nickase and their unactivated forms ("dCasDNA binding agents"). As used herein, "Cas nuclease" encompasses Cas lyase, Cas nickase, and dCas DNA-binding agents. Cas lyase/nickase and dCas DNA binding agents include the Csm or Cmr complex of a type III CRISPR system, its Cas10, Csm1, or Cmr2 subunits; a cascade complex of a type I CRISPR system, Cas3 subunit thereof; and a class 2 Cas nuclease. As used herein, a "class 2 Cas nuclease" is a single-stranded polypeptide having RNA-guided DNA binding activity. Class 2 Cas nucleases include class 2 Cas lyases/nickases (e.g., H840A, D10A, or N863A variants) that also have RNA-guided DNA lyase or nickase activity; and class 2 dCas DNA binding agents, wherein the lyase/nickase activity is not activated. Class 2 Cas nucleases include, for example, Cas9, Cpf1, C2C1, C2C2, C2C3, HF Cas9 (e.g., N497A, R661A, Q695A, Q926A variants), HypaCas9 (e.g., N692A, M694A, Q695A, H698A variants), eSPCas9(1.0) (e.g., K810A, K1003A, R1060A variants) and eSPCas9(1.1) (e.g., K848A, K1003A, R1060A variants) proteins and modified forms thereof. Cpf1 protein (Zetsche et al, Cell, 163:1-13(2015)) is homologous to Cas9 and contains a RuvC-like nuclease domain. The Cpf1 sequence from Zetsche is incorporated by reference in its entirety. See, e.g., Zetsche, table S1, and table S3. See, e.g., Makarova et al, Nat Rev Microbiol, 13(11):722-36 (2015); shmakov et al, Molecular Cell, 60: 385-.

In some embodiments, the RNA-guided DNA-binding agent is a class 2 Cas nuclease. In some embodiments, the RNA-guided DNA-binding agent has a lyase activity, which may also be referred to as a double-stranded endonuclease activity. In some embodiments, the RNA-guided DNA-binding agent comprises a Cas nuclease, such as a class 2 Cas nuclease (which may be, for example, a type II, V, or VI Cas nuclease). Class 2 Cas nucleases include, for example, Cas9, Cpf1, C2C1, C2C2 and C2C3 proteins and modified forms thereof. Examples of Cas9 nucleases include those of the type II CRISPR system of streptococcus pyogenes, staphylococcus aureus and other prokaryotes (see, e.g., the list in the next paragraph), and modified (e.g., engineered or mutated) versions thereof. See, e.g., US2016/0312198a 1; US 2016/0312199 a 1. Other examples of Cas nickases include the Csm or Cmr complex of a type III CRISPR system, or Cas10, Csm1, or Cmr2 subunits thereof; and the cascade complex of the type I CRISPR system, or Cas3 subunit thereof. In some embodiments, the Cas nuclease may be from a type IIA, type IIB, or type IIC system. See, e.g., Makarova et al, nat. rev. microbiol.9: 467-; makarova et al, nat. Rev. Microbiol, 13:722-36 (2015); shmakov et al, Molecular Cell, 60: 385-.

Non-limiting exemplary species from which the Cas nuclease can be derived include Streptococcus pyogenes (Streptococcus pyogenes), Streptococcus thermophilus (Streptococcus thermophilus), Streptococcus species (Streptococcus sp.), Staphylococcus aureus (Staphylococcus aureus), Listeria innocua (Listeria innocus), Lactobacillus gasseri (Lactobacillus gasseri), franciscella novaculeatus (francisella anovularia), wolframus succinogenes (wolframaria succinogenes), klebsiella lavandula (sutterella wadensis), proteus gammaproteinea (gammoproteobacterium), Neisseria meningitidis (Neisseria meningitidis), Campylobacter jejuni (Campylobacter jejunii), Pasteurella multocida (Pasteurella multocida), Streptomyces cellulosae (rhodobacter roseus), Streptomyces viridis (rhodobacter carotovorus), Streptomyces viridis (Streptococcus thermophilus), Streptomyces carotovorus), Streptomyces viridis (Streptococcus lactis), Streptomyces rhodobacter viridis (Streptococcus lactis), Streptomyces strain, Streptomyces carotovorans, Streptomyces carotovorus, Streptomyces strain, etc., strain, strain, strain, strain, Bacillus pseudomycoides (Bacillus pseudomycoides), Bacillus arsenic reducing bacteria (Bacillus selinitriensis), Bacillus sibiricus (Exiguobacterium sibiricum), Lactobacillus delbrueckii (Lactobacillus delbrueckii), Lactobacillus salivarius (Lactobacillus salivarius), Lactobacillus buchneri (Lactobacillus buchneri), Treponema denticola (Treponema pallidum), Microcilaria marinus (Microcilaria marinus), Micrococcus rhodochrous (Polaromonas naphylenesus), Micrococcus rhodochrous (Polaromyces sp), Micrococcus pyogenes (Micrococcus pyogenes), Micrococcus thermophilus (Micrococcus pyogenes), Micrococcus pyogenes (Clostridium thermoacidophilus), Clostridium thermocola (Clostridium thermoacidophilus), Micrococcus (Clostridium thermobacillus acidophylum), Micrococcus pyogenes (Clostridium thermobacillus acidovorans), Clostridium thermoascus, Clostridium thermonitricola (Clostridium thermobacillus acidophylum), Clostridium thermoacidophilus (Clostridium thermoacidophilus), Clostridium thermoacidophilus (Clostridium thermoacidophilus, Clostridium thermobacillus acidophylium, Clostridium thermobacillus acidophylum, Clostridium thermoacidophilus, Clostridium thermoacidophil, Thiobacillus acidophilus (Acidithiobacillus caldus), Thiobacillus acidophilus (Acidithiobacillus ferrooxidans), Allolobacter vinaceus (Allochinomatous vinosus), Haematococcus species (Marinobacter sp.), Nitrosomonas halophilus (Nitrosococcus halophilus), Nitrosococcus vannamei (Nitrosococcus watsoni), Pseudoalteromonas mobilis (Psudoallomonas halophilus), Micrococcus racemosus (Kteodobacter asiaticus), Methanobacterium investigatans (Methanohalophilus), Anabaena varia (Anabaena variabilis), Synechococcus foamoides (Nodularia), Streptococcus faecalis (Anabaena varia), Streptococcus faecalis (Nodulcosis), Streptococcus faecalis (Streptococcus faecalis), Streptococcus faecalis sp), Streptococcus faecalis (Streptococcus faecalis sp), Streptococcus faecalis strain (Streptococcus faecalis sp), Streptococcus faecalis strain (Streptococcus faecalis sp), Streptococcus faecalis strain (Streptococcus faecalis sp), Streptococcus faecalis strain (Streptococcus), Streptococcus faecalis strain (Streptococcus), Streptococcus faecalis strain (Streptococcus), Streptococcus faecalis strain (Streptococcus, Corynebacterium diphtheriae (Corynebacterium diphtheria), Aminococcus sp, Lachnospiraceae (Lachnospiraceae) ND2006 and Acarylychloride marianum (Acarylchloride marina).

In some embodiments, the Cas nuclease is Cas9 nuclease from streptococcus pyogenes. In some embodiments, the Cas nuclease is Cas9 nuclease from streptococcus thermophilus. In some embodiments, the Cas nuclease is Cas9 nuclease from neisseria meningitidis. In some embodiments, the Cas nuclease is Cas9 nuclease from staphylococcus aureus. In some embodiments, the Cas nuclease is Cpf1 nuclease from francisella novacellularis. In some embodiments, the Cas nuclease is a Cpf1 nuclease from a rhodococcus species. In some embodiments, the Cas nuclease is Cpf1 nuclease from lachnospiraceae ND 2006. In other embodiments, the Cas nuclease is a Cpf1 nuclease from: francisella tularensis (Francisella reticulata), Muricidae, Vibrio proteolyticus (Butyrivibrio proteoclasius), Heterophaera species (Peregrinibacter), Pakura bacteria (Parcuberia bacterium), Smith bacteria (Smithlla), Aminococcus, Methylobacterium termitarium (Candidatus Methanoplastrum), Mycobacterium phlei (Eubacterium elegiensis), Moraxella bovis (Moraxella vacuoli), Leptospira oryzae (Leptospira inadi), Porphyromonas canis (Porphyromonas cremoris), Prevotella saccharolytica (Prevotella), or Porphyromonas actinidiae (Porphyromonas). In certain embodiments, the Cas nuclease is a Cpf1 nuclease from the family aminoacetococcus or lachnospiraceae.

Wild-type Cas9 has two nuclease domains: RuvC and HNH. The RuvC domain cleaves non-target DNA strands and the HNH domain cleaves target DNA strands. In some embodiments, the Cas9 nuclease comprises more than one RuvC domain and/or more than one HNH domain. In some embodiments, the Cas9 nuclease is wild-type Cas 9. In some embodiments, Cas9 is capable of inducing double strand breaks in the target DNA. In certain embodiments, the Cas nuclease can cleave dsDNA, it can cleave one strand of dsDNA, or it can have no DNA cleaving enzyme or nickase activity. An exemplary Cas9 amino acid sequence is provided in the form of SEQ ID NO: 3. An exemplary Cas9 mRNA ORF sequence including a start codon and a stop codon is provided in the form of SEQ ID No. 4. An exemplary Cas9 mRNA coding sequence suitable for inclusion in the fusion protein is provided in SEQ ID NO: 10.

In some embodiments, a chimeric Cas nuclease is used in which one domain or region of a protein is replaced with a portion of a different protein. In some embodiments, the Cas nuclease domain may be replaced with a domain from a different nuclease such as Fok 1. In some embodiments, the Cas nuclease may be a modified nuclease.

In other embodiments, the Cas nuclease may be from a type I CRISPR/Cas system. In some embodiments, the Cas nuclease may be a component of a cascade complex of a type I CRISPR/Cas system. In some embodiments, the Cas nuclease may be a Cas3 protein. In some embodiments, the Cas nuclease may be from a type III CRISPR/Cas system. In some embodiments, the Cas nuclease may have RNA cleavage activity.

In some embodiments, the RNA-guided DNA binding agent has single-strand nickase activity, i.e., can cleave one DNA strand to produce a single-strand break, also referred to as a "nick. In some embodiments, the RNA-guided DNA-binding agent comprises a Cas nickase. Nicking enzymes are enzymes that make nicks in dsDNA, i.e., that cleave one strand but not the other strand of the DNA duplex. In some embodiments, the Cas nickase is a version of a Cas nuclease (e.g., the Cas nucleases discussed above) in which the endonuclease active site is inactivated, e.g., by one or more alterations (e.g., point mutations) of the catalytic domain. See, e.g., U.S. patent No. 8,889,356 for a discussion of Cas nickases and exemplary catalytic domain changes. In some embodiments, a Cas nickase, such as a Cas9 nickase, has an unactivated RuvC or HNH domain.

In some embodiments, the RNA-guided DNA binding agent is modified to contain only one functional nuclease domain. For example, the agent protein may be modified such that one of the nuclease domains is mutated or deleted in whole or in part to reduce its nucleolytic activity. In some embodiments, a nickase having a RuvC domain with reduced activity is used. In some embodiments, a nickase having an inactive RuvC domain is used. In some embodiments, a nickase having an HNH domain with reduced activity is used. In some embodiments, a nickase having an inactive HNH domain is used.

In some embodiments, a conserved amino acid in a Cas protein nuclease domain is substituted to reduce or alter nuclease activity. In some embodiments, the Cas nuclease may comprise an amino acid substitution in a RuvC or RuvC-like nuclease domain. Exemplary amino acid substitutions in the RuvC or RuvC-like nuclease domain include D10A (based on the streptococcus pyogenes Cas9 protein). See, e.g., Zetsche et al (2015) Cell Oct 22:163(3): 759-. In some embodiments, the Cas nuclease may comprise an amino acid substitution in an HNH or HNH-like nuclease domain. Exemplary amino acid substitutions in HNH or HNH-like nuclease domains include E762A, H840A, N863A, H983A, and D986A (based on streptococcus pyogenes Cas9 protein). See, e.g., Zetsche et al (2015). Other exemplary amino acid substitutions include D917A, E1006A, and D1255A (based on the franccpf 1(FnCpf1) sequence of franciscella foeniculis U112Cpf1 (UniProtKB-A0Q7Q2(Cpf1_ FRATN)).

In some embodiments, a pair of guide RNAs complementary to the sense and antisense strands, respectively, of the target sequence are combined to provide an mRNA encoding a nicking enzyme. In this embodiment, the guide RNA directs the nicking enzyme to the target sequence and introduces the DSB by making a nick on the opposite strand of the target sequence (i.e., double nicking). In some embodiments, the use of double nicks can improve specificity and reduce off-target effects. In some embodiments, a nickase is used in conjunction with two independent guide RNAs that target opposite strands of DNA to create a double nick in the target DNA. In some embodiments, a nicking enzyme is used in conjunction with two independent guide RNAs that are selected to be in close proximity to create a double nick in the target DNA.

In some embodiments, the RNA-guided DNA binding agent lacks lyase and nickase activity. In some embodiments, the RNA-guided DNA binding agent comprises a dCas DNA binding polypeptide. dCas polypeptides have DNA binding activity and essentially lack catalytic (lyase/nickase) activity. In some embodiments, the dCas polypeptide is a dCas9 polypeptide. In some embodiments, an RNA-guided DNA-binding agent or dCas DNA-binding polypeptide lacking lyase and nickase activity is a version of a Cas nuclease (e.g., the Cas nucleases discussed above) in which the endonuclease active site is inactivated, e.g., by one or more alterations (e.g., point mutations) of the catalytic domain. See, e.g., US 2014/0186958 a 1; US 2015/0166980 a 1.

In some embodiments, the RNA-guided DNA binding agent comprises one or more heterologous functional domains (e.g., is or comprises a fusion polypeptide).

In some embodiments, the heterologous functional domain may facilitate the delivery of an RNA-guided DNA binding agent into the nucleus of a cell. For example, the heterologous functional domain can be a Nuclear Localization Signal (NLS). In some embodiments, the RNA-guided DNA binding agent can be fused to 1-10 NLS. In some embodiments, the RNA-guided DNA binding agent can be fused to 1-5 NLS. In some embodiments, the RNA-guided DNA binding agent may be fused to one NLS. In the case of using one NLS, the NLS can be linked at the N-terminus or C-terminus of the RNA-guided DNA-binding agent sequence. NLS can also be inserted into RNA-guided DNA-binding agent sequences. In other embodiments, the RNA-guided DNA binding agent may be fused to more than one NLS. In some embodiments, the RNA-guided DNA binding agent can be fused to 2, 3,4, or 5 NLS. In some embodiments, the RNA-guided DNA binding agent can be fused to two NLSs. In certain cases, the two NLSs may be the same (e.g., two SV40 NLSs) or different. In some embodiments, the RNA-guided DNA binding agent is fused to two SV40 NLS sequences linked at the carboxy terminus. In some embodiments, the RNA-guided DNA-binding agent may be fused to two NLSs, one at the N-terminus and one at the C-terminus. In some embodiments, the RNA-guided DNA binding agent can be fused to 3 NLS. In some embodiments, the RNA-guided DNA binding agent may not be fused to the NLS. In some embodiments, the NLS may be a single sequence, such as, for example, SV40 NLS, PKKKRKV, or PKKKRRV. In some embodiments, the NLS can be a binary sequence, such as NLS of nucleoplasmin, krpaatkkagqakkkkkkk. In particular embodiments, a single PKKKRKV NLS may be attached at the C-terminus of an RNA-guided DNA binding agent. One or more linkers are optionally included at the fusion site.

In some embodiments, the heterologous functional domain may be capable of modifying the intracellular half-life of the RNA-guided DNA-binding agent. In some embodiments, the half-life of the RNA-guided DNA binding agent may be increased. In some embodiments, the half-life of the RNA-guided DNA binding agent can be decreased. In some embodiments, a heterologous functional domain may be capable of improving the stability of an RNA-guided DNA binding agent. In some embodiments, the heterologous functional domain may be capable of reducing the stability of an RNA-guided DNA binding agent. In some embodiments, the heterologous functional domain may serve as a signal peptide for protein degradation. In some embodiments, protein degradation may be mediated by proteolytic enzymes, such as, for example, proteasome, lysosomal proteases, or calpain proteases. In some embodiments, the heterologous functional domain may comprise a PEST sequence. In some embodiments, the RNA-guided DNA binding agent may be modified by the addition of ubiquitin or polyubiquitin chains. In some embodiments, the ubiquitin can be a ubiquitin-like protein (UBL). Non-limiting examples of ubiquitin-like proteins include ubiquitin-like modifier (SUMO), ubiquitin cross-reactive protein (UCRP, also known as interferon-stimulated gene-15 (ISG15)), ubiquitin-related modifier-1 (URM1), neuronal-precursor-cell expressed developmentally down-regulated protein-8 (NEDD8, also known as Rub1 in saccharomyces cerevisiae (s. cerevisiae), human leukocyte antigen F-related (FAT10), autophagy-8 (ATG8) and autophagy-12 (ATG12), Fau ubiquitin-like protein (FUB1), membrane anchor (UBL mub), folding modifier-1 (UFM1) and ubiquitin-like protein-5 (UBL 5).

In some embodiments, the heterologous functional domain can be a marker domain. Non-limiting examples of marker domains include fluorescent proteins, purification tags, epitope tags, and reporter gene sequences. In some embodiments, the marker domain may be a fluorescent protein. Non-limiting examples of suitable fluorescent proteins include Green fluorescent proteins (e.g., GFP-2, tagGFP, turboGFP, sfGFP, EGFP, Emerald (Emerald), Azami Green (Azami Green), Single Azami Green (MonomericAzami Green), CopGFP, AceGFP, ZsGreen1), yellow fluorescent proteins (e.g., YFP, EYFP, Citrine, Venus, YPet, PhiYFP, ZsYellow1), blue fluorescent proteins (e.g., EBFP2, Azite, mKalamal, GFPuv, Sapphire (Sapphire), T-Sapphire (T-Sapphire)), blue fluorescent proteins (e.g., ECFP, sRhsleman, Cypett, AmCyan 5, Midoisis blue (Midoisis blue), red fluorescent proteins (red-Cyrite), red fluorescent proteins (red-yellow), red fluorescent proteins (red-red), red fluorescent proteins (red-red fluorescent proteins), red fluorescent proteins (red fluorescent proteins), red fluorescent proteins (red fluorescent proteins), red fluorescent proteins) (Monomered fluorescent proteins) (e.g., red fluorescent proteins) (RCB 3, red fluorescent proteins) (rDNA red fluorescent proteins (red fluorescent proteins), red fluorescent proteins) (rDNA, red fluorescent proteins) (, Single Kusabira Orange (monomer Kusabira-Orange), mTangerine, tdTomato) or any other suitable fluorescent protein. In other embodiments, the tagging domain may be a purification tag and/or an epitope tag. Non-limiting exemplary tags include glutathione-S-transferase (GST), chitin-binding protein (CBP), maltose-binding protein (MBP), Thioredoxin (TRX), poly (NANP), Tandem Affinity Purification (TAP) tag, myc, AcV5, AU1, AU5, E, ECS, E2, FLAG, HA, nus, Softag1, Softag 3, streptomycin, SBP, Glu-Glu, HSV, KT3, S, S1, T7, V5, VSV-G, 6XHis, 8XHis, Biotin Carboxyl Carrier Protein (BCCP), polyhis, and calmodulin. Non-limiting exemplary reporter genes include glutathione-S-transferase (GST), horseradish peroxidase (HRP), Chloramphenicol Acetyltransferase (CAT), beta-galactosidase, beta-glucuronidase, luciferase, or fluorescent protein.

In other embodiments, the heterologous functional domain can target the RNA-guided DNA binding agent to a particular organelle, cell type, tissue, or organ. In some embodiments, the heterologous functional domain can target an RNA-guided DNA binding agent to the mitochondria.

In other embodiments, the heterologous functional domain may be an effector domain. When an RNA-guided DNA binding agent is directed to its target sequence, e.g., when a Cas nuclease is directed to the target sequence through a gRNA, the effector domain may modify or affect the target sequence. In some embodiments, the effector domain can be selected from a nucleic acid binding domain, a nuclease domain (e.g., a non-Cas nuclease domain), an epigenetic modification domain, a transcriptional activation domain, or a transcriptional repression domain. In some embodiments, the heterologous functional domain is a nuclease, such as fokl nuclease. See, for example, U.S. patent No. 9,023,649. In some embodiments, the heterologous functional domain is a transcriptional activator or repressor. See, e.g., Qi et al, "reproducing CRISPR as an RNA-guided platform for sequence-specific control gene expression," Cell 152:1173-83 (2013); Perez-Pinera et al, "RNA-guided genetic analysis by CRISPR-Cas9-based transformation factors," nat. methods 10:973-6 (2013); mali et al, "CAS 9 transgenic activators for target specific information and targeted information for collaborative genome engineering," nat. Biotechnol.31:833-8 (2013); gilbert et al, "CRISPR-mediated modular RNA-guided regulation of transformation in eukaryotes," Cell 154:442-51 (2013). Thus, an RNA-guided DNA binding agent essentially becomes a transcription factor that can be guided using a guide RNA to bind a desired target sequence. In certain embodiments, the DNA modification domain is a methylation domain, such as a demethylation or methyltransferase domain. In certain embodiments, the effector domain is a DNA modification domain, such as a base editing domain. In particular embodiments, a DNA modification domain is a nucleic acid editing domain, such as a deaminase domain, that introduces specific modifications into DNA. See, e.g., WO 2015/089406; US 2016/0304846. The nucleic acid editing domain, deaminase domain and Cas9 variants described in WO 2015/089406 and u.s.2016/0304846 are incorporated herein by reference.

The nuclease may comprise at least one domain that interacts with a guide RNA ("gRNA"). In addition, nucleases can be directed to the target sequence through the gRNA. In class 2 Cas nuclease systems, the gRNA interacts with a nuclease and a target sequence such that it directs binding to the target sequence. In some embodiments, the grnas provide specificity for targeted cleavage, and the nucleases can be universal and paired with different grnas to cleave different target sequences. Class 2 Cas nucleases can be paired with gRNA scaffold structures of the types, orthologs, and exemplary species listed above.

Guide RNA (gRNA)

In some embodiments of the disclosure, the cargo of the LNP formulation includes at least one gRNA. The gRNA can direct a Cas nuclease or class 2 Cas nuclease to a target sequence on a target nucleic acid molecule. In some embodiments, the gRNA binds to a class 2 Cas nuclease and provides cleavage specificity by the class 2 Cas nuclease. In some embodiments, the gRNA and Cas nuclease may form a Ribonucleoprotein (RNP), e.g., a CRISPR/Cas complex, such as a CRISPR/Cas9 complex. In some embodiments, the CRISPR/Cas complex can be a type II CRISPR/Cas9 complex. In some embodiments, the CRISPR/Cas complex may be a type V CRISPR/Cas complex, such as a Cpf 1/guide RNA complex. Cas nuclease and homologous gRNA can pair. The gRNA scaffold structure that pairs with each class 2 Cas nuclease varies with the specific CRISPR/Cas system.

"guide RNA," "gRNA," and simply "guide" are used interchangeably herein to refer to crRNA (also referred to as CRISPR RNA), or a combination of crRNA and trRNA (also referred to as tracrRNA). The crRNA and trRNA may associate as a single RNA molecule (single guide RNA, sgRNA) or as two separate RNA molecules (double guide RNA, dgRNA). "guide RNA" or "gRNA" refers to each type. the trRNA may be a naturally occurring sequence or a trRNA sequence having modifications or alterations compared to the naturally occurring sequence.

As used herein, "guide sequence" refers to a sequence in a guide RNA that is complementary to a target sequence and serves to direct the guide RNA to the target sequence for binding or modification (e.g., cleavage) by an RNA-guided DNA binding agent. A "guide sequence" may also be referred to as a "target sequence" or a "spacer sequence". The guide sequence can be 20 base pairs in length, for example in the case of streptococcus pyogenes (i.e., SpyCas9) and related Cas9 homologs/orthologs. Shorter or longer sequences may also be used as guides, for example 15, 16, 17, 18, 19, 21, 22, 23, 24 or 25 nucleotides in length. In some embodiments, the target sequence is, for example, in a gene or on a chromosome, and is complementary to the guide sequence. In some embodiments, the degree of complementarity or identity between a guide sequence and its corresponding target sequence may be about 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%. In some embodiments, the guide sequence may be 100% complementary or identical to the target region. In other embodiments, the guide sequence and target region may contain at least one mismatch. For example, the guide sequence and target sequence may contain 1,2, 3, or 4 mismatches, with the total length of the target sequence being at least 17, 18, 19, 20, or more base pairs. In some embodiments, the guide sequence and target region may contain 1-4 mismatches, wherein the guide sequence comprises at least 17, 18, 19, 20 or more nucleotides. In some embodiments, the guide sequence and target region may contain 1,2, 3, or 4 mismatches, wherein the guide sequence comprises 20 nucleotides.

Target sequences for Cas proteins include both the positive and negative strands of genomic DNA (i.e., the given sequence and the reverse complement of the sequence) because the nucleic acid substrate of Cas protein is a double-stranded nucleic acid. Thus, where a guide sequence is said to be "complementary to" a target sequence, it is understood that the guide sequence can direct the binding of a guide RNA to the reverse complement of the target sequence. Thus, in some embodiments, where the guide sequence binds to the reverse complement of the target sequence, the guide sequence is identical to certain nucleotides of the target sequence (e.g., a target sequence that does not include PAM) except that U replaces T in the guide sequence.

The length of the targeting sequence may depend on the CRISPR/Cas system and components used. For example, different class 2 Cas nucleases from different bacterial species have varying optimal targeting sequence lengths. Thus, the targeting sequence may comprise 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, or greater than 50 nucleotides in length. In some embodiments, the targeting sequence length is 0, 1,2, 3,4, or 5 nucleotides longer or shorter than the guide sequence of the naturally occurring CRISPR/Cas system. In certain embodiments, the Cas nuclease and gRNA scaffold will be derived from the same CRISPR/Cas system. In some embodiments, the targeting sequence may comprise or consist of 18-24 nucleotides. In some embodiments, the targeting sequence may comprise or consist of 19-21 nucleotides. In some embodiments, the targeting sequence may comprise or consist of 20 nucleotides.

In some embodiments, the sgRNA is a "Cas 9 sgRNA" capable of RNA-guided DNA cleavage mediated by a Cas9 protein. In some embodiments, the sgRNA is a "Cpf 1 sgRNA" capable of RNA-directed DNA cleavage mediated by a Cpf1 protein. In certain embodiments, the gRNA comprises crRNA and tracr RNA sufficient to form an active complex with Cas9 protein and mediate RNA-guided DNA cleavage. In certain embodiments, the gRNA comprises crRNA sufficient to form an active complex with the Cpf1 protein and mediate RNA-guided DNA cleavage. See Zetsche 2015.

Certain embodiments of the invention also provide nucleic acids, e.g., expression cassettes, encoding grnas described herein. A "guide RNA nucleic acid" is used herein to refer to a guide RNA (e.g., sgRNA or dgRNA) and a guide RNA expression cassette, which is a nucleic acid encoding one or more guide RNAs.

In some embodiments, the nucleic acid may be a DNA molecule. In some embodiments, the nucleic acid may comprise a nucleotide sequence encoding a crRNA. In some embodiments, the nucleotide sequence encoding the crRNA comprises a targeting sequence flanked by all or a portion of a repeat sequence from a naturally occurring CRISPR/Cas system. In some embodiments, the nucleic acid may comprise a nucleotide sequence encoding a tracr RNA. In some embodiments, the crRNA and tracr RNA may be encoded by two separate nucleic acids. In other embodiments, the crRNA and tracr RNA may be encoded by a single nucleic acid. In some embodiments, the crRNA and tracr RNA may be encoded by opposite strands of a single nucleic acid. In other embodiments, the crRNA and tracr RNA may be encoded by the same strand of a single nucleic acid. In some embodiments, the gRNA nucleic acid encodes an sgRNA. In some embodiments, the gRNA nucleic acid encodes a Cas9 nuclease sgRNA. In some embodiments, the gRNA nucleic acid encodes a Cpf1 nuclease sgRNA.

The nucleotide sequence encoding the guide RNA can be operably linked to at least one transcriptional or regulatory control sequence, such as a promoter, a 3'UTR or a 5' UTR. In one example, the promoter can be a tRNA promoter, e.g., a tRNA^Lys3Or a tRNA chimera. See Mefferd et al, RNA.201521: 1683-9; scherer et al, Nucleic Acids Res.200735: 2620-2628. In certain embodiments, the promoter is recognized by RNA polymerase iii (pol iii). Non-limiting examples of Pol III promoters also include the U6 and H1 promoters. In some embodiments, the nucleotide sequence encoding the guide RNA may be operably linked to a mouse or human U6 promoter. In some embodiments, a gRNA nucleic acid is a modified nucleic acid. In certain embodiments, a gRNA nucleic acid includes a modified nucleoside or nucleotide. In some embodiments, a gRNA nucleic acid includes a 5' end modification, e.g., a modified nucleoside or nucleotide toStabilize and prevent nucleic acid integration. In some embodiments, a gRNA nucleic acid comprises a double-stranded DNA having a 5' end modification on each strand. In certain embodiments, a gRNA nucleic acid includes an inverted dideoxy-T or inverted abasic nucleoside or nucleotide as a 5' end modification. In some embodiments, gRNA nucleic acids include labels such as biotin, desthiobiotin-TEG (desthiobioten-TEG), digoxigenin, and fluorescent labels including, for example, FAM, ROX, TAMRA, and AlexaFluor.

In certain embodiments, more than one gRNA nucleic acid, such as a gRNA, can be used in a CRISPR/Cas nuclease system. Each gRNA nucleic acid can contain a different targeting sequence, such that the CRISPR/Cas system cleaves more than one target sequence. In some embodiments, one or more grnas can have the same or different properties, such as activity or stability, within the CRISPR/Cas complex. Where more than one gRNA is used, each gRNA may be encoded on the same or different gRNA nucleic acid. The promoters used to drive expression of more than one gRNA may be the same or different.

Modified RNA

In certain embodiments, the LNP composition comprises a modified RNA.

The modified nucleoside or nucleotide can be present in an RNA, such as a gRNA or mRNA. Grnas or mrnas comprising one or more modified nucleosides or nucleotides, for example, are referred to as "modified" RNAs, and are used to describe the presence of one or more non-natural and/or naturally occurring components or configurations used in place of or in addition to the typical A, G, C and U residues. In some embodiments, the modified RNA is synthesized from atypical nucleosides or nucleotides, referred to herein as "modified.

Modified nucleosides and nucleotides can include one or more of the following: (i) changes, such as substitutions (exemplary backbone modifications), of one or both of the non-bonded phosphate oxygens and/or one or more of the bonded phosphate oxygens in the phosphodiester backbone linkage; (ii) alterations in the ribose moiety, e.g., the 2' hydroxyl group on ribose, such as substitutions (exemplary sugar modifications); (iii) bulk replacement of phosphate moieties with "dephosphorylated" linkers (exemplary backbone modifications); (iv) modifications or substitutions of naturally occurring nucleobases, including with atypical nucleobases (exemplary base modifications); (v) substitution or modification of the ribose-phosphate backbone (exemplary backbone modifications); (vi) modification of the 3 'end or 5' end of the oligonucleotide, such as removal, modification or substitution of a terminal phosphate group or conjugation of a moiety, cap or linker (such 3 'or 5' cap modifications may include sugar and/or backbone modifications); and (vii) modifications or substitutions of sugars (exemplary sugar modifications). Certain embodiments comprise 5' end modifications of mRNA, gRNA, or nucleic acids. Certain embodiments comprise a 3' end modification of the mRNA, gRNA, or nucleic acid. The modified RNA can contain 5 'and 3' end modifications. The modified RNA may contain one or more modified residues in non-terminal positions. In certain embodiments, the gRNA includes at least one modified residue. In certain embodiments, the mRNA includes at least one modified residue.

As used herein, a first sequence is considered to be "comprising a sequence that is at least X% identical to a second sequence" if an alignment of the first sequence to the second sequence shows that X% or more of the positions of all of the second sequence match the first sequence. For example, sequence AAGA comprises a sequence that is 100% identical to sequence AAG, since an alignment will result in 100% identity because there is a match for all three positions of the second sequence. Differences between RNA and DNA (generally, uridine is replaced by thymidine or vice versa) and the presence of nucleoside analogues, such as modified uridine, do not cause differences in identity or complementarity between polynucleotides, as long as the relevant nucleotides (such as thymidine, uridine or modified uridine) have the same complementary sequence (e.g. adenosine for all thymidine, uridine or modified uridine; another example is cytosine and 5-methylcytosine, both having guanosine or modified guanosine as the complementary sequence). Thus, for example, the sequence 5'-AXG (where X is any modified uridine such as pseudouridine, N1-methylpseuduridine or 5-methoxyuridine) is considered to be 100% identical to AUG, since both are fully complementary to the same sequence (5' -CAU). Exemplary contrast algorithms are the Smith-waterman algorithm (Smith-waterman algorithm) and the Needleman-Wunsch algorithm (Needleman-Wunsch algorithm), which are well known in the art. Those skilled in the art will understand which algorithm choices and parameter settings are appropriate for a given pair of sequences to be aligned; for sequences of generally similar length and > 50% expected identity for amino acids or > 75% expected identity for nucleotides, the niemann-winche algorithm with default settings of the niemann-winche algorithm interface provided by the EBI at www.ebi.ac.uk website server is generally suitable.

mRNA

In some embodiments, a composition or formulation disclosed herein comprises an mRNA comprising an Open Reading Frame (ORF), such as, e.g., an ORF encoding an RNA-guided DNA binding agent, such as a Cas nuclease, or a class 2 Cas nuclease as described herein. In some embodiments, an mRNA comprising an ORF encoding an RNA-guided DNA-binding agent, such as a Cas nuclease or class 2 Cas nuclease, is provided, used, or administered. In some embodiments, the ORF encoding the RNA-guided DNA-binding agent is a "modified RNA-guided DNA-binding agent ORF" or simply a "modified ORF" that is used as a shorthand to indicate that the ORF is modified in one or more of the following ways: (1) the uridine content of the modified ORF is in the range of its minimum uridine content to 150% of the minimum uridine content; (2) the uridine dinucleotide content of the modified ORF is in the range of its minimum uridine dinucleotide content to 150% of its minimum uridine dinucleotide content; (3) the modified ORF is at least 90% identical to any one of SEQ ID NOs 1, 4, 10, 14, 15, 17, 18, 20, 21, 23, 24, 26, 27, 29, 30, 50, 52, 54, 65, or 66; (4) the modified ORF consists of a set of codons, wherein at least 75% of the codons are the smallest uridine codons of a given amino acid, e.g. codons with the least uridine (typically 0 or 1 in addition to codons for phenylalanine (wherein the smallest uridine codon has 2 uridines)); or (5) the modified ORF comprises at least one modified uridine. In some embodiments, the modified ORF is modified in at least two, three, or four of the aforementioned ways. In some embodiments, the modified ORF comprises at least one modified uridine and is modified with at least one, two, three, or all of (1) - (4) above.

"modified uridine" is used herein to refer to nucleosides other than thymidine that have the same hydrogen bond acceptor as uridine and one or more structural differences from uridine. In some embodiments, the modified uridine is a substituted uridine, i.e., a uridine in which one or more aprotic substituents (e.g., alkoxy groups, such as methoxy groups) replace a proton. In some embodiments, the modified uridine is a pseudouridine. In some embodiments, the modified uridine is a substituted pseudouridine, i.e., a pseudouridine in which one or more aprotic substituents (e.g., alkyl groups, such as methyl groups) replace a proton. In some embodiments, the modified uridine is any one of a substituted uridine, a pseudouridine, or a substituted pseudouridine.

As used herein, "uridine position" refers to a position in a polynucleotide occupied by uridine or a modified uridine. Thus, for example, a polynucleotide in which "100% of the uridine positions are modified uridine" contains a modified uridine at each position of the uridine in a conventional RNA (in which all bases are the standard A, U, C or G bases) that will be the same sequence. Unless otherwise indicated, U in the polynucleotide sequences of the sequence table (sequence listing) in or accompanying the present disclosure may be uridine or modified uridine.

TABLE 1 minimum uridine codon

In any of the preceding embodiments, the modified ORF may consist of a set of codons, wherein at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% of the codons are those listed in the table of smallest uridine codons. In any of the preceding embodiments, the modified ORF may comprise a sequence having at least 90%, 95%, 98%, 99% or 100% identity to any of SEQ ID NOs 1, 4, 10, 14, 15, 17, 18, 20, 21, 23, 24, 26, 27, 29, 30, 50, 52, 54, 65 or 66.

In any of the foregoing embodiments, the uridine content of the modified ORF may range from its minimum uridine content to 150%, 145%, 140%, 135%, 130%, 125%, 120%, 115%, 110%, 105%, 104%, 103%, 102%, or 101% of the minimum uridine content.

In any of the foregoing embodiments, the uridine dinucleotide content of the modified ORF may range from its minimum uridine dinucleotide content to 150%, 145%, 140%, 135%, 130%, 125%, 120%, 115%, 110%, 105%, 104%, 103%, 102%, or 101% of its minimum uridine dinucleotide content.

In any of the preceding embodiments, the modified ORF may comprise a modified uridine at least at one, more or all uridine positions. In some embodiments, the modified uridine is a uridine modified at position 5, for example with halogen, methyl or ethyl. In some embodiments, the modified uridine is a pseudouridine modified at position 1, for example with halogen, methyl or ethyl. The modified uridine may be, for example, pseudouridine, N1-methylpseudouridine, 5-methoxyuridine, 5-iodouridine or a combination thereof. In some embodiments, the modified uridine is 5-methoxyuridine. In some embodiments, the modified uridine is 5-iodouridine. In some embodiments, the modified uridine is a pseudouridine. In some embodiments, the modified uridine is N1-methylpseuduridine. In some embodiments, the modified uridine is a combination of pseudouridine and N1-methylpseuduridine. In some embodiments, the modified uridine is a combination of pseudouridine and 5-methoxyuridine. In some embodiments, the modified uridine is a combination of N1-methylpseudouridine and 5-methoxyuridine. In some embodiments, the modified uridine is a combination of 5-iodouridine and N1-methylpseuduridine. In some embodiments, the modified uridine is a combination of pseudouridine and 5-iodouridine. In some embodiments, the modified uridine is a combination of 5-iodouridine and 5-methoxyuridine.

In some embodiments, at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% of the uridine positions in an mRNA according to the present disclosure are modified uridine. In some embodiments, 10% -25%, 15% -25%, 25% -35%, 35% -45%, 45% -55%, 55% -65%, 65% -75%, 75% -85%, 85% -95%, or 90% -100% of the uridine positions in an mRNA according to the present disclosure are modified uridine, such as 5-methoxyuridine, 5-iodouridine, N1-methylpseudouridine, pseudouridine, or a combination thereof. In some embodiments, 10% -25%, 15% -25%, 25% -35%, 35% -45%, 45% -55%, 55% -65%, 65% -75%, 75% -85%, 85% -95%, or 90% -100% of the uridine positions in mRNA according to the present disclosure are 5-methoxyuridine. In some embodiments, 10% -25%, 15% -25%, 25% -35%, 35% -45%, 45% -55%, 55% -65%, 65% -75%, 75% -85%, 85% -95%, or 90% -100% of the uridine positions in mRNA according to the present disclosure are pseudouridine. In some embodiments, 10% -25%, 15% -25%, 25% -35%, 35% -45%, 45% -55%, 55% -65%, 65% -75%, 75% -85%, 85% -95%, or 90% -100% of the uridine positions in mRNA according to the present disclosure are N1-methylpseudouridine. In some embodiments, 10% -25%, 15% -25%, 25% -35%, 35% -45%, 45% -55%, 55% -65%, 65% -75%, 75% -85%, 85% -95%, or 90% -100% of the uridine positions in mRNA according to the present disclosure are 5-iodouridine. In some embodiments, 10% -25%, 15% -25%, 25% -35%, 35% -45%, 45% -55%, 55% -65%, 65% -75%, 75% -85%, 85% -95%, or 90% -100% of the uridine positions in mRNA according to the present disclosure are 5-methoxyuridine, and the remainder are N1-methylpseudouridine. In some embodiments, 10% -25%, 15% -25%, 25% -35%, 35% -45%, 45% -55%, 55% -65%, 65% -75%, 75% -85%, 85% -95%, or 90% -100% of the uridine positions in mRNA according to the present disclosure are 5-iodouridine, and the remainder are N1-methylpseudouridine.

In any of the preceding embodiments, the modified ORF may comprise a reduced uridine dinucleotide content, such as the lowest possible uridine dinucleotide (UU) content, e.g., (a) using the smallest uridine codon at each position (as discussed above) and (b) an ORF encoding the same amino acid sequence as the given ORF. The uridine dinucleotide (UU) content can be expressed in absolute terms as a count of UU dinucleotides in the ORF or, based on ratios, as a percentage of the positions occupied by the uridine of the uridine dinucleotide (e.g., the uridine dinucleotide content of AUUAU will be 40%, since the uridine of the uridine dinucleotide occupies 2 of the 5 positions). For the purpose of assessing the minimum uridine dinucleotide content, modified uridine residues are considered to be equivalent to uridine.

In some embodiments, the mRNA comprises at least one UTR from an expressed mammalian mRNA, such as a constitutively expressed mRNA. An mRNA is considered to be constitutively expressed in a mammal if it is transcribed continuously in at least one tissue of a healthy adult mammal. In some embodiments, the mRNA comprises a 5'UTR, a 3' UTR, or 5 'and 3' UTRs from an expressed mammalian RNA, such as a constitutively expressed mammalian mRNA. Actin mRNA is an example of a constitutively expressed mRNA.

In some embodiments, the mRNA comprises at least one UTR from hydroxysteroid 17-beta dehydrogenase 4(HSD17B4 or HSD), for example the 5' UTR from HSD. In some embodiments, the mRNA comprises at least one UTR from a globin mRNA, such as a human alpha globin (HBA) mRNA, a human beta globin (HBB) mRNA, or a Xenopus laevis (Xenopus laevis) beta globin (xgg) mRNA. In some embodiments, the mRNA comprises a 5'UTR, a 3' UTR, or 5 'and 3' UTRs from a globin mRNA, such as HBA, HBB, or xgg. In some embodiments, the mRNA comprises a 5' UTR from bovine growth hormone, Cytomegalovirus (CMV), mouse Hba-a1, HSD, albumin gene, Hba, HBB, or xgg. In some embodiments, the mRNA comprises a 3' UTR from bovine growth hormone, cytomegalovirus, mouse Hba-a1, HSD, albumin gene, Hba, HBB, or xgg. In some embodiments, the mRNA comprises 5 'and 3' UTRs from bovine growth hormone, cytomegalovirus, mouse Hba-a1, HSD, albumin gene, Hba, HBB, xgg, heat shock protein 90(Hsp90), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), β -actin, α -tubulin, tumor protein (p53), or Epidermal Growth Factor Receptor (EGFR).

In some embodiments, the mRNA comprises 5 'and 3' UTRs from the same source, e.g., a constitutively expressed mRNA, such as actin, albumin, or globulin (such as HBA, HBB, or xgg).

In some embodiments, the mRNA does not comprise a 5'UTR, e.g., there are no additional nucleotides between the 5' cap and the start codon. In some embodiments, the mRNA comprises a Kozak sequence (described below) between the 5 'cap and the start codon, but without any additional 5' UTR. In some embodiments, the mRNA does not comprise a 3' UTR, e.g., no additional nucleotides are present between the stop codon and the poly a (poly-a) tail.

In some embodiments, the mRNA comprises a Kozak sequence. The Kozak sequence can affect translation initiation and overall production of the polypeptide translated from the mRNA. The Kozak sequence includes a methionine codon that can serve as a start codon. The minimum Kozak sequence is NNNRUGN, where at least one of the following is true: the first N is A or G and the second N is G. In the case of the nucleotide sequence, R means purine (A or G). In some embodiments, the Kozak sequence is RNNRUGN, NNNRUGG, RNNRUGG, RNNAUGN, NNNAUGG, or RNNAUGG. In some embodiments, the Kozak sequence is rcrugg with zero mismatches or at most one or two mismatches at positions in lower case form. In some embodiments, the Kozak sequence is rccAUGg with zero mismatches or at most one or two mismatches at positions in lower case form. In some embodiments, the Kozak sequence is gccRccAUGG with zero mismatches or at most one, two, or three mismatches at positions in lowercase form. In some embodiments, the Kozak sequence is gcccaccaug with zero mismatches or at most one, two, three, or four mismatches at positions in lower case form. In some embodiments, the Kozak sequence is GCCACCAUG. In some embodiments, the Kozak sequence is gccgccrcccaugg with zero mismatches or at most one, two, three, or four mismatches at positions in lowercase.

In some embodiments, the mRNA comprising an ORF encoding an RNA-directed DNA binding agent comprises a sequence having at least 90% identity to SEQ ID NO:43, optionally wherein the ORF of SEQ ID NO:43 (i.e., SEQ ID NO:4) is substituted with a replacement ORF. In some embodiments, the mRNA comprises any one of SEQ ID NOs 10, 14, 15, 17, 18, 20, 21, 23, 24, 26, 27, 29, 30, 50, 52, 54, 65, or 66.

In some embodiments, the degree of identity to the optionally substituted sequence of SEQ ID NO 43 is 95%. In some embodiments, the degree of identity to the optionally substituted sequence of SEQ ID NO 4 is 98%. In some embodiments, the degree of identity to the optionally substituted sequence of SEQ ID NO 43 is 99%. In some embodiments, the degree of identity to the optionally substituted sequence of SEQ ID NO 43 is 100%.

In some embodiments, an mRNA disclosed herein comprises a 5' Cap, such as Cap0, Cap1, or Cap 2. The 5' cap is typically a 7-methylguanosine nucleotide (which may be further modified, as discussed below, e.g., with regard to ARCA) linked to the first nucleotide of the 5' to 3' strand of the mRNA by a 5' -triphosphate, i.e., the 5' position of the proximal nucleotide of the first cap. In Cap0, the ribose sugars of the proximal nucleotides of both the first and second caps of the mRNA contain a 2' -hydroxyl group. In Cap1, the ribose sugars of the first and second transcribed nucleotides of mRNA contain a 2 '-methoxy group and a 2' -hydroxy group, respectively. In Cap2, the ribose sugars of the proximal nucleotides of both the first and second caps of the mRNA contain a 2' -methoxy group. See, e.g., Katibah et al (2014) Proc Natl Acad Sci USA111(33): 12025-30; abbas et al (2017) Proc Natl Acad Sci USA114(11): E2106-E2115. Most endogenous higher eukaryote mrnas, including mammalian mrnas (such as human mrnas), comprise Cap1 or Cap 2. Due to recognition as "non-self" by components of the innate immune system, such as IFIT-1 and IFIT-5, Cap0, as well as other Cap structures other than Cap1 and Cap2, may be immunogenic in mammals, such as humans, which may result in elevated levels of cytokines, including type I interferons. Components of the innate immune system, such as IFIT-1 and IFIT-5, can also compete with eIF4E for binding to mrnas with caps other than Cap1 or Cap2, potentially inhibiting mRNA translation.

The cap may be included in a co-transcribed form. For example, ARCA (anti-inversion cap analog; Thermo Fisher scientific Catalogue No. AM8045) is a cap analog comprising 7-methylguanine 3' -methoxy-5 ' -triphosphate linked to the 5' position of a guanine ribonucleotide, which can be initially incorporated into a transcript in vitro. ARCA produces a Cap of Cap0 in which the 2' position of the proximal nucleotide of the first Cap is a hydroxyl group. See, e.g., Stepinski et al, (2001) "Synthesis and properties of mRNAs modifying the novel 'anti-reverse' cap analogs 7-methyl (3'-O-methyl) GpppG and 7-methyl (3' deoxy) GpppG," RNA 7: 1486-. The ARCA structure is shown below.

CleanCap^TMAG (m7G (5') ppp (5') (2' OMeA) pG; TriLink Biotech nologices Cat No. N-7113) or CleanCap^TMGG (m7G (5') ppp (5') (2' OMeG) pG; TriLink Biotechnologies Cat No. N-7133) can be used to provide the Cap1 structure in co-transcriptional mode. CleanCap^TMAG and CleanCap^TMThe 3' -O-methylated version of GG is also available from TriLink Biotechnologies under catalog numbers N-7413 and N-7433, respectively. CleanCap^TMThe AG structure is shown below.

Alternatively, caps can be added to RNA post-transcriptionally. For example, vaccinia capping enzyme is commercially available (NewEngland Biolabs catalog number M2080S) and has RNA triphosphatase and guanosine acyltransferase activities provided by its D1 subunit, and guanine methyltransferase provided by its D12 subunit. Thus, 7-methylguanine can be added to RNA in the presence of S-adenosylmethionine and GTP to produce Cap 0. See, e.g., Guo, p. and Moss, B. (1990) proc.natl.acad.sci.usa 87, 4023-; mao, X, and Shuman, S. (1994) J.biol.chem.269, 24472-24479.

In some embodiments, the mRNA further comprises a poly-adenylated (poly-a) tail. In some embodiments, the poly-a tail comprises at least 20, 30, 40, 50, 60, 70, 80, 90, or 100 adenines, optionally up to 300 adenines. In some embodiments, the poly-A tail comprises 95, 96, 97, 98, 99, or 100 adenine nucleotides. In some cases, the poly-A tail is "interrupted" by one or more non-adenine nucleotide "anchors" at one or more positions in the poly-A tail. The poly-A tail may comprise at least 8 consecutive adenine nucleotides, but also one or more non-adenine nucleotides. As used herein, "non-adenine nucleotide" refers to any natural or non-natural nucleotide that does not contain adenine. Guanine, thymine, and cytosine nucleotides are exemplary non-adenine nucleotides. Thus, a poly-a tail on an mRNA described herein can comprise consecutive adenine nucleotides located 3' to the nucleotides encoding the RNA-guided DNA binding agent or target sequence. In some cases, the poly-a tail on the mRNA comprises non-contiguous adenine nucleotides located 3' to the nucleotides encoding the RNA-guided DNA binding agent or target sequence, wherein the non-adenine nucleotides interrupt the adenine nucleotides at regular or irregular intervals.

As used herein, "non-adenine nucleotide" refers to any natural or non-natural nucleotide that does not contain adenine. Guanine, thymine, and cytosine nucleotides are exemplary non-adenine nucleotides. Thus, a poly-a tail on an mRNA described herein can comprise consecutive adenine nucleotides located 3' to the nucleotides encoding the RNA-guided DNA binding agent or target sequence. In some cases, the poly-a tail on the mRNA comprises non-contiguous adenine nucleotides located 3' to the nucleotides encoding the RNA-guided DNA binding agent or target sequence, wherein the non-adenine nucleotides interrupt the adenine nucleotides at regular or irregular intervals.

In some embodiments, the mRNA is purified. In some embodiments, mRNA is purified using a precipitation method (e.g., LiCl precipitation, alcohol precipitation, or equivalent methods, e.g., as described herein). In some embodiments, mRNA is purified using a chromatography-based method, such as an HPLC-based method or equivalent method (e.g., as described herein). In some embodiments, mRNA is purified using both precipitation methods (e.g., LiCl precipitation) and HPLC-based methods.

In some embodiments, at least one gRNA is provided in combination with an mRNA disclosed herein. In some embodiments, the gRNA is provided in the form of a molecule that is isolated from mRNA. In some embodiments, the gRNA is provided as a portion of an mRNA disclosed herein, such as a portion of a UTR.

gRNA

In one aspect, the present disclosure provides methods of delivering a genome editing system (e.g., a zinc finger nuclease system, TALEN system, meganuclease system, or CRISPR/Cas system) to a cell (or population of cells), e.g., a HSPC (or population of HSPCs), e.g., a CD34+ cell (or population of CD34+ cells), wherein a cell (or progeny thereof) with increased fetal hemoglobin expression is produced (e.g., when the cell differentiates into red blood cells). Guide sequences useful for achieving this effect are disclosed herein. In embodiments, the genome editing system comprises one or more vectors, e.g., mRNA, encoding components of the genome editing system. In other embodiments, the genome editing system comprises one or more polypeptides. In a preferred aspect, the method comprises delivering a CRISPR/Cas system. In various embodiments, the CRISPR/Cas system comprises a gRNA complexed, e.g., in the form of a ribonucleoprotein complex (RNP), and a Cas nuclease. In other embodiments, the CRISPR/Cas system comprises one or more vectors encoding grnas and/or Cas nucleases. In other embodiments, the CRISPR/Cas system comprises one or more vectors (e.g., mrnas) encoding Cas nucleases (e.g., class 2 Cas nucleases) and one or more grnas. In various aspects, the CRISPR/Cas system comprises a gRNA described in WO2017/115268, the contents of which are incorporated herein by reference in their entirety. In various aspects, the CRISPR/Cas system comprises a gRNA comprising a guide sequence complementary to a target sequence within the BCL11a gene or a regulatory element thereof. In other aspects, the CRISPR/Cas system comprises a gRNA comprising a guide sequence complementary to a target sequence within intron 2 of BCL11a gene (e.g., within the region of intron 2 of BCL11a gene that is located at or adjacent to the GATA1 binding site). In various aspects, the CRISPR/Cas system comprises a gRNA comprising a guide sequence complementary to a target sequence within a region from ch2:60494000 to ch2:60498000 (according to hg38) in intron 2 of the BCL11a gene, for example within a region from ch2:60494250 to ch2:60496300 (according to hg38) in intron 2 of the BCL11a gene. In various embodiments, the CRISPR/Cas system comprises a gRNA comprising the guide sequences listed in table 2 of U.S. provisional application No. 62/566,232, filed 2017, 9, 29, which is incorporated herein by reference.

Exemplary guide sequences for grnas that are complementary to a target sequence within intron 2 of the BCL11a gene. +58, +62, and +55 refer to DNase hypersensitivity sites in the erythroid specificity enhancer region, e.g., Bauer et al, Science 2013; 342(6155) 253 and 257.

In other aspects, the CRISPR/Cas system comprises a gRNA comprising a guide sequence complementary to a target sequence within the globin locus on chromosome 11. In one aspect, the CRISPR/Cas system comprises a gRNA comprising a guide sequence complementary to a sequence within the HPFH region. As used herein, the term "HPFH region" refers to the following genomic loci: which when modified (e.g., mutated or deleted) causes increased HbF production in adult erythrocytes and includes the HPFH region identified in the literature (see, e.g., one line Mendelian Inheritance in Man: http:// www.omim.org/entry/141749, incorporated herein by reference). In an exemplary embodiment, the HPFH region is a region located within the beta globin gene cluster on chromosome 11p15 or a region encompassing the beta globin gene cluster on chromosome 11p 15. In an exemplary embodiment, the HPFH region is located within or encompasses at least a portion of the globin gene and its regulatory elements. In an exemplary embodiment, the HPFH region is a region of the promoter of HBG 1. In an exemplary embodiment, the HPFH region is a region of the promoter of HBG 2. In an exemplary embodiment, the HPFH region is the region described in Sankarangg et al NEJM (2011)365: 807-. In an exemplary embodiment, the HPFH region is a French breakpoint deletion HPFH as described in Sankaran VG et al NEJM (2011)365: 807-. In an exemplary embodiment, the HPFH region is an Algorian and Liya HPFH as described in Sankaran VG et al NEJM (2011)365: 807-. In an exemplary embodiment, the HPFH region is a Srilankan HPFH as described in Sankaran VG et al NEJM (2011)365: 807-. In an exemplary embodiment, the HPFH region is HPFH-3 as described in Sankaran VG et al NEJM (2011)365: 807-. In an exemplary embodiment, the HPFH region is HPFH-2 as described in Sankaran VG et al NEJM (2011)365: 807-. In one embodiment, the HPFH-1 region is HPFH-3 as described in Sankaran VG et al NEJM (2011)365: 807-. In an exemplary embodiment, the HPFH region is a Sterlan (. beta.) 0 thalassemia HPFH as described in Sankaran VG et al NEJM (2011)365: 807-. In an exemplary embodiment, the HPFH region is Sicily (. beta.) 0 thalassemia HPFH as described in Sankaran VG et al NEJM (2011)365: 807-. In an exemplary embodiment, the HPFH region is Marston (. beta.) 0 thalassemia HPFH as described in Sankaran VG et al NEJM (2011)365: 807-. In an exemplary embodiment, the HPFH region is a Korld β 0 thalassemia HPFH as described in Sankaran VG et al NEJM (2011)365: 807-. In an exemplary embodiment, the HPFH region is a region located at Chr11:5213874-5214400(hg 18). In an exemplary embodiment, the HPFH region is a region located at Chr11:5215943-5215046(hg 18). In an exemplary embodiment, the HPFH zone is a zone located at Chr11:5234390 and 5238486(hg 38). In various embodiments, the CRISPR/Cas system comprises a gRNA comprising a guide sequence comprising a sequence as described in WO2017/077394, the contents of which are incorporated herein by reference in their entirety. In various embodiments, the CRISPR/Cas system comprises a gRNA comprising a guide sequence selected from the sequences of the guide sequences of WO 2017/077394. In various embodiments, the CRISPR/Cas system comprises a gRNA comprising the guide sequences listed in table 3 of U.S. provisional application No. 62/566,232, filed 2017, 9, 29, which is incorporated herein by reference.

Exemplary guidance sequences were directed to French HPFH (French HPFH; Sankaran VG et al A functional homology for total hemoglobin cloning. NEJM (2011)365:807-

In various embodiments, the CRISPR/Cas system comprises a gRNA comprising the guide sequences listed in table 4 of U.S. provisional application No. 62/566,232, filed 2017, 9, 29, which is incorporated herein by reference.

Exemplary guide sequences may be directed to the HBG1 and/or HBG2 promoter regions.

Chemically modified gRNA

In some embodiments, the gRNA is chemically modified. Grnas comprising one or more modified nucleosides or nucleotides are referred to as "modified" grnas or "chemically modified" grnas to describe the presence of one or more non-natural and/or naturally occurring components or configurations used in place of or in addition to the typical A, G, C and U residues. In some embodiments, a modified gRNA is synthesized from atypical nucleosides or nucleotides, referred to herein as "modified. Modified nucleosides and nucleotides can include one or more of the following: (i) changes, such as substitutions (exemplary backbone modifications), of one or both of the non-bonded phosphate oxygens and/or one or more of the bonded phosphate oxygens in the phosphodiester backbone linkage; (ii) alterations in the ribose moiety, e.g., the 2' hydroxyl group on ribose, such as substitutions (exemplary sugar modifications); (iii) bulk replacement of phosphate moieties with "dephosphorylated" linkers (exemplary backbone modifications); (iv) modifications or substitutions of naturally occurring nucleobases, including with atypical nucleobases (exemplary base modifications); (v) substitution or modification of the ribose-phosphate backbone (exemplary backbone modifications); (vi) modification of the 3 'end or 5' end of the oligonucleotide, such as removal, modification or substitution of a terminal phosphate group or conjugation of a moiety, cap or linker (such 3 'or 5' cap modifications may include sugar and/or backbone modifications); and (vii) modifications or substitutions of sugars (exemplary sugar modifications).

In some embodiments, the gRNA comprises a modified uridine at some or all of the uridine positions. In some embodiments, the modified uridine is a uridine modified at position 5, for example with halogen or C1-C6 alkoxy. In some embodiments, the modified uridine is a pseudouridine modified at position 1, for example with a C1-C6 alkyl group. The modified uridine may be, for example, pseudouridine, N1-methylpseudouridine, 5-methoxyuridine, 5-iodouridine or a combination thereof. In some embodiments, the modified uridine is 5-methoxyuridine. In some embodiments, the modified uridine is 5-iodouridine. In some embodiments, the modified uridine is a pseudouridine. In some embodiments, the modified uridine is N1-methylpseuduridine. In some embodiments, the modified uridine is a combination of pseudouridine and N1-methylpseuduridine. In some embodiments, the modified uridine is a combination of pseudouridine and 5-methoxyuridine. In some embodiments, the modified uridine is a combination of N1-methylpseudouridine and 5-methoxyuridine. In some embodiments, the modified uridine is a combination of 5-iodouridine and N1-methylpseuduridine. In some embodiments, the modified uridine is a combination of pseudouridine and 5-iodouridine. In some embodiments, the modified uridine is a combination of 5-iodouridine and 5-methoxyuridine.

In some embodiments, at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% of the uridine positions in a gRNA according to the present disclosure are modified uridines. In some embodiments, 10% -25%, 15% -25%, 25% -35%, 35% -45%, 45% -55%, 55% -65%, 65% -75%, 75% -85%, 85% -95%, or 90% -100% of the uridine positions in a gRNA according to the present disclosure are modified uridine, such as 5-methoxyuridine, 5-iodouridine, N1-methylpseuduridine, pseudouridine, or a combination thereof. In some embodiments, 10% -25%, 15% -25%, 25% -35%, 35% -45%, 45% -55%, 55% -65%, 65% -75%, 75% -85%, 85% -95%, or 90% -100% of the uridine positions in a gRNA according to the present disclosure are 5-methoxyuridine. In some embodiments, 10% -25%, 15% -25%, 25% -35%, 35% -45%, 45% -55%, 55% -65%, 65% -75%, 75% -85%, 85% -95%, or 90% -100% of the uridine positions in a gRNA according to the present disclosure are pseudouridine. In some embodiments, 10% -25%, 15% -25%, 25% -35%, 35% -45%, 45% -55%, 55% -65%, 65% -75%, 75% -85%, 85% -95%, or 90% -100% of the uridine positions in a gRNA according to the present disclosure are N1-methylpseudouridine. In some embodiments, 10% -25%, 15% -25%, 25% -35%, 35% -45%, 45% -55%, 55% -65%, 65% -75%, 75% -85%, 85% -95%, or 90% -100% of the uridine positions in a gRNA according to the present disclosure are 5-iodouridine. In some embodiments, 10% -25%, 15% -25%, 25% -35%, 35% -45%, 45% -55%, 55% -65%, 65% -75%, 75% -85%, 85% -95%, or 90% -100% of the uridine positions in a gRNA according to the present disclosure are 5-methoxyuridine, and the remainder is N1-methylpseudouridine. In some embodiments, 10% -25%, 15% -25%, 25% -35%, 35% -45%, 45% -55%, 55% -65%, 65% -75%, 75% -85%, 85% -95%, or 90% -100% of the uridine positions in a gRNA according to the present disclosure are 5-iodouridine, and the remainder is N1-methylpseudouridine.

Chemical modifications, such as those listed above, can be combined to yield modified grnas comprising nucleosides and nucleotides (collectively, "residues") that can have two, three, four, or more modifications. For example, a modified residue can have a modified sugar and a modified nucleobase. In some embodiments, each base of the gRNA is modified, e.g., all bases have a modified phosphate group, such as a phosphorothioate group. In certain embodiments, all or substantially all of the phosphate groups of the gRNA molecule are replaced with phosphorothioate groups. In some embodiments, the modified gRNA comprises at least one modified residue at or near the 5' end of the RNA. In some embodiments, the modified gRNA comprises at least one modified residue at or near the 3' end of the RNA.

In some embodiments, the gRNA comprises one, two, three, or more modified residues. In some embodiments, at least 5% (e.g., at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100%) of the positions in the modified gRNA are modified nucleosides or nucleotides.

Unmodified nucleic acids can be readily degraded by nucleases such as those found in intracellular nucleases or in serum. For example, nucleases can hydrolyze nucleic acid phosphodiester bonds. Thus, in one aspect, a gRNA described herein can contain one or more modified nucleosides or nucleotides, e.g., to introduce stability towards intracellular nucleases or serum-based nucleases. In some embodiments, the modified gRNA molecules described herein can exhibit a reduced innate immune response when introduced into a population of cells in vivo and ex vivo. The term "innate immune response" encompasses cellular responses to exogenous nucleic acids (including single-stranded nucleic acids), involving the induction of cytokine (especially interferon) expression and release, and cell death.

In some embodiments of backbone modifications, the phosphate group of the modified residue may be modified by replacing one or more oxygens with different substituents. In addition, a modified residue, e.g., a modified residue present in a modified nucleic acid, can comprise a bulk replacement of an unmodified phosphate moiety with a modified phosphate group as described herein. In some embodiments, backbone modifications of the phosphate backbone may include alterations that result in charged linkers with no electrical linkers or with asymmetric charge distributions.

Examples of modified phosphate groups include phosphorothioates, phosphoroselenoates, boranophosphates, hydrogenphosphonates, phosphoramidates, alkyl or aryl phosphonates, and phosphotriesters. The phosphorus atom in the unmodified phosphate group is achiral. However, replacement of one of the non-bridging oxygens with one of the above atom or group of atoms may render the phosphorus atom chiral. The stereosymmetric phosphorus atom may have an "R" configuration (herein Rp) or an "S" configuration (herein Sp). The backbone may also be modified by replacing the bridging oxygen (i.e., the oxygen linking the phosphate to the nucleoside) with nitrogen (bridging phosphoramidate), sulfur (bridging phosphorothioate), and carbon (bridging methylenephosphonate). The displacement may occur at either or both of the connecting oxygens.

The phosphate group may be replaced in certain backbone modifications by a phosphorus-free linking group. In some embodiments, the charged phosphate groups may be replaced by neutral moieties. Examples of moieties that can replace a phosphate group can include, but are not limited to, methyl phosphonates, hydroxyamines, siloxanes, carbonates, carboxymethyl, carbamates, amides, thioethers, ethylene oxide linkers, sulfonates, sulfonamides, thiometals, formals, oximes, methyleneimino, methylenemethylimino, methylenehydrazine, methylenedimethylhydrazine, and methyleneoxymethylimino, for example.

In some embodiments, the invention includes sgrnas that comprise one or more modifications in one or more of the following regions: a nucleotide at the 5' end; a lower stem region; a raised region; a stalk region; a connecting area; a hairpin 1 region; a hairpin 2 region; and a nucleotide at the 3' end. In some embodiments, the modification comprises a 2 '-O-methyl (2' -O-Me) modified nucleotide. In some embodiments, the modifications comprise 2 '-fluoro (2' -F) modified nucleotides. In some embodiments, the modifications comprise Phosphorothioate (PS) linkages between nucleotides.

In some embodiments, the first three or four nucleotides at the 5 'end and the last three or four nucleotides at the 3' end are modified. In some embodiments, the first four nucleotides at the 5 'end and the last four nucleotides at the 3' end are linked via a Phosphorothioate (PS) linkage. In some embodiments, the modification comprises 2' -O-Me. In some embodiments, the modification comprises 2' -F.

In some embodiments, the first four nucleotides at the 5' end and the last four nucleotides at the 3' end are connected by PS linkages, and the first three nucleotides at the 5' end and the last three nucleotides at the 3' end comprise a 2' -O-Me modification.

In some embodiments, the first four nucleotides at the 5' end and the last four nucleotides at the 3' end are connected by a PS linkage, and the first three nucleotides at the 5' end and the last three nucleotides at the 3' end comprise a 2' -F modification.

In some embodiments, the sgRNA comprises a modified version of SEQ ID NO: 74: (mN NNNNNNNNNNNNNNNNNNNNNNNNNNNGUUUUUAGAGAmmAmAmAmAmAmmGmCAAGUUAAAUAAGGCUAGUCCGUACAmmAmmGmUmUmGmGmGmGmGmGmGmGmGmMmGmMmU mU), wherein N is any natural or non-natural nucleotide. In some embodiments, the sgRNA comprises SEQ ID NO: 74. In certain embodiments, the sgRNA includes 2 'O-methyl modifications at the first three residues of its 5' end with phosphorothioate linkages between residues 1-2, 2-3, and 3-4 of the RNA.

Template nucleic acid

The compositions and methods disclosed herein can include a template nucleic acid. The template can be used to alter or insert a nucleic acid sequence at or near a target site of a Cas nuclease. In some embodiments, the method comprises introducing the template into the cell. In some embodiments, a single template may be provided. In other embodiments, two or more templates may be provided such that editing may occur at two or more target sites. For example, different templates may be provided to edit a single gene in a cell, or two different genes in a cell.

In some embodiments, the template may be used in homologous recombination. In some embodiments, homologous recombination can result in the integration of a template sequence or a portion of a template sequence into a target nucleic acid molecule. In other embodiments, the template may be used for homology directed repair involving DNA strand invasion at a cleavage site in a nucleic acid. In some embodiments, homology directed repair can result in the inclusion of a template sequence in the edited target nucleic acid molecule. In other embodiments, the template may be used for gene editing mediated by non-homologous end joining. In some embodiments, the template sequence does not have similarity to a nucleic acid sequence near the cleavage site. In some embodiments, a template or a portion of a sequence of templates is incorporated. In some embodiments, the template comprises a flanking Inverted Terminal Repeat (ITR) sequence.

In some embodiments, the template may comprise a first homology arm and a second homology arm (also referred to as a first nucleotide sequence and a second nucleotide sequence) that are complementary to sequences located upstream and downstream of the cleavage site, respectively. When the template contains two homology arms, each arm may be the same length or a different length, and the sequence between the homology arms may be substantially similar or identical to the target sequence between the homology arms, or the sequences may be completely unrelated. In some embodiments, the degree of complementarity or percent identity between the first nucleotide sequence on the template and the sequence upstream of the cleavage site, and between the second nucleotide sequence on the template and the sequence downstream of the cleavage site, can permit homologous recombination, such as high fidelity homologous recombination, between the template and the target nucleic acid molecule. In some embodiments, the degree of complementarity may be about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100%. In some embodiments, the degree of complementarity may be about 95%, 97%, 98%, 99%, or 100%. In some embodiments, the degree of complementarity may be at least 98%, 99%, or 100%. In some embodiments, the degree of complementarity may be 100%. In some embodiments, the percent identity may be about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100%. In some embodiments, the percent identity may be about 95%, 97%, 98%, 99%, or 100%. In some embodiments, the percent identity may be at least 98%, 99%, or 100%. In some embodiments, the percent identity may be 100%.

In some embodiments, the template sequence may correspond to, comprise, or consist of an endogenous sequence of the target cell. The template sequence may additionally or alternatively correspond to, comprise or consist of a sequence foreign to the target cell. As used herein, the term "endogenous sequence" refers to a sequence that is native to a cell. The term "exogenous sequence" refers to a sequence that is not native to a cell, or a sequence that is at a different location in the genome of a cell from the native location. In some embodiments, the endogenous sequence can be a genomic sequence of the cell. In some embodiments, the endogenous sequence can be a chromosomal or extra-chromosomal sequence. In some embodiments, the endogenous sequence can be a plasmid sequence of the cell. In some embodiments, the template sequence may be substantially identical to a portion of the endogenous sequence at or near the site of cleavage in the cell, but comprise at least one nucleotide change. In some embodiments, cleavage of a target nucleic acid molecule with template editing can result in a mutation comprising an insertion, deletion, or substitution of one or more nucleotides of the target nucleic acid molecule. In some embodiments, the mutation may result in one or more amino acid changes in a protein expressed by a gene comprising the target sequence. In some embodiments, the mutation may result in one or more nucleotide changes in the RNA expressed by the target gene. In some embodiments, the mutation can alter the expression level of the target gene. In some embodiments, the mutation may result in increased or decreased expression of the target gene. In some embodiments, the mutation can result in a gene knock-out. In some embodiments, the mutation can result in a gene knockout. In some embodiments, the mutation may result in restoring gene function. In some embodiments, cleavage of a target nucleic acid molecule with template editing can result in changes in the exonic sequences, intronic sequences, regulatory sequences, transcriptional control sequences, translational control sequences, splice sites, or non-coding sequences of the target nucleic acid molecule (such as DNA).

In other embodiments, the template sequence may comprise an exogenous sequence. In some embodiments, the exogenous sequence can comprise a protein or RNA coding sequence operably linked to an exogenous promoter sequence such that when the exogenous sequence is integrated into the target nucleic acid molecule, the cell is capable of expressing the protein or RNA encoded by the integrated sequence. In other embodiments, when the exogenous sequence is integrated into the target nucleic acid molecule, expression of the integrated sequence can be regulated by an endogenous promoter sequence. In some embodiments, the exogenous sequence can provide a cDNA sequence encoding a protein or a portion of a protein. In other embodiments, the exogenous sequence may comprise or consist of an exonic sequence, an intronic sequence, a regulatory sequence, a transcriptional control sequence, a translational control sequence, a splice site, or a non-coding sequence. In some embodiments, integration of the exogenous sequence can result in restoring gene function. In some embodiments, integration of the exogenous sequence can result in a gene knock-in. In some embodiments, integration of the exogenous sequence can result in gene knock-out.

The template may have any suitable length. In some embodiments, the length of the template may comprise 10, 15, 20, 25, 50, 75, 100, 150, 200, 500, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000, 5500, 6000 or more nucleotides. The template may be a single stranded nucleic acid. The template may be a double-stranded or partially double-stranded nucleic acid. In certain embodiments, the single stranded template is 20, 30, 40, 50, 75, 100, 125, 150, 175, or 200 nucleotides in length. In some embodiments, a template can comprise a nucleotide sequence that is complementary to a portion of a target nucleic acid molecule comprising a target sequence (i.e., a "homology arm"). In some embodiments, the template may comprise a homology arm that is complementary to a sequence located upstream or downstream of the cleavage site on the target nucleic acid molecule.

In some embodiments, the template contains ssDNA or dsDNA having flanking Inverted Terminal Repeat (ITR) sequences. In some embodiments, the template is provided in the form of a vector, plasmid, minicircle, nanoring, or PCR product.

Purification of nucleic acids

In some embodiments, the nucleic acid is purified. In some embodiments, nucleic acids are purified using a precipitation method (e.g., LiCl precipitation, alcohol precipitation, or equivalent methods, e.g., as described herein). In some embodiments, nucleic acids are purified using a chromatography-based method, such as an HPLC-based method or equivalent method (e.g., as described herein). In some embodiments, nucleic acids are purified using both precipitation methods (e.g., LiCl precipitation) and HPLC-based methods.

Target sequence

In some embodiments, the CRISPR/Cas system of the present disclosure can target and cleave a target sequence on a target nucleic acid molecule. For example, the target sequence may be recognized and cleaved by a Cas nuclease. In certain embodiments, the target sequence of the Cas nuclease is located near the homologous PAM sequence of the nuclease. In some embodiments, a class 2 Cas nuclease can be guided to a target sequence of a target nucleic acid molecule by a gRNA, wherein the gRNA hybridizes to the target sequence and the class 2 Cas protein cleaves the target sequence. In some embodiments, the guide RNA hybridizes to a target sequence adjacent to or comprising its cognate PAM and the class 2 Cas nuclease cleaves the target sequence. In some embodiments, the target sequence may be complementary to the targeting sequence of the guide RNA. In some embodiments, the degree of complementarity between the targeting sequence of the guide RNA and the portion of the corresponding target sequence that hybridizes to the guide RNA may be about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100%. In some embodiments, the percentage of identity between the targeting sequence of the guide RNA and the portion of the corresponding target sequence that hybridizes to the guide RNA can be about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100%. In some embodiments, the homologous regions of the target are adjacent to homologous PAM sequences. In some embodiments, the target sequence may comprise a sequence that is 100% complementary to the targeting sequence of the guide RNA. In other embodiments, the target sequence may comprise at least one mismatch, deletion, or insertion as compared to the targeting sequence of the guide RNA.

The length of the target sequence may depend on the nuclease system used. For example, the length of the targeting sequence of the guide RNA of the CRISPR/Cas system can comprise 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, or more than 50 nucleotides and the target sequence is the corresponding length, optionally adjacent to a PAM sequence. In some embodiments, the length of the target sequence may comprise 15-24 nucleotides. In some embodiments, the length of the target sequence may comprise 17-21 nucleotides. In some embodiments, the target sequence may comprise 20 nucleotides in length. When a nicking enzyme is used, the target sequence may comprise a pair of target sequences recognized by a pair of nicking enzymes that cleave opposite strands of the DNA molecule. In some embodiments, the target sequence may comprise a pair of target sequences recognized by a pair of nicking enzymes that cleave the same strand of a DNA molecule. In some embodiments, the target sequence may comprise a portion of the target sequence recognized by one or more Cas nucleases.

The target nucleic acid molecule can be any DNA or RNA molecule that is endogenous or exogenous to the cell. In some embodiments, the target nucleic acid molecule can be episomal DNA, plasmid, genomic DNA, viral genome, mitochondrial DNA, or chromosomal DNA from or in the cell. In some embodiments, the target sequence of a target nucleic acid molecule can be a genomic sequence from or in a cell (including a human cell).

In other embodiments, the target sequence may be a viral sequence. In other embodiments, the target sequence may be a pathogen sequence. In other embodiments, the target sequence may be a synthetic sequence. In other embodiments, the target sequence may be a chromosomal sequence. In certain embodiments, the target sequence may comprise a translocation junction, such as a translocation associated with cancer. In some embodiments, the target sequence may be on a eukaryotic chromosome, such as a human chromosome. In certain embodiments, the target sequence is a liver-specific sequence in that it is expressed in liver cells.

In some embodiments, the target sequence may be located in a coding sequence of a gene, an intron sequence of a gene, a regulatory sequence, a transcriptional control sequence of a gene, a translational control sequence of a gene, a splice site, or a non-coding sequence between genes. In some embodiments, the gene may be a protein-encoding gene. In other embodiments, the gene may be a non-coding RNA gene. In some embodiments, the target sequence may comprise all or a portion of a disease-associated gene. In some embodiments, the target sequence may be located in a non-gene functional site in the genome, for example, a site that controls aspects of chromatin organization, such as a scaffold site or a locus control region.

In embodiments involving Cas nucleases, such as class 2 Cas nucleases, the target sequence may be adjacent to a protospacer adjacent motif ("PAM"). In some embodiments, the PAM may be adjacent to or within 1,2, 3 or 4 nucleotides of the 3' end of the target sequence. The length and sequence of the PAM may depend on the Cas protein used. For example, PAM can be selected from a common sequence of a particular Cas9 protein or Cas9 ortholog or a particular PAM sequence, including those disclosed in FIG. 1 of Ran et al, Nature, 520: 186-containing 191(2015), and in FIG. S5 of Zetsche 2015, the relevant disclosures of which are each incorporated herein by reference. In some embodiments, the PAM can be 2, 3,4, 5, 6, 7, 8, 9, or 10 nucleotides in length. Non-limiting exemplary PAM sequences include NGG, NGGNG, NG, NAAAAN, nnaaw, NNNNACA, GNNNCNNA, TTN, and NNNNGATT (where N is defined as any nucleotide and W is defined as a or T). In some embodiments, the PAM sequence may be NGG. In some embodiments, the PAM sequence may be NGGNG. In some embodiments, the PAM sequence may be TTN. In some embodiments, the PAM sequence can be nnaaaw.

Lipid formulations

Disclosed herein are various embodiments of LNP formulations of biologically active agents such as RNAs (including CRISPR/Cas cargo). Such LNP formulations comprise "amine lipids" or "biodegradable lipids", optionally together with one or more of helper lipids, neutral lipids, and stealth lipids (such as PEG lipids). By "lipid nanoparticle" is meant a particle comprising a plurality (i.e., more than one) of lipid molecules physically associated with each other by intermolecular forces.

Amine lipids

In certain embodiments, LNP compositions for delivering bioactive agents comprise "amine lipids," which are defined as lipid a or its equivalent, including acetal analogs of lipid a.

In some embodiments, the amine lipid is lipid a, which is octadeca-9, 12-dienoic acid (9Z,12Z) -3- ((4, 4-bis (octyloxy) butyryl) oxy) -2- ((((3- (diethylamino) propoxy) carbonyl) oxy) methyl) propyl ester, also known as (9Z,12Z) -octadeca-9, 12-dienoic acid 3- ((4, 4-bis (octyloxy) butyryl) oxy) -2- ((((3- (diethylamino) propoxy) carbonyl) oxy) methyl) propyl ester. Lipid a can be depicted as:

lipid a can be synthesized according to WO2015/095340 (e.g. pages 84-86). In certain embodiments, the amine lipid is the equivalent of lipid a.

In certain embodiments, the amine lipid is an analog of lipid a. In certain embodiments, the lipid a analog is an acetal analog of lipid a. In certain LNP compositions, the acetal analogs are C4-C12 acetal analogs. In some embodiments, the acetal analog is a C5-C12 acetal analog. In another embodiment, the acetal analog is a C5-C10 acetal analog. In additional embodiments, the acetal analog is selected from the group consisting of C4, C5, C6, C7, C9, C10, C11, and C12 acetal analogs.

Amine lipids and other "biodegradable lipids" suitable for use in the LNPs described herein are biodegradable in vivo. Amine lipids have low toxicity (e.g., tolerated in animal models without adverse effects in amounts greater than or equal to 10 mg/kg). In certain embodiments, the LNPs comprising amine lipids comprise LNPs that: wherein at least 75% of the amine lipids are cleared from the plasma within 8, 10, 12, 24, or 48 hours or 3,4, 5, 6, 7, or 10 days. In certain embodiments, the LNPs comprising amine lipids comprise LNPs that: wherein at least 50% of the mRNA or gRNA is cleared from the plasma within 8, 10, 12, 24, or 48 hours or within 3,4, 5, 6, 7, or 10 days. In certain embodiments, the LNPs comprising amine lipids comprise LNPs that: wherein at least 50% of the LNP is cleared from plasma within 8, 10, 12, 24, or 48 hours or 3,4, 5, 6, 7, or 10 days, e.g., by measuring lipids (e.g., amine lipids), RNA (e.g., mRNA), or other components. In certain embodiments, lipid-encapsulated versus free lipid, RNA, or nucleic acid components of LNPs are measured.

Biodegradable lipids include biodegradable lipids such as those of WO/2017/173054, WO2015/095340 and WO 2014/136086.

Lipid clearance can be measured as described in the literature. See Maier, m.a. et al, Biodegradable, lipids engineering, Lipid Nanoparticles for systematic Delivery of rnai therapeutics, mol. ther.2013, 21(8), 1570-78 ("Maier"). For example, in Maier, six to eight week old male C57Bl/6 mice are administered the LNP-siRNA system containing siRNA targeting luciferase by intravenous bolus injection via the lateral tail vein at 0.3 mg/kg. Blood, liver and spleen samples were collected at 0.083, 0.25, 0.5, 1,2, 4, 8, 24, 48, 96 and 168 hours post-dose. Mice were perfused with saline and blood samples were processed to obtain plasma prior to tissue collection. All samples were processed and analyzed by LC-MS. In addition, Maier describes a procedure for assessing toxicity after administration of LNP-siRNA formulations. For example, sirnas targeting luciferase were administered to male Sprague-duller rats (Sprague-Dawley rat) at 0, 1,3, 5 and 10mg/kg (5 animals/group) via single intravenous bolus injection at a dose volume of 5 mL/kg. After 24 hours, about 1mL of blood was obtained from the jugular vein of the awakened animal and serum was isolated. At 72 hours post-dose, all animals were euthanized for necropsy. Clinical signs, body weight, serum chemistry, organ weight and histopathological assessments were performed. Although Maier describes methods for assessing siRNA-LNP formulations, these methods may also be applicable to assessing clearance, pharmacokinetics, and toxicity of administration of the LNP compositions of the present disclosure.

Lipids can lead to increased clearance. In some embodiments, the clearance rate is lipid clearance rate, e.g., the rate at which lipids are cleared from blood, serum, or plasma. In some embodiments, the clearance rate is an RNA clearance rate, e.g., the rate of clearance of mRNA or gRNA from blood, serum, or plasma. In some embodiments, the clearance rate is the rate at which LNP is cleared from blood, serum, or plasma. In some embodiments, the clearance rate is the rate at which LNP is cleared from a tissue, such as liver tissue or spleen tissue. In certain embodiments, a high removal rate results in a security profile that does not have a significant adverse effect. Amine lipids and biodegradable lipids can reduce LNP accumulation in the circulation and in tissues. In some embodiments, reduced LNP accumulation in circulation and in tissue produces a safety profile that does not have significant adverse effects.

The lipid may be ionizable depending on the pH of the medium in which the lipid is located. For example, in a weakly acidic medium, lipids such as amine lipids may be protonated and thus positively charged. Conversely, in weakly basic media, such as blood at a pH of about 7.35, lipids such as amine lipids may not be protonated and therefore uncharged.

The ability of lipids to charge is related to their intrinsic pKa. In some embodiments, the amine lipids of the present disclosure may each independently have a pKa in the range of about 5.1 to about 7.4. In some embodiments, the biodegradable lipids of the present disclosure may each independently have a pKa in the range of about 5.1 to about 7.4. For example, the amine lipids of the present disclosure may each independently have a pKa in the range of about 5.8 to about 6.5. Lipids having a pKa in the range of about 5.1 to about 7.4 are effective for delivering the cargo in vivo (e.g., to a tumor). Furthermore, it has been found that lipids having a pKa in the range of about 5.3 to about 6.4 are effective for in vivo delivery (e.g., to a tumor). See, for example, WO 2014/136086.

Additional lipids

"neutral lipids" suitable for use in the lipid compositions of the present disclosure include, for example, a variety of neutral lipids, uncharged lipids, or zwitterionic lipids. Examples of neutral phospholipids suitable for use in the present disclosure include, but are not limited to, 5-heptadecylbenzene-1, 3-diol (resorcinol), Dipalmitoylphosphatidylcholine (DPPC), Distearoylphosphatidylcholine (DSPC), phosphorylcholine (DOPC), Dimyristoylphosphatidylcholine (DMPC), phosphatidylcholine (PLPC), 1, 2-distearoylsn-glycero-3-phosphorylcholine (DAPC), Phosphatidylethanolamine (PE), Egg Phosphatidylcholine (EPC), Dilauroylphosphatidylcholine (DLPC), Dimyristoylphosphatidylcholine (DMPC), 1-Myristoylphosphatidylcholine (MPPC), 1-palmitoyl-2-myristoylphosphatidylcholine (PMPC), 1-palmitoyl-2-stearoylphosphatidylcholine (PSPC), 1, 2-dianeoyl-sn-glycero-3-phosphocholine (DBPC), 1-stearoyl-2-palmitoyl phosphatidylcholine (SPPC), 1, 2-eicosenoyl-sn-glycero-3-phosphocholine (DEPC), Palmitoyl Oleoyl Phosphatidylcholine (POPC), lysophosphatidylcholine, dioleoyl phosphatidylethanolamine (DOPE), Dilinoleoylphosphatidylethanolamine (DSPE), dimyristoyl phosphatidylethanolamine (DMPE), dipalmitoyl phosphatidylethanolamine (DPPE), Palmitoyl Oleoyl Phosphatidylethanolamine (POPE), lysophosphatidylethanolamine, and combinations thereof. In one embodiment, the neutral phospholipid may be selected from the group consisting of: distearoylphosphatidylcholine (DSPC) and Dimyristoylphosphatidylethanolamine (DMPE). In another embodiment, the neutral phospholipid may be Distearoylphosphatidylcholine (DSPC).

"helper lipids" include steroids, sterols, and alkylresorcinols. Helper lipids suitable for use in the present disclosure include, but are not limited to, cholesterol, 5-heptadecylresorcinol, and cholesterol hemisuccinate. In one embodiment, the helper lipid may be cholesterol. In one embodiment, the helper lipid may be cholesterol hemisuccinate.

"stealth lipids" are lipids that alter the length of time that a nanoparticle can be present in vivo (e.g., in blood). Stealth lipids can aid the formulation process by, for example, reducing particle aggregation and controlling particle size. The stealth lipids used herein may modulate the pharmacokinetic properties of LNP. Stealth lipids suitable for use in the lipid compositions of the present disclosure include, but are not limited to, stealth lipids having a hydrophilic head group attached to a lipid moiety. Stealth lipids suitable for use in the lipid compositions of the present disclosure, as well as information regarding the biochemistry of such lipids, can be found in Romberg et al, Pharmaceutical Research, Vol.25, No. 1, 2008, pp.55-71 and Hoekstra et al, Biochimica et Biophysica Acta 1660(2004) 41-52. Further suitable PEG lipids are disclosed in e.g. WO 2006/007712.

In one embodiment, the hydrophilic head group of the stealth lipid comprises a polymer moiety selected from a PEG-based polymer. Stealth lipids may comprise a lipid moiety. In some embodiments, the stealth lipid is a PEG lipid.

In one embodiment, the stealth lipid comprises a polymer moiety selected from the group consisting of: PEG (sometimes referred to as poly (ethylene oxide)), poly (oxazoline), poly (vinyl alcohol), poly (glycerol), poly (N-vinyl pyrrolidone), polyamino acids, and poly [ N- (2-hydroxypropyl) methacrylamide ].

In one embodiment, the PEG lipid comprises a PEG-based (sometimes referred to as poly (ethylene oxide)) polymer moiety.

The PEG lipid also comprises a lipid moiety. In some embodiments, the lipid moiety may be derived from diacylglycerols or diacyloleamides (diacylglycylamides), including those comprising a dialkylglycerol or dialkylglyceramide group having an alkyl chain length independently comprising from about C4 to about C40 saturated or unsaturated carbon atoms, wherein the chain may comprise one or more functional groups, such as an amide or an ester. In some embodiments, the alkyl chain length comprises from about C10 to C20. The dialkylglycerol or dialkylglyceroamide group may further comprise one or more substituted alkyl groups. The chain length may be symmetrical or asymmetrical.

As used herein, unless otherwise indicated, the term "PEG" means any polyethylene glycol or other polyalkylene ether polymer. In one embodiment, the PEG is an optionally substituted linear or branched polymer of ethylene glycol or ethylene oxide. In one embodiment, the PEG is unsubstituted. In one embodiment, the PEG is substituted, for example, with one or more alkyl, alkoxy, acyl, hydroxy, or aryl groups. In one embodiment, the term includes PEG copolymers, such as PEG-polyurethane or PEG-polypropylene (see, e.g., J.Milton Harris, Poly (ethylene glycol) chemistry: biotechnical and biomedical applications (1992)); in another embodiment, the term does not include PEG copolymers. In one embodiment, the molecular weight of the PEG is from about 130 to about 50,000, in sub-embodiments from about 150 to about 30,000, in sub-embodiments from about 150 to about 20,000, in sub-embodiments from about 150 to about 15,000, in sub-embodiments from about 150 to about 10,000, in sub-embodiments from about 150 to about 6,000, in sub-embodiments from about 150 to about 5,000, in sub-embodiments from about 150 to about 4,000, in sub-embodiments from about 150 to about 3,000, in sub-embodiments from about 300 to about 3,000, in sub-embodiments from about 1,000 to about 3,000, and in sub-embodiments from about 1,500 to about 2,500.

In certain embodiments, the PEG (e.g., conjugated to a lipid moiety or lipid, such as a stealth lipid) is "PEG-2K," also referred to as "PEG 2000," which has an average molecular weight of about 2,000 daltons. PEG-2K is represented herein by the following formula (I), wherein n is 45, meaning that the number average degree of polymerization comprises about 45 subunitsHowever, other PEG embodiments known in the art may be used, including for example, median thereofThose having a mean degree of polymerization comprising about 23 subunits (n-23) and/or 68 subunits (n-68). In some embodiments, n may range from about 30 to about 60. In some embodiments, n may range from about 35 to about 55. In some embodiments, n may range from about 40 to about 50. In some embodiments, n may range from about 42 to about 48. In some embodiments, n may be 45. In some embodiments, R may be selected from H, substituted alkyl, and unsubstituted alkyl. In some embodiments, R may be unsubstituted alkyl. In some embodiments, R may be methyl.

In any of the embodiments described herein, the PEG lipid may be selected from PEG-dilauroyl glycerol, PEG-dimyristoyl glycerol (PEG-DMG) (cat # GM-020, available from NOF, Tokyo, Japan), PEG-dipalmitoyl glycerol, PEG-distearoyl glycerol (PEG-DSPE) (cat # DSPE-020CN, NOF, Tokyo, Japan), PEG-dilauryl glycinamide, PEG-dimyristylyl glycinamide, PEG-dipalmitoyl glycinamide, and PEG-distearoyl glycinamide, PEG-cholesterol (1- [8' - (cholest-5-ene-3 [ β ] -oxy) carboxamido-3 ',6' -dioxaoctyl ] carbamoyl- [ ω ] -methyl-poly (ethylene glycol), PEG-DMB (3, 4-ditetradecylphenylmethyl- [ omega ] -methyl-poly (ethylene glycol) ether), 1, 2-dimyristoyl-sn-glycerol-3-phosphoethanolamine-N- [ methoxy (polyethylene glycol) -2000] (PEG2k-DMG) (cat # 880150P, available from Avanti Polar Lipids, Alabaster, Alabama, USA), 1, 2-distearoyl-sn-glycerol-3-phosphoethanolamine-N- [ methoxy (polyethylene glycol) -2000] (PEG2k-DSPE) (cat # 880120C, available from Avanti pollipids, Alabaster, Alabama, USA), 1, 2-distearoyl-sn-glycerol, methoxypolyethylene glycol (PEG2 k-DSG; GS-020, NOF Tokyo, Japan), poly (ethylene glycol) -2000-dimethacrylate (PEG2k-DMA) and 1, 2-distearoyloxypropyl-3-amine-N- [ methoxy (polyethylene glycol) -2000] (PEG2 k-DSA). In one embodiment, the PEG lipid can be PEG2 k-DMG. In some embodiments, the PEG lipid may be PEG2 k-DSG. In one embodiment, the PEG lipid can be PEG2 k-DSPE. In one embodiment, the PEG lipid can be PEG2 k-DMA. In one embodiment, the PEG lipid can be PEG2 k-C-DMA. In one embodiment, the PEG lipid may be compound S027, which is disclosed in WO2016/010840 (paragraphs [00240] to [00244 ]). In one embodiment, the PEG lipid can be PEG2 k-DSA. In one embodiment, the PEG lipid can be PEG2 k-C11. In some embodiments, the PEG lipid may be PEG2 k-C14. In some embodiments, the PEG lipid may be PEG2 k-C16. In some embodiments, the PEG lipid may be PEG2 k-C18.

LNP formulations

LNPs can contain (i) biodegradable lipids; (ii) optionally a neutral lipid; (iii) a helper lipid; and (iv) stealth lipids, such as PEG lipids. LNPs can contain biodegradable lipids as well as one or more of neutral lipids, helper lipids, and stealth lipids (such as PEG lipids).

LNPs can contain (i) amine lipids for encapsulation and for endosomal escape; (ii) neutral lipids for stabilization; (iii) helper lipids that are also used for stabilization; and (iv) stealth lipids, such as PEG lipids. LNPs can contain biodegradable lipids as well as one or more of neutral lipids, helper lipids and stealth lipids (such as PEG lipids) that are also used for stabilization.

In some embodiments, the LNP composition can comprise an RNA component comprising one or more of an RNA-guided DNA-binding agent, Cas nuclease mRNA, class 2 Cas nuclease mRNA, Cas9 mRNA, and a gRNA. In some embodiments, the LNP composition can include a class 2 Cas nuclease and a gRNA as RNA components. In certain embodiments, the LNP composition can comprise an RNA component, an amine lipid, a helper lipid, a neutral lipid, and a stealth lipid. In certain LNP compositions, the helper lipid is cholesterol. In other compositions, the neutral lipid is DSPC. In further embodiments, the stealth lipid is PEG2k-DMG or PEG2 k-C11. In certain embodiments, the LNP composition comprises lipid a or an equivalent of lipid a; a helper lipid; a neutral lipid; stealth lipids; and a guide RNA. In certain compositions, the amine lipid is lipid a. In certain compositions, the amine lipid is lipid a or an acetal analog thereof; the helper lipid is cholesterol; the neutral lipid is DSPC; and the stealth lipid is PEG2 k-DMG.

In certain embodiments, the lipid composition is described in terms of the respective molar ratios of the lipid components in the formulation. Embodiments of the present disclosure provide lipid compositions described in terms of the respective molar ratios of the lipid components in the formulation. In one embodiment, the mol% of the amine lipid may be from about 30 mol% to about 60 mol%. In one embodiment, the mol% of the amine lipid may be from about 40 mol% to about 60 mol%. In one embodiment, the mol% of the amine lipid may be from about 45 mol% to about 60 mol%. In one embodiment, the mol% of the amine lipid may be from about 50 mol% to about 60 mol%. In one embodiment, the mol% of the amine lipid may be from about 55 mol% to about 60 mol%. In one embodiment, the mol% of the amine lipid may be from about 50 mol% to about 55 mol%. In one embodiment, the mol% of the amine lipid may be about 50 mol%. In one embodiment, the mol% of the amine lipid may be about 55 mol%. In some embodiments, the mol% of amine lipids of the LNP batch will be ± 30%, ± 25%, ± 20%, ± 15%, ± 10%, ± 5% or ± 2.5% of the target mol%. In some embodiments, the amine lipid mol% of the LNP batch will be ± 4 mol%, ± 3 mol%, ± 2 mol%, ± 1.5 mol%, ± 1 mol%, ± 0.5 mol%, or ± 0.25 mol% of the target mol%. All mol% numbers are given as the fraction of the lipid component of the LNP composition. In certain embodiments, the LNP batch-to-batch variation rate for amine lipid mol% will be less than 15%, less than 10%, or less than 5%.

In one embodiment, the mol% of neutral lipids may be from about 5 mol% to about 15 mol%. In one embodiment, the mol% of neutral lipids may be from about 7 mol% to about 12 mol%. In one embodiment, the mol% of neutral lipids may be about 9 mol%. In some embodiments, the mol% neutral lipids of the LNP batch will be ± 30%, ± 25%, ± 20%, ± 15%, ± 10%, ± 5% or ± 2.5% of the mol% of the target neutral lipids. In certain embodiments, the LNP batch-to-batch rate of change will be less than 15%, less than 10%, or less than 5%.

In one embodiment, the mol% of helper lipids may be from about 20 mol% to about 60 mol%. In one embodiment, the mol% of helper lipids may be from about 25 mol% to about 55 mol%. In one embodiment, the mol% of helper lipids may be from about 25 mol% to about 50 mol%. In one embodiment, the mol% of helper lipids may be from about 25 mol% to about 40 mol%. In one embodiment, the mol% of helper lipids may be from about 30 mol% to about 50 mol%. In one embodiment, the mol% of helper lipids may be from about 30 mol% to about 40 mol%. In one embodiment, the mol% of helper lipids is adjusted based on amine lipid, neutral lipid, and PEG lipid concentrations to achieve 100 mol% lipid component. In some embodiments, the mol% helper lipids of the LNP batch will be ± 30%, ± 25%, ± 20%, ± 15%, ± 10%, ± 5% or ± 2.5% of the target mol%. In certain embodiments, the LNP batch-to-batch rate of change will be less than 15%, less than 10%, or less than 5%.

In one embodiment, the mol% of PEG lipid may be from about 1 mol% to about 10 mol%. In one embodiment, the mol% of PEG lipid may be from about 2 mol% to about 10 mol%. In one embodiment, the mol% of PEG lipid may be from about 2 mol% to about 8 mol%. In one embodiment, the mol% of PEG lipid may be from about 2 mol% to about 4 mol%. In one embodiment, the mol% of PEG lipid may be from about 2.5 mol% to about 4 mol%. In one embodiment, the mol% of PEG lipid may be about 3 mol%. In one embodiment, the mol% of PEG lipid may be about 2.5 mol%. In some embodiments, the PEG lipid mol% of the LNP batch will be ± 30%, ± 25%, ± 20%, ± 15%, ± 10%, ± 5% or ± 2.5% of the target PEG lipid mol%. In certain embodiments, the LNP batch-to-batch rate of change will be less than 15%, less than 10%, or less than 5%.

In certain embodiments, the cargo comprises an mRNA encoding an RNA-guided DNA-binding agent (e.g., Cas nuclease, class 2 Cas nuclease, or Cas9), and a gRNA or a nucleic acid encoding a gRNA, or a combination of an mRNA and a gRNA. In one embodiment, the LNP composition can comprise lipid a or an equivalent thereof. In some aspects, the amine lipid is lipid a. In some aspects, the amine lipid is a lipid a equivalent, e.g., an analog of lipid a. In certain aspects, the amine lipid is an acetal analog of lipid a. In various embodiments, the LNP composition comprises an amine lipid, a neutral lipid, a helper lipid, and a PEG lipid. In certain embodiments, the helper lipid is cholesterol. In certain embodiments, the neutral lipid is DSPC. In a particular embodiment, the PEG lipid is PEG2 k-DMG. In some embodiments, the LNP composition can comprise lipid a, a helper lipid, a neutral lipid, and a PEG lipid. In some embodiments, the LNP composition comprises an amine lipid, DSPC, cholesterol, and a PEG lipid. In some embodiments, the LNP composition comprises a PEG lipid comprising DMG. In certain embodiments, the amine lipid is selected from lipid a, and equivalents of lipid a, including acetal analogs of lipid a. In further embodiments, the LNP composition comprises lipid a, cholesterol, DSPC, and PEG2 k-DMG.

Embodiments of the present invention also provide lipid compositions described in terms of the molar ratio between the positively charged amine groups (N) of amine lipids and the negatively charged phosphate groups (P) of the nucleic acid to be encapsulated. This can be mathematically represented by the equation N/P. In some embodiments, the LNP composition can comprise a lipid component comprising an amine lipid, a helper lipid, a neutral lipid, and a helper lipid; and a nucleic acid component, wherein the N/P ratio is about 3 to 10. In some embodiments, the LNP composition can comprise a lipid component comprising an amine lipid, a helper lipid, a neutral lipid, and a helper lipid; and an RNA component, wherein the N/P ratio is about 3 to 10. In one embodiment, the N/P ratio may be from about 5 to 7. In one embodiment, the N/P ratio may be from about 4.5 to 8. In one embodiment, the N/P ratio may be about 6. In one embodiment, the N/P ratio may be 6. + -.1. In one embodiment, the N/P ratio may be about 6. + -. 0.5. In some embodiments, the N/P ratio will be ± 30%, ± 25%, ± 20%, ± 15%, ± 10%, ± 5% or ± 2.5% of the target N/P ratio. In certain embodiments, the LNP batch-to-batch rate of change will be less than 15%, less than 10%, or less than 5%.

In some embodiments, the RNA component can comprise mRNA, such as mRNA encoding a Cas nuclease. In one embodiment, the RNA component can comprise Cas9 mRNA. In some compositions comprising Cas nuclease-encoding mRNA, the LNP further comprises a gRNA nucleic acid, such as a gRNA. In some embodiments, the RNA component comprises Cas nuclease mRNA and a gRNA. In some embodiments, the RNA component comprises a class 2 Cas nuclease mRNA and a gRNA.

In certain embodiments, the LNP composition can comprise mRNA encoding a Cas nuclease (such as a class 2 Cas nuclease), amine lipids, helper lipids, neutral lipids, and PEG lipids. In certain LNP compositions comprising an mRNA encoding a Cas nuclease (such as a class 2 Cas nuclease), the helper lipid is cholesterol. In other compositions comprising mRNA encoding a Cas nuclease (such as a class 2 Cas nuclease), the neutral lipid is DSPC. In other embodiments comprising an mRNA encoding a Cas nuclease (such as a class 2 Cas nuclease), the PEG lipid is PEG2k-DMG or PEG2 k-C11. In particular compositions comprising mRNA encoding a Cas nuclease (such as a class 2 Cas nuclease), the amine lipid is selected from lipid a and equivalents thereof, such as an acetal analog of lipid a.

In some embodiments, the LNP composition can comprise grnas. In certain embodiments, the LNP composition can comprise an amine lipid, a gRNA, a helper lipid, a neutral lipid, and a PEG lipid. In certain LNP compositions comprising grnas, the helper lipid is cholesterol. In some compositions comprising grnas, the neutral lipid is DSPC. In other embodiments comprising a gRNA, the PEG lipid is PEG2k-DMG or PEG2 k-C11. In certain embodiments, the amine lipid is selected from lipid a and equivalents thereof, such as an acetal analog of lipid a.

In one embodiment, the LNP composition can comprise sgrnas. In one embodiment, the LNP composition can comprise Cas9 sgRNA. In one embodiment, the LNP composition may comprise Cpf1 sgRNA. In some compositions comprising sgrnas, the LNPs include amine lipids, helper lipids, neutral lipids, and PEG lipids. In certain compositions comprising sgrnas, the helper lipid is cholesterol. In other compositions comprising sgRNA, the neutral lipid is DSPC. In other embodiments comprising sgrnas, the PEG lipid is PEG2k-DMG or PEG2 k-C11. In certain embodiments, the amine lipid is selected from lipid a and equivalents thereof, such as an acetal analog of lipid a.

In certain embodiments, the LNP composition comprises mRNA encoding a Cas nuclease and a gRNA, which can be an sgRNA. In one embodiment, the LNP composition can comprise an amine lipid, an mRNA encoding a Cas nuclease, a gRNA, a helper lipid, a neutral lipid, and a PEG lipid. In certain compositions comprising Cas nuclease-encoding mRNA and gRNA, the helper lipid is cholesterol. In some compositions comprising Cas nuclease-encoding mRNA and gRNA, the neutral lipid is DSPC. In other embodiments comprising mRNA encoding a Cas nuclease and a gRNA, the PEG lipid is PEG2k-DMG or PEG2 k-C11. In certain embodiments, the amine lipid is selected from lipid a and equivalents thereof, such as an acetal analog of lipid a.

In certain embodiments, the LNP composition comprises a Cas nuclease mRNA, such as a class 2 Cas mRNA and at least one gRNA. In certain embodiments, the LNP composition comprises grnas and Cas nuclease mrnas, such as class 2 Cas nuclease mrnas, in a ratio of about 25:1 to about 1: 25. In certain embodiments, the LNP formulation comprises a gRNA to Cas nuclease mRNA, such as a class 2 Cas nuclease mRNA, in a ratio of about 10:1 to about 1: 10. In certain embodiments, the LNP formulation comprises a gRNA to Cas nuclease mRNA, such as a class 2 Cas nuclease mRNA, in a ratio of about 8:1 to about 1: 8. As measured herein, the ratio is by weight. In some embodiments, the LNP formulation comprises a gRNA to Cas nuclease mRNA, such as a class 2 Cas mRNA, in a ratio of about 5:1 to about 1: 5. In some embodiments, the ratio ranges from about 3:1 to 1:3, about 2:1 to 1:2, about 5:1 to 1:1, about 3:1 to 1:2, about 3:1 to 1:1, about 3:1, about 2:1 to 1: 1. In some embodiments, the ratio of gRNA to mRNA is about 3:1 or about 2:1 in some embodiments, the ratio of gRNA to Cas nuclease mRNA (such as a class 2 Cas nuclease) is about 1: 1. The ratio may be about 25:1, 10:1, 5:1, 3:1, 1:3, 1:5, 1:10, or 1: 25.

LNP compositions disclosed herein can include a template nucleic acid. The template nucleic acid can be co-formulated with Cas nuclease-encoding mRNA, such as class 2 Cas nuclease mRNA. In some embodiments, the template nucleic acid may be co-formulated with the guide RNA. In some embodiments, the template nucleic acid can be co-formulated with both mRNA encoding the Cas nuclease and the guide RNA. In some embodiments, the template nucleic acid can be formulated separately from the mRNA or guide RNA encoding the Cas nuclease. The template nucleic acid can be delivered with the LNP composition or separately therefrom. In some embodiments, the template nucleic acid may be single-stranded or double-stranded, depending on the repair mechanism desired. The template may have regions of homology to the target DNA or to sequences adjacent to the target DNA.

In some embodiments, the LNP is formed by mixing an aqueous RNA solution with an organic solvent-based lipid solution, e.g., 100% ethanol. Suitable solutions or solvents include or may contain: water, PBS, Tris buffer, NaCl, citrate buffer, ethanol, chloroform, diethyl ether, cyclohexane, tetrahydrofuran, methanol, isopropanol. Pharmaceutically acceptable buffers can be used, for example, for in vivo administration of LNP. In certain embodiments, the buffer is used to maintain the pH of the composition comprising LNP at or above pH 6.5. In certain embodiments, the buffer is used to maintain the pH of the composition comprising LNP at or above pH 7.0. In certain embodiments, the pH of the composition is in the range of about 7.2 to about 7.7. In further embodiments, the pH of the composition is in the range of about 7.3 to about 7.7 or in the range of about 7.4 to about 7.6. In other embodiments, the pH of the composition is about 7.2, 7.3, 7.4, 7.5, 7.6, or 7.7. The pH of the composition can be measured using a micro pH probe. In certain embodiments, a cryoprotectant is included in the composition. Non-limiting examples of cryoprotectants include sucrose, trehalose, glycerol, DMSO, and ethylene glycol. Exemplary compositions may include up to 10% of a cryoprotectant, such as sucrose. In certain embodiments, the LNP composition can include about 1,2, 3,4, 5, 6, 7, 8, 9, or 10% cryoprotectant. In certain embodiments, the LNP composition can include about 1,2, 3,4, 5, 6, 7, 8, 9, or 10% sucrose. In some embodiments, the LNP composition can comprise a buffer. In some embodiments, the buffer may comprise Phosphate Buffered Saline (PBS), Tris buffer, citrate buffer, and mixtures thereof. In certain exemplary embodiments, the buffer comprises NaCl. In certain embodiments, NaCl is omitted. Exemplary amounts of NaCl may range from about 20mM to about 45 mM. Exemplary amounts of NaCl may range from about 40mM to about 50 mM. In some embodiments, the amount of NaCl is about 45 mM. In some embodiments, the buffer is a Tris buffer. Exemplary amounts of Tris may range from about 20mM to about 60 mM. Exemplary amounts of Tris may range from about 40mM to about 60 mM. In some embodiments, the amount of Tris is about 50 mM. In some embodiments, the buffer comprises NaCl and Tris. Certain exemplary embodiments of the LNP composition contain 5% sucrose and Tris buffer containing 45mM NaCl. In other exemplary embodiments, the composition contains sucrose in an amount of about 5% w/v, about 45mM NaCl and about 50mM Tris (pH 7.5). The amount of salt, buffer and cryoprotectant can be varied to maintain osmolality of the total formulation. For example, the final osmolality can be maintained at less than 450 mOsm/L. In other embodiments, the osmolality is between 350 and 250 mOsm/L. Certain embodiments have a final osmolality of 300+/-20 mOsm/L.

In some embodiments, microfluidic mixing, T-type mixing, or cross-mixing is used. In certain aspects, flow rates, linker sizes, linker geometries, linker shapes, tube diameters, solutions, and/or RNA and lipid concentrations can vary. The LNP or LNP composition can be concentrated or purified, for example, via dialysis, tangential flow filtration, or chromatography. LNP can be stored, for example, in the form of a suspension, emulsion or lyophilized powder. In some embodiments, the LNP composition is stored at 2 ℃ -8 ℃, in certain aspects the LNP composition is stored at room temperature. In further embodiments, the LNP composition is stored frozen, e.g., at-20 ℃ or-80 ℃. In other embodiments, the LNP composition is stored at a temperature in the range of from about 0 ℃ to about-80 ℃. The frozen LNP composition can be thawed prior to use, e.g., on ice, at 4 ℃, at room temperature, or at 25 ℃. The frozen LNP composition can be maintained at various temperatures, for example, on ice, at 4 ℃, at room temperature, at 25 ℃, or at 37 ℃.

Methods of engineering stem cells (e.g., HSPCs); engineered stem cells (e.g., HSPC)

LNP compositions disclosed herein are useful in methods of engineering stem cells (e.g., HSPCs) in vitro, for example, by CRISPR/Cas system gene editing. In some embodiments, the genetically engineered cell population is a CD34+ cell population. In some embodiments, a method is provided for generating a population of genetically engineered HSPC or CD34+ cells in vitro, the method comprising (a) preincubating a serum factor with an LNP composition for delivery of Cas nuclease mRNA and gRNA; (b) contacting a population of HSPC or CD34+ cells with a pre-incubated LNP composition in vitro; and (c) culturing the population of HSPC or CD34+ cells in vitro, thereby producing the genetically engineered HSPC. In some embodiments, the methods comprise contacting a HSPC or CD34+ cell with an LNP composition described herein according to the delivery methods described herein.

In some embodiments, engineered stem cells (e.g., HSPCs) are provided, e.g., engineered HSPCs or HSPC populations. Such engineered cells are prepared according to the methods described herein. In some embodiments, the engineered HSPCs reside in a tissue or organ, such as bone marrow, blood, or other tissue in a subject, e.g., after transplantation of the engineered HSPCs.

In some of the methods and cells described herein, the cell comprises a modification, such as an insertion or deletion ("index") or substitution of a nucleotide in the target sequence. In some embodiments, the modification comprises an insertion of 1,2, 3,4, or 5 or more nucleotides in the target sequence. In some embodiments, the modification comprises an insertion of 1 or 2 nucleotides in the target sequence. In other embodiments, the modification comprises a deletion of 1,2, 3,4, 5, 6, 7, 8, 9, 10, 15, 20, or 25 or more nucleotides in the target sequence. In some embodiments, the modification comprises a deletion of 1 or 2 nucleotides in the target sequence. In some embodiments, the modification comprises an insertion or deletion that results in a frame shift mutation in the target sequence. In some embodiments, the modification comprises a substitution of 1,2, 3,4, 5, 6, 7, 8, 9, 10, 15, 20, or 25 or more nucleotides in the target sequence. In some embodiments, the modification comprises a substitution of 1 or 2 nucleotides in the target sequence. In some embodiments, the modification comprises one or more nucleotide insertions, deletions, or substitutions resulting from incorporation into a template nucleic acid, e.g., any of the template nucleic acids described herein.

In some embodiments, a cell population comprising engineered cells is provided, e.g., a cell population comprising cells engineered according to the methods described herein. In some embodiments, the population comprises engineered cells cultured in vitro. In some embodiments, the population resides within a tissue or organ, e.g., a liver of the subject. In some embodiments, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% or more of the cells within a population are engineered. In certain embodiments, the methods disclosed herein result in an editing efficiency (or "percent editing") of at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95%, as defined by detecting an insertion or deletion. In other embodiments, the methods disclosed herein result in at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% DNA modification efficiency, defined by detecting sequence changes (by insertions, deletions, substitutions, or otherwise). In certain embodiments, the methods disclosed herein result in an editing efficiency level or DNA modification efficiency level of between about 5% to about 100%, about 10% to about 50%, about 20% to about 100%, about 20% to about 80%, about 40% to about 100%, or about 40% to about 80% in a population of cells.

In some of the methods and cells described herein, the cells within the population comprise a modification, such as an insertion or deletion or substitution, at the target sequence. In some embodiments, the modification comprises an insertion of 1,2, 3,4, or 5 or more nucleotides in the target sequence. In some embodiments, the modification comprises an insertion of 1 or 2 nucleotides in the target sequence. In other embodiments, the modification comprises a deletion of 1,2, 3,4, 5, 6, 7, 8, 9, 10, 15, 20, or 25 or more nucleotides in the target sequence. In some embodiments, the modification comprises a deletion of 1 or 2 nucleotides in the target sequence. In some embodiments, the modification results in a frameshift mutation in the target sequence. In some embodiments, the modification comprises an insertion or deletion that results in a frame shift mutation in the target sequence. In some embodiments, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% or more of the engineered cells in the population comprise a frameshift mutation. In some embodiments, the modification comprises a substitution of 1,2, 3,4, 5, 6, 7, 8, 9, 10, 15, 20, or 25 or more nucleotides in the target sequence. In some embodiments, the modification comprises a substitution of 1 or 2 nucleotides in the target sequence. In some embodiments, the modification comprises one or more nucleotide insertions, deletions, or substitutions resulting from incorporation into a template nucleic acid, e.g., any of the template nucleic acids described herein.

Gene editing method

The methods disclosed herein can be used for gene editing in stem cells, HSPCs, or HSPC populations in vitro. In one embodiment, one or more LNP compositions described herein can be administered to a stem cell, HSPC, or HSPC population. In one embodiment, one or more LNP compositions described herein can contact stem cells, HSPCs and HSCs or HPCs. In one embodiment, genetically engineered cells can be produced by contacting cells with an LNP composition according to the methods described herein. In some gene editing methods, the HSPC or HSPC population is maintained in culture. In some gene editing methods, the HSPC or HSPC population is transplanted into a patient. In some embodiments, the genetically engineered HSPCs reside in a tissue or organ, such as bone marrow, blood, or other tissue in a patient, e.g., after transplantation of the engineered HSPCs.

In some embodiments, the method comprises a population of stem cells, HSPCs, or HSPCs that are autologous with respect to the patient to whom the cells are administered. In some embodiments, the method comprises a HSPC or HSPC population that is allogeneic with respect to the patient to whom the cells are to be administered.

In various embodiments, the methods described herein achieve CRISPR-Cas gene editing in a population of stem cells, HSPCs, or HSPCs. In some embodiments, the method further comprises detecting gene editing in the HSPC or HSPC population. In some embodiments, gene editing is measured as a percentage of editing. In some embodiments, gene editing is measured as a percentage of DNA modification. The method can achieve at least 40%, 50%, 60%, 70%, 80%, 90% or 95% editing. The methods can achieve at least 40%, 50%, 60%, 70%, 80%, 90%, or 95% DNA modification.

In one embodiment, an LNP composition comprising mRNA encoding a class 2 Cas nuclease and a gRNA can be administered to a stem cell, HSPC, or HSPC population. In further embodiments, a template nucleic acid is also introduced into the cell. In certain instances, an LNP composition comprising a class 2 Cas nuclease and an sgRNA can be administered to a cell.

In one embodiment, the LNP composition can be used to edit a gene in a stem cell, HSPC or HSPC population, resulting in a gene knockout. In one embodiment, the LNP composition can be used to edit genes in a HSPC or HSPC population, resulting in gene knockouts, for example, in a cell population. Knockouts or knockouts can be detected by measuring target protein levels. Knockouts or knockouts can be detected by detecting the target DNA. In another embodiment, the LNP composition can be used to edit a gene in an HSPC or HSPC population, resulting in gene correction. In another embodiment, the LNP compositions can be used to edit cells, resulting in gene insertion.

The LNP compositions can be administered in a formulation in combination with one or more pharmaceutically acceptable excipients. The term "excipient" includes any ingredient other than the compounds of the present disclosure, other lipid components, and bioactive agents. Excipients may impart functional (e.g., drug release rate control) and/or non-functional (e.g., processing aids or diluents) characteristics to the formulation. The choice of excipient will depend to a large extent on factors such as the particular mode of administration, the effect of the excipient on stem cell or HSPC cultures and on solubility and stability, and the nature of the dosage form.

When the formulation is aqueous, excipients such as sugars (including but not limited to glucose, mannitol, sorbitol, and the like), salts, carbohydrates, and buffering agents (preferably a pH of 3 to 9) may be used, but for some applications the formulation may be more suitably formulated as a sterile non-aqueous solution or in dry form to be used in combination with a suitable vehicle such as sterile pyrogen-free Water (WFI).

While the invention will be described in conjunction with the illustrated embodiments, it will be understood that it is not intended to limit the invention to those embodiments. On the contrary, the invention is intended to cover alternatives, modifications and equivalents, including equivalents of the specific features, which may be included within the invention as defined by the appended claims.

The foregoing general and detailed description, as well as the following examples, are exemplary and explanatory only and are not limiting of the teachings. The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described in any way. In the event that any document incorporated by reference contradicts any term defined in the specification, the specification controls. Unless otherwise stated, all ranges given in this application are inclusive of the endpoints.

It should be noted that, as used in this application, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a composition" includes a plurality of compositions and reference to "a cell" includes a plurality of cells and the like. Unless stated otherwise, the use of "or" is inclusive and means "and/or.

Numerical ranges include the numbers defining the range. The measured and measurable values are to be understood as approximate, taking into account the significant figures and the errors associated with the measurement. The term "about" or "approximately" means an acceptable error for a particular value as determined by one of ordinary skill in the art, which depends in part on how the value is measured or determined. A modifier such as "about" is used before a range or before a list of values to modify each end point of the range or each value in the list. For example, "about 50-55" encompasses "about 50 to about 55". In addition, the use of "comprising", "comprises", "comprising", "containing", "including", and "including" is not restrictive.

Unless expressly indicated in the foregoing specification, embodiments in which "comprising" various components is recited in the specification are also considered to "consist of" or "consist essentially of" the components; the statement in this specification that "consists of" various components "is also considered to be" comprising "or" consisting essentially of "the components; embodiments in the present specification that recite "about" various components are also contemplated as "in" the component; and the statement in this specification that "consists essentially of" each component "is also to be taken as" consisting of "or" comprising "the component(s) (this interchangeability does not apply to the use of these terms in the claims).

Examples

Example 1 Process

Cell culture

Cryopreserved human CD34+ bone marrow cells were obtained from AllCells (catalog No. ABM017F) or StemCellTechnologies (catalog No. 70008). After thawing and washing twice in 20ml StemBan SFEM (Stem Cell technologies, Cat. No. 09650), the cells were cultured for 48 hours in StemBan SFEM (StemShell technologies, Cat. No. 09650) containing: thrombopoietin (TPO, 50ng/ml, StemShell Technologies, Cat. No. 02922), human Flt3 ligand (Flt3l, 50ng/ml, StemShell Technologies, Cat. No. 78137.2), human interleukin-6 (Il-6, 50ng/ml, StemShell Technologies, Cat. No. 78148.2), human stem cell factor (SCF, 50ng/ml, StemShell Technologies, Cat. No. 78155.2) and StemRegein-1 (SR1, 0.75uM) and penicillin/streptomycin (P/S, 100U/ml penicillin and 100ug/ml streptomycin, Life Technologies, Cat. No. 15140122).

Lipid nanoparticle ("LNP") formulations

LNP was formulated by dissolving the lipid nanoparticle component in 100% ethanol in the following molar ratios: 45 mol% (12.7mM) lipid amine (e.g., lipid A); 44 mol% (12.4mM) helper lipid (e.g., cholesterol); 9 mol% (2.53mM) neutral lipid (e.g., DSPC); 2 mol% (.563mM) PEG lipid (e.g., PEG2k-DMG or PEG2k-C11), unless otherwise noted. The N/P ratio (moles of lipid amine to moles of RNA) was 4.5. The ID number of the LNP formulation is as follows: LNP522, LNP525(GFPmRNA) and LNP670, LNP926(B2M single guide, Cas9 mRNA) and LNP899(AAVS1 single guide, Cas9 mRNA). The RNA cargo was dissolved in 50mM acetate buffer (pH 4.5) or 25mM sodium citrate, 100mM NaCl, pH 5.0, resulting in an RNA cargo concentration of approximately 0.45 mg/mL.

LNPs were formed by microfluidically mixing lipid and RNA solutions using Precision Nanosystems nanoasssemblr (tm) bench top instruments according to the manufacturer's protocol. A 2:1 ratio of aqueous phase to organic solvent was maintained during mixing using differential flow rates. After mixing, LNP was collected and diluted in phosphate buffered saline, pH 7.4(PBS) or 50mM Tris, pH 7.5(Tris) (approximately 1:1) to reduce ethanol content prior to further processing. By using a 10kDa Slide-a-Lyzer at 4 ℃ under gentle stirring^TMThe G2 dialysis cartridge (ThermoFisher Scientific) was dialyzed overnight against PBS or Tris (100-fold excess of sample volume) to complete the final buffer exchange. Tris treated formulations were diluted 1:1 into 100mM Tris, 90mM saline, 5% (w/v) sucrose, pH 7.5(2 × TSS). Alternatively, LNP is collected after mixing, diluted in water, left at room temperature for 1 hour, and then diluted a second time with 1:1 water. The final buffer exchange to TSS was done using a PD-10 desalting column (GE). If desired, the preparation obtained by either treatment method is concentrated by centrifugation using an Amicon 100kDa centrifugal filter (Millipore). The resulting mixture was then filtered using a 0.2 μm sterile filter. If the final buffer is PBS, storing the obtained filtrate at 2-8 ℃; if the final buffer is TSS, the resulting filtrate is stored at-80 ℃.

In vitro transcription of nuclease mRNA and Single guide RNA (sgRNA) ("IVT")

The capped and polyadenylated Cas9 mRNA was generated by in vitro transcription using a linearized plasmid DNA template and T7 RNA polymerase. Plasmid DNA containing the T7 promoter and a 100-residue poly (A/T) region was linearized by incubation with XbaI at 37 ℃ for 2 hours under the following conditions: 200 ng/. mu.L plasmid, 2U/. mu.L XbaI (NEB) and 1 Xreaction buffer. XbaI was inactivated by heating the reaction at 65 ℃ for 20 minutes. Linearized plasmids were purified from enzyme and buffer salts using silica spin columns (Epoch Lifesciences) and analyzed by agarose gel to confirm linearization. IVT reactions used to generate Cas9 modified mRNA were incubated for 4 hours at 37 ℃ under the following conditions: 50 ng/. mu.L linearized plasmid; 2mM of each of GTP, ATP, CTP and N1-methyl pseudo UTP (Trilink); 10mM ARCA (Trilink); 5U/. mu. L T7 RNA polymerase (NEB); 1U/. mu.L of murine RNase inhibitor (NEB); 0.004U/. mu.L of inorganic E.coli pyrophosphatase (NEB); and 1x reaction buffer. After 4 hours incubation, TURBO DNase (ThermoFisher) was added to a final concentration of 0.01U/. mu.L, and the reaction was incubated for an additional 30 minutes to remove the DNA template. Cas9 mRNA was purified using LiCl precipitation.

For all methods, transcript concentration was determined by measuring absorbance at 260nm (nanodrop) and transcripts were analyzed by capillary electrophoresis using a Bioanalyzer (Agilent). SgRNA was chemically synthesized.

LNP transfection of human CD34+ bone marrow cells

LNP containing GFP mRNA or Cas9 mRNA and a single guide targeting β 2-microglobulin (B2M) was added to 30,000 human CD34+ bone marrow cells at various concentrations ranging from 50.0Ng to 800.0Ng, for a total volume of 100.0 ul. The sequence of the sgRNA targeting GGCCACGGAGCGAGACATCT B2M target sequence (SEQ ID NO:75) is: mG mC CACGGAGCGAGACAUUCUGUUUUUAGAGAmmCmAmmGmAmAmAmAmmGmCAAGUAAUAAGGCUAGUCCGUACAMMmCmUmUmGmAmmGmGmGmGmGmGmUmGmGmGmMmU mU (SEQ ID NO: 76). In this nucleic acid sequence, A, U, G and C indicate adenine, uracil, cytosine and guanine, respectively; m represents 2' -O-methyl nucleotide; and indicates phosphorothioate linkages.

For the species-specific serum study (Triple S study), LNP was incubated in 6.0% serum from the following for 5 minutes at 37 ℃ prior to cell transfection: mice (BiorecomationIVT, Cat No. MSESRM, batch No. MSE 245847), cynomolgus monkey (M.fascicularis) (BiorecomationIVT, Cat No. CYNSRM, batch No. CYN197451) and homo sapiens (Sigma, pooled, H4522-20ml, batch No. SLBR 7629V; BiorecomationIVT, Cat No. HMSRM, batch No. BRH 1278638; BiorecomationIVT, Cat No. HMSRM, batch No. BRH 1227947). Human recombinant apolipoprotein E3(ApoE3, R & D Systems, Cat. No. 4144-AE) was used in the recommended buffer at a range of concentrations (01.ug/ml, 1.0ug/ml, 10.0ug/ml and 50.0ug/ml) under the same incubation conditions as described above.

Flow cytometric readout of LNP transfected human CD34+ bone marrow cells

Cells were collected for antibody staining at 24 hours after LNP-GFP transfection or 5 days after LNP-B2M transfection. After washing the cells in sample medium (PBS + 2% FBS +2mM EDTA), the cells were blocked with Human TruStain FcX (Biolegend, cat # 422302) for 5 minutes at Room Temperature (RT).

We stained the cells with the following antibodies and markers as shown in table 2.

Table 2 antibodies and labels.

Cells were run on a Beckman Coulter cytoflex s and analyzed using the FlowJo software package.

Cell survival was assessed by 7-AAD staining. Live cells were normalized based on the 7-AAD insertion in the GC-rich DNA region and subsequently detected in a flow cytometry assay. Cell viability was calculated using the following formula:

[ (sample 1)_{Number of cellular events}Sample 1_{Number of bead events}) Sample 1_{Total number of beads added}]Average { [ (control 1)_{Number of cellular events}Control 1_{Number of bead events}) Control 1_{Total number of beads added}][ (control)2_{Number of cellular events}Control 1_{Number of bead events}) Control 2_{Total number of beads added}]…, control N }

In this formula, a "sample" is defined as any population of human CD34+ HSPCs treated during the course of the experiment with LNP, mRNA, gRNA, or any combination of the foregoing, and a "control" is defined as any population of human CD34+ HSPCs not treated during the course of the experiment with LNP, mRNA, gRNA, or any combination of the foregoing.

Next generation sequencing ("NGS") and lysis efficiency analysis

To quantitatively determine the efficiency of editing at a target location in a genome, the presence of insertions and deletions introduced by gene editing is identified using deep sequencing.

Cells were harvested on day 5 post-transfection and DNA was extracted using a PureLink Genomic DNA Mini kit (ThermoFisher scientific, Cat. No. K182002). Primers for the B2M target locus containing Illumina P5 and P7 adaptor sequences were used to amplify the genomic locus of interest in a standard PCR reaction.

PCR primers were designed around the B2M target site and the genomic region of interest was amplified. Samples were submitted for sample preparation (Illumina MiSeq v2 kit, 300 cycles, catalog No. 15033624) and sequencing was run on the Illumina MiSeq instrument. The editing frequency at the target site of interest is analyzed using a custom pipeline (beshook pipeline). Briefly, additional PCR was performed according to the manufacturer's protocol (Illumina) to add the necessary chemistry for sequencing. Amplicons were sequenced on the illumina miseq instrument. After eliminating reads with low quality scores, the reads were aligned to a human reference genome (e.g., hg 38). The resulting file containing reads is mapped to a reference genome (BAM file), where reads that overlap the target region of interest are selected and the number of wild type reads is counted relative to the number of reads containing insertions, substitutions or deletions.

The percent edit (e.g., "edit efficiency" or "edit%") is provided as the total number of sequence reads with insertions or deletions compared to the total number of sequence reads comprising the wild-type.

Formulation analysis

LNP preparations were analyzed for mean particle size, polydispersity (pdi), total RNA content, and RNA encapsulation efficiency. The average particle size and polydispersity were measured by Dynamic Light Scattering (DLS) using a Malvern Zetasizer instrument. LNP samples were diluted 30X in PBS prior to measurement by DLS. The Z-average diameter, which is an intensity-based measure of the average particle size, is reported along with the number average diameter and pdi.

Using fluorescence-based assays (Thermo fisher Scientific) to determine total RNA concentration and free RNA. Encapsulation efficiency was calculated as (total RNA-free RNA)/total RNA. LNP samples were diluted with 1XTE buffer containing 0.2% Triton-X100 in an appropriate manner to determine total RNA or 1XTE buffer to determine free RNA. Standard curves were prepared by using starting RNA solutions used to make up the formulation and diluted in 1 × TE buffer +/-0.2% Triton-X100. Then dilutedDye (100X in 1xTE buffer, according to the manufacturer's instructions) was added to each of the standards and samples and they were incubated in the absence of light at room temperature for approximately 10 minutes. Samples were read using a SpectraMax M5 microplate reader (Molecular Devices) with excitation, auto-cut-off and emission wavelengths set to 488nm, 515nm and 525nm, respectively. Total and free RNA were determined according to appropriate standard curves. Encapsulation efficiency was calculated as (total RNA-free RNA)/total RNA. The same procedure can be used to determine the encapsulation efficiency of DNA-based cargo components. For single stranded DNA, Oligreen dye may be used, and for double stranded DNA, Picogreen dye may be used.

Example 2 delivery of GFP to CD34+ bone marrow cells

LNP and GFP mRNA were formulated in final PBS buffer and added to 30,000 human CD34+ bone marrow cells in a total volume of 100.0ul as described in example 1 to provide 0, 50.0ng, 100.0ng and 200.0ng of GFP mRNA in each reaction. LNP was preincubated with 6% (v/v) serum from mice (bioremodeling ivt, catalogue number msesm, lot number MSE 24821) for 5 minutes at 37 ℃ prior to administration to cells. Cells were cultured as described in example 1.

GFP + cells were quantified by flow cytometry 24 hours after LNP was added to human CD34+ cells. The GFP + cell population was determined in the FITC channel (maximum excitation 490, maximum emission 525, laser line 488) relative to the GFP-control (labeled "control" in figure 1). The percentage of GFP + cells in all human CD34+ live bone marrow cells 24 hours after LNP-mediated GFP mRNA delivery is shown in figure 1. LNP composition is as follows, fig. 1 (a): 45% lipid a, 44% cholesterol, 9% DSPC, 2% PEG; FIG. 1 (B): 45% lipid a, 45% cholesterol, 9% DSPC, 1% PEG. The biological sample size n is 3.

LNP compositions demonstrate dose-dependent in vitro delivery of mRNA to CD34+ bone marrow cells.

Example 3 LNP Pre-incubation facilitates delivery

Tests have demonstrated that LNP requires incubation with 6% mouse serum (v/v) prior to transfection to effectively deliver GFPmRNA to human CD34⁺Bone marrow cells. Cells were cultured and transfected with LNP compositions as described in example 2 with the following modifications:

FIG. 2A shows human CD34 with LNP applied immediately on day 0 after thawing of cryopreserved cell vials⁺GFP in all live cells of bone marrow samples⁺Percentage of cells. LNP with 50.0ng, 100.0ng or 200.0ng GFP mRNA was added to the cells with or without serum incubation prior to transfection.

FIG. 2B shows human CD34 with LNP on day 2 after thawing of cryopreserved cell vials⁺GFP in all live cells of bone marrow samples⁺Percentage of cells. LNP with 50.0ng, 100.0ng or 200.0ng GFP mRNA was added to the cells with or without serum incubation prior to transfection. The biological sample size n is 3.

Example 4-delivery of Cas9 and guide RNA via LNP; gene editing in CD34+ bone marrow cells

LNP was formulated with sgRNA (G529) and Cas9 mRNA as described in example 1 and in a 1:1 weight ratio in final TSS buffer as described in example 1. LNP consists of 45% lipid a, 44% cholesterol, 9% DSPC, 2% PEG; the N/P ratio was 4.5.

Transfection of human CD34 Using the LNP delivery method⁺Bone marrow cells, which can efficiently deliver Cas9 mRNA and B2M sgRNA to cells by pre-incubation with increasing percentage (v/v) of mice or cynomolgus monkey serum. Active Cas9-sgRNA complexes were delivered via LNP pre-incubated with various sera.

Fig. 3A depicts FACS analysis results of transfected cells showing the percentage of B2M negative cells after application of LNP (400.0ng Cas9 mRNA and sgRNA (1:1 by weight)) incubated with 6%, 30% and 60% (v/v) mouse serum ("mouse-S") or 6%, 30% and 60% (v/v) non-human primate serum ("cynomolgus monkey-S"). LNP without serum pre-incubation ("LNP only") and untreated cells ("control (Ctrl)") showed no effective delivery (measured as B2M expression knockouts). Pre-incubation with mouse or primate serum facilitates efficient knock-out of CD34⁺B2M expression in bone marrow cells.

FIG. 3B depicts human CD34 transfected with LNPs (400.0ng Cas9 mRNA and sgRNA (1:1 by weight)) incubated with 6%, 30%, and 60% (v/v) mouse serum or 6%, 30%, and 60% (v/v) non-human primate serum, as determined by NGS⁺Editing efficiency at the genomic level of bone marrow cells. As shown in figure 3(a), LNP administration without serum pre-incubation and untreated cells did not show efficient delivery (% measure edit). Insertions ("In", light grey) and deletions ("Del", black) are plotted on the Y-axis, showing CD34⁺An editing efficiency in the cell of greater than about 60%, greater than about 70%, greater than about 80%, and greater than about 90%. The "LNP only" and "control" samples did not exhibit detectable levels of insertions or deletions at the B2M locus. The biological sample size n is 3.

Example 5 Pre-incubation with isolated serum factor ApoE3

To investigate whether the serum pre-incubation step could be replaced by recombinant protein, LNP delivering Cas9 and B2M sgRNA was pre-incubated with human recombinant apolipoprotein E3(ApoE3), mouse serum, or non-human primate serum during the LNP incubation step as described in example 4 before cell transfection.

The percentage of B2M negative cells after application of LNP (400ng Cas9 mRNA and sgRNA (1:1)) pre-incubated with 6% (v/v) mouse serum ("mouse-S") or 6% (v/v) non-human primate serum ("cynomolgus monkey-S") or 0.1. mu.g/ml, 1.0. mu.g/ml, 10.0. mu.g/ml and 50.0. mu.g/ml ApoE3 is shown in FIG. 4A. Untreated human CD34⁺Bone marrow cells were used as a negative control ("control"). ApoE3 showed a dose-dependent increase in delivery to CD34+ cells and it could be used for the pre-incubation step.

Also, gene editing showed a dose-dependent response to ApoE 3. FIG. 4B depicts human CD34 transfected with 400.0ng LNP incubated with 6% (v/v) mouse serum or 6% (v/v) non-human primate serum or 0.1 μ g/ml, 1.0 μ g/ml, 10.0 μ g/ml and 50.0 μ g/ml ApoE3 as determined by NGS⁺Edit percentage of B2M target of bone marrow cells. Untreated human CD34⁺Bone marrow cells were used as a negative control that did not exhibit detectable levels of insertions or deletions at the B2M locus. The biological sample size n is 3.

Example 6 Pre-incubation with serum factors

This experiment tested preincubation of LNP with a variety of different apolipoproteins, showing in vitro LNP uptake of ApoE isoforms as measured by B2M knock-out levels and editing frequency in HSPC populations. Prior to transfection, LNP (ID LNP926) was incubated with 6% of mice serum (v/v) or various concentrations of apolipoproteins below for 5 minutes at 37 ℃: recombinant human ApoA-I (Millipore Sigma, cat # SRP4693), ApoB from human plasma (Millipore Sigma, cat # A5353), ApoC-I from human plasma (Millipore Sigma, cat # A7785), human recombinant ApoE2(Millipore Sigma, cat # SRP4760), human recombinant ApoE3(Millipore Sigma, cat # SRP4696), human recombinant ApoE4(Millipore Sigma, cat # A3234). LNP was added to human CD34+ bone marrow cells at a concentration of 200ng total RNA cargo (Cas 9 mRNA and single guide in a 1:1w/w ratio). On day 5 post-transfection, B2M expression at the protein level was determined by flow cytometry using the same antibodies as described above. Data analysis was performed using FlowJo software package. Data represent mean +/-SD of one biological sample (N ═ 1), technical replicates.

Figure 5 shows B2M knockdown in CD34+ HSPC populations following transfection with LNP pre-incubated with cynomolgus monkey serum, ApoE2, ApoE3 and ApoE 4. Untreated, non-pre-incubated and pre-incubated with ApoA-I, ApoB and ApoC-I did not result in B2M knockouts. In this experiment, pre-incubation of LNP with ApoE2 showed less B2M knockouts compared to the other two ApoE isoforms.

Example 7 time course of LNP Exposure

This experiment tested the effect of LNP exposure duration on survival and edit rates. LNP899, which delivers Cas9 mRNA and AAVS1 targeting G562, was preincubated with 6% (v/v) non-human primate serum at 37 ℃ for about 5 minutes. LNP was added to human CD34+ bone marrow cells at a concentration of 300ng total RNA cargo (Cas 9 mRNA and single guide in a 1:1w/w ratio). At 2 hours, 6 hours or 24 hours post-transfection, cells were centrifuged and resuspended in fresh medium without LNP. CountBright measured on a Cytoflexs flow cytometer (Beckman Coulter) was used^TMAbsolute count beads (Invitrogen, Cat. No. C36950) assessed cell viability for 3 and 8 days. Compiled by NGS measurements as described in example 1. Table 3 and fig. 6B show the editing frequency 8 days after transduction. Table 3 and fig. 6A show cell viability 3 and 8 days after transduction.

Table 3 survival rates and edits under different LNP exposures

Brief description of the published sequence listing

For the sequences themselves, see the sequence listing below. The transcript sequence typically includes GGG in the form of the first three nucleotides used with ARCA or in the form of a protein associated with CleanCap^TMAGG in the form of the first three nucleotides used together. Thus, the first three nucleotides can be modified for use with other capping methods, such as vaccinia capping enzymes. Promoters and poly-A sequences are not included in the transcript sequence. Promoters, such as the T7 promoter (SEQ ID NO:31) and poly-A sequences, such as SEQ ID NO:63, may be attached to the disclosed transcript sequences at the 5 'and 3' ends, respectively. Most nucleotide sequences are provided in DNA form, but can be readily converted to RNA by changing T to U.

Sequence listing

The following sequence listing provides a listing of the sequences disclosed herein. It will be understood that if a DNA sequence (comprising T) is referred to with respect to RNA, then T should be replaced by U (which may be modified or unmodified depending on the context), and vice versa.

PS linkage; m-2' -O-Me nucleotide; n-any natural or non-natural nucleotide

GFP sequence:

Claims

1. a method of delivering mRNA to a hematopoietic stem and/or progenitor cell (HSPC) or HSPC population, the method comprising:

a. preincubating serum factors with an LNP composition comprising the mRNA, amine lipids, helper lipids, neutral lipids, and PEG lipids;

b. contacting the HSPC or the population of HSPCs with the pre-incubated LNP composition in vitro; and

c. culturing the HSPC or the population of HSPCs in vitro;

thereby delivering the mRNA to the HSPC or the HSPC population.

2. A method of delivering mRNA to HSPCs, the method comprising:

a. preincubating serum factors with an LNP composition comprising the mRNA and amine lipids;

b. contacting the cells in vitro with the pre-incubated LNP composition; and

c. culturing the HSPCs in vitro;

thereby delivering the mRNA to the HSPC.

3. A method of delivering mRNA to a stem cell or stem cell population, the method comprising:

a. preincubating serum factors with an LNP composition comprising the mRNA;

b. contacting the population of stem cells with the pre-incubated LNP composition in vitro; and

c. culturing the population of stem cells in vitro;

thereby delivering the mRNA to the stem cell population.

4. The method of any one of claims 1-3, wherein the mRNA encodes a Cas nuclease.

5. A method of introducing Cas nuclease mRNA and gRNA into HSPC, the method comprising:

a. preincubating a serum factor with an LNP composition comprising the Cas nuclease mRNA, gRNA, amine lipids, helper lipids, neutral lipids, and PEG lipids;

b. contacting the HSPCs with the pre-incubated LNP composition in vitro; and

c. culturing the HSPCs;

thereby introducing the Cas nuclease mRNA and the gRNA into the HSPC.

6. A method of producing genetically engineered HSPCs in vitro, the method comprising:

a. preincubating a serum factor with an LNP composition comprising Cas nuclease mRNA, gRNA, amine lipids, helper lipids, neutral lipids, and PEG lipids;

b. contacting the HSPCs with the pre-incubated LNP composition in vitro; and

c. culturing the HSPCs in vitro;

thereby producing genetically engineered HSPCs.

7. A method of introducing Cas nuclease mRNA and gRNA into a stem cell, the method comprising:

a. preincubating a serum factor with an LNP composition comprising the Cas nuclease mRNA, gRNA, and amine lipids;

b. contacting the stem cells in vitro with the pre-incubated LNP composition; and

c. culturing the stem cells;

thereby introducing the Cas nuclease mRNA and the gRNA into the stem cell.

8. A method of producing genetically engineered stem cells such as HSPCs in vitro, the method comprising:

a. preincubating a serum factor with an LNP composition comprising Cas nuclease mRNA, gRNA, and biodegradable lipids;

b. contacting the cells in vitro with the pre-incubated LNP composition; and

c. culturing the cell in vitro;

thereby producing genetically engineered stem cells, such as HSPCs.

9. The method of claim 4, wherein the LNP composition further comprises a gRNA.

10. The method of any one of claims 4-9, wherein the Cas nuclease is a class 2 Cas nuclease.

11. The method of claim 10, wherein the class 2 Cas nuclease is Cas9 nuclease.

12. The method of claim 11, wherein the Cas9 nuclease is streptococcus pyogenes Cas 9.

13. The method of claim 10, wherein the class 2 Cas nuclease is Cpf1 nuclease.

14. The method of any one of claims 5-13, wherein the gRNA is a dual guide rna (dgrna).

15. The method of any one of claims 5-13, wherein the gRNA is a single guide rna (sgrna).

16. The method of any one of the preceding claims, further comprising a washing step after the contacting step.

17. The method of any one of the preceding claims, wherein the contacting step is between about 1 minute and about 72 hours long.

18. The method of any one of the preceding claims, wherein the contacting step is between about 1 minute and about 24 hours long.

19. The method of claim 17 or 18, wherein the contacting step is between about 2 hours and about 24 hours.

20. The method of any one of claims 17-19, wherein the contacting step is between about 4 hours and about 12 hours.

21. The method of any one of claims 17-20, wherein the contacting step is between about 6 hours and about 12 hours.

22. The method of any one of the preceding claims, wherein cell viability is at least 60% post-transfection.

23. The method of claim 22, wherein cell viability is at least 70% post-transfection.

24. The method of claim 22, wherein cell viability is at least 80% post-transfection.

25. The method of claim 22, wherein cell viability is at least 90% post-transfection.

26. The method of claim 22, wherein cell viability is at least 95% post-transfection.

27. The method of any one of the preceding claims, further comprising pre-incubating the serum factor with the LNP composition for about 30 seconds to overnight.

28. The method of claim 27, comprising pre-incubation for about 1 minute to 1 hour.

29. The method of claim 27, comprising pre-incubation for about 1-30 minutes.

30. The method of claim 27, comprising pre-incubation for about 1-10 minutes.

31. The method of claim 27, comprising pre-incubation for about 5 minutes.

32. The method of claim 27 or claim 31, comprising pre-incubation for 5 minutes ± 2 minutes.

33. The method of any one of the preceding claims, wherein the pre-incubation occurs at about 4 ℃.

34. The method of any one of the preceding claims, wherein the pre-incubation occurs at about 25 ℃.

35. The method of any one of the preceding claims, wherein the pre-incubation occurs at about 37 ℃.

36. The method of any one of the preceding claims, wherein the pre-incubation step comprises a buffer.

37. The method of claim 36, wherein the buffer comprises or consists of a HSPC medium.

38. The method of any one of the preceding claims, wherein the LNP composition is pre-incubated with serum.

39. The method of claim 38, wherein the serum is mammalian, mouse, primate, or human serum.

40. The method of any one of claims 1-37, wherein the LNP composition is pre-incubated with isolated serum factors.

41. The method of claim 40, wherein the serum factor is ApoE.

42. The method of claim 40, wherein the serum factor is selected from ApoE2, ApoE3 and ApoE 4.

43. The method of any one of claims 40 to 42, wherein the ApoE is a recombinant human protein.

44. The method of any one of the preceding claims, wherein the culturing step comprises expanding the stem cell, the HSPCs, or the HSPC population in a HSPC culture buffer.

45. The method of any one of the preceding claims, further comprising replacing the culture medium between the contacting step and the culturing step.

46. The method of any one of the preceding claims, wherein the culturing step comprises a stem cell expansion agent.

47. The method of any one of claims 1-2, 4-6, or 8-46, wherein the HSPC is a Hematopoietic Stem Cell (HSC).

48. The method of any one of the preceding claims, wherein the stem cell, the HSPC or the HSPC population is a human cell or sample.

49. The method of any one of claims 5-48, wherein the mRNA and the guide RNA nucleic acid are formulated in a single LNP composition.

50. The method of any one of claims 5-48, wherein the mRNA and the gRNA are co-encapsulated in the LNP composition.

51. The method of any one of claims 5-48, wherein the mRNA and the gRNA are encapsulated separately in LNP.

52. The method of any one of claims 5-48, wherein the mRNA is formulated in a first LNP composition and the guide RNA nucleic acid is formulated in a second LNP composition.

53. The method of claim 52, wherein the first LNP composition and the second LNP composition are administered simultaneously.

54. The method of claim 52, wherein the first LNP composition and the second LNP composition are administered sequentially.

55. The method of any one of claims 52-54, wherein the first LNP composition and the second LNP composition are combined prior to the pre-incubation step.

56. The method of any one of claims 52-54, wherein the first LNP composition and the second LNP composition are pre-incubated separately.

57. The method of any one of the preceding claims, further comprising introducing a template nucleic acid into the cell.

58. The method of any one of the preceding claims, wherein the LNP composition comprises: an RNA component and a lipid component, wherein the lipid component comprises an amine lipid, a neutral lipid, a helper lipid, and a stealth lipid; and wherein the N/P ratio is from about 1 to about 10.

59. The method of claim 58, wherein the lipid component comprises lipid A or an acetal analog thereof.

60. The method of claim 58, wherein the lipid component comprises:

about 40-60 mol% amine lipid;

about 5-15 mol% neutral lipid; and

about 1.5-10 mol% PEG lipid,

wherein the remainder of the lipid component is a helper lipid, and

wherein the N/P ratio of the LNP composition is about 3-10.

61. The method of claim 58, wherein the lipid component comprises:

about 50-60 mol% amine lipid;

about 8-10 mol% neutral lipid; and

about 2.5-4 mol% PEG lipid,

wherein the remainder of the lipid component is a helper lipid, and

wherein the N/P ratio of the LNP composition is about 3-8.

62. The method of claim 56, wherein the lipid component comprises:

about 50-60 mol% amine lipid;

about 5 to 15 mol% DSPC; and

about 2.5-4 mol% PEG lipid,

wherein the remainder of the lipid component is cholesterol, and

wherein the N/P ratio of the LNP composition is about 3-8.

63. The method of claim 58, wherein the lipid component comprises:

48-53 mol% lipid A;

about 8 to 10 mol% DSPC; and

1.5-10 mol% PEG lipid,

wherein the remainder of the lipid component is cholesterol, and

wherein said N/P ratio of said LNP composition is from 3 to 8 + -0.2.

64. The method of any one of the preceding claims, wherein the RNA is a modified RNA.

65. The method of claim 64, wherein the modified RNA is a modified mRNA.

66. The method of any one of the preceding claims, wherein said RNA comprises an open reading frame encoding an RNA-guided DNA binding agent, wherein the uridine content of said open reading frame is in the range of its minimum uridine content to 150% of said minimum uridine content.

67. The composition of any one of the preceding claims, wherein said RNA comprises an open reading frame comprising a polynucleotide encoding an RNA-directed DNA binding agent, wherein said open reading frame has a uridine dinucleotide content in the range of its minimum uridine dinucleotide content to 150% of said minimum uridine dinucleotide content.

68. The composition of any one of the preceding claims, wherein the RNA comprises a sequence at least 90% identical to any one of SEQ ID NOs 1, 4, 10, 14, 15, 17, 18, 20, 21, 23, 24, 26, 27, 29, 30, 50, 52, 54, 65, or 66, wherein the mRNA comprises an open reading frame encoding an RNA-guided DNA binding agent.

69. The method of any one of claims 5-68, wherein the gRNA is a modified gRNA.

70. The method of claim 69, wherein the gRNA comprises a modification selected from: 2 '-O-methyl (2' -O-Me) -modified nucleotides, Phosphorothioate (PS) linkages between nucleotides; and 2 '-fluoro (2' -F) -modified nucleotides.

71. The method of claim 69 or 70, wherein the gRNA comprises a modification at one or more of the first five nucleotides of the 5' end.

72. The method of any one of claims 69-71, wherein the gRNA comprises a modification at one or more of the last five nucleotides of the 3' end.

73. The method of any one of claims 69-72, wherein the gRNA includes a PS bond between the first four nucleotides.

74. The method of any one of claims 69-73, wherein the gRNA includes a PS bond between the last four nucleotides.

75. The method of any one of claims 69-74, further comprising a 2'-O-Me modified nucleotide at the first three nucleotides of the 5' end.

76. The method of any one of claims 69-75, further comprising a 2'-O-Me modified nucleotide at the last three nucleotides of the 3' end.

77. The method of claims 1-2, 4-6 or 8-76, wherein the HSPCs or the HSPC population are CD34 +.

78. The method of claims 1-2, 4-6 or 8-77, wherein the HSPCs or the population of HSPCs are CD34+ CD90 +.

79. An engineered stem cell or stem cell population produced by the method of any one of the preceding claims.

80. An engineered HSPC or HSPC population produced by the method of any one of the preceding claims.

81. A HSPC or HSPC population of claim 78 wherein the engineered HSPC resides in a tissue or organ, such as bone marrow, blood or other tissue, in a patient, e.g., after transplantation of the engineered HSPC.

82. The method of any one of the preceding claims, wherein the stem cells, the HSPCs, or the HSPC population are autologous with respect to the patient to whom the cells are administered.

83. The method of any one of the preceding claims, wherein the stem cells, the HSPCs, or the HSPC population are allogeneic with respect to the patient to whom the cells are to be administered.

84. The method of any one of the preceding claims, further comprising effecting CRISPR-Cas gene editing in the stem cell, the HSPC or the population of HSPCs.

85. The method of any one of the preceding claims, further comprising detecting gene editing in the stem cell, the HSPC, or the HSPC population.

86. The method of claim 84 or 85, wherein the gene editing is measured as a percentage of editing or a percentage of DNA modification.

87. The method of claim 86, wherein the edit percentage is at least 40%.

88. The method of claim 86, wherein the edit percentage is at least 60%.

89. The method of claim 86, wherein the edit percentage is at least 70%.

90. The method of claim 86, wherein the edit percentage is at least 80%.

91. The method of claim 86, wherein the edit percentage is at least 90%.

92. The method of claim 86, wherein the edit percentage is at least 95%.

93. The method of claim 86, wherein the percentage of DNA modification is at least 40%.

94. The method of claim 86, wherein the percentage of DNA modification is at least 60%.

95. The method of claim 86, wherein the percentage of DNA modification is at least 70%.

96. The method of claim 86, wherein the percentage of DNA modification is at least 80%.

97. The method of claim 86, wherein the percentage of DNA modification is at least 90%.

98. The method of claim 86, wherein the percentage of DNA modification is at least 95%.

99. The method of any one of the preceding claims, wherein the stem cell, the HSPC or the HSPC population are from a bone marrow sample.