Genetic modification of hepatocytes

Abstract

The invention provides a method for producing genetically modified human hepatocytes suitable for hepatocyte transplantation, the method comprising disrupting one or more major histocompatibility complex (MHC) class I or class II genes in isolated human hepatocytes or hepatocyte progenitor cells by introducing a base editor and one or more gRNAs that hybridize to target sequences in one or more major histocompatibility complex (MHC) class I or class II genes, thereby producing genetically modified human hepatocytes.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Patent Application No. 63 / 176,104, filed April 16, 2021, which is incorporated by reference in its entirety for all purposes. [Background technology]

[0002] Orthotopic liver transplantation (OLT) is the gold standard treatment for end-stage liver disease, acute liver failure, and liver-based metabolic disorders. OLT has several major drawbacks, including a shortage of donor organs, the risk of surgery-related complications, the high cost of the procedure, and the need for lifelong immunosuppression.

[0003] Hepatocyte transplantation (HT) is a highly attractive and clinically safe alternative to OLT, as it is less invasive, less expensive, and can be performed repeatedly if necessary. The limitations of HT are related to the limited supply of high-quality hepatocytes and poor engraftment / long-term acceptance of the allograft. Encouraging clinical improvements have been seen in patients transplanted with allogeneic hepatocytes, but long-term efficacy remains hampered by limited long-term acceptance of the allogeneic cell graft, despite immunosuppression. Summary of the Invention

[0004] Human primary hepatocytes are highly immunogenic, and therefore alternative strategies for their immunomodulation prior to transplantation are desirable to improve hepatocyte engraftment. There are currently several obstacles to using hepatocytes to treat liver disease. These are generally: 1) a limited supply of human hepatocytes, and 2) poor engraftment of hepatocytes into subjects. The limited supply of high-quality hepatocytes is due, at least in part, to a limited supply of donor livers from which high-quality hepatocytes can be isolated. The creation and use of humanized animal models that function as hepatocyte bioreactors has enabled the procurement and expansion of human hepatocytes for program-scale development. The second obstacle referenced above is poor engraftment, which has thus far limited the long-term acceptance of allogeneic cell grafts, despite immunosuppression. The inventors have surprisingly discovered a unique methodology for genetically modifying hepatocytes that renders the genetically modified hepatocytes suitable for administration to subjects in need thereof.

[0005] In some aspects, a method of producing genetically modified human hepatocytes suitable for hepatocyte transplantation is provided, the method comprising disrupting one or more major histocompatibility complex (MHC) class I or class II genes in isolated human hepatocytes or hepatocyte progenitor cells by introducing a base editor and one or more gRNAs that hybridize to a target sequence in one or more class I or class II genes, thereby producing genetically modified human hepatocytes. In some embodiments, disrupting one or more MHC class I or class II genes occurs in isolated human hepatocytes. The isolated human hepatocytes can be freshly isolated or previously expanded. MHC class I and class II genes are known in the art. For example, MHC class I genes include HLA-A, HLA-B, HLA-C, HLA-E, HLA-F, HLA-G, HLA-K, and HLA-L. For example, MHC class II genes include HLA-DP, HLA-DM, HLA-DOA, HLA-DOB, HLA-DQ, HLA-DR.

[0006] In some embodiments, the base editor comprises a CRISPR protein fused to a deaminase.

[0007] In some embodiments, the genetically modified human hepatocytes have one or more nucleobase edits in the target sequence. For example, the genetic modification can have 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 10 nucleobase edits.

[0008] In some embodiments, the genetically modified human hepatocytes have a disrupted target sequence. In some embodiments, the disrupted target sequence results in decreased expression of the target gene. In some embodiments, the disrupted target sequence results in increased expression of the target gene.

[0009] In some embodiments, the genetically modified human hepatocytes have reduced or abolished alloreactivity. Thus, in some embodiments, the genetically modified human hepatocytes have reduced alloreactivity. In some embodiments, the genetically modified human hepatocytes have abolished alloreactivity. By "abolished" it is meant that there is no detectable alloreactivity using methods known in the art.

[0010] In some embodiments, the class I or class II gene is selected from one or more of B2M, CD142, CIITA, HLA-A, or HLA-B genes. Thus, in some embodiments, the class I or class II gene is B2M. In some embodiments, the class I or class II gene is CD142. In some embodiments, the class I or class II gene is CIITA. In some embodiments, the class I or class II gene is HLA-A. In some embodiments, the class I or class II gene is HLA-B.

[0011] In some embodiments, a stop codon or splice site is introduced into one or more of the B2M, CD142, CIITA, HLA-A, or HLA-B genes. Thus, in some embodiments, a stop codon is introduced into one or more of the B2M, CD142, CIITA, HLA-A, or HLA-B genes. In some embodiments, a splice site is introduced into one or more of the B2M, CD142, CIITA, HLA-A, or HLA-B genes.

[0012] In some embodiments, the splice site is introduced at nucleotide position 19 of the B2M gene.

[0013] In some embodiments, a stop codon is introduced at nucleotide position 5 of the B2M gene.

[0014] In some embodiments, the splice site is introduced at nucleotide position 28 of the CD142 gene.

[0015] In some embodiments, a stop codon is introduced at nucleotide position 19 of the CD142 gene.

[0016] In some embodiments, a splice site is introduced at nucleotide position 147 of the CIITA gene.

[0017] In some embodiments, a stop codon is introduced at nucleotide position 130 of the CIITA gene.

[0018] In some embodiments, the CRISPR protein is Cas9 or Cas12. In some embodiments, the CRISPR protein is a Cas9 protein. In some embodiments, the CRISPR protein is a Cas12 protein.

[0019] In some embodiments, Cas9 is Streptococcus pyogenes (SpCas9) or Staphylococcus aureus (SaCas9). Thus, in some embodiments, Cas9 is from Streptococcus pyogenes (SpCas9). In some embodiments, Cas9 is from Staphylococcus aureus (SaCas9). Various Cas9 proteins have been described in the art and are obtained or modified from various bacteria, including Cas9 with mutations. Cas9 and its mutants have been described in various publications, including, for example, WO2013 / 176772, US10,266,850, WO2014 / 093661, WO2014 / 093655, WO2014 / 093595, the contents of which are incorporated herein by reference.

[0020] Various Cas12 proteins are known in the art, including, for example, class 2 type V and type VI proteins. For example, class 2 type V Cas12 includes, among others, Cas12a, Cas12b, Cas12c. Various names for Cas12 have been used, including Cpf1, C2c1, C2c1p, C2c3, C2cp3, C2c2p. Some embodiments of the methods disclosed herein use Cas12 proteins from class 2 type V or type VI proteins. For example, in some embodiments, a Cas12 suitable for the methods described herein comprises a Cas12a protein. In some embodiments, a Cas12 suitable for the methods described herein comprises a Cas12b protein. In some embodiments, a Cas12 suitable for the methods described herein comprises a Cas12c protein. In some embodiments, a Cas12 suitable for the methods described herein comprises a Cpf1 protein. In some embodiments, a Cas12 suitable for the methods described herein comprises a C2c1 protein. In some embodiments, a Cas12 suitable for the methods described herein comprises a C2c1p protein. In some embodiments, a Cas12 suitable for the methods described herein comprises a C2c3 protein. In some embodiments, a Cas12 suitable for the methods described herein comprises a C2cp3 protein. In some embodiments, a Cas12 suitable for the methods described herein comprises a C2c2p protein. Various Cas12s are described in WO / 2016 / 205711 and WO / 2016 / 205749, the contents of which are incorporated by reference.

[0021] In some embodiments, the Cas9 protein is a super-precision Cas9. In some embodiments, the Cas9 protein comprises mutations corresponding to N692A, M694A, Q695A, and / or H698A with reference to SpyCas9 (SEQ ID NO: 68). In some embodiments, the Cas9 protein is a high-fidelity Cas9. In some embodiments, the Cas9 protein comprises mutations corresponding to N467A, R661A, Q695A, and / or Q926A with reference to SpyCas9 (SEQ ID NO: 68). In some embodiments, the Cas9 protein is a SuperFi-Cas9. In some embodiments, the Cas9 protein comprises mutations where the Y1016, R1019, Y1010, Y1013, K1031, Q1027, and / or V1018 residues corresponding to SpyCas9 (SEQ ID NO: 68) are mutated to aspartic acid.

[0022] In some embodiments, the CRISPR protein is fused to an adenine or adenosine base editor (ABE), a cytidine or cytosine base editor (CBE), or an inosine base editor (IBE). Thus, in some embodiments, the CRISPR protein is fused to an ABE. In some embodiments, the CRISPR protein is fused to a CBE. In some embodiments, the CRISPR protein is fused to an IBE. In some embodiments, the CRISPR is fused to a base editor that includes an adenine or adenosine deaminase domain, or a cytidine or cytosine deaminase domain. In some embodiments, the CRISPR is fused to a base editor that includes an adenine or adenosine deaminase domain and a cytidine or cytosine deaminase domain.

[0023] In some embodiments, the CRISPR protein comprises a nuclear localization sequence (NLS) and / or a FLAG, HIS, or HA tag. Thus, in some embodiments, the CRISPR protein comprises an NLS. In some embodiments, the CRISPR protein comprises a FLAG tag. In some embodiments, the CRISPR protein comprises a HIS tag. In some embodiments, the CRISPR protein comprises an HA tag.

[0024] In some embodiments, the CRISPR protein comprises at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, or at least 10 mutations in SEQ ID NO:1 (SpCas9), SEQ ID NO:2 (SaCas9), or SEQ ID NO:3 (Cpf1 Cas12). The amino acid sequences of SpCas9, SaCas9, and Cpf1 Cas12 are shown in the table below.

[0025] [Table 1-1]

[0026] [Table 1-2]

[0027] [Table 1-3]

[0028] [Table 1-4]

[0029] In some embodiments, the mutation is an amino acid substitution.

[0030] In some embodiments, at least one mutation results in an inactive Cas9 (dCas9).

[0031] In some embodiments, the at least one mutation is one or more amino acid substitutions in the PAM interaction domain, the RuvC domain, and / or the HNH domain of Cas9. Thus, in some embodiments, the at least one mutation is one or more amino acid substitutions in the PAM interaction domain. In some embodiments, the at least one mutation is one or more amino acid substitutions in the RuvC domain. In some embodiments, the at least one mutation is one or more amino acid substitutions in the HNH domain. In some embodiments, the at least one or more amino acid substitutions occur in the PAM interaction domain, the RuvC domain, and the HNH domain. In some embodiments, the at least one or more amino acid substitutions occur in the PAM interaction domain and the RuvC domain. In some embodiments, the at least one or more amino acid substitutions occur in the PAM interaction domain and the HNH domain. In some embodiments, the at least one or more amino acid substitutions occur in the RuvC domain and the HNH domain.

[0032] In some embodiments, the at least one mutation is an aspartic acid to alanine substitution at amino acid 10 of SpCas9 (D10A), or the corresponding mutation in the Cas9 protein.

[0033] In some embodiments, the at least one mutation is a histidine to alanine substitution at amino acid 840 of SpCas9 (H840A), or the corresponding mutation in the Cas9 protein.

[0034] In some embodiments, the Cas9 protein has nickase activity, hi some embodiments, one or more mutations in the Cas9 protein render the Cas9 catalytically inactive, otherwise referred to as "inactive Cas9" or "dCas9."

[0035] In some embodiments, the CRISPR protein is fused to adenosine deaminase and has an amino acid sequence at least 80% identical to SEQ ID NO:65.

[0036] In some embodiments, the CRISPR protein is fused to a cytosine deaminase and has an amino acid sequence at least 80% identical to SEQ ID NOs: 4-64.

[0037] In some embodiments, the SpCas9 protein recognizes a PAM sequence that includes 5'-NGG-3', 5'-NGA-3', or 5'-NGC-3'. In some embodiments, the SpCas9 protein recognizes a PAM sequence that includes 5'-NGG-3'. In some embodiments, the SpCas9 protein recognizes a PAM sequence that includes 5'-NGA-3'. In some embodiments, the SpCas9 protein recognizes a PAM sequence that includes 5'-NGC-3'.

[0038] In some embodiments, the SaCas9 protein recognizes a PAM sequence that includes 5'-NNNRRT-3' or 5'-NNGRRT-3'. In some embodiments, the SaCas9 protein recognizes a PAM sequence that includes 5'-NNNRRT-3'. In some embodiments, the SaCas9 protein recognizes a PAM sequence that includes 5'-NNGRRT-3'.

[0039] In some embodiments, the Cas12 protein recognizes a PAM sequence comprising 5'-RTTN-3'.

[0040] In some embodiments, the isolated human hepatocytes have been previously cryopreserved and subsequently thawed. In some embodiments, the isolated human hepatocytes are primary cultures. In some embodiments, the isolated human hepatocytes are freshly isolated.

[0041] In some embodiments, the genetically modified human hepatocytes overexpress CIITA compared to non-genetically modified human hepatocytes. In some embodiments, the genetically modified human hepatocytes overexpress B2M. In some embodiments, the genetically modified human hepatocytes overexpress B2M-HLA-E fusion protein. In some embodiments, the genetically modified human hepatocytes overexpress PDL1. In some embodiments, the genetically modified human hepatocytes overexpress PDL2.

[0042] In some embodiments, the genetically modified human hepatocytes are transplanted into a humanized animal model for expansion.

[0043] In some embodiments, the humanized animal model is an FRG pig, an FRG mouse, or an FRG rat. Thus, in some embodiments, the humanized animal model is an FRG pig. In some embodiments, the humanized animal model is an FRG mouse. In some embodiments, the humanized animal model is an FRG rat.

[0044] In some embodiments, the genetically modified human hepatocytes are first transplanted into FRG mice or FRG rats for initial cell expansion. In some embodiments, the genetically modified human hepatocytes are transplanted into FRG mice for initial expansion. In some embodiments, the genetically modified human hepatocytes are transplanted into FRG rats for initial expansion.

[0045] In some embodiments, after initial cell expansion, the genetically modified cells are then implanted into FRG pigs for further cell expansion.

[0046] In some embodiments, the initially expanded cells or the further expanded cells are isolated from the animal.

[0047] In some embodiments, the initially expanded cells or the further expanded cells are isolated by fluorescence activated cell sorting, immunomagnetic cell separation, density gradient centrifugation, and / or immunodensity cell separation. Any type of isolation strategy that maintains cell viability can be used in the methods herein. In some embodiments, the cells are isolated by fluorescence activated cell sorting. In some embodiments, the cells are isolated by immunomagnetic cell separation. In some embodiments, the cells are isolated by density gradient centrifugation. In some embodiments, the cells are isolated by immunodensity cell separation.

[0048] In some embodiments, the genetically modified human hepatocytes have one, two, three, or more nucleobase edits. Thus, in some embodiments, the genetically modified human hepatocytes have one nucleobase edit. In some embodiments, the genetically modified human hepatocytes have two nucleobase edits. In some embodiments, the genetically modified human hepatocytes have three nucleobase edits. In some embodiments, the genetically modified human hepatocytes have four nucleobase edits. In some embodiments, the genetically modified human hepatocytes have five nucleobase edits. In some embodiments, the genetically modified human hepatocytes have six nucleobase edits. In some embodiments, the genetically modified human hepatocytes have seven nucleobase edits. In some embodiments, the genetically modified human hepatocytes have eight nucleobase edits. In some embodiments, the genetically modified human hepatocytes have nine nucleobase edits. In some embodiments, the genetically modified human hepatocytes have ten nucleobase edits. In some embodiments, the genetically modified human hepatocytes have more than ten nucleobase edits.

[0049] In some embodiments, a single base editor used in combination with more than one guide generates two, three, or more nucleobase edits. Thus, in some embodiments, a single base editor used in combination with more than one guide generates two nucleobase edits. In some embodiments, a single base editor used in combination with more than one guide generates three nucleobase edits. In some embodiments, a single base editor used in combination with more than one guide generates more than three nucleobase edits. Thus, this method allows for multiplexing of nucleobase edits.

[0050] In some embodiments, the more than one base editor generates one, two, three, or more nucleobase edits.

[0051] In some embodiments, a nucleic acid encoding a base editor and one or more gRNAs that hybridize to a target sequence as described herein are provided.

[0052] In some embodiments, the nucleic acid is codon optimized for expression in a mammalian cell.

[0053] In some embodiments, the nucleic acid is codon optimized for expression in a human cell.

[0054] In some aspects, vectors encoding the nucleic acids described herein are provided.

[0055] In some embodiments, a eukaryotic cell is provided that comprises a base editor and one or more gRNAs that hybridize to a target sequence described herein.

[0056] In some embodiments, the cell is a mammalian cell. In some embodiments, the cell is a human cell. In some embodiments, the cell is a lymphocyte.

[0057] In some aspects, a method of treating liver disease is provided, the method comprising administering to a subject in need thereof genetically modified human hepatocytes produced according to the methods described herein.

[0058] In some embodiments, the genetically modified human hepatocytes are injected into the portal vein of a subject in need thereof.

[0059] In some embodiments, about 10 to 15 billion genetically modified human hepatocytes are administered to a subject in need thereof. In some embodiments, the genetically modified human hepatocytes are injected into the portal vein of a subject in need thereof. In some embodiments, about 5 to 20 billion genetically modified human hepatocytes are injected into the portal vein of a subject in need thereof. In some embodiments, about 10 to 12 billion genetically modified human hepatocytes are injected into the portal vein of a subject in need thereof. In some embodiments, about 12 to 15 billion genetically modified human hepatocytes are injected into the portal vein of a subject in need thereof.

[0060] In some embodiments, a base editor and one or more guide RNAs targeting the B2M gene are provided, wherein the base editor and the corresponding one or more guide RNAs are selected from Table 2.

[0061] In some aspects, a base editor and one or more guide RNAs targeting the CD142 gene are provided, wherein the base editor and corresponding one or more guide RNAs are selected from Table 3.

[0062] In some embodiments, a base editor and one or more guide RNAs targeting the CIITA gene are provided, wherein the base editor and the corresponding one or more guide RNAs are selected from Table 4.

[0063] In some embodiments, a base editor and one or more guide RNAs targeting an HLA-A gene, wherein the base editor and corresponding one or more guide RNAs are selected from Table 5.

[0064] In some embodiments, a base editor and one or more guide RNAs targeting an HLA-B gene, wherein the base editor and corresponding one or more guide RNAs are selected from Table 6.

[0065] In some embodiments, a base editor and one or more guide RNAs are provided to make one, two, three, or more than three edits to a target gene.

[0066] In some embodiments, the guide RNA sequence comprises 1-4 mismatches to the guide targeting sequence. In some embodiments, the guide RNA sequence comprises 1-4 mismatches corresponding to any one of the sequences listed in Tables 2A-6A or the RNA version of any one of the protospacer sequences listed in Tables 2-6.

[0067] In some aspects, a cell is provided comprising a base editor and one or more guide RNAs.

[0068] In some aspects, genetically modified human hepatocytes having one or more edits in an MHC gene are provided as described herein.

[0069] In some embodiments, the MHC gene is selected from B2M, CD142, CIITA, HLA-A, and / or HLA-B. Thus, in some embodiments, the MHC gene is a B2M gene. In some embodiments, the MHC gene is a CD142 gene. In some embodiments, the MHC gene is a CIITA gene. In some embodiments, the MHC gene is an HLA-A gene. In some embodiments, the MHC gene is an HLA-B gene.

[0070] In some embodiments, edits to one or more of the B2M, CD142, CIITA, HLA-A, and / or HLA-B genes result in increased expression of the B2M, CD142, CIITA, HLA-A, and / or HLA-B genes compared to non-genetically modified human hepatocytes. For example, in some embodiments, edits to the B2M gene result in increased expression of the B2M gene. In some embodiments, edits to the CD142 gene result in increased expression of the CD142 gene. In some embodiments, edits to the CIITA gene result in increased expression of the CIITA gene. In some embodiments, edits to the HLA-A gene result in increased expression of the HLA-A gene. In some embodiments, edits to the HLA-B gene result in increased expression of the HLA-B gene.

[0071] definition In order that the present invention may be more readily understood, certain terms are first defined below. Further definitions for these terms, as well as other terms, are set forth throughout the specification.

[0072] a or an: The articles "a" and "an" are used herein to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. By way of example, "an element" means one element or more than one element.

[0073] Approximately or about: As used herein, the term "approximately" or "about" when applied to one or more values ​​of interest refers to a value similar to a stated reference value. In certain embodiments, the term "approximately" or "about" refers to a range of values ​​that falls within 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1% or less in either direction (greater or less) of the stated reference value (except where such number exceeds 100% of possible values), unless otherwise stated or clear from the context.

[0074] Associated: As the term is used herein, two events or entities are "associated" with each other when the presence, level and / or form of one correlates with the presence, level and / or form of the other. For example, a particular entity (e.g., a polypeptide) is considered to be associated with a particular disease, disorder or condition when its presence, level and / or form correlates with the incidence and / or susceptibility of the disease, disorder or condition (e.g., across a relevant population). In some embodiments, two or more entities are physically "associated" with each other such that they are in and maintain physical proximity to each other when they directly or indirectly interact with each other. In some embodiments, two or more entities that are physically associated with each other are covalently bound to each other, and in some embodiments, two or more entities that are physically associated with each other are not covalently bound to each other, but are non-covalently associated, for example, by hydrogen bonds, van der Waals interactions, hydrophobic interactions, magnetism, and combinations thereof.

[0075] Base Editor "Base Editor (BE)" or "Nucleic Acid Base Editor (NBE)" refers to an agent that binds to a polynucleotide and has nucleobase modifying activity. In various embodiments, the base editor comprises a nucleobase modifying polypeptide (e.g., a deaminase) and a polynucleotide programmable nucleotide binding domain in conjunction with a guide polynucleotide (e.g., a guide RNA). In various embodiments, the agent is a biomolecular complex that comprises a protein domain with base editing activity, i.e., a domain that can modify a base (e.g., A, T, C, G, or U) in a nucleic acid molecule (e.g., DNA). In some embodiments, the polynucleotide programmable DNA binding domain is fused or linked to a deaminase domain. In one embodiment, the agent is a fusion protein that comprises one or more domains with base editing activity. In another embodiment, the protein domain with base editing activity is linked to a guide RNA (e.g., via an RNA binding motif on the guide RNA and an RNA binding domain fused to a deaminase). In some embodiments, the protein domain with base editing activity is capable of deaminating a base in a nucleic acid molecule. In some embodiments, the base editor is capable of deaminating one or more bases in a DNA molecule. In some embodiments, the base editor is capable of deaminating cytosine (C) or adenosine (A) in DNA. In some embodiments, the base editor is capable of deaminating cytosine (C) and adenosine (A) in DNA. In some embodiments, the base editor is a cytidine base editor (CBE). In some embodiments, the base editor is a cytidine base editor (CBE). In some embodiments, the base editor is an adenosine base editor (ABE). In some embodiments, the base editor is an adenosine base editor (ABE). In some embodiments, the base editor is an adenosine base editor (ABE) and a cytidine base editor (CBE). In some embodiments, the base editor is a nuclease-inactive Cas9 (dCas9) fused to an adenosine deaminase.In some embodiments, the base editor is fused to an inhibitor of base excision repair (e.g., a UGI domain, or a dISN domain). In some embodiments, the fusion protein comprises a Cas9 nickase fused to a deaminase and an inhibitor of base excision repair (e.g., a UGI domain or a dISN domain). In other embodiments, the base editor is an abasic base editor. Details of base editors are described in International PCT Application Nos. 2017 / 045381 (WO2018 / 027078) and PCT / US2016 / 058344 (WO2017 / 070632), each of which is incorporated by reference herein in its entirety. Also, Komor, AC, et al., “Programmable editing of a target base in genomic DNA without double-stranded DNA cleavage” Nature 533, 420-424 (2016), Gaudelli, NM, et al., “Programmable base editing of A·T to G·C in genomic DNA without DNA cleavage” Nature 551, 464-471 (2017), Komor, AC, et al., “Improved base excision repair inhibition and bacteriophage Mu Gam protein yields C:G-to-T:A base editors with higher efficiency and product purity” Science Advances 3:eaao4774 (2017), and Rees, HA, et al., “Base editing: precision chemistry on the genome and transcriptome of living cells.”Nat Rev Genet.2018 Dec;19(12):770-788. doi:10.1038 / s41576-018-0059-1, the entire contents of which are incorporated herein by reference.As used herein, the term "base editor" can also include CRISPR proteins, such as Cas9 or Cas12 proteins.

[0076] Base editing activity: "Base editing activity" refers to acting to chemically modify a base in a polynucleotide. In one embodiment, a first base is converted to a second base. In one embodiment, the base editing activity is a cytidine deaminase activity, e.g., converting a target C·G to T·A. In another embodiment, the base editing activity is an adenosine deaminase activity or an adenine deaminase activity, e.g., converting an A·T to G·C. In another embodiment, the base editing activity is a cytidine deaminase activity (e.g., converting a target C·G to T·A) and an adenosine deaminase activity or an adenine deaminase activity (e.g., converting an A·T to G·C).

[0077] Base editor system: The term "base editor system" refers to a system for editing nucleobases of a target nucleotide sequence. In various embodiments, the base editor (BE) system includes (1) a polynucleotide programmable nucleotide binding domain (e.g., Cas9 or Cas12), a deaminase domain, and a cytidine deaminase domain for deaminating nucleobases in a target nucleotide sequence, and (2) one or more guide polynucleotides (e.g., guide RNAs) in conjunction with the polynucleotide programmable nucleotide binding domain. In various embodiments, the base editor (BE) system includes a nucleobase editor domain selected from adenosine deaminase or cytidine deaminase, and a domain having nucleic acid sequence-specific binding activity. In some embodiments, the base editor system includes (1) a base editor (BE) including a polynucleotide programmable DNA binding domain and a deaminase domain for deaminating one or more nucleobases in a target nucleotide sequence, and (2) one or more guide RNAs in conjunction with the polynucleotide programmable DNA binding domain. In some embodiments, the polynucleotide programmable nucleotide binding domain is a polynucleotide programmable DNA binding domain. In some embodiments, the base editor is a cytidine base editor (CBE). In some embodiments, the base editor is an adenine or adenosine base editor (ABE). In some embodiments, the base editor is an adenine or adenosine base editor (ABE) or a cytidine base editor (CBE).

[0078] Biologically active: As used herein, the phrase "biologically active" refers to the characteristic of any agent that has activity in a biological system, particularly in an organism. For example, an agent that, when administered to an organism, has a biological effect on the organism is considered to be biologically active. In certain embodiments, if a peptide is biologically active, a portion of the peptide that shares at least one biological activity of the peptide is typically referred to as a "biologically active" portion.

[0079] Cleavage: As used herein, cleavage refers to the cut of the target nucleic acid made by the nuclease of the CRISPR system described herein. In some embodiments, the cleavage event is double-stranded DNA cut. In some embodiments, the cleavage event is single-stranded DNA cut. In some embodiments, the cleavage event is single-stranded RNA cut. In some embodiments, the cleavage event is double-stranded RNA cut.

[0080] Complementary: As used herein, complementary refers to a strand of nucleic acid that forms Watson-Crick base pairing or non-conventional base pairing with bases on a second nucleic acid strand, such that A bases pair with T and C bases pair with G. In other words, nucleic acids that hybridize to each other under appropriate conditions.

[0081] Clustered interspaced short palindromic repeats (CRISPR) associated (Cas) system: As used herein, CRISPR-Cas9 system refers to nucleic acids and / or proteins involved in the expression or directing the activity of CRISPR effectors, including sequences encoding CRISPR effectors, RNA guides, and other sequences and transcripts from the CRISPR locus. In some embodiments, the CRISPR system is an engineered non-naturally occurring CRISPR system. In some embodiments, the components of the CRISPR system may include nucleic acids (e.g., vectors) encoding one or more components of the system, components in protein form, or combinations thereof.

[0082] CRISPR array: The term "CRISPR array" as used herein refers to a nucleic acid (e.g., DNA) segment that includes CRISPR repeats and spacers, starting with the first nucleotide of the first CRISPR repeat and ending with the last nucleotide of the last (terminal) CRISPR repeat. Typically, each spacer in a CRISPR array is located between two repeats. The term "CRISPR repeat" or "CRISPR direct repeat" or "direct repeat" as used herein refers to multiple short direct repeat sequences that show little or no sequence variation in a CRISPR array.

[0083] CRISPR-associated protein (Cas): The term "CRISPR-associated protein", "CRISPR effector", "effector", or "CRISPR enzyme", as used herein, refers to a protein that performs an enzymatic activity or binds to a target site on a nucleic acid specified by an RNA guide. In various embodiments, the CRISPR effector has endonuclease activity, nickase activity, exonuclease activity, transposase activity, and / or excision activity. In some embodiments, the Cas is a high-precision Cas. In some embodiments, the Cas is a high-fidelity Cas. In some embodiments, the Cas is a SuperFi-Cas. In some embodiments, the high-precision, high-fidelity, and SuperFi-Cas are as described in Bravo, J. et al. Structural basis for mismatch surveillance by CRISPR-Cas9 Nature, 603, March 2022.

[0084] crRNA: The term "CRISPR RNA" or "crRNA" as used herein refers to an RNA molecule that includes a guide sequence that is used by a CRISPR effector to target a specific nucleic acid sequence. Typically, the crRNA contains a sequence that mediates target recognition and a sequence that forms a duplex with the tracrRNA. In some embodiments, the crRNA:tracrRNA duplex binds to a CRISPR effector.

[0085] Ex vivo: As used herein, the term "ex vivo" refers to events that take place in cells or tissues grown outside, rather than within, a multicellular organism.

[0086] Functional equivalent or analog: As used herein, the term "functional equivalent" or "functional analog" in the context of a functional derivative of an amino acid sequence refers to a molecule that retains substantially similar biological activity (either function or structure) as that of the original sequence. A functional derivative or equivalent may be a natural derivative or may be synthetically prepared. Exemplary functional derivatives include amino acid sequences having one or more amino acid substitutions, deletions, or additions, provided that the biological activity of the protein is preserved. The substituting amino acid desirably has similar chemical and physical properties as the amino acid that is substituted. Desirable similar chemical and physical properties include charge similarity, bulkiness, hydrophobicity, hydrophilicity, and the like.

[0087] Half-life: As used herein, the term "half-life" is the time required for a quantity, such as a protein concentration or activity, to fall to half of its value measured at the beginning of the period.

[0088] Improve, increase, or reduce: As used herein, the terms "improve," "increase," or "reduce," or grammatical equivalents, refer to a value relative to a baseline measurement, such as a measurement in the same individual prior to the initiation of a treatment described herein, or a measurement in a control subject (or control subjects) in the absence of a treatment described herein. A "control subject" is a subject suffering from the same form of disease as the subject being treated, and who is about the same age as the subject being treated.

[0089] Inhibition: As used herein, the terms "inhibit," "inhibit," and "inhibiting" refer to a process or method of decreasing or reducing the activity and / or expression of a protein or gene of interest. Typically, inhibiting a protein or gene refers to reducing the expression or associated activity of the protein or gene by at least 10% or more, e.g., 20%, 30%, 40%, or 50%, 60%, 70%, 80%, 90% or more, or a greater than 1-fold, 2-fold, 3-fold, 4-fold, 5-fold, 10-fold, 50-fold, 100-fold or more decrease in expression or associated activity as measured by one or more methods described herein or recognized in the art.

[0090] Hybridization: As used herein, the term "hybridization" refers to a reaction in which two or more nucleic acids bind to one another through hydrogen bonding by Watson-Crick pairing, Hoogsteen binding, or other sequence-specific bonds between the bases of the two nucleic acids. A sequence that can hybridize to another sequence is called the "complement" of the sequence and is said to be "complementary" or to exhibit "complementarity."

[0091] Indel: As used herein, the term "indel" refers to an insertion or deletion of a base in a nucleic acid sequence, which generally results in a mutation and is a common form of genetic variation.

[0092] In vitro: As used herein, the term "in vitro" refers to events that take place not in a multicellular organism but in an artificial environment, e.g., in a test tube or reaction vessel, in cell culture, etc.

[0093] In vivo: As used herein, the term "in vivo" refers to events that occur within multicellular organisms, such as humans and non-human animals. In the context of cell-based systems, the term can be used to refer to events that occur within living cells (as opposed to, for example, in vitro systems).

[0094] Mutation: As used herein, the term "mutation" has its ordinary meaning in the art and includes, for example, point mutations, substitutions, insertions, deletions, inversions, and deletions.

[0095] Oligonucleotide: As used herein, the term "oligonucleotide" generally refers to a polynucleotide of single- or double-stranded DNA of about 5 to about 100 nucleotides. Oligonucleotides are also known as "oligomers" or "oligos" and can be isolated from genes or chemically synthesized.

[0096] PAM: The term "PAM" or "protospacer adjacent motif" refers to a short nucleic acid sequence (usually 2-6 base pairs in length) that follows a nucleic acid region that is targeted for cleavage by a CRISPR system, such as CRISPR-Cas9. The PAM is required for a Cas nuclease to cleave and is generally found 3-4 nucleotides downstream from the cleavage site.

[0097] Polypeptide: The term "polypeptide" as used herein refers to a continuous chain of amino acids linked together via peptide bonds. Although the term is used to refer to an amino acid chain of any length, one of skill in the art will understand that the term is not limited to a long chain and can refer to a minimum chain that includes two amino acids linked together via a peptide bond. As known to those of skill in the art, polypeptides can be processed and / or modified. As used herein, the terms "polypeptide" and "peptide" are used interchangeably.

[0098] Prevent: As used herein, the terms "prevent" or "prevention," when used in relation to the occurrence of a disease, disorder, and / or condition, refer to reducing the risk of developing the disease, disorder, and / or condition.

[0099] Protein: The term "protein" as used herein refers to one or more polypeptides that function as separate units. When a single polypeptide is a separate functional unit and does not require permanent or temporary physical association with other polypeptides to form a separate functional unit, the terms "polypeptide" and "protein" may be used interchangeably. When a separate functional unit consists of more than one polypeptide that is physically associated with one another, the term "protein" refers to multiple polypeptides that are physically coupled and function together as a separate unit.

[0100] Reference: A "reference" entity, system, amount, set of conditions, etc., that is compared to a test entity, system, amount, set of conditions, etc., as described herein. For example, in some embodiments, a "reference" antibody is a control antibody that has not been engineered as described herein.

[0101] RNA guide: The term RNA guide refers to an RNA molecule that facilitates targeting of a protein to a target nucleic acid as described herein. Exemplary "RNA guide" or "guide RNA" include, but are not limited to, a crRNA or a crRNA in combination with a cognate tracrRNA. The latter may be an independent RNA or may be fused as a single RNA using a linker (sgRNA). In some embodiments, the RNA guide is engineered to include chemical or biochemical modifications, and in some embodiments, the RNA guide may include one or more nucleotides.

[0102] Subject: The term "subject," as used herein, means any subject for which diagnosis, prognosis, or therapy is desired. For example, the subject can be a mammal, such as a human or a non-human primate (such as an ape, monkey, orangutan, or chimpanzee), dog, cat, guinea pig, rabbit, rat, mouse, horse, cattle, or cow.

[0103] sgRNA: The term "sgRNA" or "single guide RNA" refers to a single guide RNA that contains (i) a guide sequence (crRNA sequence) and (ii) a Cas9 nuclease recruitment sequence (tracrRNA).

[0104] Substantial identity: The phrase "substantial identity" is used herein to refer to a comparison between amino acid or nucleic acid sequences. As will be understood by those skilled in the art, two sequences are generally considered to be "substantially identical" if they contain identical residues at corresponding positions. As is well known in the art, amino acid or nucleic acid sequences can be compared using any of a variety of algorithms, including those available in commercially available computer programs such as BLASTN for nucleotide sequences, and BLASTP, gapped BLAST, and PSI-BLAST for amino acid sequences. Exemplary such programs are described in Altschul, et al., Basic local alignment search tool, J. Mol. Biol., 215(3):403-410, 1990, Altschul, et al., Methods in Enzymology, Altschul et al., Nucleic Acids Res. 25:3389-3402, 1997, Baxevanis et al., Bioinformatics: A Practical Guide to the Analysis of Genes and Proteins, Wiley, 1998, and Misener, et al., (eds.), Bioinformatics Methods and Protocols (Methods in Molecular Biology, Vol. 132), Humana Press, 1999. In addition to identifying identical sequences, the above-mentioned programs typically provide an indication of the degree of identity. In some embodiments, two sequences are considered to be substantially identical if at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more of the corresponding residues are identical over the relevant stretch of residues. In some embodiments, the relevant stretch is the complete sequence.In some embodiments, the relevant stretch is at least 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500 or more residues.

[0105] Target nucleic acid: The term "target nucleic acid" as used herein refers to any length of nucleotide (oligonucleotide or polynucleotide), deoxyribonucleotide, ribonucleotide, or any of their analogs to which the CRISPR-Cas9 system binds. Target nucleic acids may have a three-dimensional structure that may include coding or non-coding regions, and may include exons, introns, mRNA, tRNA, rRNA, siRNA, shRNA, miRNA, ribozymes, cDNA, plasmids, vectors, exogenous sequences, endogenous sequences. Target nucleic acids may include modified nucleotides, methylated nucleotides, or nucleotide analogs. Target nucleic acids may be interspersed with non-nucleic acid components. Target nucleic acids are, but are not limited to, single-stranded, double-stranded, or multi-stranded DNA or RNA, genomic DNA, cDNA, DNA-RNA hybrids, or polymers that include purine and pyrimidine bases, or other natural, chemically or biochemically modified, non-natural, or derivatized nucleotide bases.

[0106] Therapeutically effective amount: As used herein, the term "therapeutically effective amount" refers to an amount of a therapeutic molecule (e.g., an engineered antibody described herein) that confers a therapeutic effect on a treated subject at a reasonable benefit / risk ratio applicable to any medical treatment. The therapeutic effect may be objective (i.e., measurable by some test or marker) or subjective (i.e., the subject gives an indication of or feels an effect). In particular, a "therapeutically effective amount" refers to an amount of a therapeutic molecule or composition that is effective to treat, ameliorate, or prevent a particular disease or condition, or that is effective to exhibit a detectable therapeutic or prophylactic effect, such as by improving symptoms associated with the disease, preventing or delaying the onset of the disease, and / or reducing the severity or frequency of symptoms of the disease. Therapeutically effective amounts can be administered in a dosing regimen that may include multiple unit doses. For any particular therapeutic molecule, the therapeutically effective amount (and / or the appropriate unit dose within an effective dosing regimen) may vary depending, for example, on the route of administration, combination with other drugs. In addition, the particular therapeutically effective amount (and / or unit dose) for any particular subject may depend on a variety of factors, including the disorder being treated and the severity of the disorder; the activity of the particular agent used; the particular composition used; the age, weight, general health, sex, and diet of the subject; the time of administration, route of administration, and / or excretion or metabolic rate of the particular therapeutic molecule used; the duration of treatment; and similar factors well known in the medical arts.

[0107] tracrRNA: The term "tracrRNA" or "transactivating crRNA" as used herein refers to an RNA that contains a sequence that forms the structure required for a CRISPR-associated protein to bind to a specified target nucleic acid.

[0108] Treatment: As used herein, the term "treatment" (also "treat" or "treating") refers to any administration of a therapeutic molecule (e.g., a CRISPR-Cas therapeutic protein or system described herein) that partially or completely alleviates, ameliorates, relieves, inhibits, delays the onset of, reduces the severity of, and / or reduces the incidence of, one or more symptoms or characteristics of a particular disease, disorder, and / or condition. Such treatment may be of subjects who do not show signs of the relevant disease, disorder, and / or condition and / or who show only early signs of the disease, disorder, and / or condition. Alternatively or additionally, such treatment may be of subjects who show one or more established signs of the relevant disease, disorder, and / or condition. [Brief description of the drawings]

[0109] [Figure 1A] FIG. 1 is a schematic diagram showing B2M gene BE4 and ABE compatible target sequences and associated PAM and protospacer region sites. [Figure 1B]

[0023] Figure 1 is a graph showing base editing of the B2M gene. Data from these studies show the editing efficiency of the B2M gene using either the ABE editor (ABE7.10) or the BE4 editor. [Figure 2A] FIG. 1 is a schematic diagram showing CIITA BE4 and ABE compatible target sequences and associated PAM and protospacer region sites. [Figure 2B] 1 is a graph showing base editing of the CIITA gene. Data from these studies show the editing efficiency of the CIITA gene using either the ABE editor (ABE8.2m) or the BE4 editor. [Figure 3A] Graph of base editing efficiency of an exemplary B2M target gene after 4 and 6 days of culture, post-editing response, and base editing efficiency of the CIITA gene on day 6. [Figure 3B] Flow cytometry data to analyze the protein level KO of the CIITA gene and evaluate the editing efficiency. [Figure 4]13 is a graph of base editing efficiency of B2M and CIITA target genes relative to HEK2 S2 control by flow cytometry. [Figure 5A] 1 is a table depicting exemplary reaction conditions and mRNA:gRNA ratios for nucleofection and transfection of exemplary base editors. [Figure 5B] Graph showing exemplary base editing efficiencies and cell viability at the BE4 locus at exemplary ratios of mRNA:sgRNA of 1:1, 2:1, 3:1, and 4:1. [Figure 6] Graph showing base editing efficiency and cell viability at the B2M locus. Bars represent editing efficiency at the B2M locus, dots represent the percentage of B2M-negative cells as assessed by flow cytometry, and "HEK2-2" indicates the targeted control locus. [Figure 7] Graph showing comparative gene editing efficiency at the B2M locus in combination with introduction of a transgene (Tg#1 or Tg#2) for integration in an ex vivo procedure. [Figure 8] 1 is a graph showing the efficiency of dual engineering involving base editing at the BE4 B2M locus by introduction of a transgene. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0110] Described herein is the production of genetically modified human hepatocytes suitable for use in the treatment of disease. Also described are suitable compositions comprising vectors, nucleic acids, and / or cells to achieve the genetically modified human hepatocytes. Additionally, various methods of using the genetically modified hepatocytes to treat subjects in need thereof are described.

[0111] Methods for Producing Genetically Modified Human Hepatocytes A method for producing genetically modified human hepatocytes suitable for hepatocyte transplantation is provided, the method comprising disrupting one or more major histocompatibility complex (MHC) class I or class II genes in isolated human hepatocytes or hepatocyte progenitor cells by introducing a base editor and one or more gRNAs that hybridize with target sequences in one or more MHC class I or class II genes, thereby producing genetically modified human hepatocytes. The genetically modified human hepatocytes can have one or more nucleic acid base edits that alter the expression of the corresponding MHC class I or class II genes. Alternatively, or complementary, the genetically modified human hepatocytes have reduced or suppressed expression of one or more MHC class I or class II genes. In this way, the genetically modified hepatocytes, when transplanted into a subject in need thereof, will not cause a rejection reaction that leads to selective death of the transplanted genetically modified hepatocytes. Thus, the genetically modified human hepatocytes have reduced or eliminated alloreactivity.

[0112] Any type or MHC class I or class II genes can be targeted to either reduce, eliminate, or suppress gene expression. For example, MHC class I genes include HLA-A, HLA-B, HLA-C, HLA-E, HLA-F, HLA-G, HLA-K, and HLA-L. For example, MHC class II genes include HLA-DP, HLA-DM, HLA-DOA, HLA-DOB, HLA-DQ, HLA-DR. In some embodiments, one or more MHC class I or class II genes are targeted to increase gene expression. In some embodiments, one or more MHC class I or class II genes are targeted to decrease gene expression. In some embodiments, the genetically modified human hepatocytes overexpress CD47 and / or CD142 compared to non-genetically modified human hepatocytes.

[0113] The isolated human hepatocytes can be obtained from any suitable donor. In some embodiments, the donor does not have liver disease. In some embodiments, the donor has liver disease. The method can be used with freshly isolated hepatocytes or hepatocytes that have been frozen and thawed. In some embodiments, the method uses hepatocytes obtained from progenitor cells or stem cells. For example, the progenitor cells or stem cells can be any suitable pluripotent cells, such as induced pluripotent cells (iPS cells) or embryonic stem (ES) cells.

[0114] In some embodiments, the methods comprise a base editor and one or more guide RNAs targeting a B2M gene, and a corresponding one or more guide RNAs are selected that comprise a base editor and an RNA version of any one of the protospacer sequences listed in Table 2.

[0115] In some embodiments, the methods comprise a base editor and one or more guide RNAs targeting the CD142 gene, wherein the base editor and corresponding one or more guide RNAs comprising an RNA version of any one of the protospacer sequences listed in Table 3 are selected.

[0116] In some embodiments, the methods comprise a base editor and one or more guide RNAs targeting the CIITA gene, wherein the base editor and corresponding one or more guide RNAs comprising an RNA version of any one of the protospacer sequences listed in Table 4 are selected.

[0117] In some embodiments, the methods comprise a base editor and one or more guide RNAs targeting an HLA-A gene, wherein a base editor and corresponding one or more guide RNAs comprising an RNA version of any one of the protospacer sequences listed in Table 5 are selected.

[0118] In some embodiments, the methods comprise a base editor and one or more guide RNAs targeting an HLA-B gene, wherein a base editor and corresponding one or more guide RNAs comprising an RNA version of any one of the protospacer sequences listed in Table 6 are selected.

[0119] In some embodiments, provided herein is a guide RNA comprising an RNA version of any one of the protospacer sequences listed in Table 2. In some embodiments, the guide RNA comprises an RNA version of any one of the protospacer sequences listed in Table 3. In some embodiments, the guide RNA comprises an RNA version of any one of the protospacer sequences listed in Table 4. In some embodiments, the guide RNA comprises an RNA version of any one of the protospacer sequences listed in Table 5. In some embodiments, the guide RNA comprises an RNA version of any one of the protospacer sequences listed in Table 6.

[0120] In some embodiments, provided herein is a guide RNA comprising any one of the sequences listed in Table 2A. In some embodiments, the guide RNA comprises any one of the sequences listed in Table 3A. In some embodiments, the guide RNA comprises any one of the sequences listed in Table 4A. In some embodiments, the guide RNA comprises any one of the sequences listed in Table 5A. In some embodiments, the guide RNA comprises any one of the sequences listed in Table 6A.

[0121] In some embodiments, the methods include a base editor and one or more guide RNAs targeting a B2M gene, wherein a base editor and corresponding one or more guide RNAs are selected that comprise any one of the protospacer sequences listed in Table 2A.

[0122] In some embodiments, the methods comprise a base editor and one or more guide RNAs targeting the CD142 gene, and the corresponding one or more guide RNAs are selected that comprise any one of the base editor and protospacer sequences listed in Table 3A.

[0123] In some embodiments, the methods comprise a base editor and one or more guide RNAs targeting the CIITA gene, wherein a base editor and corresponding one or more guide RNAs comprising any one of the protospacer sequences listed in Table 4A are selected.

[0124] In some embodiments, the methods comprise a base editor and one or more guide RNAs targeting an HLA-A gene, wherein a base editor and corresponding one or more guide RNAs are selected that comprise any one of the protospacer sequences listed in Table 5A.

[0125] In some embodiments, the methods comprise a base editor and one or more guide RNAs targeting an HLA-B gene, wherein a base editor and corresponding one or more guide RNAs are selected that comprise any one of the protospacer sequences listed in Table 6A.

[0126] A variety of base editors can be used in the methods for generating genetically modified human hepatocytes.

[0127] CRISPR proteins and base editors, including any one or more of an adenine base editor (ABE), a cytidine base editor (CBE), or an inosine base editor (IBE), are suitable for the methods described herein. In some embodiments, the methods described herein can be accomplished by using CRISPR proteins to achieve targeted inhibition of a gene of interest (e.g., one or more of the MHC class I or class II genes).

[0128] CRISPR proteins suitable for the methods described herein are described throughout, and include any Cas9 or Cas12 CRISPR protein. For example, Cas9 can be selected from any suitable bacterium, including the described Cas9 isolated from Streptococcus pyogenes (SpCas9) or Staphylococcus aureus (SaCas9). Cas12 CRISPR proteins suitable for the methods described herein include any class 2 V or VI Cas12 protein (including, for example, class 2 V Cas12). Among others, include Cas12a, Cas12b, Cas12c.

[0129] CRISPR protein suitable for the method described herein may have one or more mutations. One or more mutations may result in CRISPR protein being a nickase or catalytically inactive CRISPR protein. "Mutation" refers to any point mutation, substitution, deletion, inversion, or fusion, or any combination thereof. Fusion may occur anywhere in CRISPR protein, for example, at either N-terminus, C-terminus, or between N-terminus and C-terminus. To achieve a nickase or catalytically inactive CRISPR protein, one or more mutations may be made in either PAM interaction domain, RuvC domain, and / or HNH domain, or any combination thereof. Various mutations are described in the art, including, for example, those described in US9,790,490 (the contents of which are incorporated herein by reference).

[0130] In some embodiments, the Cas9 is a high-fidelity Cas9. In some embodiments, the high-fidelity Cas9 variant comprises enhanced specificity, which minimizes off-target cleavage. In some embodiments, the Cas9 is an ultra-high-fidelity Cas9. In some embodiments, engineered variants are used, such as "ultra-high-fidelity Cas9" (N692A, M694A, Q695A, and / or H698A mutations corresponding to SpyCas9) and / or "high-fidelity Cas9" (N467A, R661A, Q695A, and / or Q926A mutations corresponding to SpyCas9) that comprise mutations primarily within the REC3 domain to achieve higher specificity and fidelity. High-fidelity variants reduce the ability of Cas9 to stabilize mismatches and reduce off-target DNA cleavage. In some embodiments, the increase in specificity is accompanied by an approximately 100-fold loss in the efficiency of on-target cleavage. In some embodiments, SuperFi-Cas9 is used, a high-fidelity variant that maintains on-target cleavage rates comparable to wild-type Cas9. In some embodiments, SuperFi-Cas9 contains a mutation in the RuvC loop. In some embodiments, the mutation inhibits the formation of a kinked conformation that promotes subsequent cleavage of the gRNA-TS duplex. In some embodiments, the Y1016, R1019, Y1010, Y1013, K1031, Q1027, and / or V1018 residues corresponding to SpyCas9 are mutated, for example, to aspartic acid. (Bravo, J. et al. Structural basis for mismatch surveillance by CRISPR-Cas9 Nature, 603, March 2022).

[0131] In some embodiments, the CRISPR protein is fused with a deaminase, such as adenosine deaminase, cytosine deaminase, or inosine deaminase, as described herein. Multiple configurations of base editors can achieve multiple types of multiplexing of base editing. For example, in some embodiments, a single base editor is used in combination with more than one guide to generate two, three, or more nucleobase edits. Alternatively, in some embodiments, multiple base editors paired with suitable guides are used to generate two, three, or more nucleobase edits. Multiple base editors and associated guides are shown in Tables 2, 3, 4, 5, and 6. Thus, in some embodiments, base editors and suitable guides are provided that target one or more specific genes, such as B2M gene, CD142 gene, CIITA gene, HLA-A gene, and HLA-B gene.

[0132] In some embodiments, the base editing system is provided in one or more vectors. For example, the base editing system can be provided in a single vector or a "split vector" consisting of more than one vector delivering the components of the base editing system. The corresponding nucleic acid can be codon optimized. Such codon optimization is performed to optimize the nucleic acid for expression in human cells.

[0133] Following production of genetically modified hepatocytes, the genetically modified cells are expanded in a suitable humanized animal model. This expansion allows for the production of a suitable number of cells sufficient for transplantation into a subject in need thereof. Various humanized animal models are known in the art, including, for example, FRG pig, FRG mouse, and FRG rat animals. In some embodiments, the genetically modified hepatocytes undergoing a first expansion in FRG mice and / or FRG mouse animals are followed by a second expansion in a larger humanized FRG animal (e.g., pig). Generally, about 500,000 to 1 million cells generate about 80 million to 150 million hepatocytes per FRG mouse. Generally, about 500,000 to 1 million cells generate about 480 million to 900 million hepatocytes per FRG rat. FRG pigs can generally generate about 100 times more in terms of cell expansion than FRG rats.

[0134] After the expansion step, the genetically modified human hepatocytes are then isolated from the FRG animals according to methods known in the art, such as, for example, fluorescence activated cell sorting, immunomagnetic cell separation, density gradient centrifugation, and / or immunodensity cell separation.

[0135] How to Treat Liver Disease Described herein are methods of using the described genetically modified human hepatocytes to treat subjects with liver disease. The genetically modified human hepatocytes can be used to treat a variety of liver diseases, including, for example, alpha-1 antitrypsin deficiency, Crigler-Najjar syndrome type 1, familial hypercholesterolemia, congenital coagulation factor VII deficiency, hemophilia A, glycogen storage disease type I, infantile Refsum disease, maple syrup urine disease, neonatal hemochromatosis, progressive familial intrahepatic cholestasis type 2 (PFIC2), urea cycle disorders (e.g., ornithine transcarbamylase (OTC) deficiency, argininosuccinate lyase deficiency, carbamoyl phosphate synthase type 1 deficiency, citrullinemia, Wilson's disease), acute liver failure, fatty liver of pregnancy, and acute exacerbation of chronic liver failure. Thus, the methods described herein can be used to treat either congenital or acquired liver disease. Thus, in some embodiments, the genetically modified human hepatocytes are used to treat alpha-1 antitrypsin deficiency. In some embodiments, the genetically modified human hepatocytes are used to treat Crigler-Najjar syndrome type 1. In some embodiments, the genetically modified human hepatocytes are used to treat familial hypercholesterolemia. In some embodiments, the genetically modified human hepatocytes are used to treat congenital coagulation factor VII deficiency. In some embodiments, the genetically modified human hepatocytes are used to treat hemophilia A. In some embodiments, the genetically modified human hepatocytes are used to treat glycogen storage disease type I. In some embodiments, the genetically modified human hepatocytes are used to treat infantile Refsum's disease. In some embodiments, the genetically modified human hepatocytes are used to treat maple syrup urine disease. In some embodiments, the genetically modified human hepatocytes are used to treat neonatal hemochromatosis. In some embodiments, the genetically modified human hepatocytes are used to treat progressive familial intrahepatic cholestasis type 2 (PFIC2). In some embodiments, genetically modified human hepatocytes are used to treat progressive familial intrahepatic cholestasis type 2 (PFIC2).In some embodiments, the genetically modified human hepatocytes are used to treat urea cycle disorders such as ornithine transcarbamylase (OTC) deficiency, argininosuccinate lyase deficiency, carbamoyl phosphate synthase type 1 deficiency, citrullinemia, and Wilson's disease. In some embodiments, the genetically modified human hepatocytes are used to treat acute liver failure. In some embodiments, the genetically modified human hepatocytes are used to treat fatty liver of pregnancy during pregnancy. In some embodiments, the genetically modified human hepatocytes are used to treat acute exacerbations of chronic liver failure.

[0136] A method of treating a subject in need of treatment includes administration of the genetically modified human hepatocytes described herein. Various modes of administration (e.g., intraportal injection or injection of cells) are suitable for treating a subject in need thereof. In some embodiments, the genetically modified human hepatocytes are administered into the portal vein of a subject in need thereof. For dosing purposes, the amount of genetically modified human hepatocytes administered to a subject in need thereof is about 5-20 billion cells. In some embodiments, about 5-20 billion genetically modified human hepatocytes are injected into the portal vein of a subject in need thereof. In some embodiments, about 10-12 billion genetically modified human hepatocytes are injected into the portal vein of a subject in need thereof. In some embodiments, about 12-15 billion genetically modified human hepatocytes are injected into the portal vein of a subject in need thereof.

[0137] In some embodiments, the genetically modified hepatocytes are administered to the subject in an amount of about 2-15% of the total liver mass. In some embodiments, the genetically modified hepatocytes are administered to the subject in an amount of about 5-10% of the total liver mass. Thus, in some embodiments, the genetically modified hepatocytes are administered to the subject in an amount of about 2% of the total liver mass. In some embodiments, the genetically modified hepatocytes are administered to the subject in an amount of about 3% of the total liver mass. In some embodiments, the genetically modified hepatocytes are administered to the subject in an amount of about 4% of the total liver mass. In some embodiments, the genetically modified hepatocytes are administered to the subject in an amount of about 5% of the total liver mass. In some embodiments, the genetically modified hepatocytes are administered to the subject in an amount of about 6% of the total liver mass. In some embodiments, the genetically modified hepatocytes are administered to the subject in an amount of about 7% of the total liver mass. In some embodiments, the genetically modified hepatocytes are administered to the subject in an amount of about 8% of the total liver mass. In some embodiments, the genetically modified hepatocytes are administered to the subject in an amount of about 9% of the total liver mass. In some embodiments, the genetically modified hepatocytes are administered to the subject in an amount of about 10% of the total liver mass. In some embodiments, the genetically modified hepatocytes are administered to the subject in an amount of about 11% of the total liver mass. In some embodiments, the genetically modified hepatocytes are administered to the subject in an amount of about 12% of the total liver mass. In some embodiments, the genetically modified hepatocytes are administered to the subject in an amount of about 13% of the total liver mass. In some embodiments, the genetically modified hepatocytes are administered to the subject in an amount of about 14% of the total liver mass. In some embodiments, the genetically modified hepatocytes are administered to the subject in an amount of about 15% of the total liver mass.

[0138] In some embodiments, the genetically modified hepatocytes are up to about 2×10 per kg of body weight. 8 In some embodiments, the genetically modified hepatocytes are administered to a subject at a dose of up to about 1.5×10 cells per kg of body weight. 8 In some embodiments, the genetically modified hepatocytes are administered to a subject at a dose of up to about 1.2×10 cells per kg of body weight. 8 In some embodiments, the genetically modified hepatocytes are administered to a subject at a dose of up to about 1.0×10 cells per kg of body weight. 8In some embodiments, the genetically modified hepatocytes are administered to a subject at a dose of up to about 0.8×10 cells per kg of body weight. 8 In some embodiments, the genetically modified hepatocytes are administered to a subject at a dose of up to about 0.5×10 cells per kg of body weight. 8 The cells are administered to a subject.

[0139] CRISPR Fusion Proteins In some embodiments, the Cas9 or Cas12 protein is fused to one or more heterologous protein domains. In some embodiments, the Cas9 or Cas12 enzyme is fused to about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more protein domains. In some embodiments, the heterologous protein domain is fused to the C-terminus of the Cas9 or Cas12 enzyme. In some embodiments, the heterologous protein domain is fused to the N-terminus of the Cas9 or Cas12 enzyme. In some embodiments, the heterologous protein domain is fused internally between the C-terminus and the N-terminus of the Cas9 or Cas12 enzyme. In some embodiments, the internal fusion is made within the Cas9 RuvCI, RuvC II, RuvCIII, HNH, REC I, or PAM interaction domain.

[0140] Cas9 or Cas12 protein can be directly or indirectly linked to another protein domain. In some embodiments, a suitable CRISPR system contains a linker or spacer that connects Cas9 protein and a heterologous protein. The amino acid linker or spacer is generally designed to be flexible or to interpose a structure such as an alpha-helix between the two protein moieties. The linker or spacer can be relatively short or can be longer. Typically, the linker or spacer contains, for example, 1 to 100 (e.g., 1 to 100, 5 to 100, 10 to 100, 20 to 100, 30 to 100, 40 to 100, 50 to 100, 60 to 100, 70 to 100, 80 to 100, 90 to 100, 5 to 55, 10 to 50, 10 to 45, 10 to 40, 10 to 35, 10 to 30, 10 to 25, 10 to 20) amino acids in length. In some embodiments, the linker or spacer is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 amino acids in length or more. Typically, a longer linker may reduce steric hindrance. In some embodiments, the linker will comprise a mixture of glycine and serine residues. In some embodiments, the linker may further comprise threonine, proline, and / or alanine residues.

[0141] In some embodiments, the Cas9 or Cas12 protein is fused to a cellular localization signal, an epitope tag, a reporter gene, and a protein domain having enzymatic activity, epigenetic modification activity, RNA cleavage activity, nucleic acid binding activity, transcriptional regulation activity, hi some embodiments, the Cas9 protein is fused to a nuclear localization sequence (NLS), a FLAG tag, a HIS tag, and / or an HA tag.

[0142] Suitable fusion partners include, but are not limited to, polypeptides that provide methyltransferase activity, demethylase activity, acetyltransferase activity, deacetylase activity, kinase activity, phosphatase activity, ubiquitin ligase activity, deubiquitinating activity, adenylating activity, deadenylating activity, sumoylating activity, desumoylating activity, ribosylation activity, deribosylation activity, myristoylating activity, demyristoylating activity, integrase activity, transposase activity, recombinase activity, polymerase activity, ligase activity, helicase activity, or nuclease activity, any of which can modify DNA or DNA-associated polypeptides (e.g., histones or DNA-binding proteins). In some embodiments, the Cas9 protein is fused to a histone demethylase, transcriptional activator, or deaminase.

[0143] Additional suitable fusion partners include, but are not limited to, boundary elements (e.g., CTCF), proteins and fragments thereof that provide peripheral recruitment (e.g., Lamin A, Lamin B, etc.), and protein docking elements (e.g., FKBP / FRB, Pill / Abyl, etc.).

[0144] In certain embodiments, Cas9 is fused to a cytidine or adenosine deaminase domain, e.g., for use in base editing. In some embodiments, Cas9 is fused to an adenine and cytosine base editor (ACBE or CABE), which is generated by fusing a heterodimer of TadA and activation-induced cytidine deaminase (AID) to the N-terminus and C-terminus of Cas9 nickase (nCas9). In some embodiments, ACBE or CABE simultaneously induces C-to-T and A-to-G base editing at the same target site. Xie, J et al. ACBE, a new base editor for simultaneous C-to-T and A-to-G substitutions in mammalian systems. BMC Biology (18:131), 2020)

[0145] In certain embodiments, Cas9 or Cas12 is fused to a cytidine or adenosine deaminase domain, for example, for use in base editing. In some embodiments, the terms "cytidine deaminase" and "cytosine deaminase" can be used interchangeably. In certain embodiments, the cytidine deaminase domain can have 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity with any cytidine deaminase described herein. In some embodiments, the cytidine deaminase domain has cytidine deaminase activity (e.g., converting C to U). In certain embodiments, the adenosine deaminase domain may have 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to any adenosine deaminase described herein. In some embodiments, the adenosine deaminase domain has adenosine deaminase activity (e.g., converts A to I). In some embodiments, the terms "adenosine deaminase" and "adenine deaminase" can be used interchangeably.

[0146] In some embodiments, the cytidine deaminase can comprise all or part of an apolipoprotein B mRNA editing complex (APOBEC) family deaminase. APOBEC is an evolutionarily conserved family of cytidine deaminases. Members of this family are C to U editing enzymes. The N-terminal domain of APOBEC-like proteins is the catalytic domain, and the C-terminal domain is the pseudocatalytic domain. More specifically, the catalytic domain is a zinc-dependent cytidine deaminase domain, which is important for the deamination of cytidine. APOBEC family members include APOBEC1, APOBEC2, APOBEC3A, APOBEC3B, APOBEC3C, APOBEC3D (now referred to as "APOBEC3E"), APOBEC3F, APOBEC3G, APOBEC3H, APOBEC4, and activation-induced (cytidine) deaminase. In some embodiments, the deaminase incorporated into the fusion protein comprises all or part of APOBEC1 deaminase. In some embodiments, the deaminase incorporated into the fusion protein comprises all or a portion of an APOBEC2 deaminase. In some embodiments, the deaminase incorporated into the fusion protein comprises all or a portion of an APOBEC3 deaminase. In some embodiments, the deaminase incorporated into the fusion protein comprises all or a portion of an APOBEC3A deaminase. In some embodiments, the deaminase incorporated into the fusion protein comprises all or a portion of an APOBEC3B deaminase. In some embodiments, the deaminase incorporated into the fusion protein comprises all or a portion of an APOBEC3C deaminase. In some embodiments, the deaminase incorporated into the fusion protein comprises all or a portion of an APOBEC3D deaminase. In some embodiments, the deaminase incorporated into the fusion protein comprises all or a portion of an APOBEC3E deaminase. In some embodiments, the deaminase incorporated into the fusion protein comprises all or a portion of an APOBEC3F deaminase. In some embodiments, the deaminase incorporated into the fusion protein comprises all or a portion of an APOBEC3G deaminase.In some embodiments, the deaminase incorporated into the fusion protein comprises all or a portion of an APOBEC3H deaminase. In some embodiments, the deaminase incorporated into the fusion protein comprises all or a portion of an APOBEC4 deaminase. In some embodiments, the deaminase incorporated into the fusion protein comprises all or a portion of an activation-induced deaminase (AID). In some embodiments, the deaminase incorporated into the fusion protein comprises all or a portion of a cytidine deaminase 1 (CDA1). It should be understood that the fusion protein may comprise a deaminase from any suitable organism (e.g., human or rat). In some embodiments, the deaminase domain of the fusion protein is from a human, chimpanzee, gorilla, monkey, cow, dog, rat, or mouse. In some embodiments, the deaminase domain of the fusion protein is from a rat (e.g., rat APOBEC1). In some embodiments, the deaminase domain is human APOBEC1. In some embodiments, the deaminase domain is pmCDA1.

[0147] Exemplary cytidine deaminase sequences are provided below. pmCDA1 (Petromyzon marinus) MTDAEYVRIHEKLDIYTFKKQFFNNKKSVSHRCYVLFELKRRGERRACFWGYAVNKPQSGTERGIHAEIFSIRKVEEYLRDNPGQFTINWYSSWSPCADCAEKILEWYNQELRGNGHTLKIWACKLYYEKNARNQIGLWNLRDNGVGLNVMVSEHYQCCRKIFIQSSHNQLNENRWLEKTLKRAEKRRSELSIMIQVKILHTTKSPAV (SEQ ID NO: 4) Human AID: MDSLLMNRRKFLYQFKNVRWAKGRRETYLCYVVKRRDSATSFSLDFGYLRNKNGCHVELLFLRYISDWDLDPGRCYRVTWFTSWSPCYDCARHVADFLRGNPNLSLRIFTARLYFCEDRKAEPEGLRRLHRAGVQIAIMTFKAPV (SEQ ID NO: 5) Human AID: JPEG2024514649000005.jpg26148 (SEQ ID NO: 6) (underlined: nuclear localization sequence, double underlined: nuclear export signal) Mouse AID: JPEG2024514649000006.jpg26148 (SEQ ID NO: 7) (underlined: nuclear localization sequence, double underlined: nuclear export signal) Canine AID: JPEG2024514649000007.jpg26151 (SEQ ID NO: 8) (underlined: nuclear localization sequence, double underlined: nuclear export signal) Bovine AID: JPEG2024514649000008.jpg26148 (SEQ ID NO: 9) (underlined: nuclear localization sequence, double underlined: nuclear export signal) Rat AID: JPEG2024514649000009.jpg34150 (SEQ ID NO:10) (Underlined: nuclear localization sequence, double underlined: nuclear export signal) clAID(Canis lupus familiaris): MDSLLMKQRKFLYHFKNVRWAKGRHETYLCYVVKRRDSATSFSLDFGHLRNKSGCHVELLFLRYISDWDLDPGRCYRVTWFTSWSPCYDCARHVADFLRGYPNLSLRIFAARLYFCEDRKAEPEGLRRLHRAGVQIAIMTFKDYFYCWNTFVENREKTFKAWEGLHENSVRLSRQLRRILLPLYEVDDLRDAFRTLGL (SEQ ID NO: 11) btAID(Bos Taurus): MDSLLKKQRQFLYQFKNVRWAKGRHETYLCYVVKRRDSPTSFSLDFGHLRNKAGCHVELLFLRYISDWDLDPGRCYRVTWFTSWSPCYDCARHVADFLRGYPNLSLRIFTARLYFCDKERKAEPEGLRRLHRAGVQIAIMTFKDYFYCWNTFVENHERTFKAWEGLHENSVRLSRQLRRILLPLYEVDDLRDAFRTLGL(SEQ ID NO: 12) mAID (Mus musculus): MDSLLMNRRKFLYQFKNVRWAKGRRETYLCYVVKRRDSATSFSLDFGYLRNKNGCHVELLFLRYISDWDLDPGRCYRVTWFTSWSPCYDCARHVADFLRGNPNLSLRIFTARLYFCEDRKAEPEGLRRLHRAGVQIAIMTFKDYFYCWNTFVENHERTFKAWEGLHENSVRLSRQLRRILLPLYEVDDLRDAFRTLGL(SEQ ID NO: 13) rAPOBEC-1 (Rattus norvegicus): MSSETGPVAVDPTLRRRIEPHEFEVFFDPRELRKETCLLYEINWGGRHSIWRHTSQNTNKHVEVNFIEKFTTERYFCPNTRCSITWFLSWSPCGECSRAITEFLSRYPHVTLFIYIARLYHHADPRNRQGLRDLISSGVTIQIMTEQESGYCWRNFVNYSPSNEAHWPRYPHLWVRLYVLELYCIILGLPPCLNILRRKQPQLTFFTIALQSCHYQRLPPHILWATGLK(SEQ ID NO: 14) maAPOBEC-1 (Mesocricetus auratus): MSSETGPVVVDPTLRRRIEPHEFDAFFDQGELRKETCLLYEIRWGGRHNIWRHTGQNTSRHVEINFIEKFTSERYFYPSTRCSIVWFLSWSPCGECSKAITEFLSGHPNVTLFIYAARLYHHTDQRNRQGLRDLISRGVTIRIMTEQEYCYCWRNFVNYPPSNEVYWPRYPNLWMRLYALELYCIHLGLPPCLKIKRRHQYPLTFFRLNLQSCHYQRIPPHILWATGFI(SEQ ID NO: 15) ppAPOBEC-1(Pongo pygmaeus): MTSEKGPSTGDPTLRRRIESWEFDVFYDPRELRKETCLLYEIKWGMSRKIWRSSGKNTTNHVEVNFIKKFTSERRFHSSISCSITWFLSWSPCWECSQAIREFLSQHPGVTLVIYVARLFWHMDQRNRQGLRDLVNSGVTIQIMRASEYYHCWRNFVNYPPGDEAHWPQYPPLWMMLYALELHCIILSLPPCLKISRRWQNHLAFFRLHLQNCHYQTIPPHILLATGLIHPSVTWR(SEQ ID NO: 16) ocAPOBEC1(Oryctolagus cuniculus): MASEKGPSNKDYTLRRRIEPWEFEVFFDPQELRKEACLLYEIKWGASSKTWRSSGKNTTNHVEVNFLEKLTSEGRLGPSTCCSITWFLSWSPCWECSMAIREFLSQHPGVTLIIFVARLFQHMDRRNRQGLKDLVTSGVTVRVMSVSEYCYCWENFVNYPPGKAAQWPRYPPRWMLMYALELYCIILGLPPCLKISRRHQKQLTFFSLTPQYCHYKMIPPYILLATGLLQPSVPWR(SEQ ID NO: 17) mdAPOBEC-1(Monodelphis domestica): MNSKTGPSVGDATLRRRIKPWEFVAFFNPQELRKETCLLYEIKWGNQNIWRHSNQNTSQHAEINFMEKFTAERHFNSSVRCSITWFLSWSPCWECSKAIRKFLDHYPNVTLAIFISRLYWHMDQQHRQGLKELVHSGVTIQIMSYSEYHYCWRNFVDYPQGEEDYWPKYPYLWIMLYVLELHCIILGLPPCLKISGSHSNQLALFSLDLQDCHYQKIPYNVLVATGLVQPFVTWR (SEQ ID NO: 18) ppAPOBEC-2 (Pongo pygmaeus): MAQKEEAAAATEAASQNGEDLENLDDPEKLKELIELPPFEIVTGERLPANFFKFQFRNVEYSSGRNKTFLCYVVEAQGKGGQVQASRGYLEDEHAAAHAEEAFFNTILPAFDPALRYNVTWYVSSSPCAACADRIIKTLSKTKNLRLLILVGRLFMWEELEIQDALKKLKEAGCKLRIMKPQDFEYVWQNFVEQEEGESKAFQPWEDIQENFLYYEEKLADILK (SEQ ID NO: 19) btAPOBEC-2 (Bos Taurus): MAQKEEAAAAAEPASQNGEEVENLEDPEKLKELIELPPFEIVTGERLPAHYFKFQFRNVEYSSGRNKTFLCYVVEAQSKGGQVQASRGYLEDEHATNHAEEAFFNSIMPTFDPALRYMVTWYVSSSPCAACADRIVKTLNKTKNLRLLILVGRLFMWEEPEIQAALRKLKEAGCRLRIMKPQDFEYIWQNFVEQEEGESKAFEPWEDIQENFLYYEEKLADILK (SEQ ID NO: 20) mAPOBEC-3-(1) (Mus musculus): MQPQRLGPRAGMGPFCLGCSHRKCYSPIRNLISQETFKFHFKNLGYAKGRKDTFLCYEVTRKDCDSPVSLHHGVFKNKDNIHAEICFLYWFHDKVLKVLSPREEFKITWYMS WSPCFECAEQIVRFLATHHNLSLDIFSSRLYNVQDPETQQNLCRLVQEGAQVAAMDLYEFKKCWKKFVDNGGRRFRPWKRLLTNFRYQDSKLQEILRPCYISVPSSSSSTLS NICLTKGLPETRFWVEGRRMDPLSEEEFYSQFYNQRVKHLCYYHRMKPYLCYQLEQFNGQAPLKGCLLSEKGKQHAEILFLDKIRSMELSQVTITCYLTWSPCPNCAWQLAAFKRDRPDLILHIYTSRLYFHWKRPFQKGLCSLWQSGILVDVMDLPQFTDCWTNFVNPKRPFWPWKGLEIISRRTQRRLRRIKESWGLQDLVNDFGNLQLGPPMS (SEQ ID NO: 21) Mouse APOBEC-3-(2): JPEG2024514649000010.jpg52150 (SEQ ID NO: 22) (italics: nucleic acid editing domain) Rat APOBEC-3: JPEG2024514649000011.jpg53154 (SEQ ID NO: 23) (italics: nucleic acid editing domain) hAPOBEC-3A (Homo sapiens): MEASPASGPRHLMDPHIFTSNFNNGIGRHKTYLCYEVERLDNGTSVKMDQHRGFLHNQAKNLLCGFYGRHAELRFLDLVPSLQLDPAQIYRVTWFISWSPCFSWGCAGEVRAFLQENTHVRLRIFAARIYDYDPLYKEALQMLRDAGAQVSIMTYDEFKHCWDTFVDHQGCPFQPWDGLDEHSQALSGRLRAILQNQGN (SEQ ID NO: 24) hAPOBEC-3F (Homo sapiens): MKPHFRNTVERMYRDTFSYNFYNRPILSRRNTVWLCYEVKTKGPSRPRLDAKIFRGQVYSQPEHHAEMCFLSWFCGNQLPAYKCFQITWFVSWTPCPDCVAKLAEFLAEHPNVTLTISAARLYYYWERDYRRALCRLSQAGARVKIMDDEEFAYCWENFVYSEGQPFMPWYKFDDNYAFLHRTLKEILRNPMEAMYPHIFYFHFKNLRKAYGRNESWLCFTMEVVKHHSPVSWKRGVFRNQVDPETHCHAERCFLSWFCDDILSPNTNYEVTWYTSWSPCPEECAGEVAEFLARHSNVNLTIFTARLYYFWDTDYQEGLRSLSQEGASVEIMGYKDFKYCWENFVYNDDEPFKPWKGLKYNFLFLDSKLQEILE (SEQ ID NO: 25) Rhesus APOBEC-3G: JPEG2024514649000012.jpg45149 (SEQ ID NO: 26) (italics: nucleic acid editing domain, underline: cytoplasmic localization signal) Chimpanzee APOBEC-3G: JPEG2024514649000013.jpg46149 (SEQ ID NO:27) (Italics: nucleic acid editing domain, underline: cytoplasmic localization signal) Green Monkey APOBEC-3G: JPEG2024514649000014.jpg44145 (SEQ ID NO:28) (Italics: nucleic acid editing domain, underline: cytoplasmic localization signal) Human APOBEC-3G: JPEG2024514649000015.jpg45148 (SEQ ID NO:29) (Italics: nucleic acid editing domain, underline: cytoplasmic localization signal) Human APOBEC-3F: JPEG2024514649000016.jpg45146 (SEQ ID NO:30) (Italics: nucleic acid editing domain) Human APOBEC-3B: JPEG2024514649000017.jpg45146 (SEQ ID NO:31) (Italics: nucleic acid editing domain) Rat APOBEC-3B: MQPQGLGPNAGMGPVCLGCSHRRPYSPIRNPLKKLYQQTFYFHFKNVRYAWGRKNNFLCYEVNGMDCALPVPLRQGVFRKQGHIHAELCFIYWFHDKVLRVLSPMEEFKVTWYMSWSPCSKCAEQVARFLAAHRNLSLAIFSSRLYYYLRNPNYQQKLCRLIQEGVHVAAMDLPEFKKCWNKFVDNDGQPFRPWMRLRINFSFYDCKLQEIFSRMNLLREDVFYLQFNNSHRVKPVQNRYYRRKSYLCYQLERANGQEPLKGYLLYKKGEQHVEILFLEKMRSMELSQVRITCYLTWSPCPNCARQLAAFKKDHPDLILRIYTSRLYFWRKKFQKGLCTLWRSGIHVDVMDLPQFADCWTNFVNPQRPFRPWNELEKNSWRIQRRLRRIKESWGL (SEQ ID NO: 32) Bovine APOBEC-3B: MDGWEVAFRSGTVLKAGVLGVSMTEGWAGSGHPGQGACVWTPGTRNTMNLLREVLFKQQFGNQPRVPAPYYRRKTYLCYQLKQRNDLTLDRGCFRNKKQRHAERFIDKINSLDLNPSQSYKIICYITWSPCPNCANELVNFITRNNHLKLEIFASRLYFHWIKSFKMGLQDLQNAGISVAVMTHTEFEDCWEQFVDNQSRPFQPWDKLEQYSASIRRRLQRILTAPI (SEQ ID NO: 33) Chimpanzee APOBEC-3B: MNPQIRNPMEWMYQRTFYYNFENEPILYGRSYTWLCYEVKIRRGHSNLLWDTGVFRGQMYSQPEHHAEMCFLSWFCGNQLSAYKCFQITWFVSWTPCPDCVAKLAKFLAEHPNVTLTISAARLY YYWERDYRRALCRLSQAGARVKIMDDEEFAYCWENFVYNEGQPFMPWYKFDDNYAFLHRTLKEIIRHLMDPDTFTFNFNNDPLVLRRHQTYLCYEVERLDNGTWVLMDQHMGFLCNEAKNLLCGF YGRHAELRFLDLVPSLQLDPAQIYRVTWFISWSPCFSWGCAGQVRAFLQENTHVRLRIFAARIYDYDPLYKEALQMLRDAGAQVSIMTYDEFEYCWDTFVYRQGCPFQPWDGLEEHSQALSGRLRAILQVRASSLCMVPHRPPPPPQSPGPCLPLCSEPPLGSLLPTGRPAPSLPFLLTASFSFPPPASLPPLPSLSLSPGHLPVPSFHSLTSCSIQPPCSSRIRETEGWASVSKEGRDLG (SEQ ID NO: 34) Human APOBEC-3C: JPEG2024514649000018.jpg26153 (SEQ ID NO:35) (Italics: nucleic acid editing domain) Gorilla APOBEC-3C JPEG2024514649000019.jpg27154 (SEQ ID NO:36) (Italics: nucleic acid editing domain) Human APOBEC-3A: JPEG2024514649000020.jpg28161 (SEQ ID NO:37) (Italics: nucleic acid editing domain) Rhesus APOBEC-3A: JPEG2024514649000021.jpg27154 (SEQ ID NO:38) (Italics: nucleic acid editing domain) Bovine APOBEC-3A: JPEG2024514649000022.jpg26154 (SEQ ID NO:39) (Italics: nucleic acid editing domain) Human APOBEC-3H: JPEG2024514649000023.jpg25150 (SEQ ID NO:40) (Italics: nucleic acid editing domain) Rhesus APOBEC-3H: MALLTAKTFSLQFNNKRRVNKPYYPRKALLCYQLTPQNGSTPTRGHLKNKKKDHAEIRFINKIKSMGLDETQCYQVTCYLTWSPCPSCAGELVDFIKAHRHLNLRIFASRLYYHWRPNYQEGLLLLCGSQVPVEVMGLPEFTDCWENFVDHKEPPSFNPSEKLEELDKNSQAIKRRLERIKSRSVDVLENGLRSLQLGPVTPSSSIRNSR (SEQ ID NO: 41) Human APOBEC-3D: JPEG2024514649000024.jpg49161 (SEQ ID NO:42) (Italics: nucleic acid editing domain) Human APOBEC-1: MTSEKGPSTGDPTLRRRIEPWEFDVFYDPRELRKEACLYEIKWGMSRKIWRSSGKNTTNHVEVNFIKKFTSERDFHPSMSCSITWFLSWSPCWECSQAIREFLSRHPGVTLVIYVARLFWHMDQQNRQGLRDLVNSGVTIQIMRASEYYHCWRNFVNYPPGDEAHWPQYPPLWMMLYALELHCIILSLPPCLKISRRWQNHLTFFRLHLQNCHYQTIPPHILLATGLIHPSVAWR (SEQ ID NO: 43) Mouse APOBEC-1: MSSETGPVAVDPTLRRRIEPHEFEVFFDPRELRKETCLLYEINWGGRHSVWRHTSQNTSNHVEVNFLEKFTTERYFRPNTRCSITWFLSWSPCGECSRAITEFLSRHPYVTLFIYIARLYHHTDQRNRQGLRDLISSGVTIQIMTEQEYCYCWRNFVNYPPSNEAYWPRYPHLWVKLYVLELYCIILGLPPCLKILRRKQPQLTFFTITLQTCHYQRIPPHLLWATGLK (SEQ ID NO: 44) Rat APOBEC-1: MSSETGPVAVDPTLRRRIEPHEFEVFFDPRELRKETCLLYEINWGGRHSIWRHTSQNTNKHVEVNFIEKFTTERYFCPNTRCSITWFLSWSPCGECSRAITEFLSRYPHVTLFIYIARLYHHADPRNRQGLRDLISSGVTIQIMTEQESGYCWRNFVNYSPSNEAHWPRYPHLWVRLYVLELYCIILGLPPCLNILRRKQPQLTFFTIALQSCHYQRLPPHILWATGLK (SEQ ID NO: 45) Human APOBEC-2: MAQKEEAAVATEAASQNGEDLENLDDPEKLKELIELPPFEIVTGERLPANFFKFQFRNVEYSSGRNKTFLCYVVEAQGKGGQVQASRGYLEDEHAAAHAEEAFFNTILPAFDPALRYNVTWYVSSSPCAACADRIIKTLSKTKNLRLLILVGRLFMWEEPEIQAALKKLKEAGCKLRIMKPQDFEYVWQNFVEQEEGESKAFQPWEDIQENFLYYEEKLADILK (SEQ ID NO: 46) Mouse APOBEC-2: MAQKEEAAEAAAPASQNGDDLENLEDPEKLKELIDLPPFEIVTGVRLPVNFFKFQFRNVEYSSGRNKTFLCYVVEVQSKGGQAQATQGYLEDEHAGAHAEEAFFNTILPAFDPALKYNVTWYVSSSPCAACADRILKTLSKTKNLRLLILVSRLFMWEEPEVQAALKKLKEAGCKLRIMKPQDFEYIWQNFVEQEEGESKAFEPWEDIQENFLYYEEKLADILK (SEQ ID NO: 47) Rat APOBEC-2: MAQKEEAAEAAAPASQNGDDLENLEDPEKLKELIDLPPFEIVTGVRLPVNFFKFQFRNVEYSSGRNKTFLCYVVEAQSKGGQVQATQGYLEDEHAGAHAEEAFFNTILPAFDPALKYNVTWYVSSSPCAACADRILKTLSKTKNLRLLILVSRLFMWEEPEVQAALKKLKEAGCKLRIMKPQDFEYLWQNFVEQEEGESKAFEPWEDIQENFLYYEEKLADILK (SEQ ID NO: 48) Bovine APOBEC-2: MAQKEEAAAAAEPASQNGEEVENLEDPEKLKELIELPPFEIVTGERLPAHYFKFQFRNVEYSSGRNKTFLCYVVEAQSKGGQVQASRGYLEDEHATNHAEEAFFNSIMPTFDPALRYMVTWYVSSSPCAACADRIVKTLNKTKNLRLLILVGRLFMWEEPEIQAALRKLKEAGCRLRIMKPQDFEYIWQNFVEQEEGESKAFEPWEDIQENFLYYEEKLADILK (SEQ ID NO: 49) Petromyzon marinus CDA1(pmCDAl): MTDAEYVRIHEKLDIYTFKKQFFNNKKSVSHRCYVLFELKRRGERRACFWGYAVNKPQSGTERGIHAEIFSIRKVEEYLRDNPGQFTINWYSSWSPCADCAEKILEWYNQELRGNGHTLKIWACKLYYEKNARNQIGLWNLRDNGVGLNVMVSEHYQCCRKIFIQSSHNQ LNENRWLEKTLKRAEKRRSELSFMIQVKILHTTKSPAV (SEQ ID NO: 50) Human APOBEC3G D316R D317R: MKPHFRNTVERMYRDTFSYNFYNRPILSRRNTVWLCYEVKTKGPSRPPLDAKIFRGQVYSELKYHPEMRFFHWFSKWRKLHRDQEYEVTWYISWSPCTKCTRDMATFLAEDPKVTLTIFVARLYYFWDPDYQEALRSLCQKRDGPRATMKFNYDEFQHCWSKFVYSQRELFEPWNNLPKYYILLHFMLGEILRHSMDPPTFTFNFNNEPWVRGRHETYLCYEVERMHNDTWVLLNQRRGFLCNQAPHKHGFLEGRHAELCFLDVIPFWKLDLDQDYRVTCFTSWSPCFSCAQEMAKFISKKHVSLCIFTARIYRRQGRCQEGLRTLAEAGAKISFTYSEFKHCWDTFVDHQGCPFQPWDGLDEHSQDLSGRLRAILQNQEN (SEQ ID NO: 51) Human APOBEC3G A chain: MDPPTFTFNFNNEPWWGRHETYLCYEVERMHNDTWVLLNQRRGFLCNQAPHKHGFLEGRHAELCFLDVIPFWKLDLDQDYRVTCFTSWSPCFSCAQEMAKFISKNKHVSLCIFTARIYDDQGRCQEGLRTLAEAGAKISFTYSEFKHCWDTFVDHQGCPFQPWDGLD EHSQDLSGRLRAILQ (SEQ ID NO: 52) Human APOBEC3G A chain D120R D121R: MDPPTFTFNFNNEPWVRGRHETYLCYEVERMHNDTWVLLNQRRGFLCNQAPHKHGFLEGRHAELCFLDVIPFWKLDLDQDYRVTCFTSWSPCFSCAQEMAKFISKNKHVSLCIFTARIYRRQGRCQEGLRTLAEAGAKISFMTYSEFKHCWDTFVDHQGCPFQPWDGLDEHSQDLSGRLRAILQ(SEQ ID NO: 53) hAPOBEC-4 (Homo sapiens): MEPIYEEYLANHGTIVKPYYWLSFSLDCSNCPYHIRTGEEARVSLTEFCQIFGFPYGTTFPQTKHLTFYELKTSSGSLVQKGHASSCTGNYIHPESMLFEMNGYLDSAIYNNDSIRHIILYSNNSPCNEANHCCISKMYNFLITYPGITLSIYFSQLYHTEMDFPASAWNREALRSLASLWPRVVLSPISGGIWHSVLHSFISGVSGSHVFQPILTGRALADRHNAYEINAITGVKPYFTDVLLQTKRNPNTKAQEALESYPLNNAFPGQFFQMPSGQLQPNLPPDLRAPVVFVLVPLRDLPPMHMGQNPNKPRNIVRHLNMPQMSFQETKDLGRLPTGRSVEIVEITEQFASSKEADEKKKKKGKK(SEQ ID NO: 54) mAPOBEC-4 (Mus musculus): MDSLLMKQKKFLYHFKNVRWAKGRHETYLCYVVKRRDSATSCSLDFGHLRNKSGCHVELLFLRYISDWDLDPGRCYRVTWFTSWSPCYDCARHVAEFLRWNPNLSLRIFTARLYFCEDRKAEPEGLRRLHRAGVQIGIMTFKDYFYCWNTFVENRERTFKAWEGLHENSVRLTRQLRRILLPLYEVDDLRDAFRMLGF(SEQ ID NO: 55) rAPOBEC-4 (Rattus norvegicus): MEPLYEEYLTHSGTIVKPYYWLSVSLNCTNCPYHIRTGEEARVPYTEFHQTFGFPWSTYPQTKHLTFYELRSSSGNLIQKGLASNCTGSHTHPESMLFERDGYLDSLIFHDSNIRHIILYSNNSPCDEANHCCISKMYNFLMNYPEVTLSVFFSQLYHTENQFPTSAWNREALRGLASLWPQVTLSAISGGIWQSILETFVSGISEGLTAVRPFTAGRTLTDRYNAYEINCITEVKPYFTDALHSWQKENQDQKVWAASENQPLHNTTPAQWQPDMSQDCRTPAVFMLVPYRDLPPIHVNPSPQKPRTVVRHLNTLQLSASKVKALRKSPSGRPVKKEEARKGSTRSQEANETNKSKWKKQTLFIKSNICHLLEREQKKIGILSSWSV(SEQ ID NO: 56) mfAPOBEC-4 (Macaca fascicularis): MEPTYEEYLANHGTIVKPYYWLSFSLDCSNCPYHIRTGEEARVSLTEFCQIFGFPYGTTYPQTKHLTFYELKTSSGSLVQKGHASSCTGNYIHPESMLFEMNGYLDSAIYNNDSIRHIILYCNNSPCNEANHCCISKVYNFLITYPGITLSIYFSQLYHTEMDFPASAWNREALRSLASLWPRVVLSPISGGIWHSVLHSFVSGVSGSHVFQPILTGRALTDRYNAYEINAITGVKPFFTDVLLHTKRNPNTKAQMALESYPLNNAFPGQSFQMTSGIPPDLRAPVVFVLLPLRDLPPMHMGQDPNKPRNIIRHLNMPQMSFQETKDLERLPTRRSVETVEITERFASSKQAEEKTKKKKGKK(SEQ ID NO: 57) pmCDA-1 (Petromyzon marinus): MAGYECVRVSEKLDFDTFEFQFENLHYATERHRTYVIFDVKPQSAGGRSRRLWGYIINNPNVCHAELILMSMIDRHLESNPGVYAMTWYMSWSPCANCSSKLNPWLKNLLEEQGHTLTMHFSRIYDRDREGDHRGLRGLKHVSNSFRMGVVGRAEVKECLAEYVEASRRTLTWLDTTESMAAKMRRKLFCILVRCAGMRESGIPLHLFTLQTPLLSGRVVWWRV (SEQ ID NO: 58) pmCDA-2 (Petromyzon marinus): MELREVVDCALASCVRHEPLSRVAFLRCFAAPSQKPRGTVILFYVEGAGRGVTGGHAVNYNKQGTSIHAEVLLLSAVRAALLRRRRCEDGEEATRGCTLHCYSTYSPCRDCVEYIQEFGASTGVRVVIHCCRLYELDVNRRRSEAEGVLRSLSRLGRDFRLMGPRDAIALLLGGRLANTADGESGASGNAWVTETNVVEPLVDMTGFGDEDLHAQVQRNKQIREAYANYASAVSLMLGELHVDPDKFPFLAEFLAQTSVEPSGTPRETRGRPRGASSRGPEIGRQRPADFERALGAYGLFLHPRIVSREADREEIKRDLIVVMRKHNYQGP (SEQ ID NO: 59) pmCDA-5 (Petromyzon marinus): MAGDENVRVSEKLDFDTFEFQFENLHYATERHRTYVIFDVKPQSAGGRSRRLWGYIINNPNVCHAELILMSMIDRHLESNPGVYAMTWYMSWSPCANCSSKLNPWLKNLLEEQGHTLMMHFSRIYDRDREGDHRGLRGLKHVSNSFRMGVVGRAEVKECLAEYVEASRRTLTWLDTTESMAAKMRRKLFCILVRCAGMRESGMPLHLFT (SEQ ID NO: 60) yCD (Saccharomyces cerevisiae): MVTGGMASKWDQKGMDIAYEEAALGYKEGGVPIGGCLINNKDGSVLGRGHNMRFQKGSATLHGEISTLENCGRLEGKVYKDTTLYTTLSPCDMCTGAIIMYGIPRCVVGENVNFKSKGEKYLQTRGHEVVVVDDERCKKIMKQFIDERPQDWFEDIGE (SEQ ID NO: 61) rAPOBEC-1(Δ177-186): MSSETGPVAVDPTLRRRIEPHEFEVFFDPRELRKETCLLYEINWGGRHSIWRHTSQNTNKHVEVNFIEKFTTERYFCPNTRCSITWFLSWSPCGECSRAITEFLSRYPHVTLFIYIARLYHHADPRNRQGLRDLISSGVTIQIMTEQESGYCWRNFVNYSPSNEAHWPRYPHLWVRGLPPCLNILRRKQPQLTFFTIALQSCHYQRLPPHILWATGLK (SEQ ID NO: 62) rAPOBEC-1(Δ202-213): MSSETGPVAVDPTLRRRIEPHEFEVFFDPRELRKETCLLYEINWGGRHSIWRHTSQNTNKHVEVNFIEKFTTERYFCPNTRCSITWFLSWSPCGECSRAITEFLSRYPHVTLFIYIARLYHHADPRNRQGLRDLISSGVTIQIMTEQESGYCWRNFVNYSPSNEAHWPRYPHLWVRLYVLELYCIILGLPPCLNILRRKQPQHYQRLPPHILWATGLK (SEQ ID NO: 63) Mouse APOBEC-3: JPEG2024514649000025.jpg53152 (SEQ ID NO:64) (Italics: nucleic acid editing domain)

[0148] In some embodiments, the adenosine deaminase can include all or a portion of adenosine deaminase ADAR (e.g., ADAR1 or ADAR2). In another embodiment, the adenosine deaminase can include all or a portion of adenosine deaminase ADAT. In some embodiments, the adenosine deaminase can include all or a portion of ADAT (EcTadA) from Escherichia coli, including one or more of the following mutations: D108N, A106V, D147Y, E155V, L84F, H123Y, I157F, or a corresponding mutation in another adenosine deaminase. The adenosine deaminase can be derived from any suitable organism (e.g., E. coli). In some embodiments, the adenosine deaminase is from Escherichia coli, Staphylococcus aureus, Salmonella typhi, Shewanella putrefaciens, Haemophilus influenzae, Caulobacter crescentus, or Bacillus subtilis. In some embodiments, the adenosine deaminase is from E. coli. In some embodiments, the adenosine deaminase is a naturally occurring adenosine deaminase that includes one or more mutations corresponding to any of the mutations provided herein (e.g., mutations in ecTadA). Corresponding residues in any homologous protein can be identified, for example, by sequence alignment and determining homologous residues. Mutations in any naturally occurring adenosine deaminase (e.g., with homology to ecTadA) that correspond to any of the mutations described herein (e.g., any of the mutations identified in ecTadA) can be generated accordingly. In certain embodiments, the TadA is any one of the TadAs described in PCT / US2017 / 045381 (WO2018 / 027078), the entire contents of which are incorporated herein by reference. Mutations with the desired adenosine deaminase activity on single-stranded DNA as shown in Table 7 were identified through rounds of evolution and selection (e.g., TadA*7.10=variant 10 from the 7th round of evolution).

[0149] [Table 2-1]

[0150] [Table 2-2]

[0151] [Table 2-3]

[0152] In some embodiments, TadA is provided as a monomer or a dimer (e.g., a heterodimer of wild-type E. coli TadA and an engineered TadA variant). In some embodiments, the adenosine deaminase is an 8th generation TadA*8 variant as shown in Table 8 below.

[0153] [Table 3]

[0154] In some embodiments, the adenosine deaminase is a 9th generation TadA*9 variant containing an alteration at an amino acid position selected from the following: 21, 23, 25, 38, 51, 54, 70, 71, 72, 72, 94, 124, 133, 138, 139, 146, and 158 of the TadA variants shown in the following reference sequence: JPEG2024514649000030.jpg57137 (SEQ ID NO:65)

[0155] In one embodiment, the adenosine deaminase variant contains modifications at two or more amino acid positions selected from the following of the TadA reference sequence above: 21, 23, 25, 38, 51, 54, 70, 71, 72, 94, 124, 133, 138, 139, 146, and 158. In another embodiment, the adenosine deaminase variant contains one or more (e.g., two, three, four) modifications selected from the following: R21N, R23H, E25F, N38G, L51W, P54C, M70V, Q71M, N72K, Y73S, M94V, P124W, T133K, D139L, D139M, C146R, and A158K of SEQ ID NO:1. In other embodiments, the adenosine deaminase variant further contains one or more of the following modifications: Y147T, Y147R, Q154S, Y123H, and Q154R. In yet other embodiments, the adenosine deaminase variant contains a combination of modifications to the above TadA reference sequence selected from: E25F+V82S+Y123H,T133K+Y147R+Q154R; E25F+V82S+Y123H+Y147R+Q154R;L51W+V82S+Y123H+C146R+Y147R+Q154R; Y73S+V82S+Y123H+Y147R+Q154R; P54C+V82S+Y123H+Y147R+Q154R; N38G+V82T+Y123H+Y147R+Q154R; N72K+V82S+Y123H+D139L+Y147R+Q154R; E25F+V82S+Y123H+D139M+Y147R+Q154R; Q71M+V82S+Y123H+Y147R+Q154R; E25F+V82S+Y123H+T133K+Y147R+Q154R; E25F+V82S+Y123H+Y147R+Q154R; V82S+Y123H+P124W+Y147R+Q154R; L51W+V82S+Y123H+C146R+Y147R+Q154R; P54C+V82S+Y123H+Y147R+Q154R; Y73S+V82S+Y123H+Y147R+Q154R; N38G+V82T+Y123H+Y147R+Q154R; R23H+V82S+Y123H+Y147R+Q154R; R21N+V82S+Y123H+Y147R+Q154R; V82S+Y123H+Y147R+Q154R+A158K; N72K+V82S+Y123H+D139L+Y147R+Q154R; E25F+V82S+Y123H+D139M+Y147R+Q154R; M70V+V82S+M94V+Y123H+Y147R+Q154R; Q71M+V82S+Y123H+Y147R+Q154R;E25F+I76Y+V82S+Y123H+Y147R+Q154R;I76Y+V82T+Y123H+Y147R+Q154R;N38G+I76Y+V82S+Y123H+Y147R+Q154R; R23H+I76Y+V82S+Y123H+Y147R+Q154R; P54C+I76Y+V82S+Y123H+Y147R+Q154R; R21N+I76Y+V82S+Y123H+Y147R+Q154R; I76Y+V82S+Y123H+D138M+Y147R+Q154R; Y72S+I76Y+V82S+Y123H+Y147R+Q154R;E25F+I76Y+V82S+Y123H+Y147R+Q154R; I76Y+V82T+Y123H+Y147R+Q154R; N38G+I76Y+V82S+Y123H+Y147R+Q154R; R23H+I76Y+V82S+Y123H+Y147R+Q154R; P54C+I76Y+V82S+Y123H+Y147R+Q154R; R21N+I76Y+V82S+Y123H+Y147R+Q154R; I76Y+V82S+Y123H+D138M+Y147R+Q154R; Y72S+I76Y+V82S+Y123H+Y147R+Q154R; and V82S+Q154R; N72K+V82S+Y123H+Y147R+Q154R; Q71M+V82S+Y123H+Y147R+Q154R; V82S+Y123H+T133K+Y147R+Q154R; V82S+Y123H+T133K+Y147R+Q154R+A158K; M70V+Q71M+N72K+V82S+Y123H+Y147R+Q154R; N72K_V82S+Y123H+Y147R+Q154R; Q71M_V82S+Y123H+Y147R+Q154R;M70V+V82S+M94V+Y123H+Y147R+Q154R; V82S+Y123H+T133K+Y147R+Q154R; V82S+Y123H+T133K+Y147R+Q154R+A158K; and M70V+Q71M+N72K+V82S+Y123H+Y147R+Q154R.

[0156] In some embodiments, the deaminase or other polypeptide sequence, for example when included as a component of a fusion protein, lacks a methionine. This may result in a change in the numbering of positions. However, one of skill in the art will understand that such corresponding mutations refer to the same mutation (e.g., Y73S and Y72S, and D139M and D138M).

[0157] In some embodiments, Cas9 or Cas12 is fused to a nuclear localization sequence, including the NLS of SV40 large T antigen, nucleoplasmin, c-myc, hRNPA1M9, the IBB domain from importin-alpha, the NLS of sarcoma T protein, human p53, c-ablIV, influenza virus NS1, hepatitis virus delta antigen, mouse Mx1, human poly(ADP-ribose) polymerase, steroid hormone receptor (human) glucocorticoid.

[0158] In some embodiments, the Cas9 or Cas12 protein is fused to an epitope tag, including, but not limited to, a hemagglutinin (HA) tag, a histidine (His) tag, a FLAG tag, a Myc tag, a V5 tag, a VSV-G tag, a SNAP tag, or a thioredoxin (Trx) tag.

[0159] In some embodiments, Cas9 or Cas12 are fused to a reporter gene, including, but not limited to, glutathione-S-transferase (GST), horseradish peroxidase (HRP), chloramphenicol transferase (CAT), HcRed, DsRed, cyan fluorescent protein, yellow fluorescent protein, and blue fluorescent protein, green fluorescent protein (GFP), including enhanced versions or superfolder GFP, and other modified versions of reporter genes.

[0160] In some embodiments, the serum half-life of the engineered Cas9 or Cas12 protein is increased by fusion with a heterologous protein, such as a carboxy-terminal peptide (CTP of chorionic gonadotropin beta chain), human serum albumin protein, transferrin protein, human IgG, and / or a sialylated peptide.

[0161] In some embodiments, the serum half-life of the engineered Cas9 or Cas12 protein is decreased by fusion to a destabilization domain, including, but not limited to, geminin, ubiquitin, FKBP12-L106P, and / or dihydrofolate reductase.

[0162] Suitable fusion partners that provide increased or decreased stability include, but are not limited to, degron sequences. Degrons are readily understood by those skilled in the art to be amino acid sequences that control the stability of the protein of which they are a part. For example, the stability of a protein that includes a degron sequence is controlled at least in part by the degron sequence. In some cases, a suitable degron is constitutive such that the degron exerts its effect on protein stability independent of experimental controls (i.e., the degron is not drug-inducible, temperature-inducible, etc.). In some cases, the degron provides a variant Cas9 polypeptide with controllable stability such that the variant Cas9 polypeptide can be turned "on" (i.e., stable) or "off" (i.e., unstable, degraded) depending on desired conditions. For example, if the degron is a temperature-sensitive degron, the variant Cas9 polypeptide may be functional (i.e., "on," stable) below a threshold temperature (e.g., 42°C, 41°C, 40°C, 39°C, 38°C, 37°C, 36°C, 35°C, 34°C, 33°C, 32°C, 31°C, 30°C, etc.), but may be non-functional (i.e., "off," degraded) above the threshold temperature. As another example, if the degron is a drug-inducible degron, the presence or absence of a drug may switch the protein from an "off (i.e., unstable) state" to an "on" (i.e., stable) state, or vice versa. An exemplary drug-inducible degron is derived from the FKBP12 protein. The stability of the degron is controlled by the presence or absence of a small molecule that binds to the degron.

[0163] Examples of suitable degrons include, but are not limited to, those degrons regulated by Shield-1, DHFR, auxin, and / or temperature. Non-limiting examples of suitable degrons are known in the art (e.g., Dohmen et al., Science, 1994.263(5151):p.1273-1276:Heat-inducible degron: a method for constructing temperature-sensitive mutants; Schoeber et al., Am J Physiol Renal Physiol.2009 Jan;296(l):F204-l l:Conditional fast expression and function of multimeric TRPV5 channels using Shield-1; Chu et al., Bioorg Med Chem Lett.2008 Nov 15;18(22):5941-4:Recent progress with FKBP-derived destabilizing domains; Kanemaki, Pflugers Arch.2012 Dec 28:Frontiers of protein expression control with conditional degrons; Yang et al., Mol Cell.2012 Nov 30;48(4):487-8:Titivated for destruction:the methyl degron, Barbour et al.,Biosci Rep.2013 Jan 18;33(1).:Characterization of the bipartite degron that regulates ubiquitin-independent degradation of thymidylate synthase, and Greussing et al.,J Vis Exp.2012 Nov 10;(69):Monitoring of ubiquitin-proteasome activity in living cells using a Degron (dgn)-destabilized green fluorescent protein (GFP)-based reporter protein, all of which are incorporated by reference in their entireties.

[0164] Exemplary degron sequences have been well characterized and tested in both cells and animals, and thus, fusing an inactive Cas9 or Cas12 to a degron sequence produces a "regulatable" and "inducible" inactive Cas9 or Cas12 polypeptide.

[0165] Any of the fusion partners described herein can be used in any desired combination. As one non-limiting example to illustrate this point, Cas9 or Cas12 fusion protein can include a YFP sequence for detection, a degron sequence for stability, and a transcription activator sequence for increasing transcription of target DNA. Furthermore, the number of fusion partners that can be used in dCas9 fusion protein is unlimited. In some cases, Cas9 fusion protein includes one or more (e.g., two or more, three or more, four or more, or five or more) heterologous sequences.

[0166] Recombinant Gene Technology In accordance with the present disclosure there may be employed conventional molecular biology, microbiology, and recombinant DNA techniques within the skill of the art. Such techniques are described in the literature (e.g., Sambrook, Fritsch & Maniatis, Molecular Cloning: A Laboratory Manual, Second Edition (1989) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY; DNA Cloning: A Practical Approach, Volumes I and II (DN Glover ed. 1985); Oligonucleotide Synthesis (MJ Gait ed. 1984); Nucleic Acid Hybridization (B.D. Hames & S.J. Higgins eds. (1985)); Transcription And Translation (B.D. Hames & S.J. Higgins, eds. (1984)); Animal Cell Culture (R.I. Freshney, ed. (1986)); Immobilized Cells and Enzymes (IRL Press, (1986)); B. Perbal, A Practical Guide To Molecular Cloning (1984); F.M. Ausubel et al. (eds.), Current See Protocols in Molecular Biology, John Wiley & Sons, Inc. (1994).

[0167] Recombinant expression of a gene, such as a nucleic acid encoding a polypeptide, such as the engineered Cas9 or Cas12 enzyme described herein, can include the construction of an expression vector containing the nucleic acid encoding the polypeptide. Once a polynucleotide is obtained, a vector for the production of the polypeptide can be produced by recombinant DNA technology using techniques known in the art. Using known methods, an expression vector containing a polypeptide coding sequence and appropriate transcriptional and translational control signals can be constructed. These methods include, for example, in vitro recombinant DNA techniques, synthetic techniques, and in vivo genetic recombination.

[0168] The expression vector can be transferred to a host cell by conventional techniques and the transfected cells can then be cultured by conventional techniques to produce the polypeptide.

[0169] In some embodiments, the nucleotide sequence encoding the DNA-targeting RNA and / or the Cas9 or Cas12 protein is operably linked to a control element, e.g., a transcription control element, such as a promoter. The transcription control element can be functional in either a eukaryotic cell, e.g., a mammalian cell, or a prokaryotic cell (e.g., a bacterial or archaeal cell). In some embodiments, the eukaryotic cell is a human cell. In some embodiments, the nucleotide sequence encoding the DNA-targeting RNA and / or the Cas9 or Cas12 protein is operably linked to multiple control elements that allow expression of the encoded nucleotide sequence in both prokaryotic and eukaryotic cells.

[0170] The promoter may be a constitutively active promoter (i.e., a promoter that is constitutively active / "ON" state), but may also be an inducible promoter (i.e., a promoter whose state, active / "ON" or inactive / "OFF", is controlled by an external stimulus, e.g., a particular temperature, compound, or the presence of a protein), may be a spatially restricted promoter (i.e., a transcriptional control element, enhancer, etc.) (e.g., a tissue-specific promoter, a cell type-specific promoter, etc.), or may be a temporally restricted promoter (i.e., the promoter is in the "ON" or "OFF" state during a particular stage of embryonic development or during a particular stage of a biological process, e.g., the hair follicle cycle in mice).

[0171] Suitable promoters may be derived from a virus, and therefore may be referred to as viral promoters, or they may be derived from any organism, including prokaryotes or eukaryotes. Suitable promoters can be used to drive expression by any RNA polymerase (e.g., pol I, pol II, pol III). Exemplary promoters include, but are not limited to, the SV40 early promoter, the mouse mammary tumor virus long terminal repeat (LTR) promoter, the adenovirus major late promoter (Ad MLP), the herpes simplex virus (HSV) promoter, the cytomegalovirus (CMV) promoter, e.g., the CMV immediate early promoter region (CMVIE), the Rous sarcoma virus (RSV) promoter, the human U6 micronucleus promoter (U6) (Miyagishi et al., Nature Biotechnology 20, 497-500 (2002)), the enhanced U6 promoter (e.g., Xia et al., Nucleic Acids Res. 2003 Sep 1; 31 (17)), and / or the human HI promoter (HI).

[0172] Examples of inducible promoters include, but are not limited to, T7 RNA polymerase promoter, T3 RNA polymerase promoter, isopropyl-beta-D-thiogalactopyranoside (IPTG) regulated promoter, lactose inducible promoter, heat shock promoter, tetracycline regulated promoter (e.g., Tet-ON, Tet-OFF, etc.), steroid regulated promoter, metal regulated promoter, estrogen receptor regulated promoter, etc. Thus, an inducible promoter can be regulated by molecules including, but not limited to, doxycycline, RNA polymerase (e.g., T7 RNA polymerase), estrogen receptor, and / or estrogen receptor fusions.

[0173] In some embodiments, the promoter is a spatially restricted promoter (i.e., a cell type specific promoter, a tissue specific promoter, etc.), and in a multicellular organism, the promoter is active (i.e., "ON") in a specific subset of cells. A spatially restricted promoter may also be referred to as an enhancer, a transcriptional control element, a control sequence, etc. Any convenient spatially restricted promoter may be used, and the selection of a suitable promoter (e.g., a brain-specific promoter, a promoter that drives expression in a subset of neurons, a promoter that drives expression in the germline, a promoter that drives expression in the lung, a promoter that drives expression in muscle, a promoter that drives expression in islet cells of the pancreas, etc.) will depend on the organism. Thus, a spatially restricted promoter can be used to regulate the expression of a nucleic acid encoding a site-specific polypeptide of interest in a wide variety of different tissues and cell types, depending on the organism. Some spatially restricted promoters are also temporally restricted, such that the promoter is in an "ON" or "OFF" state during a particular stage of embryonic development or during a particular stage of a biological process (e.g., the hair follicle cycle).

[0174] Nucleic acid base editor In some embodiments, any of the base editors provided herein result in less than 50%, less than 40%, less than 30%, less than 20%, less than 19%, less than 18%, less than 17%, less than 16%, less than 15%, less than 14%, less than 13%, less than 12%, less than 11%, less than 10%, less than 9%, less than 8%, less than 7%, less than 6%, less than 5%, less than 4%, less than 3%, less than 2%, less than 1%, less than 0.9%, less than 0.8%, less than 0.7%, less than 0.6%, less than 0.5%, less than 0.4%, less than 0.3%, less than 0.2%, less than 0.1%, less than 0.09%, less than 0.08%, less than 0.07%, less than 0.06%, less than 0.05%, less than 0.04%, less than 0.03%, less than 0.02%, or less than 0.01% indel formation in the target polynucleotide sequence.

[0175] Some aspects of the present disclosure are based on the recognition that any of the base editors provided herein can efficiently generate intended mutations, e.g., point mutations, in a nucleic acid (e.g., a nucleic acid in a genome of a subject) without generating a significant number of unintended mutations, such as unintended point mutations. In some embodiments, any of the base editors provided herein can generate at least 0.01% intended mutations (i.e., at least 0.01% base editing efficiency). In some embodiments, any of the base editors provided herein can generate at least 0.01%, 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 40%, 45%, 50%, 60%, 70%, 80%, 90%, 95%, or 99% of intended mutations.

[0176] In some embodiments, the base editors provided herein are capable of generating a ratio of intended point mutations to indels of greater than 1: 1. In some embodiments, the base editors provided herein are capable of generating a ratio of intended point mutations to indels of greater than 1: 1. In some embodiments, the base editors provided herein are capable of generating a ratio of intended point mutations to indels of greater than 1: 1. In some embodiments, a ratio of intended point mutations to indels can be generated that is at least 13:1, at least 14:1, at least 15:1, at least 20:1, at least 25:1, at least 30:1, at least 40:1, at least 50:1, at least 100:1, at least 200:1, at least 300:1, at least 400:1, at least 500:1, at least 600:1, at least 700:1, at least 800:1, at least 900:1, or at least 1000:1 or more.

[0177] The number of intended mutations and indels can be determined, for example, from International PCT Application Nos. PCT / 2017 / 045381 (WO2018 / 027078) and PCT / US2016 / 058344 (WO2017 / 070632), Komor, AC, et al., “Programmable editing of a target base in genomic DNA without double-stranded DNA cleavage” Nature 533, 420-424 (2016), Gaudelli, NM, et al., “Programmable base editing of A·T to G·C in genomic DNA without DNA cleavage” Nature 551, 464-471 (2017), and Komor, AC, et al., “Improved base excision repair inhibition and bacteriophage Mu Gam protein yields C:G-to-T:A base editors with higher efficiency and product purity” Science Advances 3:eaao4774 (2017), the contents of which are incorporated herein by reference in their entireties.

[0178] In some embodiments, to calculate the indel frequency, sequencing reads are scanned for exact matches with two 10 bp sequences flanking the window in which an indel may occur. If an exact match is not found, the read is excluded from the analysis. If the length of this indel window is an exact match with the reference sequence, the read is classified as not containing an indel. If the indel window is more than one base longer or shorter than the reference sequence, the sequencing read is classified as an insertion or deletion, respectively. In some embodiments, the base editors provided herein can limit the formation of indels in a region of a nucleic acid. In some embodiments, the region is at the nucleotide targeted by the base editor or within 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides of the nucleotide targeted by the base editor.

[0179] The number of indels formed at a target nucleotide region can depend on the amount of time a nucleic acid (e.g., a nucleic acid in the genome of a cell) is exposed to a base editor. In some embodiments, the number or percentage of indels is determined at least 1 hour, at least 2 hours, at least 6 hours, at least 12 hours, at least 24 hours, at least 36 hours, at least 48 hours, at least 3 days, at least 4 days, at least 5 days, at least 7 days, at least 10 days, or at least 14 days after exposing a target nucleotide sequence (e.g., a nucleic acid in the genome of a cell) to a base editor. It is understood that the features of base editors described herein can be applied to any of the fusion proteins or methods of using the fusion proteins provided herein.

[0180] therapeutic use The methods and compositions described herein can have a variety of therapeutic applications (eg, in the treatment of liver disease).

[0181] In some embodiments, the CRISPR methods or systems described herein can be used to edit a target nucleic acid to modify the target nucleic acid (e.g., by inserting, deleting, or mutating one or more nucleic acid residues). For example, in some embodiments, the CRISPR systems described herein include an exogenous donor template nucleic acid (e.g., a DNA molecule or an RNA molecule) that includes a desired nucleic acid sequence. After resolution of the cleavage event induced by the CRISPR systems described herein, the molecular machinery of the cell will utilize the exogenous donor template nucleic acid in repairing and / or resolving the cleavage event. Alternatively, the molecular machinery of the cell can utilize an endogenous template in repairing and / or resolving the cleavage event. In some embodiments, the CRISPR systems described herein can be used to modify a target nucleic acid to result in an insertion, deletion, and / or point mutation. In some embodiments, the insertion is a scarless insertion (i.e., insertion of an intended nucleic acid sequence into a target nucleic acid that does not result in additional unintended nucleic acid sequences upon resolution of the cleavage event). The donor template nucleic acid can be a double-stranded or single-stranded nucleic acid molecule (e.g., DNA or RNA). In some embodiments, the CRISPR methods or systems described herein comprise a nucleobase editor.

[0182] In applications where it is desired to insert a polynucleotide sequence into a target DNA sequence, a polynucleotide comprising the donor sequence to be inserted is also provided to the cell. By "donor sequence" or "donor polynucleotide" is meant a nucleic acid sequence to be inserted at the cleavage site induced by the site-directed modifying polypeptide. The donor polynucleotide contains sufficient homology to the genomic sequence at the cleavage site, e.g., 70%, 80%, 85%, 90%, 95%, or 100% homology to the nucleotide sequence adjacent to the cleavage site, e.g., within about 50 bases or less, e.g., within about 30 bases, within about 15 bases, within about 10 bases, within about 5 bases, or immediately adjacent to the cleavage site, to support homology-directed repair between the genomic sequence with which it has homology. Approximately 25, 50, 100, or 200 nucleotides, or more than 200 nucleotides of sequence homology between the donor and the genomic sequence (or any integer value between 10 and 200 nucleotides or more) will support homology-directed repair. The donor sequence can be of any length, e.g., 10 nucleotides or more, 50 nucleotides or more, 100 nucleotides or more, 250 nucleotides or more, 500 nucleotides or more, 1000 nucleotides or more, 5000 nucleotides or more, etc.

[0183] The donor sequence is typically not identical to the genomic sequence it replaces. Rather, the donor sequence may contain at least one or more single base changes, insertions, deletions, inversions, or translocations with respect to the genomic sequence, so long as there is sufficient homology to support homology-directed repair. In some embodiments, the donor sequence contains two homologous regions and flanking non-homologous sequences, such that homology-directed repair between the target DNA region and the two flanking sequences results in the insertion of the non-homologous sequence into the target region. The donor sequence may also include a vector backbone that contains sequences that are not homologous to the DNA region of interest and are not intended for insertion into the DNA region of interest. In general, the homologous regions of the donor sequence will have at least 50% sequence identity with the genomic sequence with which recombination is desired. In certain embodiments, there is 60%, 70%, 80%, 90%, 95%, 98%, 99%, or 99.9% sequence identity. Depending on the length of the donor polynucleotide, there may be any value of sequence identity between 1% and 100%.

[0184] The donor sequence may contain certain sequence differences compared to the genomic sequence, such as restriction sites, nucleotide polymorphisms, selectable markers (e.g., drug resistance genes, fluorescent proteins, enzymes, etc.), which may be used to evaluate the successful insertion of the donor sequence at the cleavage site, or in some cases, for other purposes (e.g., to indicate expression at the targeted genomic locus). In some cases, when located in a coding region, such nucleotide sequence differences will not change the amino acid sequence or will make silent amino acid changes (i.e., changes that do not affect the structure or function of the protein). Alternatively, these sequence differences may include adjacent recombination sequences, such as FLP, loxP sequences, which can be activated later to remove the marker sequence.

[0185] The donor sequence may be provided to the cell as single-stranded DNA, single-stranded RNA, double-stranded DNA, or double-stranded RNA. It may be introduced into the cell in linear or circular form. If introduced in linear form, the ends of the donor sequence may be protected (e.g., from exonucleolytic degradation) by methods known to those skilled in the art. For example, one or more dideoxynucleotide residues may be added to the 3' end of the linear molecule and / or a self-complementary oligonucleotide may be ligated to one or both ends. Additional methods for protecting exogenous polynucleotides from degradation include, but are not limited to, the addition of terminal amino groups and the use of modified internucleotide linkages, such as, for example, phosphorothioates, phosphoramidates, and O-methyl ribose or deoxyribose residues. As an alternative to protecting the ends of linear donor sequences, additional lengths of sequence may be included outside the regions of homology that may be degraded without affecting recombination. The donor sequence may be introduced into the cell as part of a vector molecule with additional sequences, such as, for example, an origin of replication, a promoter, and a gene encoding antibiotic resistance. Additionally, the donor sequence can be introduced as naked nucleic acid, as nucleic acid complexed with an agent such as a liposome or poloxamer, or delivered by a virus (e.g., adenovirus, AAV), as described above for the nucleic acid encoding the DNA-targeting RNA and / or the site-directed modifying polypeptide and / or donor polynucleotide.

[0186] According to the methods described above, the DNA region of interest can be cleaved and modified, i.e., "genetically modified", ex vivo. In some embodiments, as in the case where a selectable marker is inserted into the DNA region of interest, the population of cells can be enriched for those containing the genetic modification by separating the genetically modified cells from the remaining population. Prior to enrichment, the "genetically modified" cells may only constitute about 1% or more (e.g., 2% or more, 3% or more, 4% or more, 5% or more, 6% or more, 7% or more, 8% or more, 9% or more, 10% or more, 15% or more, or 20% or more) of the cell population. Separation of the "genetically modified" cells can be achieved by any convenient separation technique appropriate for the selectable marker used. For example, if a fluorescent marker is inserted, the cells can be separated by fluorescence activated cell sorting, and if a cell surface marker is inserted, the cells can be separated from the heterogeneous population by affinity separation techniques, such as magnetic separation, affinity chromatography, "panning" with affinity reagents bound to a solid matrix, or other convenient techniques. Techniques that provide accurate separation include fluorescence-activated cell sorters, which can have various degrees of sophistication, such as multiple color channels, low-angle and obtuse-angle light scattering detection channels, impedance channels, etc. Cells can be selected against dead cells by using a dye that is associated with dead cells (e.g., propidium iodide). Any technique that is not overly detrimental to the viability of the genetically modified cells can be used. A cell composition highly enriched for cells containing modified DNA can be achieved in this manner. By "highly enriched" it is meant that the genetically modified cells are 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, e.g., about 95% or more, or 98% or more of the cell composition. In other words, the composition can be a substantially pure composition of genetically modified cells.

[0187] The genetically modified cells (e.g., genetically modified human hepatocytes) produced by the methods described herein can be used immediately. Alternatively, the cells can be frozen at liquid nitrogen temperatures, stored for extended periods, and thawed and reused. In such cases, the cells are typically frozen in 10% dimethylsulfoxide (DMSO), 50% serum, 40% buffered medium, or some other such solution commonly used in the art to preserve cells at such freezing temperatures, and thawed in a manner commonly known in the art to thaw frozen cultured cells.

[0188] The genetically modified cells may be cultured in vitro under a variety of culture conditions. The cells may be grown in culture, i.e., under conditions that promote the growth of the cells. The culture medium may be liquid or semi-solid (e.g., containing agar, methylcellulose, etc.). The cell population may be suspended in a suitable nutrient medium, such as Iscove's modified DMEM or RPMI 1640, usually supplemented with fetal bovine serum (about 5%-10%), L-glutamine, thiols (especially 2-mercaptoethanol), and antibiotics (penicillin and streptomycin). The culture may contain growth factors to which the regulatory T cells are responsive. Growth factors, as defined herein, are molecules that can promote the survival, proliferation and / or differentiation of cells, either in culture or in intact tissue, through specific effects on transmembrane receptors. Growth factors include polypeptide and non-polypeptide factors.

[0189] Such genetically modified cells can be implanted into a subject, for example, to treat a disease or as an antiviral, antipathogenic, or anticancer therapeutic, for purposes such as gene therapy, for the production of genetically modified organisms in agriculture, or for biological research. The subject can be a neonate, juvenile, or adult. Of particular interest are mammalian subjects. Mammalian species that can be treated with the method include dogs and cats, horses, cows, sheep, etc., as well as primates, especially humans. Animal models, especially small mammals (e.g., mice, rats, guinea pigs, hamsters, lagomorphs (e.g., rabbits), etc.), can be used for experimental investigations.

[0190] The cells may be provided to a subject alone or with a suitable substrate or matrix, for example to support the growth and / or organization of the cells within the tissue into which they are implanted. In some embodiments, the cells may be introduced into a subject via any of the following routes: parenteral, subcutaneous, intravenous, intracranial, intraspinal, intraocular, or in the spinal fluid. The cells may be introduced by injection, catheter, etc.

[0191] The number of times that the treatment is administered to a subject may vary. Introducing genetically modified cells into a subject may be a one-time event, but in certain circumstances, such treatment may induce improvement over a limited period of time and require a series of ongoing repeated treatments. In other circumstances, multiple administrations of genetically modified cells may be required before an effect is observed. The exact protocol depends on the disease or condition, the stage of the disease, and the parameters of the individual subject being treated.

[0192] A pharmaceutical preparation is a composition comprising one or more of the base editors or base editor systems described herein in a pharma- ceutically acceptable vehicle. A "pharma-ceutically acceptable vehicle" can be a vehicle approved by a federal or state government regulatory agency or listed in the United States.

[0193] Pharmacopoeias or other generally accepted pharmacopoeias for use in mammals, such as humans. The term "vehicle" refers to a diluent, adjuvant, excipient, or carrier in which the compound of the present invention is formulated for administration to a mammal. Such pharmaceutical vehicles can be lipids, such as liposomes, e.g., liposomal dendrimers; oils, including those of petroleum, animal, vegetable, or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil, and the like, liquids, such as saline; gum acacia, gelatin, starch paste, talc, keratin, colloidal silica, urea, and the like. In addition, auxiliary agents, stabilizers, thickeners, lubricants, and colorants can be used. The pharmaceutical compositions can be formulated into solid, semi-solid, liquid, or gaseous form of preparations, such as tablets, capsules, powders, granules, ointments, solutions, suppositories, injections, inhalants, gels, microparticles, and aerosols. Thus, administration of DNA-targeting RNA and / or site-directed modified polypeptides and / or donor polynucleotides can be accomplished in a variety of ways, including oral, buccal, rectal, parenteral, intraperitoneal, intradermal, transdermal, intratracheal, intraocular, etc. The active agent can be systemic following administration, or localized by use of topical administration, intramural administration, or the use of an implant that acts to retain the active dose at the site of implantation. The active agent can be formulated for immediate activity or for sustained release.

[0194] The effective amount given to a particular patient will depend on a variety of factors, some of which will vary from patient to patient. A competent clinician will be able to determine the effective amount of therapeutic agent to administer to a patient to halt or reverse the progression of a disease state, if necessary. Using LD50 animal data, and other available information about the agent, the clinician will be able to determine the maximum safe dose for an individual, depending on the route of administration. For example, a dose administered intravenously may be higher than a dose administered intrathecally, given the larger volume of fluid into which the therapeutic composition is administered. Similarly, compositions that are rapidly cleared from the body may be administered at higher doses or in repeated doses to maintain therapeutic concentrations. Using routine techniques, a competent clinician will be able to optimize the dosage of a particular therapeutic agent during the course of routine clinical trials.

[0195] The pharmaceutical composition may contain a pharma- ceutically acceptable non-toxic carrier of a diluent, which is defined as a vehicle commonly used to formulate pharmaceutical compositions for animal or human administration, depending on the desired formulation. The diluent is selected so as not to affect the biological activity of the combination. Examples of such diluents are distilled water, buffered water, physiological saline, PBS, Ringer's solution, dextrose solution, and Hank's solution. In addition, the pharmaceutical composition or formulation may contain other carriers, adjuvants, or non-toxic, non-therapeutic, non-immunogenic stabilizers, excipients, and the like. The composition may also contain additional substances that approximate physiological conditions, such as pH adjusting and buffering agents, toxicity adjusting agents, wetting agents, and detergents.

[0196] The composition may also include any of a variety of stabilizing agents, such as, for example, antioxidants. When the pharmaceutical composition includes a polypeptide, the polypeptide may be complexed with a variety of well-known compounds that enhance the in vivo stability of the polypeptide or otherwise enhance its pharmacological properties (e.g., increase the half-life of the polypeptide, reduce its toxicity, enhance solubility or uptake). Examples of such modifying or complexing agents include sulfates, gluconates, citrates, and phosphates. The nucleic acids or polypeptides of the composition may also be complexed with molecules that enhance their in vivo attributes. Such molecules include, for example, carbohydrates, polyamines, amino acids, other peptides, ions (e.g., sodium, potassium, calcium, magnesium, manganese), and lipids.

[0197] The pharmaceutical composition can be administered for prophylactic and / or therapeutic treatment. Toxicity and therapeutic efficacy of the active ingredient can be determined according to standard pharmaceutical procedures in cell cultures and / or experimental animals, including, for example, determining LD50 (the dose lethal to 50% of the population) and ED50 (the dose therapeutically effective for 50% of the population). The dose ratio between toxic effect and therapeutic effect is the therapeutic index, which can be expressed as the ratio LD50 / ED50. Therapies that exhibit large therapeutic indices are preferred.

[0198] Data obtained from cell culture and / or animal studies can be used in formulating a range of dosages for humans.The dosage of active ingredient is typically within the range of circulating concentrations that include the ED50 with low toxicity.Dosages can vary within this range depending on the dosage form used and the route of administration utilized.

[0199] Ingredients used to formulate pharmaceutical compositions are preferably of high purity and substantially free of potentially harmful contaminants (e.g., at least National Food (NF) grade, generally at least analytical grade, and more typically at least pharmaceutical grade). Moreover, compositions intended for in vivo use are usually sterile. To the extent that a given compound must be synthesized prior to use, the resulting product is typically substantially free of any potentially toxic agents, particularly any endotoxins, that may be present during the synthesis or purification process. Compositions for parental administration are also sterile, substantially isotonic, and made under GMP conditions.

[0200] Delivery System The base editors or base editor systems described herein, or components thereof, nucleic acid molecules thereof, and / or nucleic acid molecules encoding or providing the components thereof, CRISPR-associated proteins, or RNA guides can be delivered by various delivery systems, such as vectors (such as plasmids and delivery vectors). Exemplary embodiments are described below. The base editors or base editor systems (e.g., including Cas9 or Cas12, and optionally including the nucleic acid base editors described herein) can be encoded by nucleic acids contained in viral vectors. Viral vectors can include lentiviruses, adenoviruses, retroviruses, and adeno-associated viruses (AAVs). Viral vectors can be selected based on the application. For example, AAVs are commonly used for in vivo gene delivery due to their mild immunogenicity. Adenoviruses are commonly used as vaccines because they induce a strong immunogenic response. The packaging capacity of the viral vector can limit the size of the base editor that can be packaged into the vector. For example, the packaging capacity of AAV is approximately 4.5 kb, including two 145-base inverted terminal repeats (ITRs).

[0201] AAV is a small, single-stranded DNA-dependent virus belonging to the Parvoviridae family. The 4.7 kb wild-type (wt) AAV genome consists of two genes encoding four replication proteins and three capsid proteins, respectively, flanked on either side by 145 bp inverted terminal repeats (ITRs). Virions are composed of three capsid proteins, Vp1, Vp2, and Vp3, which are generated in a 1:1:10 ratio from the same open reading frame, but from differential splicing (Vp1) and alternative translation initiation sites (Vp2 and Vp3, respectively). Vp3 is the most abundant subunit in the virion and is responsible for receptor recognition at the cell surface that defines the viral tropism. A phospholipase domain that functions in viral infectivity has been identified in the unique N-terminus of Vp1.

[0202] Similar to wtAAV, recombinant AAV (rAAV) utilizes 145 bp cis-acting ITRs to flank the vector transgene cassette, providing up to 4.5 kb for packaging of foreign DNA. After infection, rAAV can express the fusion protein of the invention and persist without integrating into the host genome by existing episomally in circular head-to-tail concatemers. Although there are many successful examples of rAAV using this system in vitro and in vivo, packaging capacity limitations limit the use of AAV-mediated gene delivery when the length of the gene coding sequence is comparable to or greater than the wtAAV genome.

[0203] The small packaging capacity of AAV vectors makes it difficult to deliver some genes that exceed this size and / or use large physiological regulatory elements. These challenges can be addressed, for example, by splitting the protein to be delivered into two or more fragments, with the N-terminal fragment fused to a split intein-N and the C-terminal fragment fused to a split intein-C. These fragments are then packaged into two or more AAV vectors. As used herein, "intein" refers to a self-splicing protein intron (e.g., a peptide) that ligates adjacent N-terminal and C-terminal exteins (e.g., fragments that join). The use of certain inteins to link heterologous protein fragments is described, for example, in Wood et al., J.Biol.Chem.289(21);14512-9(2014). The inteins IntN and IntC, for example, when fused to separate protein fragments, recognize each other and splice them out while simultaneously ligating the adjacent N- and C-terminal exteins of the protein fragments to which they are fused, thereby reconstituting a full-length protein from the two protein fragments. Other suitable inteins will be apparent to those skilled in the art.

[0204] In some embodiments, the CRISPR systems of the present invention can vary in length. In some embodiments, the protein fragments range from 2 amino acids to about 1000 amino acids in length. In some embodiments, the protein fragments range from about 5 amino acids to about 500 amino acids in length. In some embodiments, the protein fragments range from about 20 amino acids to about 200 amino acids in length. In some embodiments, the protein fragments range from about 10 amino acids to about 100 amino acids in length. Other lengths of suitable protein fragments will be apparent to one of skill in the art.

[0205] In some embodiments, a portion or fragment of a nuclease (e.g., Cas9 or Cas12) is fused to an intein. The nuclease may be fused to the N-terminus or C-terminus of the intein. In some embodiments, a portion or fragment of a fusion protein is fused to an intein and fused to an AAV capsid protein. The intein, nuclease, and capsid protein may be fused together in any configuration (e.g., nuclease-intein-capsid, intein-nuclease-capsid, capsid-intein-nuclease, etc.). In some embodiments, the N-terminus of the intein is fused to the C-terminus of the fusion protein and the C-terminus of the intein is fused to the N-terminus of the AAV capsid protein.

[0206] In one embodiment, dual AAV vectors are generated by splitting a large transgene expression cassette into two separate halves (5' and 3' ends, or head and tail), and each half of the cassette is packaged into a single AAV vector (less than 5 kb). Reconstitution of the full-length transgene expression cassette is then achieved upon coinfection of the same cells with both dual AAV vectors followed by (1) homologous recombination (HR) between the 5' and 3' genomes (dual AAV overlapping vectors), (2) ITR-mediated tail-to-head concatemerization of the 5' and 3' genomes (dual AAV trans-splicing vectors), or (3) a combination of these two mechanisms (dual AAV hybrid vectors). The use of dual AAV vectors in vivo results in the expression of full-length proteins. The use of a dual AAV vector platform represents an efficient and viable gene transfer strategy for transgenes greater than 4.7 kb in size.

[0207] The disclosed strategies for designing base editors described herein may be useful for generating base editors that can be packaged into viral vectors. The use of RNA or DNA virus-based systems for delivery of base editors takes advantage of the highly evolved processes for targeting viruses to specific cells in culture or within a host and transporting the viral payload to the nucleus or host cell genome. Viral vectors can be administered directly to cells in culture, to patients (in vivo), or they can be used to treat cells in vitro, and the modified cells can optionally be administered to patients (ex vivo). Traditional virus-based systems can include retroviral, lentiviral, adenoviral, adeno-associated viral, and herpes simplex viral vectors for gene transfer. Integration into the host genome is possible with retroviral, lentiviral, and adeno-associated viral gene transfer methods, often resulting in long-term expression of the inserted transgene. Furthermore, high transduction efficiencies have been observed in many different cell types and target tissues.

[0208] The tropism of retroviruses can be altered by incorporating foreign envelope proteins and expanding the potential target population of target cells. Lentiviral vectors are retroviral vectors that can transduce or infect non-dividing cells and typically generate high viral titers. Thus, the choice of retroviral gene transfer system will depend on the target tissue. Retroviral vectors are composed of cis-acting long terminal repeats with packaging capacity for up to 6-10 kb of foreign sequence. The minimal cis-acting LTRs are sufficient for vector replication and packaging, which are then used to integrate therapeutic genes into target cells to provide persistent transgene expression. Widely used retroviral vectors include those based on murine leukemia virus (MuLV), gibbon ape leukemia virus (GaLV), simian immunodeficiency virus (SIV), human immunodeficiency virus (HIV), and combinations thereof (see, e.g., Buchscher et al., J. Virol. 66:2731-2739 (1992); Johann et al., J. Virol. 66:1635-1640 (1992); Sommnerfelt et al., Virol. 176:58-59 (1990); Wilson et al., J. Virol. 63:2374-2378 (1989); Miller et al., J. Virol. 65:2220-2224 (1991); PCT / US94 / 05700).

[0209] Retroviral vectors, particularly lentiviral vectors, may require a polynucleotide sequence smaller than a given length for efficient integration into target cells.For example, retroviral vectors longer than 9kb may result in low viral titers compared to smaller sizes.In some aspects, the CRISPR system of the present disclosure (e.g., including Cas9 as disclosed herein) is of sufficient size to allow efficient packaging and delivery to target cells via retroviral vectors.In some cases, Cas9 is of such size that it allows efficient packaging and delivery even when expressed together with guide nucleic acid and / or other components of targetable nuclease system.

[0210] For applications where transient expression is preferred, adenovirus-based systems can be used. Adenovirus-based vectors can have very high transduction efficiency in many cell types and do not require cell division. High titers and expression levels have been obtained using such vectors. The vectors can be produced in large quantities in a relatively simple system. Adeno-associated virus ("AAV") vectors can also be used to transduce cells with target nucleic acids, for example, in in vitro production of nucleic acids and peptides, for in vivo and ex vivo gene therapy procedures (see, e.g., West et al., Virology 160:38-47 (1987); U.S. Patent No. 4,797,368; WO93 / 24641; Kotin, Human Gene Therapy 5:793-801 (1994); Muzyczka, J. Clin. Invest. 94:1351 (1994)). The construction of recombinant AAV vectors has been described in several publications, including U.S. Pat. No. 5,173,414, Tratschin et al., Mol. Cell. Biol. 5:3251-3260 (1985), Tratschin, et al., Mol. Cell. Biol. 4:2072-2081 (1984), Hermonat & Muzyczka, PNAS 81:6466-6470 (1984), and Samulski et al., J. Virol. 63:03822-3828 (1989).

[0211] Thus, a base editor or base editor system (including, for example, Cas9 or Cas12 as disclosed herein) can be delivered in a viral vector. One or more components of the base editor system can be encoded on one or more viral vectors. For example, the base editor and guide nucleic acid can be encoded on a single viral vector. In other cases, the base editor and guide nucleic acid are encoded on different viral vectors. In either case, the base editor and guide nucleic acid can each be operably linked to a promoter and a terminator.

[0212] The combination of components encoded on the viral vector can be determined by the cargo size constraints of the selected viral vector.

[0213] Non-viral delivery of base editors Non-viral delivery approaches for base editors and base editor systems are also available. One important category of non-viral nucleic acid vectors are nanoparticles, which can be organic or inorganic. Nanoparticles are well known in the art. Any suitable nanoparticle design can be used to deliver the components of genome editing systems, or the nucleic acids encoding such components. For example, organic (e.g., lipid and / or polymer) nanoparticles can be suitable for use as delivery vehicles in certain embodiments of the present disclosure. Exemplary lipids for use in nanoparticle formulations and / or gene transfer are shown in Table 9 (below).

[0214] [Table 4-1]

[0215] [Table 4-2]

[0216] Table 10 lists exemplary polymers for use in gene transfer and / or nanoparticle formulations.

[0217] [Table 5]

[0218] Table 11 summarizes the delivery methods for the Cas9-encoding polynucleotides described herein.

[0219] [Table 6]

[0220] In another aspect, delivery of genome editing system components or nucleic acids encoding such components, e.g., nucleic acid binding proteins, such as Cas9 or variants thereof or Cas12 or variants thereof, optionally fused to a polypeptide having biological activity (e.g., a nucleic acid base editor), and a gRNA targeting a genomic nucleic acid sequence of interest, can be achieved by delivering ribonucleoproteins (RNPs) to cells. RNPs contain a nucleic acid binding protein (e.g., Cas9) in a complex with a target gRNA. RNPs can be delivered to cells using known methods, such as electroporation, nucleofection, or cationic lipid-mediated methods, as reported, for example, by Zuris, JA et al., 2015, Nat. Biotechnology, 33(1):73-80. RNPs are advantageous for use in CRISPR base editor systems, especially in cells that are difficult to transfect (e.g., primary cells). In addition, RNPs can also alleviate the difficulties that may occur in protein expression in cells, especially when eukaryotic promoters (e.g., CMV or EF1A) that may be used in CRISPR plasmids are not fully expressed. Advantageously, the use of RNPs does not require the delivery of foreign DNA into cells. Furthermore, the use of RNPs may limit off-target effects, since the RNPs containing nucleic acid binding proteins and gRNA complexes are degraded over time. In a manner similar to that of plasmid-based technology, RNPs can be used to deliver binding proteins (e.g., Cas9 variants or Cas12 variants) and induce homology-directed repair (HDR).

[0221] The promoter used to drive the base editor or base editor system (including, for example, Cas9 or Cas12 as described herein) can include AAV ITRs. This can be advantageous to eliminate the need for additional promoter elements that may take up space in the vector. The additional space freed can be used to drive expression of additional elements (e.g., guide nucleic acids or selectable markers). Since ITR activity is relatively weak, it can be used to reduce potential toxicity due to overexpression of the selected nuclease.

[0222] Any suitable promoter may be used to drive the expression of Cas9 and, if applicable, guide nucleic acid. For ubiquitous expression, promoters that may be used include CMV, CAG, CBh, PGK, SV40, ferritin heavy or light chain, etc. For expression in brain or other CNS cells, suitable promoters may include synapsin I for all neurons, CaMKII alpha for excitatory neurons, GAD67 or GAD65 or VGAT for GABAergic neurons, etc. For expression in hepatocytes, suitable promoters may include albumin promoter. For expression in lung cells, suitable promoters may include SP-B. For endothelial cells, suitable promoters may include ICAM. For hematopoietic cells, suitable promoters may include IFN beta or CD45. For osteoblasts, suitable promoters may include OG-2.

[0223] In some cases, the Cas9 of the disclosure is of a small enough size to allow separate promoters to drive expression of a base editor and a compatible guide nucleic acid within the same nucleic acid molecule. For example, a vector or viral vector can include a first promoter operably linked to a nucleic acid encoding a base editor and a second promoter operably linked to a guide nucleic acid.

[0224] Promoters used to drive expression of the guide nucleic acid include PolIII promoters such as U6 or H1 or PolII promoters and the use of an intron cassette expressing a gRNA adeno-associated virus (AAV).

[0225] The Cas9 or Cas12 described herein, with or without one or more guide nucleic acids, can be delivered using adeno-associated virus (AAV), lentivirus, adenovirus, or other plasmid or viral vector types, particularly using formulations and doses from, for example, U.S. Patent No. 8,454,972 (formulations, doses for adenovirus), U.S. Patent No. 8,404,658 (formulations, doses for AAV), and U.S. Patent No. 5,846,946 (formulations, doses for DNA plasmids), as well as from clinical trials and publications regarding clinical trials involving lentivirus, AAV, and adenovirus. For example, in the case of AAV, the administration route, formulation, and dose can be similar to U.S. Patent No. 8,454,972 and clinical trials involving AAV. In the case of adenovirus, the administration route, formulation, and dose can be similar to U.S. Patent No. 8,404,658 and clinical trials involving adenovirus. In the case of plasmid delivery, the route of administration, formulation, and dosage may be similar to that of US Patent No. 5,846,946 and clinical trials involving plasmids. Dosages may be based on or estimated for an average 70 kg individual (e.g., adult male) and may be adjusted for patients, subjects, mammals of different weights and species. Frequency of administration is within the scope of a physician or veterinarian (e.g., physician, veterinarian) depending on the usual factors including age, sex, general health, other conditions of the patient or subject, and the specific condition or symptom being addressed. The viral vector may be injected into the tissue of interest. In the case of cell type specific base editors, expression of the base editor and optional guide nucleic acid may be driven by a cell type specific promoter.

[0226] For in vivo delivery, AAV may be more advantageous than other viral vectors.In some cases, AAV allows low toxicity, which may be due to the purification method that does not require ultracentrifugation of cell particles that may activate immune response.In some cases, AAV allows low probability of causing insertional mutagenesis because it is not integrated into host genome.

[0227] AAV has a packaging limit of 4.5 or 4.75Kb. Constructs that exceed 4.5Kb or 4.75Kb can result in a significant reduction in virus production. For example, SpCas9 is quite large, and the gene itself exceeds 4.1Kb, making it difficult to package into AAV. Therefore, embodiments of the present disclosure include the use of the disclosed Cas9, which is shorter in length than conventional Cas9.

[0228] AAV can be AAV1, AAV2, AAV5, or any combination thereof. The type of AAV can be selected according to the cells to be targeted, for example, AAV serotype 1, 2, 5, or hybrid capsid AAV1, AAV2, AAV5, or any combination thereof for targeting brain or neuronal cells, and AAV4 can be selected when targeting heart tissue. AAV8 is useful for delivery to the liver. A table of specific AAV serotypes for these cells can be found in Grimm, D. et al, J. Virol. 82:5887-5911 (2008).

[0229] Lentiviruses are complex retroviruses that have the ability to infect and express their genes in both dividing and post-dividing cells. The most commonly known lentivirus is the human immunodeficiency virus (HIV), which uses the envelope glycoproteins of other viruses to target a broad range of cell types.

[0230] Lentivirus can be prepared as follows: After cloning pCasES10 (containing the lentiviral transcription plasmid backbone), low passage (p=5) HEK293FT were seeded to 50% confluence in T-75 flasks in DMEM with 10% fetal bovine serum and no antibiotics the day before transfection. After 20 hours, the medium was replaced with OptiMEM (serum-free) medium and transfection was performed 4 hours later. Cells are transfected with 10 μg of lentiviral transfer plasmid (pCasES10) and the following packaging plasmids: 5 μg of pMD2.G (VSV-g pseudotyped) and 7.5 μg of psPAX2 (gag / pol / rev / tat). Transfection can be performed in 4 mL of OptiMEM with cationic lipid delivery agent (50 μl of Lipofectamine 2000 and 100 ul of Plus reagent). After 6 hours, the medium is replaced with antibiotic-free DMEM containing 10% fetal bovine serum. Although these methods use serum in cell culture, serum-free methods are preferred.

[0231] Lentivirus can be purified as follows: Viral supernatant is harvested after 48 hours. The supernatant is first removed from debris and filtered through a 0.45 μm low protein binding (PVDF) filter. It is then spun in an ultracentrifuge at 24,000 rpm for 2 hours. The viral pellet is resuspended in 50 μl of DMEM overnight at 4° C. They are then aliquoted and immediately frozen at −80° C.

[0232] In another embodiment, a minimal non-primate lentiviral vector based on the Equine Infectious Anemia Virus (EIAV) is also contemplated. In another embodiment, RetinoStat® is an Equine Infectious Anemia Virus-based lentiviral gene therapy vector that expresses the angiogenesis inhibitory proteins endostatin and angiostatin, which is contemplated to be delivered by subretinal injection. In another embodiment, the use of self-inactivating lentiviral vectors is contemplated.

[0233] Any RNA of the system, such as guide RNA, can be delivered in the form of RNA. Cas9 or Cas12 coding mRNA can be generated using in vitro transcription. For example, Cas9 or Cas12 mRNA can be synthesized using a PCR cassette containing the following elements: T7 promoter, optional kozak sequence (GCCACC), nuclease sequence, and 3'UTR (e.g., 3'UTR from beta globin-polyA tail). The cassette can be used for transcription by T7 polymerase. Guide polynucleotides (e.g., gRNAs) can also be transcribed using in vitro transcription from a cassette containing a T7 promoter followed by the sequence "GG" and a guide polynucleotide sequence.

[0234] To enhance expression and reduce potential toxicity, the Cas9 sequence and / or guide nucleic acid can be modified to include one or more modified nucleosides, for example, using pseudo-U or 5-methyl-C.

[0235] The present disclosure encompasses, in some embodiments, a method of modifying a cell or organism. The cell may be a prokaryotic or eukaryotic cell. The cell may be a mammalian cell. Many of the mammalian cells are non-human primate, bovine, porcine, rodent, or murine cells. The modifications introduced into the cell by the base editors, compositions, and methods of the present disclosure can cause the cell and the cell's progeny to be altered for improved production of a biological product, such as an antibody, starch, alcohol, or other desired cellular output. The modifications introduced into the cell by the methods of the present disclosure can cause the cell and the cell's progeny to contain an alteration that changes the biological product produced.

[0236] The system can include one or more different vectors. In one aspect, Cas9 or Cas12 is codon-optimized for expression in a desired cell type, preferably a eukaryotic cell, preferably a mammalian cell or a human cell. In some embodiments, the cell is a human hepatocyte.

[0237] Generally, "codon optimization" refers to the process of modifying a nucleic acid sequence to enhance expression in a host cell of interest by substituting at least one codon (e.g., about one or more than about one, 2, 3, 4, 5, 10, 15, 20, 25, 50, or more codons) of the native sequence with a codon that is more or most frequently used in the genes of that host cell while maintaining the native amino acid sequence. Different species show a certain bias for a particular codon of a particular amino acid. Codon bias (differences in codon usage between organisms) is often thought to correlate with the efficiency of translation of messenger RNA (mRNA), which in turn depends, among other things, on the properties of the codon being translated and the availability of a particular transfer RNA (tRNA) molecule. The dominance of tRNAs selected in a cell is generally a reflection of the codons most frequently used in peptide synthesis. Thus, genes can be tailored for optimal gene expression in a given organism based on codon optimization. Codon usage tables are readily available, for example, at the "Codon Usage Database" available at www.kazusa.orjp / codon / (accessed July 9, 2002), and these tables can be adapted in several ways. See Nakamura, Y., et al. "Codon usage tabulated from the international DNA sequence databases: status for the year 2000" Nucl. Acids Res. 28:292 (2000). Computer algorithms are also available that use codons to optimize a particular sequence for expression in a particular host cell, for example, Gene Forge (Aptagen, Jacobus, Pa.). In some embodiments, one or more codons (e.g., 1, 2, 3, 4, 5, 10, 15, 20, 25, 50, or more codons, or all codons) in a sequence encoding an engineered nuclease correspond to the most frequently used codon for a particular amino acid.

[0238] Packaging cells are typically used to form viral particles capable of infecting host cells. Such cells include 293 cells, which package adenovirus, and psi.2 or PA317 cells, which package retrovirus. Viral vectors used in gene therapy are usually generated by producing cell lines that package nucleic acid vectors into viral particles. The vectors typically contain minimal viral sequences necessary for packaging and subsequent integration into the host, with other viral sequences being replaced by an expression cassette for the polynucleotide to be expressed. Missing viral functions are typically supplied in trans by the packaging cell line. For example, AAV vectors used in gene therapy typically only have ITR sequences from the AAV genome necessary for packaging and integration into the host genome. Viral DNA can be packaged into a cell line that contains a helper plasmid that encodes other AAV genes (i.e., rep and cap) but lacks ITR sequences. The cell line can be infected with adenovirus as a helper. The helper virus can facilitate replication of the AAV vector and expression of the AAV genes from the helper plasmid. In some cases, the helper plasmid is not packaged in significant amounts due to the lack of ITR sequences. Adenovirus contamination can be reduced, for example, by heat treatment, to which adenovirus is more sensitive than AAV.

[0239] Pharmaceutical Compositions Other aspects of the disclosure relate to pharmaceutical compositions comprising a base editor or base editor system (e.g., comprising Cas9 or Cas12 as disclosed herein). The term "pharmaceutical composition" as used herein refers to a composition formulated for pharmaceutical use. In some embodiments, the pharmaceutical composition further comprises a pharma- ceutical acceptable carrier. In some embodiments, the pharmaceutical composition comprises an additional agent (e.g., for specific delivery, to increase half-life, or other therapeutic compounds).

[0240] As used herein, the term "pharmaceutically acceptable carrier" means a pharma- ceutically acceptable material, composition, or vehicle, such as a liquid or solid filler, diluent, excipient, manufacturing aid (e.g., lubricant, magnesium talc, calcium or zinc stearate, or steric acid), or solvent encapsulant, involved in carrying or transporting a compound from one site in the body (e.g., delivery site) to another (e.g., an organ, tissue, or body part). A pharma- ceutically acceptable carrier is "acceptable" in the sense of being compatible with the other ingredients of the formulation and not deleterious to the tissues of the subject (e.g., physiological compatibility, sterility, physiological pH, etc.).

[0241] Some non-limiting examples of materials that can serve as pharma- ceutically acceptable carriers include: (1) sugars (e.g., lactose, glucose, and sucrose); (2) starches (e.g., corn starch and potato starch); (3) cellulose and its derivatives (e.g., sodium carboxymethylcellulose, methylcellulose, ethylcellulose, microcrystalline cellulose, and cellulose acetate); (4) powdered tragacanth; (5) malt; (6) gelatin; (7) lubricants (e.g., magnesium stearate, sodium lauryl sulfate, and talc); (8) excipients (e.g., cocoa butter and suppository wax); and (9) oils (e.g., peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil, and soybean oil). , (10) glycols (e.g., propylene glycol), (11) polyols (e.g., glycerin, sorbitol, mannitol, and polyethylene glycol (PEG)), (12) esters (e.g., ethyl oleate and ethyl laurate), (13) agar, (14) buffers (e.g., magnesium hydroxide and aluminum hydroxide), (15) alginic acid, (16) pyrogen-free water, (17) isotonic saline, (18) Ringer's solution, (19) ethyl alcohol, (20) pH buffer solutions, (21) polyesters, polycarbonates, and / or polyanhydrides, (22) swelling agents (e.g., polypeptides and amino acids), (23) serum alcohols (e.g., ethanol), and (23) other non-toxic compatible substances used in pharmaceutical formulations. Wetting agents, coloring agents, release agents, coating agents, sweetening agents, flavoring agents, fragrances, preservatives, and antioxidants may also be present in the formulation. The terms "excipient," "carrier," "pharmaceutical acceptable carrier," "vehicle," and the like are used interchangeably herein.

[0242] The pharmaceutical composition may include one or more pH buffer compounds to maintain the pH of the formulation at a predetermined level that reflects physiological pH, for example, in the range of about 5.0 to about 8.0. The pH buffer compound used in the aqueous liquid formulation may be an amino acid or a mixture of amino acids (e.g., histidine, or a mixture of amino acids such as histidine and glycine). Alternatively, the pH buffer compound is preferably an agent that maintains the pH of the formulation at a predetermined level (e.g., in the range of about 5.0 to about 8.0) and does not chelate calcium ions. Illustrative examples of such pH buffer compounds include, but are not limited to, imidazole and acetate ions. The pH buffer compound may be present in any amount suitable for maintaining the pH of the formulation at a predetermined level.

[0243] The pharmaceutical composition may also contain one or more osmotic modifiers, i.e., compounds that adjust the osmotic properties (e.g., tonicity, osmolality, and / or osmolarity) of the formulation to a level that is acceptable to the bloodstream and blood cells of the recipient individual. The osmotic modifier may be an agent that does not chelate calcium ions. The osmotic modifier may be any compound known or available to those skilled in the art that adjusts the osmotic properties of the formulation. Those skilled in the art may empirically determine the suitability of a given osmotic modifier for use in the formulations of the present invention. Illustrative examples of suitable types of osmotic modifiers include, but are not limited to, salts (e.g., sodium chloride and sodium acetate), sugars (e.g., sucrose, dextrose, and mannitol), amino acids (e.g., glycine), and mixtures of one or more of these agents and / or types of agents. The osmotic modifier may be present in any concentration sufficient to adjust the osmotic properties of the formulation.

[0244] In some embodiments, the pharmaceutical composition is formulated for delivery to a subject, for example, for gene editing. Suitable routes for administering the pharmaceutical compositions described herein include, but are not limited to, topical, subcutaneous, transdermal, intradermal, intralesional, intraarticular, intraperitoneal, intravesical, transmucosal, gingival, intradental, intracochlear, intratympanic, intravisceral, epidural, intrathecal, intramuscular, intravenous, intravascular, intraosseous, periocular, intratumoral, intracerebral, and intraventricular administration.

[0245] In some embodiments, the pharmaceutical compositions described herein are administered locally to the disease site. In some embodiments, the pharmaceutical compositions described herein are administered to the subject by injection, by catheter, by suppository, or by implant, the implant being a porous, non-porous, or gel-like material, including membranes such as sialastic membranes or fibers.

[0246] In other embodiments, the pharmaceutical compositions described herein are delivered in a controlled release system.In one embodiment, pumps can be used (see, for example, Langer, 1990, Science 249:1527-1533; Sefton, 1989, CRC Crit. Ref. Biomed. Eng. 14:201; Buchwald et al., 1980, Surgery 88:507; Saudek et al., 1989, N. Engl. J. Med. 321:574).In another embodiment, polymeric materials can be used. (See, e.g., Medical Applications of Controlled Release (Langer and Wise eds., CRC Press, Boca Raton, Fla., 1974); Controlled Drug Bioavailability, Drug Product Design and Performance (Smolen and Ball eds., Wiley, New York, 1984); Ranger and Peppas, 1983, Macromol. Sci. Rev. Macromol. Chem. 23:61; see also Levy et al., 1985, Science 228:190; During et al., 1989, Ann. Neurol. 25:351; Howard et al., 1989, J. Neurosurg. 71:105.) Other controlled release systems are discussed, e.g., in Langer, supra.

[0247] In some embodiments, the pharmaceutical composition is formulated according to routine procedures as a composition adapted for intravenous or subcutaneous administration to a subject (e.g., a human). In some embodiments, the pharmaceutical composition for administration by injection is a solution in sterile isotonic use as a solubilizing agent, and a local anesthetic, such as lignocaine, to ease pain at the injection site. Generally, the ingredients are supplied either separately or mixed together in a unit dosage form, e.g., as a dry lyophilized powder or water-free concentrate in a sealed container, such as an ampoule or sachet indicating the quantity of active agent. When the drug is administered by injection, the drug can be dispensed with an infusion bottle containing sterile pharmaceutical grade water or saline. When the pharmaceutical composition is administered by injection, an ampoule of sterile water for injection or saline can be provided so that the ingredients can be mixed prior to administration.

[0248] Pharmaceutical compositions for systemic administration may be liquid (e.g., sterile saline, lactated Ringer's solution, or Hank's solution). In addition, pharmaceutical compositions may be in solid form and redissolved or suspended immediately prior to use. Lyophilized forms are also contemplated. Pharmaceutical compositions may be contained within lipid particles or vesicles (e.g., liposomes or microcrystals also suitable for parenteral administration). The particles may be of any suitable structure, such as unilamellar or plurilamellar, so long as the composition is contained therein. Compounds may be encapsulated in "stabilized plasmid lipid particles" (SPLPs) containing the fusogenic lipid dioleoylphosphatidylethanolamine (DOPE), low levels (5-10 mol%) of cationic lipids, and stabilized by polyethylene glycol (PEG) coating (see Zhang YPet al., Gene Ther. 1999, 6:1438-47). For such particles and vesicles, positively charged lipids such as N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethyl-ammonium methylsulfate or "DOTAP" are particularly preferred.The preparation of such lipid particles is well known.See, for example, U.S. Patent Nos. 4,880,635, 4,906,477, 4,911,928, 4,917,951, 4,920,016, and 4,921,757 (each of which is incorporated herein by reference).

[0249] The pharmaceutical compositions described herein may be administered or packaged, for example, as unit doses. The term "unit dose", when used in reference to the pharmaceutical compositions of the present disclosure, refers to a physically discrete unit suitable as a unitary dosage for a subject, each unit containing a predetermined amount of active material calculated to produce a desired therapeutic effect in association with a required diluent (i.e., carrier or vehicle).

[0250] Additionally, pharmaceutical compositions may be provided as pharmaceutical kits, comprising (a) a container containing a compound of the invention in lyophilized form, and (b) a second container containing a pharma- ceutically acceptable diluent (e.g., sterile, for use in reconstituting or diluting the lyophilized compound of the invention). Optionally, a notice in a form prescribed by a governmental agency regulating the manufacture, use, or sale of pharmaceutical or biological products may be associated with such container, the notice reflecting the agency's approval of the manufacture, use, or sale for human administration.

[0251] Another aspect includes an article of manufacture containing materials useful for treating the diseases described above. In some embodiments, the article of manufacture includes a container and a label. Suitable containers include, for example, bottles, vials, syringes, and test tubes. The containers can be formed from a variety of materials, such as glass or plastic. In some embodiments, the container holds a composition effective for treating a disease described herein and can have a sterile access port. For example, the container can be an intravenous infusion bag or a vial with a stopper pierceable by a hypodermic needle. The active agent in the composition is a compound of the present invention. In some embodiments, a label on or associated with the container indicates that the composition is used to treat a selected disease. The article of manufacture can further include a second container containing a pharma- ceutically acceptable buffer, such as phosphate buffered saline, Ringer's solution, or dextrose solution. It can further include other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, needles, syringes, and package inserts containing instructions for use.

[0252] In some embodiments, a base editor or base editor system (e.g., comprising Cas9 or Cas12 as described herein) is provided as part of a pharmaceutical composition. In some embodiments, a pharmaceutical composition comprises any of the fusion proteins provided herein. In some embodiments, a pharmaceutical composition comprises any of the complexes provided herein. In some embodiments, a pharmaceutical composition comprises a ribonucleoprotein complex comprising an RNA-guided nuclease (e.g., Cas9) complexed with a gRNA and a cationic lipid. In some embodiments, a pharmaceutical composition comprises a gRNA, a nucleic acid programmable DNA binding protein, a cationic lipid, and a pharma- ceutical acceptable excipient. A pharmaceutical composition can optionally comprise one or more additional therapeutically active substances.

[0253] kit In one aspect, the present invention provides a kit containing any one or more of the elements disclosed in the above methods and compositions. In some embodiments, the kit includes a vector system and instructions for using the kit. In some embodiments, the vector system includes one or more insertion sites for inserting a guide sequence, and when expressed, the guide sequence directs sequence-specific binding of a CRISPR complex to a target sequence in a eukaryotic cell, and the CRISPR complex includes a CRISPR enzyme complexed with (1) a guide sequence hybridized to a target sequence, and (2) a sequence hybridized to a tracr sequence, and / or (b) a second regulatory element operably linked to an enzyme coding sequence encoding the CRISPR enzyme, the enzyme coding sequence including a nuclear localization sequence. The elements can be provided individually or in combination, and can be provided in any suitable container, such as a vial, bottle, or tube. In some embodiments, the kit includes instructions in one or more languages, for example, in two or more languages.

[0254] In some embodiments, the kit comprises a nucleobase editor.

[0255] In some embodiments, the kit includes one or more reagents for use in a process utilizing one or more of the elements described herein. The reagents may be provided in any suitable container. For example, the kit may provide one or more reaction or storage buffers. The reagents may be provided in a form that is usable in a particular assay or that requires the addition of one or more other components prior to use (e.g., concentrated or lyophilized form). The buffer may be any buffer, including, but not limited to, sodium carbonate buffer, sodium bicarbonate buffer, borate buffer, Tris buffer, MOPS buffer, HEPES buffer, and combinations thereof. In some embodiments, the buffer is alkaline. In some embodiments, the buffer has a pH of about 7 to about 10. In some embodiments, the kit includes one or more oligonucleotides corresponding to the guide sequence for insertion into a vector such that the guide sequence and the regulatory element are operably linked. In some embodiments, the kit includes a homologous recombination template polynucleotide.

[0256] All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. Furthermore, the materials, methods, and examples are illustrative only and are not intended to be limiting. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable methods and materials are described herein. EXAMPLES

[0257] The following examples illustrate some of the preferred modes of making and practicing the invention, however, it should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the invention.

[0258] Example 1. In vitro base editing using Cas9 in primary human hepatocytes for liver transplantation This example illustrates in vitro Cas9 base editing targeting exemplary MHC class I or class II antigen genes in primary human hepatocytes.

[0259] In this example, base editing was performed to target MHC class I or class II antigen genes to reduce immune rejection of allografts containing primary hepatocytes.

[0260] Briefly, Cas9 guide RNAs targeting specific nucleotide positions within the splice sites and / or stop codons of exemplary MHC class I or class II antigen genes, B2M and CIITA, were designed for introduction into hepatocytes (Table 1).

[0261] Primary human hepatocytes were transfected 24 hours after seeding with expression vectors containing the Cas9 enzyme fused to an adenine base editor (ABE) or cytidine base editor (CBE) and guide RNA (Table 1). Cells were harvested 5 hours after transfection and total DNA was extracted.

[0262] Deep sequencing was performed to characterize A to G or C to T conversions in primary human hepatocytes. Exemplary targets were amplified using two-round PCR to add Illumina adapters and unique barcodes to the target amplicons. PCR products were run on 2% gels and gel extracted. Samples were pooled, quantified, cDNA libraries were prepared, and sequenced on a MiSeq. Base editing was observed by deep sequencing to determine the percentage of A to G and C to T conversions.

[0263] [Table 7]

[0264] The results in this example demonstrated that guide RNA and Cas9 achieved base editing of B2M and CIITA immune genes in primary human hepatocytes.

[0265] Base editing was also performed in primary cultures of human hepatocytes. For these studies, base editors were evaluated for their ability to target either B2M (HLA MHC class I) and / or CIITA (HLA MHC class II) in cultured primary human hepatocytes (PHH). Both CT editors (BE4) and AG editors (ABE) were tested. Cas9 nuclease (SpCas9) was also used as an editing control. Guide RNAs designed to disrupt splice sites in the B2M and CIITA genes were tested in combination with either BE4 or ABE. These guides were also shown to generate indels when used with Cas9 nuclease. Human primary hepatocytes were seeded and transfected (lipofected) with a mixture containing RNA encoding the base editor (or Cas9) and guide RNA. Five days after transfection, cells were harvested for genomic DNA extraction and NGS analysis.

[0266] Data from these base editing experiments are shown in Figures 1A-1B and 2A-2B. Figure 1A shows the B2M base editor target used to generate the potential splice site shown in red. Figure 1A also shows potential bystander edits outside the intended splice site region shown in grey. Figure 2A shows the CIITA base editor target used to generate the potential splice site shown in red. Figure 2A also shows potential bystander edits outside the indented splice site region shown in grey.

[0267] Data from both of these studies showed significant base editing of the B2M gene (Figure 2A) and the CIITA gene (Figure 2B). Figure 1B shows that the B2M editing efficiency using the BE4 base editor was as high as 55%. That is, BE4 resulted in 55% C to T conversions at the BE-4 junction site. Figure 1B also shows that the use of the ABE site editor (ABE7.10) had the best editing efficiency at the B2M ABE junction site, resulting in 35% A to G conversions. Cas9 was used as a direct comparison of gene disruption. Base editing shows equal or better editing efficiency at the proposed splice site than the indels generated by Cas9. Figure 2B shows the results of base editing using the ABE editor (ABE8.2m) and BE4 targeting the CIITA gene. The results showed that the ABE editor resulted in significant editing with 40% A to G conversions and BE4 resulted in 50% C to T conversions. Similar to Figure 1B, Cas9 was used as a direct comparison for gene disruption, with base editing showing equal or better editing efficiency at proposed splice sites than Cas9-generated indels.

[0268] Example 2. Multiplexed guide RNAs for base editing of multiple immune genes in primary human hepatocytes. This example illustrates multiplex gene editing to target multiple immune system genes and reduce the immunogenicity of allogeneic liver cells for liver transplantation.

[0269] Liver transplantation is prone to graft rejection due to immune responses. Cas9-mediated gene editing using multiplexed guide RNAs targeting multiple immune system genes is used in this example to reduce / eliminate the immune response and improve graft survival of transplanted hepatocytes.

[0270] Guide RNAs targeting multiple loci in the exemplary B2M, CD142, and CIITA genes are cloned into expression vectors expressing either multiple guides from multiple promoters or polycistronic transcripts. These multiplexed guide RNAs will be introduced into hepatocytes along with the Cas9 enzyme.

[0271] The efficiency of base editing using multiplexed guide RNAs is measured by determining the rate of A to G and C to T conversions by deep sequencing.

[0272] Example 3. Bioinformatics screening to identify additional guide RNAs for immune system genes This example demonstrates the identification of additional guide RNAs targeting immune system genes using bioinformatics screening.

[0273] Bioinformatics screening was used to search for additional guide RNAs that would expand the targeting scope of CRISPR to immune system genes. Exemplary immune system genes targeted include MHC class I or class II genes, including beta2 microglobulin (B2M) and class II major histocompatibility complex transactivator (CIITA). Screening utilized seed sequences of Cas9 from S. pyogenes. Bioinformatics was performed using the tblastn variant of BLAST with an e-value threshold of 1e-6 to consider BLAST hits. Additional bioinformatics screening will be performed to determine guide RNAs that target CD142 and other exemplary immune system genes, including human leukocyte antigen A (HLA-A) and human leukocyte antigen B (HLA-B).

[0274] Guide RNA sequences and their PAMs are shown for exemplary immune system genes B2M, CD142, CIITA, HLA-A, and HLA-B in Tables 2, 3, 4, 5, and 6. Exemplary spacer sequences are shown in Tables 2A, 3A, 4A, 5A, and 6A.

[0275]

Table 8-1

[0276]

Table 8-2

[0277]

Table 9-1

[0278]

Table 9-2

[0279]

Table 10-1

[0280]

Table 10-2

[0281]

Table 10-3

[0282]

Table 11-1

[0283]

Table 11-2

[0284]

Table 11-3

[0285]

Table 12-1

[0286]

Table 12-2

[0287]

Table 12-3

[0288]

Table 12-4

[0289]

Table 12-5

[0290]

Table 12-6

[0291]

Table 12-7

[0292]

Table 12-8

[0293]

Table 12-9

[0294]

Table 12-10

[0295]

Table 12-11

[0296]

Table 12-12

[0297]

Table 12-13

[0298]

Table 12-14

[0299]

Table 13-1

[0300]

Table 13-2

[0301]

Table 13-3

[0302]

Table 13-4

[0303]

Table 13-5

[0304]

Table 13-6

[0305]

Table 13-7

[0306]

Table 13-8

[0307]

Table 13-9

[0308]

Table 13-10

[0309]

Table 13-11

[0310]

Table 13-12

[0311]

Table 13-13

[0312]

Table 14

[0313]

Table 15

[0314]

Table 16

[0315]

Table 17

[0316] Example 4. Large-scale production of base-edited human hepatocytes This example illustrates the large-scale production of base-edited human hepatocytes.

[0317] Cryopreserved primary hepatocytes or seedable / transplantable primary hepatocytes will be obtained. Multiplex gene editing will be performed on the hepatocytes as described in Examples 1 and 2.

[0318] The modified human hepatocytes produced will be verified by measuring AG and CT base transversions.

[0319] The modified human hepatocytes will be introduced into FRG mice and expanded for large-scale production.

[0320] Approximately 200-500 million cells will be transplanted into the FRG pigs, either directly from primary human hepatocyte cultures or from FRG mice.

[0321] The results of this example will produce large-scale base-edited human hepatocytes that eliminate or reduce the host immune response for liver transplantation.

[0322] Example 5. Evaluation of engraftment of base-edited hepatocytes in FRG mouse models of liver failure and metabolic disease An example of this is Fah, an animal model of liver failure and metabolic disease. - / - / Rag2 - / - / Il2rg - / - Illustrates engraftment and retention of base-edited hepatocytes in (FRG) mice.

[0323] FRG mice have approximately 5 × 10 9 The mice will be pretreated by intravenous administration of urokinase-expressing adenovirus (uPA virus) at a dose of 10 plaque forming units (pfu / mouse).

[0324] Approximately one million base-edited hepatocytes will be injected intraspleenically 24-48 hours after uPA administration and will withdraw NTBC. Liver disease in fumarylacetoacetate hydrolase (Fah) mutant mice develops only upon withdrawal of the drug 2-(2-nitro-4-trifluoromethylbenzoyl)-1,3-cyclohexanedione (NTBC). Withdrawal of NTBC in FRG mice gradually causes hepatocellular damage that ultimately leads to death after 4-8 weeks unless corrected.

[0325] FAH enzyme activity will be measured to determine hepatocyte function of the transplanted cells. Additionally, human albumin levels will be measured to confirm the presence of human edited cells. Histological / IHC analysis will be performed to confirm engraftment.

[0326] The results of this example determine the in vivo efficiency of engraftment and retention of transplanted base-edited hepatocytes in a mouse model.

[0327] Example 6. Evaluation of hepatocyte engraftment in FRG porcine bioreactors This example illustrates the engraftment of base-edited cells in FRG porcine bioreactors for large-scale production of hepatocytes.

[0328] Obtaining and expanding hepatocytes in FRG porcine bioreactors overcomes the problem of a limited supply of high quality hepatocytes due to the limited supply of donor livers for organ transplantation.

[0329] To assess engraftment and proliferation of edited hepatocytes, WT and base-edited hepatocytes will be transplanted into the FRG pig model via portal vein injection.

[0330] After transplantation, the protective drug 2-(2-nitro-4-trifluoromethylbenzil)-1,3-cyclohexanedione (NTBC) is withdrawn from the recipient pigs to provide a selective advantage for the proliferation of fumaryl acetoacetate hydrolase (Fah+) cells.

[0331] Human albumin levels will be assessed at 1, 3, and 6 months after transplantation to confirm the presence of human edited cells in the FRG pigs. A small amount of blood will be collected in a heparinized blood collection capillary tube. After dilution with Tris-buffered saline, human albumin concentration will be measured using a human albumin ELISA quantification kit. The degree of liver humanization generally correlates with blood levels of human albumin, with 1 mg / mL corresponding to approximately 20% human hepatocytes.

[0332] To confirm adequate engraftment, immunohistochemistry analysis of mouse liver tissue is also performed at months 4 and 6. Immunohistochemistry is performed for expression of FAH or human albumin or cytokeratin.

[0333] At the end of approximately 12 months, expanded human hepatocytes will be isolated, sorted, and characterized by flow cytometry for the presence / absence of class I and II markers and next-generation sequencing will be used to assess editing retention post-engraftment (Figure 1).

[0334] The results of this example will demonstrate the use of the FRG porcine bioreactor for large-scale production of base-edited modified hepatocytes suitable for liver transplantation.

[0335] Example 7. Evaluation of base editing efficiency in primary human hepatocyte (PHH) seeding protocols This example evaluates the base editing efficiency of exemplary base editors (e.g., ABE8.8, ABE8.20, and BE4) targeting exemplary target loci (e.g., B2M or CIITA).

[0336] Briefly, primary human hepatocytes were dissociated after the base editing reaction on days 4 and / or 6 of culture. Base editing efficiency was assessed using EditR software using Sanger sequencing files, and knockout of the corresponding protein was assessed by flow cytometry of dissociated PHHs performed on days 4 and / or 6 of culture after the editing reaction.

[0337] The results showed the editing efficiency of B2M at days 4 and 6, and the editing efficiency of CIITA at day 6 (FIG. 3A). The KO efficiency of B2M protein level is shown as the percentage (%) of B2M-negative (B2M-) cells measured at day 6 (FIG. 3B).

[0338] Flow cytometry analysis of HLA class II expression to assess protein level KO of CIITA revealed that the PHHs used were essentially HLA class II negative, despite the use of editing reagents to target the CIITA locus ( Figure 3B ).

[0339] Results showed that efficient editing was observed at both loci and that the levels of B2M protein KO measured by flow cytometry correlated with the editing efficiency of B2M.

[0340] Overall, the results confirmed that the ABE8.20 and BE4, as well as the ABE8.8 editing reagents, efficiently reduced B2M expression in seeded PHHs, demonstrating efficient base editing. The base editors were also effective in editing the CIITA locus.

[0341] Example 8. Comparison of the efficiency of delivery of base editors into primary human hepatocytes (PHH) by nucleofection and transfection This example evaluates the efficiency of delivery of an exemplary base editing reagent in primary human hepatocytes (PHHs) by nucleofection versus transfection.

[0342] Briefly, base editors targeting an exemplary target gene, B2M, were transfected or nucleofected into PHHs and the KO efficiency of B2M at the DNA and protein levels was determined. Cell viability of edited PHHs was assessed by flow cytometry using a live / dead stain added prior to dissociation of PHHs to determine cell viability prior to dissociation.

[0343] Briefly, PHHs were engineered with base editing reagents by nucleofection (Lonza4D-Nucleofector) or transfection under the conditions listed in Figure 5A.

[0344] After manipulation, cells were cultured and assessed for KO of B2M at the DNA and protein levels, along with cell viability, 6 days after treatment.

[0345] As shown in Figure 5B, under the same experimental conditions (BE4 / gRNA3:1), transfection-based delivery resulted in significantly higher B2M gene KO efficiency in PHHs (B2M KO score: 91.5% vs. 60.0%, B2M-cells (%): 91.8% vs. 62.2%) and higher post-editing cell viability (viability (%): 60.3% vs. 49.6%) compared to nucleofection-based delivery.

[0346] Overall, the results showed that delivery by transfection into PHHs resulted in increased base editing efficiency and higher post-editing survival compared to nucleofection.

[0347] Example 9. Evaluation of base editing reagents in PHH ex vivo preparation protocols This example evaluates the efficiency of editing of target loci in seeded PHHs using exemplary base editing reagents after incorporation into an ex vivo procedure to prepare PHHs for cell expansion.

[0348] Briefly, PHHs were engineered with base editing reagents by transfection as described in Example 8.

[0349] An exemplary base editor targeting the B2M locus was delivered by transfection. Base editing efficiency at the B2M locus is shown in Figure 6, where the dots represent the percentage of B2M- cells by flow cytometry, and "HEK2-2" indicates the targeted control locus (Figure 6).

[0350] The results showed that over 80% editing efficiency at the B2M-targeted locus was observed using each of ABE8.8, ABE8.20, and BE4. Correspondingly, a high percentage of B2M-cells was also observed by flow cytometry in B2M-targeted samples edited with ABE8.8, ABE8.20, or BE4.

[0351] The ex vivo procedure involves the introduction of a transgene, and therefore, an exemplary editing reagent was used to evaluate the efficiency of the dual operation of editing the B2M locus and introducing a transgene into PHHs according to the ex vivo procedure.

[0352] Base editing at the B2M locus was performed using BE4 in combination with the introduction of one of two exemplary transgenes (Tg#1 or Tg#2). The results shown in Figure 7 show that gene editing by BE4, measured at the B2M locus or by quantification of B2M-cells by flow cytometry, was not significantly altered by the incorporation of an ex vivo procedure that included the introduction of a transgene. Overall, the results showed that the efficiency of the dual manipulation using BE4 and a transgene was high.

[0353] As shown in Figure 8, flow cytometry measurements revealed that over 80% of cells modified with both B2M and BE4 targeting Tg#1 ("BE4 B2M+Tg#1") were both B2M negative and Tg#1 positive ("B2M- / Tg#1+") compared to cells modified solely with B2M ("BE4 B2M only"). Modification with both B2M and BE4 targeting Tg#2 was also effective in generating doubly engineered cells.

[0354] Overall, the results demonstrated effective dual engineering of PHH with BE4 and a transgene for ex vivo preparation of PHH.

[0355] Equivalence and Scope Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. The scope of the invention is not intended to be limited to the above Description, but rather is as set forth in the following claims.

Claims

1. 1. A method for producing genetically modified human hepatocytes suitable for hepatocyte transplantation, comprising disrupting one or more major histocompatibility complex (MHC) class I or class II genes in isolated human hepatocytes or hepatocyte progenitor cells by introducing a base editor and one or more gRNAs that hybridize to target sequences in the one or more MHC class I or class II genes, thereby producing genetically modified human hepatocytes.

2. 2. The method of claim 1, wherein the base editor comprises a CRISPR protein fused to a deaminase, and the CRISPR protein is Cas9 or Cas12.

3. 2. The method of claim 1, wherein the genetically modified human hepatocytes (i) have one or more nucleobase edits at a target sequence; (ii) have a disrupted target sequence; and / or (iii) have reduced or eliminated alloreactivity.

4. 2. The method of claim 1, wherein the class I or class II genes are selected from one or more of B2M, CD142, CIITA, HLA-A, or HLA-B genes.

5. 5. The method of claim 4, wherein a stop codon or splice site is introduced into one or more of the B2M, CD142, CIITA, HLA-A, or HLA-B genes.

6. The method of claim 5, wherein: (i) a splice site is introduced at nucleotide position 19 of the B2M gene; (ii) a stop codon is introduced at nucleotide position 5 of the B2M gene; (iii) a splice site is introduced at nucleotide position 28 of the CD142 gene; (iv) a stop codon is introduced at nucleotide position 19 of the CD142 gene; (v) a splice site is introduced at nucleotide position 147 of the CIITA gene; or (vi) a stop codon is introduced at nucleotide position 130 of the CIITA gene.

7. 2. The method of claim 1, wherein the Cas9 is derived from Streptococcus pyogenes (SpCas9) or Staphylococcus aureus (SaCas9).

8. (i) the Cas9 protein is an ultra-high precision Cas9; (ii) the Cas9 protein includes mutations corresponding to N692A, M694A, Q695A, and / or H698A with reference to SpyCas9 (SEQ ID NO:68); (iii) the Cas9 protein is a high fidelity Cas9; (iv) the Cas9 protein includes mutations corresponding to N692A, M694A, Q695A, and / or H698A with reference to SpyCas9 (SEQ ID NO:68).

8. The method of claim 7, wherein the Cas9 protein comprises mutations corresponding to residues 467A, R661A, Q695A, and / or Q926A; (v) the Cas9 protein is SuperFi-Cas9; and / or (vi) residues Y1016, R1019, Y1010, Y1013, K1031, Q1027, and / or V1018 corresponding to SpyCas9 (SEQ ID NO:68) are mutated to aspartic acid.

9. 3. The method of claim 2, wherein the CRISPR protein is fused to an adenine base editor (ABE), a cytidine base editor (CBE), or an inosine base editor (IBE), or is fused to a base editor comprising an adenine or adenosine deaminase domain, and / or a cytidine or cytosine deaminase domain.

10. The method of claim 9, wherein: (i) the CRISPR protein comprises a nuclear localization sequence (NLS) and / or a FLAG, HIS, or HA tag; or (ii) the CRISPR protein comprises at least one mutation in SEQ ID NO:1 (SpCas9), SEQ ID NO:2 (SaCas9), or SEQ ID NO:3 (Cas12).

11. The method of claim 10, wherein: (i) the at least one mutation results in a nuclease-inactive Cas9 (dCas9); (ii) the at least one mutation is one or more amino acid substitutions in the RuvC domain and / or the HNH domain of Cas9; (iii) the at least one mutation is an aspartic acid to alanine substitution at amino acid 10 of SpCas9 (D10A), or a corresponding mutation in a Cas9 protein; or (iv) the at least one mutation is a histidine to alanine substitution at amino acid 840 of SpCas9 (H840A), or a corresponding mutation in a Cas9 protein.

12. 3. The method of claim 2, wherein the Cas9 protein has nickase activity.

13. The method described in claim 2, wherein (i) the CRISPR protein is fused to adenosine deaminase and has an amino acid sequence at least 80% identical to SEQ ID NO: 65, or (ii) the CRISPR protein is fused to cytosine deaminase and has an amino acid sequence at least 80% identical to SEQ ID NO: 4 to 64.

14. The method of claim 2, wherein: (i) the SpCas9 protein recognizes a PAM sequence comprising 5'-NGG-3', 5'-NGA-3', or 5'-NGC-3'; (ii) the SaCas9 protein recognizes a PAM sequence comprising 5'-NNNRRT-3' or 5'-NNGRRT-3'; or (iii) the Cas12 protein recognizes a PAM sequence comprising 5'-RTTN-3'.

15. The method of claim 1, wherein the genetically modified human hepatocytes overexpress CD47 and / or CD142 compared to non-genetically modified human hepatocytes.

16. the genetically modified human hepatocytes are transplanted into a humanized animal model for expansion, the humanized animal model being an FRG pig, an FRG mouse, or an FRG rat; The method of claim 1, wherein the genetically modified human hepatocytes are first transplanted into the FRG mouse or FRG rat for initial cell expansion, and / or, after the initial cell expansion, the genetically modified cells are subsequently transplanted into the FRG pig for further cell expansion.

17. The method of claim 16, wherein (i) the initially expanded cells or the further expanded cells are isolated from the animal, and (ii) the initially expanded cells or the further expanded cells are isolated by fluorescence activated cell sorting, immunomagnetic cell separation, density gradient centrifugation, and / or immunodensity cell separation.

18. 2. The method of Claim 1, wherein the genetically modified human hepatocytes have one, two, three or more nucleobase edits, wherein the nucleobase edits are generated by (i) a single base editor or (ii) two or more base editors. [0023] (i) the method comprises the use of a base editor and one or more guide RNAs targeting the B2M gene, wherein the base editor and one or more corresponding guide RNAs comprising an RNA version of any one of the protospacer sequences listed in Table 2 or any one of the sequences set forth in Table 2A are selected; (ii) the method comprises the use of a base editor and one or more guide RNAs targeting the CD142 gene, wherein the base editor and one or more corresponding guide RNAs comprising an RNA version of any one of the protospacer sequences listed in Table 3 or any one of the sequences set forth in Table 3A are selected; (iii) the method comprises the use of a base editor and one or more guide RNAs targeting the CIITA gene, wherein the base editor and one or more corresponding guide RNAs comprising an RNA version of any one of the protospacer sequences listed in Table 4 are selected.

5. The method of claim 1 , wherein: (iv) the method comprises the use of a base editor and one or more guide RNAs targeting an HLA-A gene, wherein the base editor and one or more corresponding guide RNAs comprising an RNA version of any one of the protospacer sequences listed in Table 5 or any one of the sequences listed in Table 5A are selected; and / or (v) the method comprises the use of a base editor and one or more guide RNAs targeting an HLA-B gene, wherein the base editor and one or more corresponding guide RNAs comprising an RNA version of any one of the protospacer sequences listed in Table 6 or any one of the sequences listed in Table 6A are selected.

20. A nucleic acid encoding one or more gRNAs that hybridize to any of the base editors described in Tables 2A to 6A and a target sequence.

21. 21. The nucleic acid of claim 20, wherein the nucleic acid is codon-optimized for expression in a human cell.

22. A vector encoding the nucleic acid of claim 20 or 21.

23. A eukaryotic cell comprising a base editor and one or more gRNAs comprising any one of the sequences listed in Tables 2A-6A, or an RNA version of any one of the protospacer sequences listed in Tables 2-6 that hybridizes to a target sequence.

24. 20. Use of genetically modified human hepatocytes produced by the method of any one of claims 1 to 19 in the manufacture of a medicament for treating liver disease.

25. The use described in claim 24, wherein in treating the liver disease, approximately 10 to 15 billion genetically modified human hepatocytes are injected into the portal vein of a subject in need thereof.

26. A base editor and one or more guide RNAs targeting B2M, CD142, CIITA, HLA-A, and / or HLA-B genes, wherein the base editor and corresponding one or more guide RNAs are selected that comprise an RNA version of any one of the sequences listed in Tables 2A-6A or any one of the protospacer sequences listed in Tables 2-6.

27. ​​A guide RNA comprising any one of the sequences listed in Table 2A, Table 3A, Table 4A, Table 5A, or Table 6A, or an RNA version of any one of the protospacer sequences listed in Table 2, Table 3, Table 4, Table 5, or Table 6.

28. 1. A cell comprising a base editor and one or more guide RNAs, wherein the guide RNA comprises an RNA version of any one of the sequences listed in Tables 2A-6A or any one of the protospacer sequences listed in Tables 2-6.

29. 20. A genetically modified human hepatocyte having one or more edits in an MHC gene according to the method of any one of claims 1 to 19, wherein the MHC gene is selected from B2M, CD142, CIITA, HLA-A, and / or HLA-B.

30. 30. The genetically modified human hepatocyte of claim 29, wherein edits to one or more of the B2M, CD142, CIITA, HLA-A, and / or HLA-B genes result in increased expression of the B2M, CD142, CIITA, HLA-A, and / or HLA-B genes compared to non-genetically modified human hepatocytes.