Modified immune cells having adenosine deaminase base editors for modifying nucleobases in target sequences

Abstract

The present invention features genetically modified immune cells containing novel adenosine base editors (e.g., ABE8) that have enhanced antineoplastic activity, resistance to immunosuppression, and a reduced risk of eliciting a graft-versus-host or host-versus-graft response, or a combination thereof. The present invention also features methods for the production and use of these modified immune effector cells.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application is an international PCT application and claims priority to and benefit of U.S. Provisional Patent Application Nos. 62 / 805,271, filed February 13, 2019, 62 / 852,228, filed May 23, 2019, 62 / 852,224, filed May 23, 2019, 62 / 931,722, filed November 6, 2019, 62 / 941,523, filed November 27, 2019, 62 / 941,569, filed November 27, 2019, and 62 / 966,526, filed January 27, 2020, the contents of all of which are incorporated herein by reference in their entireties.

[0002] Incorporation by Reference All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, and patent application was specifically and individually indicated to be incorporated by reference. Unless otherwise indicated, all publications, patents, and patent applications mentioned in this specification are herein incorporated by reference in their entirety. [Background technology]

[0003] Autologous and allogeneic immunotherapy are treatment approaches for neoplasia in which immune cells expressing chimeric antigen receptors are administered to a subject. To generate immune cells expressing chimeric antigen receptors (CARs), immune cells are first collected from the subject (autologous) or from a donor different from the subject receiving treatment (allogeneic) and genetically modified to express the chimeric antigen receptor. The resulting cells express the chimeric antigen receptor on their cell surface (e.g., CAR-T cells), and upon administration to a subject, the chimeric antigen receptor binds to a marker expressed by the neoplasia cells. This interaction with the neoplasia marker activates the CAR-T cells, which then kill the neoplasia cells. However, for autologous or allogeneic cell therapy to be effective and efficient, important conditions and cellular responses, such as T cell signaling inhibition, must be overcome or avoided. For allogeneic cell therapy, graft-versus-host disease (GVHD) and host rejection of CAR-T cells can pose additional challenges. While editing the genes involved in these processes can enhance CAR-T cell function and resistance to immune suppression or inhibition, current methodologies for performing such editing can induce large genomic rearrangements in CAR-T cells, thereby adversely affecting their efficiency. Thus, there is a significant need for approaches to more precisely modify immune cells, particularly CAR-T cells. The present application addresses this and other important needs. Summary of the Invention

[0004] The present invention features genetically modified immune cells containing novel adenosine base editors (e.g., ABE8) that have enhanced antineoplastic activity, resistance to immunosuppression, and a reduced risk of eliciting a graft-versus-host or host-versus-graft response, or a combination thereof. The present invention also features methods for the production and use of these modified immune effector cells.

[0005] In one aspect, the invention provides a method for producing an engineered immune cell, the method comprising expressing in or introducing into an immune cell a nucleobase editor polypeptide and contacting the cell with two or more guide RNAs that target the nucleobase editor polypeptide to effect an alteration in a nucleic acid molecule encoding at least one polypeptide selected from the group consisting of T cell receptor alpha constant (TRAC), beta-2 microglobulin (B2M), programmed cell death 1 (PD1), cluster of differentiation 7 (CD7), cluster of differentiation 5 (CD5), cluster of differentiation 33 (CD33), cluster of differentiation 123 (CD123), Cbl proto-oncogene B (CBLB), and class II major histocompatibility complex transactivator (CIITA) polypeptide, wherein the nucleobase editor polypeptide is a nucleic acid programmable DNA binding protein (napDNAbp) and MSEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWNRAIGLHDPTAHEIMALRQGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIGRVVFGVRNAKTGAAGSLMDVLHYPGMNHRVEITEGILADECAALLCYFFRMPRQVFNAQKKAQSSTD In one embodiment, the immune cell is a T cell. In one embodiment, the immune cell is obtained from a healthy subject.

[0006] In one embodiment, the adenosine deaminase variant domain comprises alterations at amino acid positions 82 and 166. In one embodiment, the adenosine deaminase variant domain comprises an alteration of V82S. In one embodiment, the adenosine deaminase variant domain comprises an alteration of T166R. In one embodiment, the adenosine deaminase variant domain comprises an alteration of V82S and T166R. In one embodiment, the adenosine deaminase variant domain further comprises one or more of the following alterations: Y147T, Y147R, Q154S, Y123H, and / or Q154R. In one embodiment, the adenosine deaminase variant domain is Y147T+Q154R; Y147T+Q154S; Y147R+Q154S; V82S+Q154S; V82S+Y147R; V82S+Q154R; V82S+Y123H; I76Y+V82S; V82S+Y123H+Y147T; V82S+Y123H+Y147R; V82S+Y1 and I76Y+V82S+Y123H+Y147R+Q154R. In one embodiment, the adenosine deaminase variant domain comprises a combination of modifications selected from the group consisting of: 23H+Q154R; Y147R+Q154R+Y123H; Y147R+Q154R+I76Y; Y147R+Q154R+T166R; Y123H+Y147R+Q154R+I76Y; V82S+Y123H+Y147R+Q154R; and I76Y+V82S+Y123H+Y147R+Q154R. In one embodiment, the adenosine deaminase variant domain comprises a combination of modifications: V82S+Q154R. In one embodiment, the adenosine deaminase variant domain comprises a combination of modifications: Y147R+Q154R+Y123H. In one embodiment, the adenosine deaminase variant domain comprises the combination of modifications: Y147R+Q154R+Y123H+I76Y. In one embodiment, the adenosine deaminase variant domain comprises the combination of modifications: I76Y+V82S+Y123H+Y147R+Q154R. In one embodiment, the adenosine deaminase variant is TadA*8.In one embodiment, TadA*8 is TadA*8.1, TadA*8.2, TadA*8.3, TadA*8.4, TadA*8.5, TadA*8.6, TadA*8.7, TadA*8.8, TadA*8.9, TadA*8.10, TadA*8.11, TadA*8.12, TadA*8.13, TadA*8.14, TadA*8.15, TadA*8.16, TadA*8.17, TadA*8.18, TadA*8.19, TadA*8.20, TadA*8.21, TadA*8.22, TadA*8.23, TadA*8.24.

[0007] In one embodiment, the adenosine deaminase variant domain comprises a C-terminal deletion beginning at a residue selected from the group consisting of 149, 150, 151, 152, 153, 154, 155, 156, and 157. In one embodiment, the base editor domain is a monomer of the adenosine deaminase variant. In one embodiment, the base editor domain is ABE8.1-m, ABE8.2-m, ABE8.3-m, ABE8.4-m, ABE8.5-m, ABE8.6-m, ABE8.7-m, ABE8.8-m, ABE8.9-m, ABE8.10-m, ABE8.11-m, ABE8.12-m, ABE8.13-m, ABE8.14-m, ABE8.15-m, ABE8.16-m, ABE8.17-m, ABE8.18-m, ABE8.19-m, ABE8.20-m, ABE8.21-m, ABE8.22-m, ABE8.23-m, ABE8.24-m.

[0008] In one embodiment, the base editor domain is an adenosine deaminase variant heterodimer comprising a wild-type adenosine deaminase domain and an adenosine deaminase variant domain. In one embodiment, the base editor domain is ABE8.1-d, ABE8.2-d, ABE8.3-d, ABE8.4-d, ABE8.5-d, ABE8.6-d, ABE8.7-d, ABE8.8-d, ABE8.9-d, ABE8.10-d, ABE8.11-d, ABE8.12-d, ABE8.13-d, ABE8.14-d, ABE8.15-d, ABE8.16-d, ABE8.17-d, ABE8.18-d, ABE8.19-d, ABE8.20-d, ABE8.21-d, ABE8.22-d, ABE8.23-d, or ABE8.24-d.

[0009] In one embodiment, the base editor domain is an adenosine deaminase variant heterodimer comprising a TadA*7.10 domain and an adenosine deaminase variant domain. In one embodiment, the adenosine deaminase variant domain is missing 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 6, 17, 18, 19, or 20 N-terminal amino acid residues compared to full-length adenosine deaminase. In one embodiment, the adenosine deaminase variant domain has the following sequence, which has adenosine deaminase activity: MSEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWNRAIGLHDPTAHEIMALRQGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIGRVVFGVRNAKTGAAGSLMDVLHYPGMNHRVEITEGILADECAALLCTFFRMPRQVFNAQKKAQSSTD or a fragment thereof.

[0010] In one embodiment, napDNAbp has the sequence TIFF0007672982000001.tif172169, where the bolded sequence indicates the Cas9-derived sequence, the italicized sequence indicates the linker sequence, and the underlined sequence indicates the bipartite nuclear localization sequence.

[0011] In various embodiments of any aspect described herein, the napDNAbp is Staphylococcus aureus Cas9 (SaCas9), Streptococcus thermophilus 1 Cas9 (St1Cas9), Streptococcus pyogenes Cas9 (SpCas9), or a variant thereof. In one embodiment, the napDNAbp comprises a variant of SpCas9 with altered protospacer adjacent motif (PAM) specificity or specificity for a non-G PAM. In one embodiment, the altered PAM has specificity for the nucleic acid sequence 5'-NGC-3'. In one embodiment, the modified SpCas9 comprises the amino acid substitutions D1135M, S1136Q, G1218K, E1219F, A1322R, D1332A, R1335E, and T1337R, or their corresponding amino acid substitutions. In various embodiments of any aspect described herein, the napDNAbp comprises a nuclease-inactive Cas9 (dCas9), a Cas9 nickase (nCas9), or the nuclease Cas9. In one embodiment, the nickase variant comprises the amino acid substitution D10A or a corresponding amino acid substitution. In various embodiments of any aspect described herein, the nucleobase editor polypeptide further comprises a zinc finger domain. In various embodiments of any aspect described herein, the nucleobase editor polypeptide further comprises one or more uracil glycosylase inhibitors. In various embodiments of any aspect described herein, the adenosine deaminase variant domain is capable of deaminating adenine in deoxyribonucleic acid (DNA). In various embodiments of any aspect described herein, the adenosine deaminase variant domain is a non-naturally occurring modified adenosine deaminase. In various embodiments of any aspect described herein, the adenosine deaminase variant is TadA*8.In some embodiments, TadA*8 is TadA*8.1, TadA*8.2, TadA*8.3, TadA*8.4, TadA*8.5, TadA*8.6, TadA*8.7, TadA*8.8, TadA*8.9, TadA*8.10, TadA*8.11, TadA*8.12, TadA*8.13, TadA*8.14, TadA*8.15, TadA*8.16, TadA*8.17, TadA*8.18, TadA*8.19, TadA*8.20, TadA*8.21, TadA*8.22, TadA*8.23, or TadA*8.24.

[0012] In various embodiments of any aspect described herein, the nucleobase editor polypeptide further comprises a linker between the napDNAbp and the adenosine deaminase variant domain. In one embodiment, the linker comprises the amino acid sequence: SGGSSGGSSGSETPGTSESATPES.

[0013] In various embodiments of any aspect described herein, the nucleobase editor polypeptide further comprises one or more nuclear localization signals (NLS). In one embodiment, the NLS is a bipartite NLS. In one embodiment, the nucleobase editor polypeptide comprises an N-terminal NLS and a C-terminal NLS. In various embodiments of any aspect described herein, the napDNAbp is a modified Staphylococcus aureus Cas9 (SaCas9). In one embodiment, the modified SaCas9 comprises the amino acid substitutions E782K, N968K, and R1015H, or corresponding amino acid substitutions. In one embodiment, the modified SaCas9 has the amino acid sequence: Includes:

[0014] In various embodiments of any aspect described herein, two or more guide RNAs are expressed in or contacted with the cell, each targeting a different polynucleotide. In various embodiments, multiplex base editing involves simultaneous modification of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more target loci. In various embodiments of any aspect described herein, two guide RNAs are expressed in or contacted with the cell, each targeting a B2M or TRAC polynucleotide. In various embodiments of any aspect described herein, three guide RNAs are expressed in or contacted with the cell. In various embodiments of any aspect described herein, three guide RNAs are expressed in or contacted with the cell, each targeting a B2M, CD7, TRAC, CIITA, PDCD1, and / or CBLB polynucleotide. In various embodiments of any aspect described herein, three guide RNAs are expressed in or contacted with the cell, all of which target B2M, TRAC, and PDCD1 polynucleotides. In various embodiments of any aspect described herein, three guide RNAs are expressed in or contacted with the cell, all of which target B2M, TRAC, and CIITA polynucleotides. In various embodiments of any aspect described herein, four guide RNAs are expressed in or contacted with the cell, all of which target B2M, CD7, TRAC, CIITA PDCD1, and / or CBLB polynucleotides. In various embodiments of any aspect described herein, two or more guide RNAs target the TRAC exon 4 splice acceptor site, the B2M exon 1 splice donor site, and / or the PDCD1 exon 1 splice donor site. In various embodiments of any aspect described herein, two or more guide RNAs target a splice acceptor site or a splice donor site in the target polynucleotide. In various embodiments of any aspect described herein, the nucleobase editor polypeptide generates a stop codon in the target polynucleotide.In various embodiments of any aspect described herein, the nucleobase editor polypeptide generates a stop codon in PDCD1 exon 2. In various embodiments, expression of one or more of the above polypeptides is reduced by 70, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99%, or more, or even 100%, relative to a reference, by introducing a base editor and one or more guide RNAs targeting the gene encoding the polypeptide.

[0015] In another aspect, the invention provides for expressing a chimeric antigen receptor (CAR) in the modified immune cell of any aspect described herein. In various embodiments of any aspect described herein, the immune cell is modified ex vivo. In various embodiments of any aspect described herein, the immune cell is a cytotoxic T cell, a regulatory T cell, or a T helper cell. In various embodiments of any aspect described herein, the modified immune cell does not comprise a detectable translocation.

[0016] In another aspect, the invention provides modified immune cells produced according to the method of any aspect described herein. In various embodiments of any aspect described herein, the cells have reduced immunogenicity and increased antineoplastic activity. In various embodiments of any aspect described herein, the immune cells express a chimeric antigen receptor.

[0017] In various embodiments of any aspect described herein, the immune cell is a T cell. In various embodiments of any aspect described herein, the cell comprises one or more mutations in polynucleotides encoding B2M, CD7, CIITA, PD1, CBLB, and / or TARC. In one embodiment, the cell comprises one or more mutations in polynucleotides encoding B2M, TRAC, and CIITA polynucleotides. In various embodiments of any aspect described herein, the cell comprises mutations in one or more polynucleotides encoding TIGIT, TGFBR2, ZAP70, NFATc1, or TET2.

[0013] In various embodiments of any aspect described herein, the cells express any of the following proteins: V-Set immunoregulatory receptor (VISTA), T cell immunoglobulin mucin 3 (Tim-3), T cell immunoreceptor with IgG and ITIM domains (TIGIT), transforming growth factor beta receptor II (TGFbRII), regulatory factor X-related ankyrin-containing protein (RFXANK), PVR-related immunoglobulin domain containing (PVRIG), lymphocyte activation gene 3 (Lag3), cytotoxic T lymphocyte-associated protein 4 (CTLA-4), chitinase 3-like 1 (Chi3l1), cluster of differentiation 96 (CD96), B and T lymphocyte-associated (BTLA), Tet methylcytosine dioxygenase 2 (TET2), Sprouty RTK signaling antagonist 1 (Spry1), Sprouty RTK signaling antagonist 2 (Spry2), class II major histocompatibility complex transactivator (CIITA), and mutations in one or more polynucleotides encoding cluster of differentiation 7 (CD7), cluster of differentiation 33 (CD33), cluster of differentiation 52 (CD52), cluster of differentiation 123 (CD123), T-cell receptor beta constant 1 (TRBC1), T-cell receptor beta constant 2 (TRBC2), cytokine-inducible SH2-containing protein (CISH), acetyl-CoA acetyltransferase (ACAT1), cytochrome P450 family 11 subfamily A member 1 (Cyp11a1), GATA-binding protein 3 (GATA3), nuclear receptor subfamily 4 group A member 1 (NR4A1), nuclear receptor subfamily 4 group A member 2 (NR4A2), nuclear receptor subfamily 4 group A member 3 (NR4A3), methylation-regulated J protein (MCJ), Fas cell surface death receptor (FAS), or selectin P ligand / P-selectin glycoprotein ligand-1 (SELPG / PSGL1).

[0018] In various embodiments of any aspect delineated herein, the chimeric antigen receptor comprises an extracellular domain with affinity for a marker associated with neoplasia. In one embodiment, the neoplasia is multiple myeloma. In various embodiments of any aspect delineated herein, the marker is B-cell maturation antigen (BCMA).

[0019] In another aspect, the invention provides a method of modulating an immune response in a subject, the method comprising administering an effective amount of modified immune cells according to any aspect described herein. In various embodiments of any aspect described herein, the method increases or decreases the immune response.

[0020] In another aspect, the invention provides a method of treating neoplasia in a subject, the method comprising administering to the subject an effective amount of modified immune cells according to any aspect described herein.

[0021] In another aspect, the invention provides a pharmaceutical composition for the treatment of neoplasia, comprising an effective amount of modified immune cells according to any of the aspects described herein.

[0022] In another aspect, the invention provides a pharmaceutical composition comprising an effective amount of modified immune cells according to any of the aspects described herein in a pharmaceutically acceptable excipient.

[0023] In another aspect, the invention provides a kit for treating neoplasia comprising the modified immune cells according to any aspect described herein. In various embodiments of any aspect described herein, the kit further comprises written instructions for using the modified immune effector cells for treating neoplasia.

[0024] In various embodiments of any aspect described herein, the modified immune cells further comprise a chimeric antigen receptor having affinity for a marker associated with neoplasia. In certain embodiments, the chimeric antigen receptor is introduced into the cells via a viral vector, e.g., a lentiviral vector. In certain embodiments, the chimeric antigen receptor is introduced into the cells via a double-stranded DNA template, inserted into a locus cleaved by a nuclease. In various embodiments of any aspect described herein, the chimeric antigen receptor comprises an extracellular domain having affinity for a marker associated with neoplasia.

[0025] In various embodiments of any aspect described herein, the neoplasia is a B-cell cancer. In various embodiments of any aspect described herein, the B-cell cancer is a lymphoma or leukemia. In various embodiments of any aspect described herein, the B-cell cancer is multiple myeloma.

[0026] In another aspect, the invention provides a method of treating a subject having or prone to suffering from graft-versus-host disease (GVHD) with an effective amount of modified immune cells according to any aspect described herein. In another aspect, the invention provides a pharmaceutical composition for treating GVHD comprising an effective amount of modified immune cells according to any aspect described herein. In another aspect, the invention provides a kit for treating GVHD comprising modified immune cells according to any aspect described herein. In various embodiments of any aspect described herein, the modified immune cells lack functional TRAC or have reduced levels of functional TRAC.

[0027] In another aspect, the invention provides a method of treating a subject having or prone to suffering from host-versus-graft disease (HVGD) with an effective amount of modified immune cells according to any aspect described herein. In another aspect, the invention provides a pharmaceutical composition for treating HVGD comprising an effective amount of modified immune cells according to any aspect described herein. In another aspect, the invention provides a kit for treating HVGD comprising modified immune cells according to any aspect described herein. In various embodiments of any aspect described herein, the modified immune cells lack functional B2M or have reduced levels of functional B2M.

[0028] In another aspect, the invention provides a method for producing an engineered immune cell, the method comprising expressing in or introducing into an immune cell a nucleobase editor polypeptide and contacting the cell with two or more guide RNAs capable of targeting a nucleic acid molecule encoding at least one polypeptide selected from the group consisting of T-cell receptor alpha constant (TRAC), beta-2 microglobulin (B2M), programmed cell death 1 (PD1), cluster of differentiation 7 (CD7), cluster of differentiation 5 (CD5), cluster of differentiation 33 (CD33), cluster of differentiation 123 (CD123), Cbl proto-oncogene B (CBLB), and class II major histocompatibility complex transactivator (CIITA) polypeptide, wherein the nucleobase editor polypeptide comprises at least one base adenosine deaminase variant domain inserted into a nucleic acid programmable DNA-binding protein (napDNAbp).

[0029] In one embodiment, the adenosine deaminase variant domain is MSEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWNRAIGLHDPTAHEIMALRQGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIGRVVFGVRNAKTGAAGSLMDVLHYPGMNHRVEITEGILADECAALLCYFFRMPRQVFNAQKKAQSSTD wherein the amino acid sequence comprises at least one alteration. In one embodiment, the adenosine deaminase variant domain comprises an alteration at amino acid position 82 and / or 166. In one embodiment, the at least one alteration comprises V82S, T166R, Y147T, Y147R, Q154S, Y123H, and / or Q154R. In one embodiment, the adenosine deaminase variant comprises the following combinations of alterations: Y147T+Q154R; Y147T+Q154S; Y147R+Q154S; V82S+Q154S; V82S+Y147R; V82S+Q154R; V82S+Y123H; I76Y+V82S; V82S+Y123H+Y147T ;V82S+Y123H+Y147R;V82S+Y123H+Q154R;Y147R+Q154R+Y123H;Y147R+Q154R+I76Y;Y147R+Q154R+T166R;Y123H+Y147R+Q154R+I76Y;V82S+Y123H+Y147R+Q154R; and I76Y + V82S + Y123H + Y147R + Q154R. In one embodiment, the adenosine deaminase variant is TadA*8.1, TadA*8.2, TadA*8.3, TadA*8.4, TadA*8.5, TadA*8.6, TadA*8.7, TadA*8.8, TadA*8.9, TadA*8.10, TadA*8.11, TadA*8.12, TadA*8.13, TadA*8.14, TadA*8.15, TadA*8.16, TadA*8.17, TadA*8.18, TadA*8.19, TadA*8.20, TadA*8.21, TadA*8.22, TadA*8.23, TadA*8.24. In one embodiment, the adenosine deaminase variant comprises a C-terminal deletion beginning at a residue selected from the group consisting of 149, 150, 151, 152, 153, 154, 155, 156, and 157. In one embodiment, the adenosine deaminase variant domain is an adenosine deaminase monomer. In one embodiment, the adenosine deaminase variant is an adenosine deaminase variant heterodimer comprising a wild-type adenosine deaminase domain and an adenosine deaminase variant domain.In one embodiment, the adenosine deaminase variant is an adenosine deaminase heterodimer comprising a TadA domain and an adenosine deaminase variant domain.

[0030] In another embodiment, the napDNAbp is a Cas9 or Cas12 polypeptide. In one embodiment, the adenosine deaminase variant is inserted into a flexible loop, an alpha-helical region, an unstructured portion, or a solvent-accessible portion of the napDNAbp. In one embodiment, the adenosine deaminase variant is flanked by an N-terminal fragment and a C-terminal fragment of the napDNAbp. In one embodiment, the nucleobase editor polypeptide comprises the structure NH2-[N-terminal fragment of napDNAbp]-[adenosine deaminase variant]-[C-terminal fragment of napDNAbp]-COOH, where each "]-[" is an optional linker. In one embodiment, the C-terminus of the N-terminal fragment or the N-terminus of said C-terminal fragment constitutes part of the flexible loop of the napDNAbp. In one embodiment, the flexible loop comprises amino acids adjacent to the target nucleobase. In one embodiment, the target nucleobase is separated from the PAM sequence in the target polynucleotide sequence by 1 to 20 nucleobases. In one embodiment, the target nucleobase is 2-12 nucleobases upstream of the PAM sequence. In one embodiment, the N-terminal or C-terminal fragment of the napDNAbp binds to the target polynucleotide sequence.

[0031] In some embodiments, the N-terminal fragment or the C-terminal fragment comprises a RuvC domain, or the N-terminal fragment or the C-terminal fragment comprises an HNH domain, or neither the N-terminal fragment nor the C-terminal fragment comprises an HNH domain, or neither the N-terminal fragment nor the C-terminal fragment comprises a RuvC domain. In some embodiments, the napDNAbp comprises a partial or complete deletion in one or more structural domains, and the deaminase is inserted into the napDNAbp at the position of the partial or complete deletion. In some embodiments, the deletion is in the RuvC domain, the deletion is in the HNH domain, or the deletion bridges the RuvC domain and the C-terminal domain, the LI domain and the HNH domain, or the RuvC domain and the LI domain.

[0032] In another embodiment, the napDNAbp is a Cas9 or Cas12 polypeptide. In one aspect, the napDNAbp comprises a Cas9 polypeptide. In one embodiment, the Cas9 polypeptide is Streptococcus pyogenes Cas9 (SpCas9), Staphylococcus aureus Cas9 (SaCas9), Streptococcus thermophilus 1 Cas9 (St1Cas9), or a variant thereof. In one embodiment, the Cas9 polypeptide has the following amino acid sequence (Cas9 reference sequence): TIFF0007672982000002.tif180169 (single underline: HNH domain, double underline: RuvC domain), (Cas9 reference sequence), or a corresponding region thereof.

[0033] In some embodiments, the Cas9 polypeptide comprises a deletion of amino acids 1017-1069, or a corresponding amino acid, as numbered in a Cas9 polypeptide reference sequence, or the Cas9 polypeptide comprises a deletion of amino acids 792-872, or a corresponding amino acid, as numbered in a Cas9 polypeptide reference sequence, or the Cas9 polypeptide comprises a deletion of amino acids 792-906, or a corresponding amino acid, as numbered in a Cas9 polypeptide reference sequence. In one embodiment, the adenosine deaminase variant is inserted into a flexible loop of the Cas9 polypeptide. In one embodiment, the flexible loop comprises a region selected from the group consisting of amino acid residues at positions 530-537, 569-579, 686-691, 768-793, 943-947, 1002-1040, 1052-1077, 1232-1248, and 1298-1300, or their corresponding amino acid positions, numbered in the Cas9 reference sequence. In one embodiment, the deaminase is inserted between amino acid positions 768-769, 791-792, 792-793, 1015-1016, 1022-1023, 1026-1027, 1029-1030, 1040-1041, 1052-1053, 1054-1055, 1067-1068, 1068-1069, 1247-1248, or 1248-1249, or their corresponding amino acid positions, numbered in the Cas9 reference sequence. In one embodiment, the deaminase is inserted between amino acid positions 768-769, 792-793, 1022-1023, 1026-1027, 1040-1041, 1068-1069, or 1247-1248, or their corresponding amino acid positions, as numbered in the Cas9 reference sequence. In one embodiment, the deaminase is inserted between amino acid positions 1016-1017, 1023-1024, 1029-1030, 1040-1041, 1069-1070, or 1247-1248, or their corresponding amino acid positions, as numbered in the Cas9 reference sequence. In one embodiment, the adenosine deaminase variant is inserted into the Cas9 polypeptide at a locus identified in Table 13A.In one embodiment, the N-terminal fragment comprises amino acid residues 1-529, 538-568, 580-685, 692-942, 948-1001, 1026-1051, 1078-1231, and / or 1248-1297 of the Cas9 reference sequence, or their corresponding residues. In one embodiment, the C-terminal fragment comprises amino acid residues 1301-1368, 1248-1297, 1078-1231, 1026-1051, 948-1001, 692-942, 580-685, and / or 538-568 of the Cas9 reference sequence, or their corresponding residues.

[0034] In another embodiment, the Cas9 polypeptide is a modified Cas9 and has specificity for an altered PAM. In one embodiment, the Cas9 polypeptide is a nickase, or the Cas9 polypeptide is nickase-inactive. In one embodiment, the Cas9 polypeptide is a modified SpCas9 polypeptide. In one embodiment, the modified SpCas9 polypeptide has amino acid substitutions D1135M, S1136Q, G1218K, E1219F, A1322R, D1332A, R1335E, and T1337R (SpCas9-MQKFRAER) and has specificity for an altered PAM 5'-NGC-3'.

[0035] In some embodiments, the adenosine deaminase variant is inserted into a Cas12 polypeptide. In one embodiment, the Cas12 polypeptide is Cas12a, Cas12b, Cas12c, Cas12d, Cas12e, Cas12g, Cas12h, or Cas12i. In one embodiment, the adenosine deaminase variant is selected from the group consisting of: a) amino acid positions 153-154, 255-256, 306-307, 980-981, 1019-1020, 534-535, 604-605, or 344-345 of BhCas12b, or the corresponding amino acid residues of Cas12a, Cas12c, Cas12d, Cas12e, Cas12g, Cas12h, or Cas12i; b) amino acid positions 147 and 148, 248 and 249, 299 and 300, 991 and 992 of BvCas12b; or 1031 and 1032, or the corresponding amino acid residues of Cas12a, Cas12c, Cas12d, Cas12e, Cas12g, Cas12h, or Cas12i; or c) 157 and 158, 258 and 259, 310 and 311, 1008 and 1009, or 1044 and 1045 of AaCas12b, or the corresponding amino acid residues of Cas12a, Cas12c, Cas12d, Cas12e, Cas12g, Cas12h, or Cas12i. In one embodiment, the adenosine deaminase variant is inserted into the Cas12 polypeptide at a locus identified in Table 13B. In one embodiment, the Cas12 polypeptide is Cas12b. In one embodiment, the Cas12 polypeptide comprises a BhCas12b domain, a BvCas12b domain, or an AACas12b domain.

[0036] In one aspect, the invention provides modified immune cells produced according to any of the methods provided herein. In one embodiment, the immune cells are T cells. In one embodiment, the immune cells express a chimeric antigen receptor. In one embodiment, the method comprises administering an effective amount of any of the modified immune cells provided herein. In another aspect, the invention provides a pharmaceutical composition comprising an effective amount of the modified immune cells provided herein in a pharmaceutically acceptable excipient. In yet another aspect, the invention provides a kit comprising any of the modified immune cells provided herein.

[0037] In one embodiment, a polynucleotide programmable DNA binding domain and MSEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWNRAIGLHDPTAHEIMALRQGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIGRVVFGVRNAKTGAAGSLMDVLHYPGMNHRVEITEGILADECAALLCYFFRMPRQVFNAQKKAQSSTD and two or more guide RNAs that target the nucleobase editor polypeptide to effect an alteration in a nucleic acid molecule encoding at least one polypeptide selected from the group consisting of T-cell receptor alpha constant (TRAC), beta-2 microglobulin (B2M), programmed cell death 1 (PD1), cluster of differentiation 7 (CD7), cluster of differentiation 5 (CD5), cluster of differentiation 33 (CD33), cluster of differentiation 123 (CD123), Cbl proto-oncogene B (CBLB), and class II major histocompatibility complex transactivator (CIITA) polypeptide. In some embodiments, the adenosine deaminase variant comprises a V82S alteration and / or a T166R alteration. In some embodiments, the adenosine deaminase variant comprises one or more of the following alterations: Y147T, Y147R, Q154S, Y123H, and Q154R. In some embodiments, the base editor domain comprises an adenosine deaminase heterodimer comprising a wild-type adenosine deaminase domain and an adenosine deaminase variant. In some embodiments, the adenosine deaminase variant is a truncated TadA8 that is missing 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 6, 17, 18, 19, or 20 N-terminal amino acid residues compared to full-length TadA8. In some embodiments, the adenosine deaminase variant is a truncated TadA8 that lacks 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 6, 17, 18, 19, or 20 C-terminal amino acid residues compared to full-length TadA8. In some embodiments, the polynucleotide-programmable DNA-binding domain is a modified Staphylococcus aureus Cas9 (SaCas9), Streptococcus thermophilus 1 Cas9 (St1Cas9), a modified Streptococcus pyogenes Cas9 (SpCas9), or a variant thereof.In some embodiments, the polynucleotide-programmable DNA-binding domain is a variant of SpCas9 with altered protospacer adjacent motif (PAM) specificity or specificity for non-G PAMs. In some embodiments, the polynucleotide-programmable DNA-binding domain is a nuclease-inactive Cas9. In some embodiments, the polynucleotide-programmable DNA-binding domain is a Cas9 nickase.

[0038] In one embodiment, two or more guide RNAs and the following sequence: a polynucleotide-programmable DNA-binding domain comprising TIFF0007672982000003.tif195169, wherein the bolded sequences represent Cas9-derived sequences, the italicized sequences represent linker sequences, and the underlined sequences represent bipartite nuclear localization sequences; and MSEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWNRAIGLHDPTAAHAEIMALRQGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIGRVVFGVRNAKTGAAGSLMDVLHYPGMNHRVEITEGILADECAALLCYFFRMPRQVFNAQKKAQSST

[0014] Provided herein is a base editor system comprising a fusion protein comprising at least one base editor domain comprising an adenosine deaminase variant comprising an alteration at amino acid positions 82 and / or 166 of T-cell receptor alpha constant (TRAC), beta-2 microglobulin (B2M), programmed cell death 1 (PD1), cluster of differentiation 7 (CD7), cluster of differentiation 5 (CD5), cluster of differentiation 33 (CD33), cluster of differentiation 123 (CD123), Cbl proto-oncogene B (CBLB), and class II major histocompatibility complex transactivator (CIITA) polypeptide.

[0039] In one aspect, a cell is provided comprising any one of the base editor systems described above. Any one of the cells is a human cell or a mammalian cell. In some embodiments, the cell is ex vivo, in vivo, or in vitro.

[0040] The description and examples herein provide detailed explanations of embodiments of the present disclosure. It should be understood that the present disclosure is not limited to the specific embodiments described herein and may therefore vary. Those skilled in the art will appreciate that the present disclosure has numerous variations and modifications that fall within the scope of the present invention.

[0041] The practice of some embodiments disclosed herein will employ, unless otherwise indicated, conventional techniques of immunology, biochemistry, chemistry, molecular biology, microbiology, cell biology, genetics, and recombinant DNA, which are within the skill of the art. See, e.g., Sambrook and Green, Molecular Cloning: A Laboratory Manual, 4th Edition (2012); the series Current Protocols in Molecular Biology (F.M. Ausubel, et al. eds.); the series Methods in Enzymology (Academic Press, Inc.), PCR 2: A Practical Approach (M.J. MacPherson, B.D. Hames, and G.R. Taylor eds. (1995)), Harlow and Lane, eds. (1988) Antibodies, A Laboratory Manual, and Culture of Animal Cells: A Manual of Basic Technique and Specialized Applications, 6th Edition (R.I. Freshney, ed. (2010)).

[0042] The headings used herein are for organizational purposes only and should not be construed as limiting the subject matter described.

[0043] Although various features of the present disclosure may be described in the context of a single embodiment, the features may also be provided individually or in any suitable combination. Conversely, although for clarity the present disclosure may be described herein in the context of separate embodiments, the present disclosure may also be implemented in a single embodiment. The headings used herein are for organizational purposes only and should not be construed as limiting the subject matter described.

[0044] The features of the present disclosure are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present invention will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the present disclosure are utilized, and by consideration of the accompanying drawings, which are described below.

[0045] [Definition] The following definitions supplement those in the art and are directed to this application and are not to be construed as attributing any related or unrelated matter, such as commonly owned patents or applications. Although any methods and materials similar or equivalent to those described herein can be used in the practice of testing of the present disclosure, the preferred materials and methods are described herein. Therefore, the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting.

[0046] Unless otherwise defined, all technical and scientific terms used herein have the meanings commonly understood by those skilled in the art to which this invention belongs. The following references provide those skilled in the art with general definitions of many of the terms used in this invention: Singleton et al., Dictionary of Microbiology and Molecular Biology (2nd ed. 1994); The Cambridge Dictionary of Science and Technology (Walker ed., 1988); The Glossary of Genetics, 5th Ed., R. Rieger et al. (eds.), Springer Verlag (1991); and Hale & Marham, The HarperCollins Dictionary of Biology (1991).

[0047] In this application, the use of the singular includes the plural unless specifically stated otherwise. It should be noted that as used herein, the singular forms "a," "an," and "the" include plural references unless the context clearly dictates otherwise. In this application, the use of "or" means "and / or" and is understood to be inclusive unless otherwise stated. Furthermore, the term "including" and other forms, such as "include," "includes," and "included," are not limiting.

[0048] As used in the specification and claims, the terms "comprising" (and any of its forms, such as "comprise" and "comprises"), "having" (and any of its forms, such as "have" and "has"), "including" (and any of its forms, such as "include" and "includes"), or "containing" (and any of its forms, such as "contains" and "contain") are inclusive or open-ended and do not exclude additional, unrecited elements or method steps. It is contemplated that any embodiment discussed herein can be implemented with respect to any method or composition of the disclosure, and vice versa. Furthermore, the compositions of the disclosure can be used to achieve the methods of the disclosure.

[0049] The term "about" or "approximately" means within an acceptable error range for a particular value, as determined by one skilled in the art, which depends in part on how the value is measured or determined, i.e., the limitations of the measurement system. For example, "about" can mean within 1 or more standard deviations, according to practice in the art. Alternatively, "about" can mean a range of up to 20%, up to 10%, up to 5%, or up to 1% of a given value. Alternatively, particularly with respect to biological systems or processes, the term can mean a value within the same order of magnitude, for example, within 5-fold or 2-fold. When a particular value is described in an application or claim, unless otherwise specified, the term "about" should be assumed to mean within an acceptable error range for that particular value.

[0050] Ranges provided herein are understood to be shorthand for all values ​​within the range. For example, a range of 1 to 50 is understood to include any number, combination of numbers, or subrange from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50.

[0051] References in the specification to "some embodiments," "an embodiment," "one embodiment," or "other embodiments" mean that the particular feature, structure, or characteristic described in connection with that embodiment is included in at least some embodiments of the present disclosure, but not necessarily in all embodiments.

[0052] "Adenosine deaminase" refers to a polypeptide or fragment thereof that can catalyze the hydrolytic deamination of adenine or adenosine. In some embodiments, the deaminase or deaminase domain is an adenosine deaminase that catalyzes the hydrolytic deamination of adenosine to inosine or deoxyadenosine to deoxyinosine. In some embodiments, the adenosine deaminase catalyzes the hydrolytic deamination of adenine or adenosine in deoxyribonucleic acid (DNA). The adenosine deaminases provided herein (e.g., engineered adenosine deaminases, evolved adenosine deaminases) can be from any organism, such as bacteria.

[0053] In some embodiments, the adenosine deaminase is TadA deaminase. In some embodiments, the TadA deaminase is a TadA variant. In some embodiments, the TadA variant is TadA*8. In some embodiments, the deaminase or deaminase domain is a variant of a naturally occurring deaminase from an organism such as a human, chimpanzee, gorilla, monkey, cow, dog, rat, or mouse. In some embodiments, the deaminase or deaminase domain is not naturally occurring. For example, in some embodiments, the deaminase or deaminase domain is at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, or at least 99.9% identical to a naturally occurring deaminase. For example, deaminase domains are described in International PCT Applications PCT / 2017 / 045381 (WO 2018 / 027078) and PCT / US2016 / 058344 (WO 2017 / 070632), each of which is incorporated herein by reference 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.

[0054] The wild-type TadA (wt) adenosine deaminase has the following sequence (also referred to as the TadA reference sequence): MSEVEFSHEYWMRHALTLAKRAWDEREVPVGAVLVHNNRVIGEGWNRPIGRHDPTAHAEIMALRQGGLVMQNYRLIDATLYVTLEPCVMCAGAMIHSRIGRVVFGARDAKTGAAGSLMDVLHHPGMNHRVEITEGILADECAALLSDFFRMRRQEIKAQKKAQSSTD It has.

[0055] In some embodiments, the adenosine deaminase has the following sequence: MSEVEFSHEY WMRHALTLAK RARDEREVPV GAVLVLNNRV IGEGWNRAIG LHDPTAHAEI MALRQGGLVM QNYRLIDATL YVTFEPCVMC AGAMIHSRIG RVVFGVRNAK TGAAGSLMDV LHYPGMNHRV EITEGILADE CAALLCYFFR MPRQVFNAQK KAQSSTD Includes changes in (also known as TadA*7.10)

[0056] In some embodiments, TadA*7.10 comprises at least one alteration. In some embodiments, TadA*7.10 comprises an alteration at amino acid 82 and / or 166. In particular embodiments, variants of the above-referenced sequences comprise one or more of the following alterations: Y147T, Y147R, Q154S, Y123H, V82S, T166R, and / or Q154R. The alteration Y123H is also referred to herein as H123H (the alteration H123Y in TadA*7.10 has been reverted to Y123H(wt)). In other embodiments, variants of the TadA*7.10 sequence are Y147T+Q154R;Y147T+Q154S;Y147R+Q154S;V82S+Q154S;V82S+Y147R;V82S+Q154R;V82S+Y123H;I76Y+V82S;V82S+Y123H+Y147T;V82 S+Y123H+Y147R; V82S+Y123H+Q154R; Y147R+Q154R+Y123H; Y147R+Q154R+I76Y; Y147R+Q154R+T166R; Y123H+Y147R+Q154R+I76Y; V82S+Y123H+Y147R+Q154R; and I76Y + V82S + Y123H + Y147R + Q154R.

[0057] In other embodiments, the invention provides adenosine deaminase variants that include a deletion, e.g., TadA*8, that includes a C-terminal deletion beginning at residues 149, 150, 151, 152, 153, 154, 155, 156, or 157 relative to TadA*7.10, the TadA reference sequence, or the corresponding mutation in another TadA. In other embodiments, the adenosine deaminase variant is a TadA (e.g., TadA*8) monomer that includes one or more of the following changes relative to TadA*7.10, the TadA reference sequence, or the corresponding mutation in another TadA: Y147T, Y147R, Q154S, Y123H, V82S, T166R, and / or Q154R. In other embodiments, the adenosine deaminase variant is Y147T+Q154R;Y147T+Q154S;Y147R+Q154S;V82S+Q154S;V82S+Y147R;V82S+Q154R;V82S+Y123H;I76Y+V82S relative to the corresponding mutation in TadA*7.10, the TadA reference sequence, or another TadA. V82S+Y123H+Y147T; V82S+Y123H+Y147R; V82S+Y123H+Q154R; Y147R+Q154R+Y123H; Y147R+Q154R+I76Y; Y147R+Q154R+T166R; Y123H+Y147R+Q154R+I76Y; V82S+Y123H+Y147R+Q154R; and I76Y + V82S + Y123H + Y147R + Q154R.

[0058] In yet other embodiments, the adenosine deaminase variant is a homodimer comprising two adenosine deaminase domains (e.g., TadA*8), each having one or more of the following alterations relative to the corresponding mutation in TadA*7.10, the TadA reference sequence, or another TadA: Y147T, Y147R, Q154S, Y123H, V82S, T166R, and / or Q154R. In other embodiments, the adenosine deaminase variant is a homodimer comprising two adenosine deaminase domains (e.g., TadA*8), each having one or more of the following alterations relative to the corresponding mutation in TadA*7.10, the TadA reference sequence, or another TadA: Y147T+Q154R; Y147T+Q154S; Y147R+Q154S; V82S+Q154S; V82S+Y147R; V82S+Q154R; V82S+Y123H; I76Y+V. and I76Y + V82S + Y123H + Y147R + Q154R.

[0059] In other embodiments, the adenosine deaminase variant is a heterodimer comprising a wild-type TadA adenosine deaminase domain and an adenosine deaminase variant domain (e.g., TadA*8) that includes one or more of the following modifications relative to TadA*7.10, the TadA reference sequence, or the corresponding mutation in another TadA: Y147T, Y147R, Q154S, Y123H, V82S, T166R, and / or Q154R. In other embodiments, the adenosine deaminase variants are Y147T+Q154R;Y147T+Q154S;Y147R+Q154S;V82S+Q154S;V82S+Y147R;V82S+Q154R;V82S+Y147R;V82S+Q154R;V82S+Y147R;V82S+Q154R;V82S+Y147R;V82S+Y ...47R;V82S+Y154R;V82S+Y147R;V82S+Y147R;V82S+Y147R;V82S+Y147R;V82S+Y147R;V82S+Y147R;V82S+Y147R;V82S+Y147R;V82S+Y147R;V82S+Y147R;V82S+Y147R;V82S+Y14 and I76Y + V82S + Y123H + Y147R + Q154R.

[0060] In other embodiments, the adenosine deaminase variant is a heterodimer comprising a TadA*7.10 domain and an adenosine deaminase variant domain (e.g., TadA*8) that includes one or more of the following alterations relative to TadA*7.10, the TadA reference sequence, or the corresponding mutation in another TadA: Y147T, Y147R, Q154S, Y123H, V82S, T166R, and / or Q154R. In other embodiments, the adenosine deaminase variant comprises the TadA*7.10 domain and a combination of the following changes relative to the corresponding mutations in TadA*7.10, the TadA reference sequence, or another TadA: Y147T+Q154R; Y147T+Q154S; Y147R+Q154S; V82S+Q154S; V82S+Y147R; V82S+Q154R; V82S+Y or a heterodimer comprising an adenosine deaminase variant domain (e.g., TadA*8) comprising: 123H; I76Y + V82S; V82S + Y123H + Y147T; V82S + Y123H + Y147R; V82S + Y123H + Q154R; Y147R + Q154R + Y123H; Y147R + Q154R + I76Y; Y147R + Q154R + T166R; Y123H + Y147R + Q154R + I76Y; V82S + Y123H + Y147R + Q154R; or I76Y + V82S + Y123H + Y147R + Q154R.

[0061] In one embodiment, the adenosine deaminase has the following sequence, which has adenosine deaminase activity: MSEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWNRAIGLHDPTAHEIMALRQGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIGRVVFGVRNAKTGAAGSLMDVLHYPGMNHRVEITEGILADECAALLCTFFRMPRQVFNAQKKAQSSTD or TadA*8, comprising or consisting essentially of a fragment thereof.

[0062] In some embodiments, TadA*8 is truncated. In some embodiments, the truncated TadA*8 lacks 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 6, 17, 18, 19, or 20 N-terminal amino acid residues compared to full-length TadA*8. In some embodiments, the truncated TadA*8 lacks 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 6, 17, 18, 19, or 20 C-terminal amino acid residues compared to full-length TadA*8. In some embodiments, the adenosine deaminase variant is full-length TadA*8.

[0063] In certain embodiments, the adenosine deaminase heterodimer comprises a TadA*8 domain and an adenosine deaminase domain selected from one of the following: Bacillus subtilis (B. subtilis) TadA: MTQDELYMKEAIKEAKKAEEKGEVPIGAVLVINGEIIARAHNLRETEQRSIAHAEMLVIDEACKALGTWRLEGATLYVTLEPCPMCAGAVVLSRVEKVVFGAFDPKGGCSGTLMNLQEERFNHQAEVVSGVLEEECGGMLSAFFRELRKKKKAARKNLSE Salmonella typhimurium (S. typhimurium) TadA: MPPAFITGVTSLSDVELDHEYWMRHALTLAKRAWDEREVPVGAVLVHNHRVIGEGWNRPIGRHDPTAPTAHAEIMALRQGGLVLQNYRLLDTTLYVTLEPCVMCAGAMVHSRIGRVVFGARDAKTGAAGSLIDVLHHPGMNHRVEIIEGVLRDECATLLSDFFRMRRQEIKALKKADRAEGAGPAV Shewanella putrefaciens (S. putrefaciens) TadA: MDEYWMQVAMQMAEKAEAAGEVPVGAVLVKDGQQIATGYNLSISQHDPTAHAEILCLRSAGKKLENYRLLDATLYITLEPCAMCAGAMVHSRIARVVYGARDEKTGAAGTVVNLLQHPAFNHQVEVTSGVLAEACSAQLSRFFKRRRDEKKALKLAQRAQQGIE Haemophilus influenzae F3031 (H. influenzae) TadA: MDAAKVRSEFDEKMMRYALELADKAEALGEIPVGAVLVDDARNIIGEGWNLSIVQSDPTΑΗAEIIALRNGAKNIQNYRLLNSTLYVTLEPCTMCAGAILHSRIKRLVFGASDYKTGAIGSRFHFFDDYKMNHTLEITSGVLAEECSQKLSTFFQKRREEKKIEKALLKSLSDK Caulobacter crescentus (C. crescentus) TadA: MRTDESEDQDHRMMRLALDAARAAAEAGETPVGAVILDPSTGEVIATAGNGPIAAHDPTAHAEIIAAMRAAAAKLGNYRLTDLTVVTLEPCAMCAGAISHARIGRVVFGADDPKGGAVVHGPKFFAQPTCHWRPEVTGGVLADESADLLRGFFRARRKAKI Geobacter sulfurreducens (G. sulfurreducens) TadA: MSSLKKTPIRDDAYWMGKAIREAAKAAARDEVPIGAVIVRDGAVIGRGHNLREGSNDPSAHAEMIAIRQAARRSANWRLTGATLYVTLEPCLMCMGAIILARLERVVFGCYDPKGGAAGSLYDLSADPRLNHQVRLSPGVCQEECGTMLSDFFRDLRRRKKAKATPALFIDERKVPPEP TadA*7.10 MSEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWNRAIGLHDPTAHEIMALRQGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIGRVVFGVRNAKTGAAGSLMDVLHYPGMNHRVEITEGILADECAALLCYFFRMPRQVFNAQKKAQSSTD

[0064] "Administering" refers herein to providing one or more compositions described herein to a patient or subject. By way of example, and not limitation, administration, e.g., injection, of a composition can be by intravenous (iv), subcutaneous (sc), intradermal (id), intraperitoneal (ip), or intramuscular (im) injection. One or more such routes can be used. Parenteral administration can be by, for example, bolus injection or by gradual perfusion over time. Alternatively, or concurrently, administration can be by the oral route.

[0065] By "agent" is meant any small molecule chemical compound, antibody, nucleic acid molecule, or polypeptide, or fragment thereof.

[0066] "Allogeneic," as used herein, refers to cells of the same species that are genetically distinct from a comparison cell.

[0067] "Alteration" means a change (e.g., an increase or decrease) in the structure, expression level, or activity of a gene or polypeptide as detected by standard art-known methods, such as those described herein. As used herein, alteration includes a 25%, 40%, 50%, or greater change in polynucleotide or polypeptide sequence or expression level.

[0068] "Ameliorate" means to lessen, inhibit, attenuate, reduce, arrest, or stabilize the occurrence or progression of a disease.

[0069] "Analog" refers to a molecule that is not identical but has similar functional or structural characteristics. For example, a polynucleotide or polypeptide analog retains the biological activity of the corresponding naturally occurring polynucleotide or polypeptide, while possessing certain modifications that enhance the analog's function compared to the naturally occurring polynucleotide or polypeptide. Such modifications can increase the analog's affinity for DNA, efficiency, specificity, protease or nuclease resistance, membrane permeability, and / or half-life, for example, without altering ligand binding. Analogs can contain non-natural nucleotides or amino acids.

[0070] "Antineoplastic activity" means preventing or inhibiting the maturation and / or proliferation of neoplastic tissue.

[0071] "Autologous," as used herein, refers to cells from the same subject.

[0072] "Base editor (BE)" or "nucleobase 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 nucleic acid-programmable nucleotide-binding domain together with a guide polynucleotide (e.g., a guide RNA). In various embodiments, the agent is a biomolecular complex comprising a protein domain with base-editing activity, i.e., a domain that can modify bases (e.g., A, T, C, G, 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 comprising a domain 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 certain embodiments, the domain with base-editing activity can deaminate a base in a nucleic acid molecule. In certain embodiments, the base editor can deaminate one or more bases in a DNA molecule. In certain embodiments, the base editor is capable of deaminating adenosine (A) in DNA. In some embodiments, the base editor is an adenosine base editor (ABE).

[0073] In some embodiments, base editors are generated by cloning an adenosine deaminase variant (e.g., TadA*8) into a scaffold comprising a circularly permuted Cas9 (e.g., SpCas9 or saCas9) and a bipartite nuclear localization sequence (e.g., ABE8). Circularly permuted Cas9s are known in the art and are described, for example, in Oakes et al., Cell 176, 254-267, 2019. Exemplary circular permutations are shown below, where bolded sequences represent sequences derived from Cas9, italicized sequences represent linker sequences, and underlined sequences represent the bipartite nuclear localization sequence. CP5 (MSP [NGC = Pam variant with mutation Regular Cas9 likes NGG], PID = Protein Interacting Domain and contains "D10A" nickase) TIFF0007672982000004.tif172169

[0074] In some embodiments, ABE8 is selected from the base editors in Tables 8, 9, 10, or 11 below. In some embodiments, ABE8 comprises an adenosine deaminase variant evolved from TadA. In some embodiments, the adenosine deaminase variant of ABE8 is a TadA*8 variant set forth in Table 9 below. In some embodiments, the adenosine deaminase variant is a TadA*7.10 variant (e.g., TadA*8) that includes one or more changes selected from the group of Y147T, Y147R, Q154S, Y123H, V82S, T166R, and / or Q154R. In various embodiments, ABE8 is Y147T+Q154R;Y147T+Q154S;Y147R+Q154S;V82S+Q154S;V82S+Y147R;V82S+Q154R;V82S+Y123H;I76Y+V82S;V82S+Y123H+Y147T;V82S+Y123

[0013] In some embodiments, the ABE8 comprises a TadA*7.10 variant (e.g., TadA*8) having a combination of alterations selected from the group consisting of: H+Y147R; V82S+Y123H+Q154R; Y147R+Q154R+Y123H; Y147R+Q154R+I76Y; Y147R+Q154R+T166R; Y123H+Y147R+Q154R+I76Y; V82S+Y123H+Y147R+Q154R; and I76Y + V82S + Y123H + Y147R + Q154R. In some embodiments, the ABE8 is a monomeric construct. In some embodiments, the ABE8 is a heterodimeric construct. In some embodiments, the ABE8 has the sequence: MSEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWNRAIGLHDPTAHEIMALRQGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIGRVVFGVRNAKTGAAGSLMDVLHYPGMNHRVEITEGILADECAALLCTFFRMPRQVFNAQKKAQSSTD Includes:

[0075] In some embodiments, the polynucleotide programmable DNA binding domain is a CRISPR-associated (e.g., Cas or Cpf1) enzyme. In some embodiments, the base editor is a catalytically dead Cas9 (dCas9) fused to a deaminase domain. In some embodiments, the base editor is a Cas9 nickase (nCas9) fused to a deaminase domain. Details of base editors are described in International PCT Application Nos. PCT / 2017 / 045381 (WO 2018 / 027078) and PCT / US 2016 / 058344 (WO 2017 / 070632), each of which is incorporated herein by reference in its entirety. 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. 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: See also 10.1038 / s41576-018-0059-1, the entire contents of which are incorporated herein by reference.

[0076] By way of example, an adenine base editor (ABE) used in the base editing compositions, systems, and methods described herein has the nucleic acid sequence (8877 base pairs) provided below (Addgene, Watertown, MA.; Gaudelli NM, et al., Nature. 2017 Nov 23;551(7681):464-471. doi: 10.1038 / nature24644; Koblan LW, et al., Nat Biotechnol. 2018 Oct;36(9):843-846. doi: 10.1038 / nbt.4172). Polynucleotide sequences having at least 95% or greater identity to the ABE nucleic acid sequence are also encompassed. ATATGCCAAGTACGCCCCCTATTGACGTCAATGACGGTAAATGGCCCGCCTGGCATTATGCCCAGTACAT GACCTTATGGGACTTTCCTACTTGGCAGTACATCTACGTATTAGTCATCGCTATTACCATGGTGATGCGG TTTTGGCAGTACATCAATGGGCGTGGATAGCGGTTTGACTCACGGGATTTCCAAGTCTCCACCCCATTG ACGTCAATGGGAGTTTGTTTTGGCACCAAAATCAACGGGACTTTCCAAAATGTCGTAACAACTCCGCCCC ATTGACGCAAATGGGCGGTAGGCGTGTACGGTGGGAGGTCTATATAAGCAGAGCTGGTTTAGTGAACCGT CAGATCCGCTAGAGATCCGCGGCCGCTAATACGACTCACTATAGGGAGAGCCGCCACCATGAAACGGACA GCCGACGGAAGCGAGTTCGAGTCACCAAAGAAGAAGCGGAAAGTCTCTGAAGTCGAGTTTAGCCACGAGT ATTGGATGAGGCACGCACTGACCCTGGCAAAGCGAGCATGGGATGAAAGAGAAGTCCCCGTGGGCGCCGT GCTGGTGCACAACAATAGAGTGATCGGAGAGGGATGGAACAGGCCAATCGGCCGCCACGACCCTACCGCA CACGCAGAGATCATGGCACTGAGGCAGGGAGGCCTGGTCATGCAGAATTACCGCCTGATCGATGCCACCC TGTATGTGACACTGGAGCCATGCGTGATGTGCGCAGGAGCAATGATCCACAGCAGGATCGGAAGAGTGGT GTTCGGAGCACGGGACGCCAAGACCGGCGCAGCAGGCTCCCTGATGGATGTGCTGCACCACCCCGGCATG AACCACCGGGTGGAGATCACAGAGGGAATCCTGGCAGACGAGTGCGCCGCCCTGCTGAGCGATTTCTTTA GAATGCGGAGACAGGAGATCAAGGCCCAGAAGAAGGCACAGAGCTCCACCGACTCTGGAGGATCTAGCGG AGGATCCTCTGGAAGCGAGACACCAGGCACAAGCGAGTCCGCCACACCAGAGAGCTCCGGCGGCTCCTCC GGAGGATCCTCTGAGGTGGAGTTTTCCCACGAGTACTGGATGAGACATGCCCTGACCCTGGCCAAGAGGG CACGCGATGAGAGGGAGGTGCCTGTGGGAGCCGTGCTGGTGCTGAACAATAGAGTGATCGGCGAGGGCTG GAACAGAGCCATCGGCCTGCACGACCCAACAGCCCATGCCGAAATTATGGCCCTGAGACAGGGCGGCCTG GTCATGCAGAACTACAGACTGATTGACGCCACCCTGTACGTGACATTCGAGCCTTGCGTGATGTGCGCCG GCGCCATGATCCACTCTAGGATCGGCCGCGTGGTGTTTGGCGTGAGGAACGCAAAAACCGGCGCCGCAGG CTCCCTGATGGACGTGCTGCACTACCCCGGCATGAATCACCGCGTCGAAATTACCGAGGGAATCCTGGCA GATGAATGTGCCGCCCTGCTGTGCTATTTCTTTCGGATGCCTAGACAGGTGTTCAATGCTCAGAAGAAGG CCCAGAGCTCCACCGACTCCGGAGGATCTAGCGGAGGCTCCTCTGGCTCTGAGACACCTGGCACAAGCGA GAGCGCAACACCTGAAAGCAGCGGGGGCAGCAGCGGGGGGTCAGACAAGAAGTACAGCATCGGCCTGGCC ATCGGCACCAACTCTGTGGGCTGGGCCGTGATCACCGACGAGTACAAGGTGCCCAGCAAGAAATTCAAGG TGCTGGGCAACACCGACCGGCACAGCATCAAGAAGAACCTGATCGGAGCCCTGCTGTTCGACAGCGGCGA AACAGCCGAGGCCACCCGGCTGAAGAGAACCGCCAGAAGAAGATACACCAGACGGAAGAACCGGATCTGC TATCTGCAAGAGATCTTCAGCAACGAGATGGCCAAGGTGGACGACAGCTTCTTCCACAGACTGGAAGAGT CCTTCCTGGTGGAAGAGGATAAGAAGCACGAGCGGCACCCCATCTTCGGCAACATCGTGGACGAGGTGGC CTACCACGAGAAGTACCCCACCATCTACCACCTGAGAAAGAAACTGGTGGACAGCACCGACAAGGCCGAC CTGCGGCTGATCTATCTGGCCCTGGCCCACATGATCAAGTTCCGGGGCCACTTCCTGATCGAGGGCGACC TGAACCCCGACAACAGCGACGTGGACAAGCTGTTCATCCAGCTGGTGCAGACCTACAACCAGCTGTTCGA GGAAAACCCCATCAACGCCAGCGGCGTGGACGCCAAGGCCATCCTGTCTGCCAGACTGAGCAAGAGCAGA CGGCTGGAAAATCTGATCGCCCAGCTGCCCGGCGGAGAAAGAATGGCCTGTTCGGAAACCTGATTGCCC TGAGCCTGGGCCTGACCCCAACTTCAAGAGCAACTTCGACCTGGCCGAGGATGCCAAACTGCAGCTGAG CAAGGACACCTACGACGACGACGACCTGGACAACCTGCTGGCCCAGATCGGCGACCAGTACGGCCGACCTGTTTT CTGGCCGCCAAGAACCTGCTCGACGCCATCCTGCTGAGCGACATCCTGAGAGTGAACACCGAGATCACCA AGGCCCCCCTGAGCGCCTCTATGATCAAGAGATACGACGAGCACCACCAGGACCTGACCCTGCTGAAAGC TCTCGTGCGGCAGCAGCTGCCTGAGAAGTACAAAGAGATTTTCTTCGACCAGAGCAAGAACGGCTACGCC GGCTACATTGACGGCGGAGCCAGCCAGGAAGAGTTCTACAAGTTCATCAAGCCCATCCTGGAAAAGATGG ACGGCACCGAGGAACTGCTCGTGAAGCTGAACAGAGGACCTGCTGCGGAAGCAGCGGACCTTCGACAA CGGCAGCATCCCCCACCAGATCCACCTGGGAGAGCTGCACGCCATTCTGCGGCGGCAGGAAGATTTTTAC CCATTCCTGAAGGACAACCGGGAAAGATCGAGAAGATCCTGACCTTCCGCATCCCCTACTACGTGGGCC CTCTGGCCAGGGGAAACAGCAGATTCGCCTGGATGACCAGAAAGAGCGAGGAAACCATCACCCCCTGGAA CTTCGAGGAAGTGGTGGACAAGGGCGCTTCCGCCCAGAGCTTCATCGAGCGGATGACCAACTTCGATAAG AACCTGCCCAACGAGAAGGTGCTGCCCAAGCACAGCCTGCTGTACGAGTACTTCACCGTGTATAACGAGC TGACCAAAGTGAAATACGTGACCGAGGGAATGAGAAAGCCCGCCTTCCTGAGCGGCGAGCAGAAAAAGGC CATCGTGGACCTGCTGTTCAAGACCAACCGGAAAGTGACCGTGAAGCAGCTGAAAGAGGACTACTTCAAG AAAATCGAGTGCTTCGACTCCGTGGAAATCTCCGGCGTGGAAGATCGGTTCAACGCCTCCCTGGGCACAT ACCACGATCTGCTGAAAATTATCAAGGACAAGGACTTCCTGGACAATGAGGAAAACGAGGACATTCTGGA AGATATCGTGCTGACCCTGACACTGTTTGAGGACAGAGAGATGATCGAGGAACGGCTGAAAACCTATGCC CACCTGTTCGACGACAAAGTGATGAAGCAGCTGAAGCGGCGGAGATACACCGGCTGGGGCAGGCTGAGCC GGAAGCTGATCAACGGCATCCGGGACAAGCAGTCCGGCAAGACAATCCTGGATTTCCTGAAGTCCGACGG CTTCGCCAACAGAAACTTCATGCAGCTGATCCACGACGACAGCCTGACCTTTAAAGAGGACATCCAGAAA GCCCAGGTGTCCGGCCAGGGCGATAGCCTGCACGAGCACATTGCCAATCTGGCCGGCAGCCCCGCCATTA AGAAGGGCATCCTGCAGACAGTGAAGGTGGTGGACGAGCTCGTGAAAGTGATGGGCCGGCACAAGCCCGA GAACATCGTGATCGAAATGGCCAGAGAGAACCAGACCACCCAGAAGGGACAGAAGAACAGCCGCGAGAGA ATGAAGCGGATCGAAGAGGGCATCAAAGAGCTGGGCAGCCAGATCCTGAAAGAACACCCCGTGGAAAACA CCCAGCTGCAGAACGAGAAGCTGTACCTGTACTACCTGCAGAATGGGCGGGATATGTACGTGGACCAGGA ACTGGACATCAACCGGCTGTCCGACTACGATGTGGACCATATCGTGCCTCAGAGCTTTCTGAAGGACGAC TCCATCGACAACAAGGTGCTGACCAGAAGCGACAAGAACCGGGGCAAGAGCGACAACGTGCCCTCCGAAG AGGTCGTGAAGAAGATGAAGAACTACTGGCGGCAGCTGCTGAACGCCAAGCTGATTACCCAGAGAAAGTT CGACAATCTGACCAAGGCCGAGAGAGGCGGCCTGAGCGAACTGGATAAGGCCGGCTTCATCAAGAGACAG CTGGTGGAAACCCGGCAGATCACAAAGCACGTGGCACAGATCCTGGACTCCCGGATGAACACTAAGTACG ACGAGAATGACAAGCTGATCCGGGAAGTGAAAGTGATCACCCTGAAGTCCAAGCTGGTGTCCGATTTCCG GAAGGATTTCCAGTTTTACAAAGTGCGCGAGATCAACAACTACCACCACGCCCACGACGCCTACCTGAAC GCCGTCGTGGGAACCGCCCTGATCAAAAAGTACCCTAAGCTGGAAAGCGAGTTCGTGTACGGCGACTACA AGGTGTACGACGTGCGGAAGATGATCGCCAAGAGCGAGCAGGAAATCGGCAAGGCTACCGCCAAGTACTT CTTCTACAGCAACATCATGAACTTTTTCAAGACCGAGATTACCCTGGCCAACGGCGAGATCCGGAAGCGG CCTCTGATCGAGACAAACGGCGAAACCGGGGAGATCGTGTGGGATAAGGGCCGGGATTTTGCCACCGTGC GGAAAGTGCTGAGCATGCCCCAAGTGAATATCGTGAAAAAGACCGAGGTGCAGACAGGCGGCTTCAGCAA AGAGTCTATCCTGCCCAAGAGGAACAGCGATAAGCTGATCGCCAGAAAGAAGGACTGGGACCCTAAGAAG TACGGCGGCTTCGACAGCCCCACCGTGGCCTATTCTGTGCTGGTGGTGGCCAAAGTGGAAAAGGGCAAGT CCAAGAAACTGAAGAGTGTGAAAGAGCTGCTGGGGATCACCATCATGGAAAGAAGCAGCTTCGAGAAGAA TCCCATCGACTTTCTGGAAGCCAAGGGCTACAAAGAAGTGAAAAAGGACCTGATCATCAAGCTGCCTAAG TACTCCCTGTTCGAGCTGGAAAACGGCCGGAAGAGAATGCTGGCCTCTGCCGGCGAACTGCAGAAGGGAA ACGAACTGGCCCTGCCCTCCAAATATGTGAACTTCCTGTACCTGGCCAGCCACTATGAGAAGCTGAAGGG CTCCCCCGAGGATAATGAGCAGAAACAGCTGTTTGTGGAACAGCACAAGCACTACCTGGACGAGATCATC GAGCAGATCAGCGAGTTCTCCAAGAGAGTGATCCTGGCCGACGCTAATCTGGACAAAGTGCTGTCCGCCT ACAACAAGCACCGGGATAAGCCCATCAGAGAGCAGGCCGAGAATATCATCCACCTGTTTACCCTGACCAA TCTGGGAGCCCCTGCCGCCTTCAAGTACTTTGACACCACCATCGACCGGAAGAGGTACACCAGCACCAAA GAGGTGCTGGACGCCACCCTGATCCACCAGAGCATCACCGGCCTGTACGAGACACGGATCGACCTGTCTC AGCTGGGAGGTGACTCTGGCGGCTCAAAAAGAACCGCCGACGGCAGCGAATTCGAGCCCAAGAAGAAGAG GAAAGTCTAACCGGTCATCATCACCATCACCATTGAGTTTAAACCCGCTGATCAGCCTCGACTGTGCCTT CTAGTTGCCAGCCATCTGTTGTTTGCCCCTCCCCCGTGCCTTCCTTGACCCTGGAAGGTGCCACTCCCAC TGTCCTTTCCTAATAAAATGAGGAAATTGCATCGCATTGTCTGAGTAGGTGTCATTCTATTCTGGGGGGT GGGGTGGGGCAGGACAGCAAGGGGGAGGATTGGGAAGACAATAGCAGGCATGCTGGGGATGCGGTGGGCT CTATGGCTTCTGAGGCGGAAAGAACCAGCTGGGGCTCGATACCGTCGACCTCTAGCTAGAGCTTGGCGTA ATCATGGTCATAGCTGTTTCCTGTGTGAAATTGTTATCCGCTCACAATTCCACACAACATACGAGCCGGA AGCATAAAGTGTAAAGCCTAGGGTGCCTAATGAGTGAGCTAACTCACATTAATTGCGTTGCGCTCACTGC CCGCTTTCCAGTCGGGAAACCTGTCGTGCCAGCTGCATTAATGAATCGGCCAACGCGCGGGGAGAGGCGG TTTGCGTATTGGGCGCTCTTCCGCTTCCTCGCTCACTGACTCGCTGCGCTCGGTCGTTCGGCTGCGGCGA GCGGTATCAGCTCACTCAAAGGCGGTAATACGGTTATCCACAGAATCAGGGGATAACGCAGGAAAGAACA TGTGAGCAAAAGGCCAGCAAAAGGCCAGGAACCGTAAAAAGGCCGCGTTGCTGGCGTTTTTCCATAGGCT CCGCCCCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAACCCGACAGGACTATAA AGATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGCCGCTTACCGGAT ACCTGTCCGCCTTTCTCCCTTCGGGAAGCGTGGCGCTTTCTCATAGCTCACGCTGTAGGTATCTCAGTTC GGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTGCACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTA TCCGGTAACTATCGTCTTGAGTCCAACCCGGTAAGACACGACTTATCGCCACTGGCAGCAGCCACTGGTA ACAGGATTAGCAGAGCGAGGTATGTAGGCGGTGCTACAGAGTTCTTGAAGTGGTGGCCTAACTACGGCTA CACTAGAAGAACAGTATTTGGTATCTGCGCTCTGCTGAAGCCAGTTACCTTCGGAAAAAGAGTTGGTAGC TCTTGATCCGGCAAACAAACCACCGCTGGTAGCGGTGGTTTTTTTGTTTGCAAGCAGCAGATTACGCGCA GAAAAAAAGGATCTCAAGAAGATCCTTTGATCTTTTCTACGGGGTCTGACACTCAGTGGAACGAAAACTC ACGTTAAGGGATTTTGGTCATGAGATTATCAAAAAGGATCTTCACCTAGATCCTTTTAAATTAAAAATGA AGTTTTAAATCAATCTAAAGTATATATGAGTAAACTTGGTCTGACAGTTACCAATGCTTAATCAGTGAGG CACCTATCTCAGCGATCTGTCTATTTCGTTCATCCATAGTTGCCTGACTCCCCGTCGTGTAGATAACTAC GATACGGGAGGGCTTACCATCTGGCCCCAGTGCTGCAATGATACCGCGAGACCCACGCTCACCGGCTCCA GATTTATCAGCAATAAACCAGCCAGCCGGAAGGGCCGAGCGCAGAAGTGGTCCTGCAACTTTATCCGCCT CCATCCAGTCTATTAATTGTTGCCGGGAAGCTAGAGTAAGTAGTTCGCCAGTTAATAGTTTGCGCAACGT TGTTGCCATTGCTACAGGCATCGTGGTGTCACGCTCGTCGTTTGGTATGGCTTCATTCAGCTCCGGTTCC CAACGATCAAGGCGAGTTACATGATCCCCCATGTTGTGCAAAAAAGCGGTTAGCTCCTTCGGTCCTCCGA TCGTTGTCAGAAGTAAGTTGGCCGCAGTGTTATCACTCATGGTTATGGCAGCACTGCATAATTCTCTTAC TGTCATGCCATCCGTAAGATGCTTTTCTGTGACTGGTGAGTACTCAACCAAGTCATTCTGAGAATAGTGT ATGCGGCGACCGAGTTGCTCTTGCCCGGCGTCAATACGGGATAATACCGCGCCACATAGCAGAACTTTAA AAGTGCTCATCATTGGAAAACGTTCTTCGGGGCGAAAACTCTCAAGGATCTTACCGCTGTTGAGATCCAG TTCGATGTAACCCACTCGTGCACCCAACTGATCTTCAGCATCTTTTACTTTCACCAGCGTTTCTGGGTGA GCAAAAACAGGAAGGCAAAATGCCGCAAAAAAGGGAATAAGGGCGACACGGAAATGTTGAATACTCATAC TCTTCCTTTTTCAATATTATTGAAGCATTTATCAGGGTTATTGTCTCATGAGCGGATACATATTTGAATG TATTTAGAAAAATAAACAAATAGGGGTTCCGCGCACATTTCCCCGAAAAGTGCCACCTGACGTCGACGGA TCGGGAGATCGATCTCCCGATCCCCTAGGGTCGACTCTCAGTACAATCTGCTCTGATGCCGCATAGTTAA GCCAGTATCTGCTCCCTGCTTGTGTGTTGGAGGTCGCTGAGTAGTGCGCGAGCAAAATTTAAGCTACAAC AAGGCAAGGCTTGACCGACAATTGCATGAAGAATCTGCTTAGGGTTAGGCGTTTTGCGCTGCTTCGCGAT GTACGGGCCAGATATACGCGTTGACATTGATTATTGACTAGTTATTAATAGTAATCAATTACGGGGTCAT TAGTTCATAGCCCATATATGGAGTTCCGCGTTACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCC CAACGACCCCCGCCCATTGACGTCAATAATGACGTATGTTCCCATAGTAACGCCAATAGGGACTTTCCAT TGACGTCAATGGGTGGAGTATTTACGGTAAACTGCCCACTTGGCAGTACATCAAGTGTATC

[0077] "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 cytidine deaminase activity, e.g., converting a target C·G to T·A. In another embodiment, the base editing activity is adenosine or adenine deaminase activity, e.g., converting A·T to G·C. In another embodiment, the base editing activity is cytidine deaminase activity, e.g., converting a target C·G to T·A, or adenosine or adenine deaminase activity, e.g., converting A·T to G·C. In some embodiments, base editing activity is assessed by editing efficiency. Base editing efficiency can be measured by a suitable means, such as Sanger sequencing or next-generation sequencing. In some embodiments, base editing efficiency is measured by the percentage of all sequencing reads that have a nucleobase conversion introduced by a base editor, e.g., the percentage of all sequencing reads that have a targeted AT base pair converted to a GC base pair. In some embodiments, base editing efficiency is measured by the percentage of all cells that have a nucleobase conversion introduced by a base editor when base editing is performed in a population of cells.

[0078] The term "base editor system" refers to a system for editing nucleobases of a target nucleotide sequence. In various embodiments, the base editor system includes (1) a polynucleotide-programmable nucleotide-binding domain (e.g., Cas9), (2) a deaminase domain (e.g., adenosine deaminase or cytidine deaminase) for deaminating the nucleobase, and (3) one or more guide polynucleotides (e.g., guide RNA). In some embodiments, the polynucleotide-programmable nucleotide-binding domain is a polynucleotide-programmable DNA-binding domain. In some embodiments, the base editor is an adenine or adenosine base editor (ABE). In some embodiments, the base editor system is ABE8.

[0079] In some embodiments, a base editor system may comprise two or more base editor components. For example, a base editor system may comprise two or more deaminases. In some embodiments, a base editor system may comprise one or more adenosine deaminases. In some embodiments, a single guide polynucleotide may be utilized to target different deaminases to a nucleic acid sequence. In some embodiments, a single pair of guide polynucleotides may be utilized to target different deaminases to a nucleic acid sequence.

[0080] The deaminase domain and polynucleotide-programmable nucleotide-binding component of the base editor system can be associated with each other covalently or non-covalently, or any combination of these associations and interactions. For example, in some embodiments, the deaminase domain can be targeted to a target nucleotide sequence by a polynucleotide-programmable nucleotide-binding domain. In some embodiments, the polynucleotide-programmable nucleotide-binding domain can be fused or linked to the deaminase domain. In some embodiments, the polynucleotide-programmable nucleotide-binding domain can target the deaminase domain to a target nucleotide sequence by non-covalently interacting or associating with the deaminase domain. For example, in some embodiments, the deaminase domain can include an additional heterologous moiety or domain that can interact, associate, or complex with an additional heterologous moiety or domain that is part of the polynucleotide-programmable nucleotide-binding domain. In some embodiments, the additional heterologous moiety can bind, interact, associate, or complex with a polypeptide. In some embodiments, the additional heterologous moiety can bind, interact, associate, or complex with a polynucleotide. In some embodiments, the additional heterologous moiety can bind to a guide polynucleotide. In some embodiments, the additional heterologous moiety can be attached to a polypeptide linker. In some embodiments, the additional heterologous moiety can be attached to a polynucleotide linker. The additional heterologous moiety can be a protein domain. In some embodiments, the additional heterologous moiety can be a K homology (KH) domain, an MS2 coat protein domain, a PP7 coat protein domain, an SfMu Com coat protein domain, a steryl alpha motif, a telomerase Ku binding motif and Ku protein, a telomerase Sm7 binding motif and Sm7 protein, or an RNA recognition motif.

[0081] The base editor system may further comprise a guide polynucleotide component. It should be recognized that the components of the base editor system may associate with each other via covalent bonds, non-covalent interactions, or any combination thereof. In some embodiments, the deaminase domain can be targeted to a target nucleotide sequence by a guide polynucleotide. For example, in some embodiments, the deaminase domain can include an additional heterologous moiety or domain (e.g., a polynucleotide binding domain such as an RNA or DNA binding protein) that can interact, associate, or complex with a portion or fragment (e.g., a polynucleotide motif) of the guide polynucleotide. In some embodiments, the additional heterologous moiety or domain (e.g., a polynucleotide binding domain such as an RNA or DNA binding protein) can be fused or linked to the deaminase domain. In some embodiments, the additional heterologous moiety can bind, interact, associate, or complex with a polypeptide. In some embodiments, the additional heterologous moiety can bind, interact, associate, or complex with a polynucleotide. In some embodiments, the additional heterologous moiety can be attached to a guide polynucleotide. In some embodiments, the additional heterologous moiety can be attached to a polypeptide linker. In some embodiments, the additional heterologous moiety can be attached to a polynucleotide linker. The additional heterologous moiety may be a protein domain, hi some embodiments, a K homology (KH) domain, an MS2 coat protein domain, a PP7 coat protein domain, an SfMu Com coat protein domain, a steryl alpha motif, a telomerase Ku binding motif and Ku protein, a telomerase Sm7 binding motif and Sm7 protein, or an RNA recognition motif.

[0082] In some embodiments, the base editor system can further comprise an inhibitor of a base excision repair (BER) component. It should be understood that the components of the base editor system can be associated with each other via covalent bonds, non-covalent interactions, or any combination of these associations and interactions. The inhibitor of a BER component can comprise a BER inhibitor. In some embodiments, the BER inhibitor can be a uracil DNA glycosylase inhibitor (UGI). In some embodiments, the BER inhibitor can be an inosine BER inhibitor. In some embodiments, the BER inhibitor can be targeted to a target nucleotide sequence by a polynucleotide programmable nucleotide binding domain. In some embodiments, the polynucleotide programmable nucleotide binding domain can be fused or linked to a BER inhibitor. In some embodiments, the polynucleotide programmable nucleotide binding domain can be fused or linked to a deaminase domain and a BER inhibitor. In some embodiments, the polynucleotide programmable nucleotide binding domain can target a BER inhibitor to a target nucleotide sequence by non-covalently interacting with or associating with the BER inhibitor. For example, in some embodiments, an inhibitor of a BER component may comprise an additional heterologous moiety or domain that may interact, associate, or complex with an additional heterologous moiety or domain that is part of the polynucleotide programmable nucleotide binding domain.

[0083] In some embodiments, the BER inhibitor can be targeted to a target nucleotide sequence by a guide polynucleotide. For example, in some embodiments, the BER inhibitor can include an additional heterologous moiety or domain (e.g., a polynucleotide binding domain such as an RNA or DNA binding protein) that can interact, associate, or complex with a portion or segment (e.g., a polynucleotide motif) of the guide polynucleotide. In some embodiments, the additional heterologous moiety or domain (e.g., a polynucleotide binding domain such as an RNA or DNA binding protein) of the guide polynucleotide can be fused or linked to the BER inhibitor. In some embodiments, the additional heterologous moiety can bind, interact, associate, or complex with the polynucleotide. In some embodiments, the additional heterologous moiety can be linked to the guide polynucleotide. In some embodiments, the additional heterologous moiety can be linked to a polypeptide linker. In some embodiments, the additional heterologous moiety can be linked to a polynucleotide linker. The additional heterologous moiety can be a protein domain. In some embodiments, the additional heterologous moiety can be a K homology (KH) domain, an MS2 coat protein domain, a PP7 coat protein domain, an SfMu Com coat protein domain, a steryl alpha motif, a telomerase Ku binding motif and Ku protein, a telomerase Sm7 binding motif and Sm7 protein, or an RNA recognition motif.

[0084] "B-cell maturation antigen, or tumor necrosis factor receptor superfamily member 17 polypeptide (BCMA)" means a protein or fragment thereof having at least about 85% amino acid sequence identity to NCBI Accession No. NP_001183, which is expressed on mature B lymphocytes. An exemplary BCMA polypeptide sequence is provided below. >NP_001183.2 Tumor necrosis factor receptor superfamily member 17 [Homo sapiens] MLQMAGQCSQNEYFDSLLHACIPCQLRCSSNTPPLTCQRYCNASVTNSVKGTNAILWTCLGLSLIISLAVFVLMFLLRKINSEPLKDEFKNTGSGLLGMANIDLEKSRTGDEIILPRGLEYTVEECTCEDCIKSKPKVDSDHCFPLPAMEEGATILVTTKTNDYCKSLPAALSATEIEKSISAR

[0085] This antigen can be targeted in relapsed or refractory multiple myeloma and other hematopoietic therapies.

[0086] "B-cell maturation antigen, or tumor necrosis factor receptor superfamily member 17 (BCMA) polynucleotide" refers to a nucleic acid molecule encoding a BCMA polypeptide. The BCMA gene encodes a cell surface receptor that recognizes B-cell activating factors. Exemplary B2M polynucleotide sequences are provided below. >NM_001192.2 Homo sapiens TNF receptor superfamily member 17 (TNFRSF17), mRNA AAGACTCAAACTTAGAAACTTGAATTAGATGTGGTATTCAAATCCTTAGCTGCCGCGAAGACACAGACAGCCCCCGTAAGAACCCACGAAGCAGGCGAAGTTCATTGTTCTCCAACATTCTAGCTGCTCTTGCTGCATTTGCTCTGGAATTCTTGTAGAGATATTACTTGTCCTTCCAGGCTGTTCTTTCTGTAGCTCCCTTGTTTTCTTTTTGTGATCATGTTGCAGATGGCTGGGCAGTGCTCCCAAAATGAATATTTTGACAGTTTGTTGCATGCTTGCATACCTTGTCAACTTCGATGTTCTTCTAATACTCCTCCTCTAACATGTCAGCGTTATTGTAATGCAAGTGTGACCAATTCAGTGAAAGGAACGAATGCGATTCTCTGGACCTGTTTGGGACTGAGCTTAATAATTTCTTTGGCAGTTTTCGTGCTAATGTTTTTGCTAAGGAAGATAAACTCTGAACCATTAAAGGACGAGTTTAAAAACACAGGA TCAGGTCTCCTGGGCATGGCTAACATTGACCTGGAAAAGAGCAGGACTGGTGATGAAATTATTCTTCCGAGAGGCCTCGAGTACACGGTGGAAGAATGCACCTTGTGAAGACTGCATCAAGAGCA AACCGAAGGTCGACTCTGACCATTGCTTTCCACTCCCAGCTATGGAGGAAGGCGCAACCATTCTTGTCACCACGAAAACGAATGACTATTGCAAGAGCCTGCCAGCTGCTTTGAGTGCTACGGA GATAGAGAAATCAATTTCTGCTAGGTAATTAACCATTTCGACTCGAGCAGTGCCACTTTAAAAATCTTTTGTCAGAATAGATGATGTGTCAGATCTCTTTAGGATGACTGTATTTTTCAGTTGC CGATACAGCTTTTTGTCCTCTAACTGTGGAAACTCTTTATGTTAGATATATTTCTCTAGGTTACTGTTGGGAGCTTAATGGTAGAAACTTCCTTGGTTTCATGATTAAACTCTTTTTTTTCCTGA

[0087] "Beta-2 microglobulin (B2M) polypeptide" means a protein or fragment thereof having at least about 85% amino acid sequence identity with UniProt Accession No. P61769 and having immunomodulatory activity. Exemplary B2M polypeptide sequences are provided below. >sp|P61769|B2MG_Human beta-2-microglobulin OS=Homo sapiens OX=9606 GN=B2M PE=1 SV=1 MSRSVALAVLALLSLSGLEAIQRTPKIQVYSRHPAENGKSNFLNCYVSGFHPSDIEVDLL KNGERIEKVEHSDLSFSKDWSFYLLYYTEFTPTEKDEYACRVNHVTLSQPKIVKWDRDM

[0088] "Beta-2-microglobulin (B2M) polynucleotide" refers to a nucleic acid molecule encoding a B2M polypeptide. The beta-2-microglobulin gene encodes a serum protein associated with the major histocompatibility complex. B2M is involved in allo-recognition by host CD8+ T cells. Exemplary B2M polynucleotide sequences are provided below. >DQ217933.1 Homo sapiens beta-2-microglobulin (B2M) gene, complete cds

[0089] The term "Cas9" or "Cas9 domain" refers to an RNA-guided nuclease containing the Cas9 protein or a fragment thereof (e.g., a protein containing an active, inactive, or partially active DNA cleavage domain of Cas9 and / or a gRNA-binding domain of Cas9). Cas9 nuclease is sometimes referred to as a casnl nuclease or a CRISPR (clustered regularly interspaced short palindromic repeat)-binding nuclease. CRISPR is an adaptive immune system that provides defense against mobile genetic elements (viruses, transposable elements, and conjugative plasmids). CRISPR clusters contain a spacer, a sequence complementary to the preceding mobile element, and a target invading nucleic acid. CRISPR clusters are transcribed and processed into CRISPR RNA (crRNA). In the type II CRISPR system, correct processing of the pre-crRNA requires a transcoding small RNA (tracrRNA), endogenous ribonuclease 3 (rnc), and the Cas9 protein. The tracrRNA guides the processing of the pre-crRNA by RNase 3. Cas9 / crRNA / tracrRNA then endonucleolytically cleaves linear or circular dsDNA targets complementary to the spacer. The target strand not complementary to the crRNA is first endonucleolytically cleaved and then exonucleolytically trimmed 3'-5'. In nature, DNA binding and cleavage typically require both a protein and both RNAs. However, single-guide RNAs ("sgRNAs," or simply "gRNAs") can be engineered to incorporate both crRNA and tracrRNA aspects into a single RNA species. See, for example, Jinek M., et al. Science 337:816-821 (2012), the entire contents of which are incorporated herein by reference. Cas9 recognizes a short motif (the PAM or protospacer-adjacent motif) in the CRISPR repeat sequence to help distinguish "self" from "non-self." The Cas9 nuclease sequence and structure are well known to those skilled in the art.(See, e.g., "Complete genome sequence of an M1 strain of Streptococcus pyogenes," Ferretti et al., Proc. Natl. Acad. Sci. USA 98:4658-4663(2001); "CRISPR RNA maturation by trans-encoded small RNA and host factor RNase III," Deltcheva E., et al., Nature 471:602-607(2011); and "A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity," Jinek M., et al., Science 337:816-821(2012), the entire contents of each of which are incorporated herein by reference.) Cas9 orthologs have been described in various species, including, but not limited to, S. pyogenes and S. thermophilus. Additional suitable Cas9 nucleases and sequences will be apparent to those of skill in the art based on the present disclosure. Such Cas9 nucleases and sequences include Cas9 sequences from the organisms and loci disclosed in Chylinski, Rhun, and Charpentier, "The tracrRNA and Cas9 families of type II CRISPR-Cas immunity systems" (2013) RNA Biology 10:5, 726-737, the entire contents of which are incorporated herein by reference.

[0090] An exemplary Cas9 is Streptococcus pyogenes Cas9 (spCas9), the amino acid sequence of which is provided below. TIFF0007672982000005.tif167169 (single underline: HNH domain, double underline: RuvC domain)

[0091] Nuclease-inactivated Cas9 proteins can be interchangeably referred to as "dCas9" proteins (meaning nuclease "dead" Cas9) or catalytically inactive Cas9. Methods for generating Cas9 proteins (or fragments thereof) with inactive DNA cleavage domains are known (see, e.g., Jinek et al., Science. 337:816-821(2012); Qi et al., "Repurposing CRISPR as an RNA-Guided Platform for Sequence-Specific Control of Gene Expression" (2013) Cell. 28;152(5):1173-83, the entire contents of which are incorporated herein by reference). For example, the DNA cleavage domain of Cas9 is known to contain two subdomains: the HNH nuclease subdomain and the RuvC1 subdomain. The HNH subdomain cleaves the strand complementary to the gRNA, while the RuvC1 subdomain cleaves the non-complementary strand. Mutations within these subdomains can suppress the nuclease activity of Cas9. For example, mutations D10A and H840A completely inactivate the nuclease activity of S. pyogenes Cas9 (Jinek et al., Science. 337:816-821(2012); Qi et al., Cell. 28;152(5):1173-83 (2013)). In some embodiments, the Cas9 nuclease has an inactive (e.g., inactivated) DNA cleavage domain, i.e., Cas9 is a nickase, referred to as an "nCas9" protein (for "nickase" Cas9). In some embodiments, proteins comprising fragments of Cas9 are provided. For example, in some embodiments, the protein comprises one of two Cas9 domains: (1) the gRNA-binding domain of Cas9, or (2) the DNA cleavage domain of Cas9. In some embodiments, proteins comprising Cas9 or fragments thereof are referred to as "Cas9 variants." Cas9 variants share homology with Cas9 or fragments thereof.For example, a Cas9 variant is at least about 70% identical, at least about 80% identical, at least about 90% identical, at least about 95% identical, at least about 96% identical, at least about 97% identical, at least about 98% identical, at least about 99% identical, at least about 99.5% identical, or at least about 99.9% identical to wild-type Cas9. In some embodiments, the Cas9 variant may have 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 21, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50 or more amino acid changes compared to wild-type Cas9. In some embodiments, Cas9 variants comprise a fragment of Cas9 (e.g., a gRNA binding domain or a DNA-cleavage domain) such that the fragment is at least about 70% identical, at least about 80% identical, at least about 90% identical, at least about 95% identical, at least about 96% identical, at least about 97% identical, at least about 98% identical, at least about 99% identical, at least about 99.5% identical, or at least about 99.9% identical to a corresponding fragment of wild-type Cas9. In some embodiments, the fragment is at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% of the amino acid length of the corresponding wild-type Cas9.

[0092] In some embodiments, fragments are at least 100 amino acids in length. In some embodiments, fragments are at least 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1050, 1100, 1150, 1200, 1250, or at least 1300 amino acids in length.

[0093] In some embodiments, the wild-type Cas9 corresponds to Cas9 from Streptococcus pyogenes (NCBI Reference Sequence: NC_017053.1, nucleotide and amino acid sequences are as follows): TIFF0007672982000006.tif168169 (single underline: HNH domain; double underline: RuvC domain)

[0094] In some embodiments, the wild-type Cas9 corresponds to or comprises the following nucleotide and / or amino acid sequence: TIFF0007672982000007.tif167169 (single underline: HNH domain, double underline: RuvC domain)

[0095] In some embodiments, the wild-type Cas9 corresponds to Cas9 from Streptococcus pyogenes (NCBI Reference Sequence: NC_002737.2 (nucleotide sequence is as follows): and Uniprot Reference Sequence: Q99ZW2 (amino acid sequence is as follows): TIFF0007672982000008.tif167169 (SEQ ID NO: 1. Single underline: HNH domain, double underline: RuvC domain)

[0096] In some embodiments, the Cas9 is a Corynebacterium ulcerans (NCBI Refs: NC_015683.1, NC_017317.1); Corynebacterium diphtheria (NCBI Refs: NC_016782.1, NC_016786.1); intermedia (NCBI Ref: NC_017861.1); Spiroplasma taiwanense (NCBI Ref: NC_021846.1); Streptococcus iniae (NCBI Ref: NC_021314.1); Belliella baltica (NCBI Ref: NC_018010.1); Psychroflexus torquisI (NCBI Ref: NC_018721.1); Streptococcus thermophilus (NCBI Ref: YP_820832.1), Listeria innocua (NCBI Ref: NP_472073.1), Campylobacter jejuni (NCBI Ref: YP_002344900.1) or Neisseria meningitidis (NCBI Ref: YP_002342100.1), or Cas9 from any other organism.

[0097] In some embodiments, the Cas9 is derived from Neisseria meningitidis (Nme). In some embodiments, the Cas9 is Nme1, Nme2, or Nme3. In some embodiments, the PAM-interacting domains of Nme1, Nme2, or Nme3 are N4GAT, N4CC, and N4CAAA, respectively (see, e.g., Edraki, A., et al., A Compact, High-Accuracy Cas9 with a Dinucleotide PAM for In Vivo Genome Editing, Molecular Cell (2018)). An exemplary Neisseria meningitidis Cas9 protein, Nme1Cas9 (NCBI Reference: WP_002235162.1; Type II CRISPR RNA-guided endonuclease Cas9), has the following amino acid sequence: 1 maafkpnpin yilgldigia svgwamveid edenpiclid lgvrvferae vpktgdslam 61 arrlarsvrr ltrrrahrll rarrllkreg vlqaadfden glikslpntp wqlraaaldr 121 kltplewsav llhlikhrgy lsqrkneget adkelgallk gvadnahalq tgdfrtpael 181 alnkfekesg hirnqrgdys htfsrkdlqa elillfekqk efgnphvsgg lkegietllm 241 tqrpalsgda vqkmlghctf epaepkaakn tytaerfiwl tklnnlrile qgserpltdt 301 eratlmdepy rkskltyaqa rkllgledta ffkglrygkd naeastlmem kayhaisral 361 ekeglkdkks plnlspelqd eigtafslfk tdeditgrlk driqpeilea llkhisfdkf 421 vqislkalrr ivplmeqgkr ydeacaeiyg dhygkkntee kiylppipad eirnpvvlra 481 lsqarkving vvrrygspar ihietarevg ksfkdrkeie krqeenrkdr ekaaakfrey 541 fpnfvgepks kdilklrlye qqhgkclysg keinlgrlne kgyveidhal pfsrtwddsf 601 nnkvlvlgse nqnkgnqtpy eyfngkdnsr ewqefkarve tsrfprskkq rillqkfded 661 gfkernlndt ryvnrflcqf vadrmrltgk gkkrvfasng qitnllrgfw glrkvraend 721 rhhaldavvv acstvamqqk itrfvrykem nafdgktidk etgevlhqkt hfpqpweffa 781 qevmirvfgk pdgkpefeea dtpeklrtll aeklssrpea vheyvtplfv srapnrkmsg 841 qghmetvksa krldegvsvl rvpltqlklk dlekmvnrer epklyealka rleahkddpa 901 kafaepfyky dkagnrtqqv kavrveqvqk tgvwvrnhng iadnatmvrv dvfekgdkyy 961 lvpiyswqva kgilpdravv qgkdeedwql iddsfnfkfs lhpndlvevi tkkarmfgyf 1021 aschrgtgni nirihdldhk igkngilegi gvktalsfqk yqidelgkei rpcrlkkrpp 1081 vr

[0098] Another exemplary Neisseria meningitidis Cas9 protein, Nme2Cas9 (NCBI Reference: WP_002230835; Type II CRISPR RNA-guided endonuclease Cas9) has the following amino acid sequence: 1 maafkpnpin yilgldigia svgwamveid eeenpirlid lgvrvferae vpktgdslam 61 arrlarsvrr ltrrrahrll rarrllkreg vlqaadfden glikslpntp wqlraaaldr 121 kltplewsav llhlikhrgy lsqrkneget adkelgallk gvannahalq tgdfrtpael 181 alnkfekesg hirnqrgdys htfsrkdlqa elillfekqk efgnphvsgg lkegietllm 241 tqrpalsgda vqkmlghctf epaepkaakn tytaerfiwl tklnnlrile qgserpltdt 301 eratlmdepy rkskltyaqa rkllgledta ffkglrygkd naeastlmem kayhaisral 361 ekeglkdkks plnlsselqd eigtafslfk tdeditgrlk drvqpeilea llkhisfdkf 421 vqislkalrr ivplmeqgkr ydeacaeiyg dhygkkntee kiylppipad eirnpvvlra 481 lsqarkving vvrrygspar ihietarevg ksfkdrkeie krqeenrkdr ekaaakfrey 541 fpnfvgepks kdilklrlye qqhgkclysg keinlvrlne kgyveidhal pfsrtwddsf 601 nnkvlvlgse nqnkgnqtpy ​​eyfngkdnsr ewqefkarve tsrfprskkq rillqkfded 661 gfkecnlndt ryvnrflcqf vadhilltgk gkrrvfasng qitnllrgfw glrkvraend 721 rhhaldavvv acstvamqqk itrfvrykem nafdgktidk etgkvlhqkt hfpqpweffa 781 qevmirvfgk pdgkpefeea dtpeklrtll aeklssrpea vheyvtplfv srapnrkmsg 841 ahkdtlrsak rfvkhnekis vkrvwlteik ladlenmvny kngreielye alkarleayg 901 gnakqafdpk dnpfykkggq lvkavrvekt qesgvllnkk naytiadngd mvrvdvfckv 961 dkkgknqyfi vpiyawqvae nilpdidckg yriddsytfc fslhkydlia fqkdekskve 1021 fayyincdss ngrfylawhd kgskeqqfri stqnlvliqk yqvnelgkei rpcrlkkrpp 1081vr

[0099] In some embodiments, the dCas9 corresponds in part or in whole to or comprises a Cas9 amino acid sequence having one or more mutations that inactivate Cas9 nuclease activity. For example, in some embodiments, the dCas9 domain comprises D10A and H840A mutations or corresponding mutations in another Cas9. In some embodiments, the dCas9 has the amino acid sequence of dCas9 (D10A and H840A): Contains TIFF0007672982000009.tif169169. (Single underline: HNH domain, double underline: RuvC domain)

[0100] In some embodiments, the Cas9 domain comprises a D10A mutation, and the residue at position 840, or the corresponding position in any of the amino acid sequences provided herein, remains a histidine in the amino acid sequences provided above.

[0101] In other embodiments, dCas9 variants are provided that have mutations other than D10A and H840A, resulting in, for example, nuclease-inactivated Cas9 (dCas9). Such mutations include, for example, other amino acid substitutions at D10 and H840, or other substitutions within the nuclease domain of Cas9 (e.g., substitutions in the HNH nuclease subdomain and / or the RuvC1 subdomain). In some embodiments, variants or homologs of dCas9 are provided that are at least about 70% identical, at least about 80% identical, at least about 90% identical, at least about 95% identical, at least about 98% identical, at least about 99% identical, at least about 99.5% identical, or at least about 99.9% identical. In some embodiments, variants of dCas9 are provided that have shorter or longer amino acid sequences of about 5 amino acids, about 10 amino acids, about 15 amino acids, about 20 amino acids, about 25 amino acids, about 30 amino acids, about 40 amino acids, about 50 amino acids, about 75 amino acids, about 100 amino acids, or more.

[0102] In some embodiments, the Cas9 fusion proteins provided herein comprise the full-length amino acid sequence of a Cas9 protein, such as one of the Cas9 sequences provided herein. However, in other embodiments, the fusion proteins provided herein do not comprise the full-length Cas9 sequence, but rather comprise only one or more fragments thereof. Exemplary amino acid sequences of suitable Cas9 domains and Cas9 fragments are provided herein. Additional suitable sequences of Cas9 domains and fragments will be apparent to those skilled in the art.

[0103] Additional Cas9 proteins, including variants and homologs thereof (e.g., nuclease-inactive Cas9 (dCas9), Cas9 nickase (nCas9), or nuclease-active Cas9), are within the scope of this disclosure. Exemplary Cas9 proteins include, but are not limited to, those provided below. In some embodiments, the Cas9 protein is a nuclease-inactive Cas9 (dCas9). In some embodiments, the Cas9 protein is a Cas9 nickase (nCas9). In some embodiments, the Cas9 protein is a nuclease-active Cas9.

[0104] Exemplary catalytically inactive Cas9 (dCas9):

[0105] Exemplary catalytic Cas9 nickases (nCas9):

[0106] Exemplary catalytically active Cas9:

[0107] In some embodiments, Cas9 refers to Cas9 from archaea (e.g., nanoarchaea), which constitute the domain and kingdom of unicellular prokaryotic microorganisms. In some embodiments, Cas refers to CasX or CasY, for example, as described in Burstein et al., "New CRISPR-Cas systems from uncultivated microbes." Cell Res. 2017 Feb 21. doi: 10.1038 / cr.2017.21, the entire contents of which are incorporated herein by reference. Using genome-resolved metagenomics, many CRISPR-Cas systems have been identified, including the first reported Cas9 in the archaeal domain of life. This divergent Cas9 protein was discovered as part of an active CRISPR-Cas system in the little-studied nanoarchaea. In bacteria, two previously unknown systems, CRISPR-CasX and CRISPR-CasY, have been discovered, which are among the most compact systems discovered to date. In some embodiments, Cas9 represents CasX or a variant of CasX. In some embodiments, Cas9 represents CasY or a variant of CasY. It is understood that other RNA-guided DNA-binding proteins can also be used as nucleic acid programmable DNA-binding proteins (napDNAbp) and are within the scope of the present disclosure.

[0108] In certain embodiments, napDNAbp useful in the methods of the invention comprise circular permutations, which are known in the art and described, for example, in Oakes et al., Cell 176, 254-267, 2019. Exemplary circular permutations are shown below, where the bolded sequence indicates the Cas9-derived sequence, the italicized sequence indicates the linker sequence, and the underlined sequence indicates the bipartite nuclear localization sequence. CP5 (MSP [NGC = Pam variant with mutation Regular Cas9 likes NGG], PID = Protein Interacting Domain and contains "D10A" nickase) TIFF0007672982000010.tif170169

[0109] Non-limiting examples of polynucleotide-programmable nucleotide-binding domains that can be incorporated into base editors include domains from CRISPR proteins, restriction nucleases, meganucleases, TAL nucleases (TALENs), and zinc finger nucleases (ZFNs).

[0110] In some embodiments, any nucleic acid programmable DNA-binding protein (napDNAbp) of a fusion protein provided herein can be a CasX or CasY protein. In some embodiments, the napDNAbp is a CasX protein. In some embodiments, the napDNAbp is a CasY protein. In some embodiments, the napDNAbp comprises an amino acid sequence that is at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identical to a naturally occurring CasX or CasY protein. In some embodiments, the napDNAbp is a naturally occurring CasX or CasY protein. In some embodiments, the napDNAbp comprises an amino acid sequence that is at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identical to any CasX or CasY protein described herein. It should be recognized that Cas12b / C2c1, CasX, and CasY from other bacterial species can also be used in accordance with the present disclosure.

[0111] Cas12b / C2c1(Cas12b / C2c1 (uniprot.org / uniprot / T0D7A2#2) sp|T0D7A2|C2C1_ALIAG CRISPR-associated endonuclease C2c1 OS= Alicyclobacillus acido- terrestris (strain ATCC 49025 / DSM 3922 / CIP 106132 / NCIMB 13137 / GD3B) GN=c2c1 PE=1 SV=1

[0112] CasX (uniprot.org / uniprot / F0NN87; uniprot.org / uniprot / F0NH53) >tr|F0NN87|F0NN87_SULIH CRISPR-related Casx protein OS = Sulfolobus islandicus (strain HVE10 / 4) GN = SiH_0402 PE=4 SV=1 MEVPLYNIFGDNYIIQVATEAENSTIYNNKVEIDDEELRNVLNLAYKIAKNNEDAAAERRGKAKKKKGEEGETTTSNIILPLSGNDKNPWTETLKCYNFPTTVALSEVFKNFSQVKECEEVSAPSFVKPEFYEFGRSPGMVERTRRVKLEVEPHYLIIAAAGWVLTRLGKAKVSEGDYVGVNVFTPTRGILYSLIQNVNGIVPGIKPETAFGLWIARKVVSSVTNPNVSVVRIYTISDAVGQNPTTINGGFSIDLTKLLEKRYLLSERLEAIARNALSISSNMRERYIVLANYIYEYLTG SKRLEDLLYFANRDLIMNLNSDDGKVRDLKLISAYVNGELIRGEG

[0113] >tr|F0NH53|F0NH53_SULIR CRISPR-related protein Casx OS = Sulfolobus islandicus (strain REY15A) GN=SiRe_0771 PE=4 SV=1 MEVPLYNIFGDNYIIQVATEAENSTIYNNKVEIDDEELRNVLNLAYKIAKNNEDAAAERRGKAKKKKGEEGETTTSNIILPLSGNDKNPWTETLKCYNFPTTVALSEVFKNFSQVKCEEVSAPSFVKPEFYKFGRSPGMVERTRRVKLEVEPHYLIMAAAGWVLTRLGKAK VSEGDYVGVNVFTPTRGILYSLIQNVNGIVPGIKPETAFGLWIARKVVSSVTNPNVSVVSIYTISDAVGQNPTTINGGFSIDLTKLLEKRDLLSERLEAIARNALSISSNMRERYIVLANYIYEYLTGSKRLEDLLYFANRDLIMNLNSDDGKVRDLKLISAYVNGELIRGEG

[0114] Deltaproteobacteria CasX MEKRINKIRKKLSADNATKPVSRSGPMKTLLVRVMTDDLKKRLEKRRKKPEVMPQVISNNAANNLRMLLDDYTKMKEAILQVYWQEFKDDHVGLMCKFAQPASKKIDQNKLKPEMDEKGNLTTAGFACSQCGQPLFVYKLEQVSEKGKAYTNYFGRCNVAEHEKLILLAQLKPVKDSDEAVTYSLGKFGQRALDFYSIHVTKESTHPVKPLAQIAGNRYASGPVGKALSDACMGTIASFLSKYQDIIIEHQKVVKGNQKRLESLRELAGKENLEYPSVTLPPQPHTKEGVDAYNEVIARVRMWVNLLWQKLKLSRDDAKPLLRLKGFPSFPVVERRENEVDWWNTINEVKKLIDAKRDMGRVFWSGVTAEKRNTILEGYNYLPNENDHKKREGSLENPKKPAKRQFGDLLLYLEKKYAGDWGKVFDEAWERIDKKIAGLTSHIEREEARNAEDAQSKAVLTDWLRAKASFVLERLKEMDEKEFYACEIQLQK WYGDLRGNPFAVEAENRVVDISGFSIGSDGHSIQYRNLLAWKYLENGKREFYLLMNYGKKGRIRFTDGTDIKKSGKWQGLLYGGGAKVIDLTFDPDDEQLIILPLAFGTRQGREFIWNDLLSLETGLIKLANGRVIEKTIYNKKIGRDEPALFVALTFERREVVDPSNIKPVNLIGVARGENIPAVIALTDPEGCPLPEFKDSSGGPTDILRIGEGYKEKQRAIQAAKEVEQRRAGGYSRKFASKSRNLADDMVRNSARDLFYHAVTHDAVLVFANLSRGFGRQGKRTFMTERQYTKMEDWLTAKLAYEGLTSKTYLSKTLAQYTSKTCSNCGFTITYADMDVMLVRLKKTSDGWATTLNNKELKAEYQITYYNRYKRQTVEKELSAELDRLSEESGNNDISKWTKGRRDEALFLLKKRFSHRPVQEQFVCLDCGHEVHAAEQAALNIARSWLFLNSNSTEFKSYKSGKQPFVGAWQAFYKRRLKEVWKPNA

[0115] CasY (ncbi.nlm.nih.gov / protein / APG80656.1) >APG80656.1 CRISPR-associated tanpak CasY [Uncultured Parcubacteria bacteria]

[0116] The term "Cas12" or "Cas12 domain" refers to an RNA-guided nuclease comprising a Cas12 protein or a fragment thereof (e.g., an active, inactive, or partially active DNA cleavage domain of Cas12 and / or a gRNA-binding domain of Cas12). Cas12 belongs to the Class 2, Type V CRISPR / Cas system. Cas12 nucleases are sometimes referred to as CRISPR (clustered regularly interspaced short palindromic repeat)-associated nucleases. The sequence of an exemplary Bacillus hisashii Cas12b (BhCas12b) Cas12 domain is provided below.

[0117] Amino acid sequences having at least 85% or more identity to the BhCas12b amino acid sequence are also useful in the methods of the invention.

[0118] "Cbl proto-oncogene B (CBLB) polypeptide" means a protein having at least about 85% amino acid sequence identity to GenBank Accession No. ABC86700.1 or a fragment thereof involved in the regulation of immune responses. Exemplary CBLB polypeptide sequences are provided below.

[0119] >ABC86700.1 CBL-B [Homo sapiens] MANSMNGRNPGGRGGNPRKGRILGIIDAIQDAVGPPKQAAADRRTVEKTWKLMDKVVRLCQNPKLQLKNSPPYILDILPDTYQHLRLILSKYDDNQKLAQLSENEYFKIYIDSLMKKSKRAIRLFKEGKERMYEEQSQDRRNLTKLSLIFSHMLAEIKAIFPNGQFQGDNFRITKADAAEFWRKFFGDKTIVPWKVFRQCLHEVHQISSGLEAMALKSTIDLTCNDYISVFEFDIFTRLFQPWGSILRNWNFLAVTHPGYMAFLTYDEVKARLQKYSTKPGSYIFRLSCTRLGQWAIGYVTGDGNILQTIPHNKPLFQALIDGSREGFYLYPDGRSYNPDLTGLCEPTPHDHIKVTQEQYELYCEMGSTFQLCKICAENDKDVKIEPCGHLMCTSCLTAWQESDGQGCPFCRCEIKGTEPIIVDPFDPRDEGSRCCSIIDPFGMPMLDLDDDDDREESLMMNRLANVRKCTDRQNSPVTSPGSSPLAQRRKPQPDPLQIPHLSLPPVPPRLDLIQKGIVRSPCGSPTGSPKSSPCMVRKQDKPLPAPPPPLRDPPPPPPERPPPIPPDNRLSRHIHHVESVPSRDPPMPLEAWCPRDVFGTNQLVGCRLLGEGSPKPGITASSNVNGRHSRVGSDPVLMRKHRRHDLPLEGAKVFSNGHLGSEEYDVPPRLSPPPPVTTLLPSIKCTGPLANSLSEKTRDPVEEDDDEYKIPSSHPVSLNSQPSHCHNVKPPVRSCDNGHCMLNGTHGPSSEKKSNIPDLSIYLKGDVFDSASDPVPLPPARPPTRDNPKHGSSLNRTPSDYDLLIPPLGEDAFDALPPSLPPPPPPARHSLIEHSKPPGSSSRPSSGQDLFLLPSDPFVDLASGQVPLPPARRLPGENVKTNRTSQDYDQLPSCSDGSQAPARPPKPRPRRTAPEIHHRKPHGPEAALENVDAKIAKLMGEGYAFEEVKRALEIAQNNVEVARSILREFAFPPPVSPRLNL

[0120] "Cbl proto-oncogene B (CBLB) polynucleotide" refers to a nucleic acid molecule that encodes a CBLB polypeptide. The CBLB gene encodes an E3 ubiquitin ligase. Exemplary CBLB nucleic acid molecules are provided below.

[0121] >DQ349203.1 Homo sapiens CBL-B mRNA, complete CDs

[0122] "Chimeric antigen receptor" or "CAR" refers to a synthetic receptor that contains an extracellular antigen-binding domain, a transmembrane domain, and an intracellular signaling domain that confers specificity for the antigen on immune cells.

[0123] "Class II major histocompatibility complex transactivator (CIITA) polypeptide" means a protein having at least about 85% amino acid sequence identity with NCBI Reference Sequence: NP_000237.2 or a fragment thereof that functions as a transcriptional coactivator. Exemplary CIITA polypeptide sequences are provided below. 1 mrclaprpag sylsepqgss qcatmelgpl eggylellns dadplclyhf ydqmdlagee 61 eielysepdt dtincdqfsr llcdmegdee treayaniae ldqyvfqdsq leglskdifk 121 higpdevige smempaevgq ksqkrpfpee lpadlkhwkp aepptvvtgs llvgpvsdcs 181 tlpclplpal fnqepasgqm rlektdqipm pfsssslscl nlpegpiqfv ptistlphgl 241 wqiseagtgv ssifiyhgev pqasqvppps gftvhglpts pdrpgstspf apsatdlpsm 301 pepaltsran mtehktsptq cpaagevsnk lpkwpepveq fyrslqdtyg aepagpdgil 361 vevdlvqarl ersssksler elatpdwaer qlaqgglaev llaakehrrp retrviavlg 421 kagqgksywa gavsrawacg rlpqydfvfs vpchclnrpg dayglqdllf slgpqplvaa 481 devfshilkr pdrvllildg feeleaqdgf lhstcgpapa epcslrglla glfqkkllrg 541 ctllltarpr grlvqslska dalfelsgfs meqaqayvmr yfessgmteh qdraltllrd 601 rplllshshs ptlcravcql seallelged aklpstltgl yvgllgraal dsppgalael 661 aklawelgrr hqstlqedqf psadvrtwam akglvqhppr aaeselafps fllqcflgal 721 wlalsgeikd kelpqylalt prkkrpydnw legvprflag lifqpparcl gallgpsaaa 781 svdrkqkvla rylkrlqpgt lrarqllell hcaheaeeag iwqhvvqelp grlsflgtrl 841 tppdahvlgk aleaagqdfs ldlrstgicp sglgslvgls cvtrfraals dtvalweslq 901 qhgetkllqa aeekftiepf kakslkdved lgklvqtqrt rsssedtage lpavrdlkkl 961 efalgpvsgp qafpklvril tafsslqhld ldalsenkig degvsqlsat fpqlksletl 1021 nlsqnnitdl gayklaealp slaasllrls lynncicdvg aeslarvlpd mvslrvmdvq 1081 ynkftaagaq qlaaslrrcp hvetlamwtp tipfsvqehl qqqdsrislr

[0124] "Class II major histocompatibility complex transactivator (CIITA) polynucleotide" means a nucleic acid molecule that encodes a CIITA polypeptide. Exemplary CIITA nucleic acid sequences are provided below. 1 ggttagtgat gaggctagtg atgaggctgt gtgcttctga gctgggcatc cgaaggcatc 61 cttggggaag ctgagggcac gaggaggggc tgccagactc cgggagctgc tgcctggctg 121 ggattcctac acaatgcgtt gcctggctcc acgccctgct gggtcctacc tgtcagagcc 181 ccaaggcagc tcacagtgtg ccaccatgga gttggggccc ctagaaggtg gctacctgga 241 gcttcttaac agcgatgctg accccctgtg cctctaccac ttctatgacc agatggacct 301 ggctggagaa gaagagattg agctctactc agaacccgac acagacacca tcaactgcga 361 ccagttcagc aggctgttgt gtgacatgga aggtgatgaa gagaccaggg aggcttatgc 421 caatatcgcg gaactggacc agtatgtctt ccaggactcc cagctggagg gcctgagcaa 481 ggacattttc aagcacatag gaccagatga agtgatcggt gagagtatgg agatgccagc 541 agaagttggg cagaaaagtc agaaaagacc cttcccagag gagcttccgg cagacctgaa 601 gcactggaag ccagctgagc cccccactgt ggtgactggc agtctcctag tgggaccagt 661 gagcgactgc tccaccctgc cctgcctgcc actgcctgcg ctgttcaacc aggagccagc 721 ctccggccag atgcgcctgg agaaaaccga ccagattccc atgccttct ccagttcctc 781 gttgagctgc ctgaatctcc ctgagggacc catccagttt gtccccacca tctccactct 841 gccccatggg ctctggcaaa tctctgaggc tggacaggg gtctccagta tattcatcta 901 ccatggtgag gtgccccagg ccagccaagt accccctccc agtggattca ctgtccacgg 961 cctcccaaca tctccagacc ggccaggctc caccagcccc ttcgctccat cagccactga 1021 cctgcccagc atgcctgac ctgccctgac ctcccgagca aacatgacag agcacaagac 1081 gtcccccacc caatgcccgg cagctggaga ggtctccaac aagcttccaa aatggcctga 1141 gccggtggag cagttctacc gctcactgca ggacacgtat ggtgccgagc ccgcaggccc 1201 ggatggcatc ctagtggagg tggatctggt gcaggccagg ctggagagga gcagcagcaa 1261 gagcctggag cgggaactgg ccaccccgga ctgggcagaa cggcagctgg cccaaggagg 1321 cctggctgag gtgctgttgg ctgccaagga gcaccggcgg ccgcgtgaga cacgagtgat 1381 tgctgtgctg ggcaaagctg gtcagggcaa gagctattgg gctggggcag tgagccgggc 1441 ctgggcttgt ggccggcttc cccagtacga ctttgtcttc tctgtcccct gccattgctt 1501 gaaccgtccg ggggatgcct atggcctgca ggatctgctc ttctccctgg gcccacagcc 1561 actcgtggcg gccgatgagg ttttcagcca catcttgaag agacctgacc gcgttctgct 1621 catcctagac ggcttcgagg agctggaagc gcaagatggc ttcctgcaca gcacgtgcgg 1681 accggcaccg gcggagccct gctccctccg ggggctgctg gccggccttt tccagaagaa 1741 gctgctccga ggttgcaccc tcctcctcac agcccggccc cggggccgcc tggtccagag 1801 cctgagcaag gccgacgccc tatttgagct gtccggcttc tccatggagc aggcccaggc 1861 atacgtgatg cgctactttg agagctcagg gatgacagag caccaagaca gagccctgac 1921 gctcctccgg gaccggccac ttcttctcag tcacagccac agccctactt tgtgccgggc 1981 agtgtgccag ctctcagagg ccctgctgga gcttggggag gacgccaagc tgccctccac 2041 gctcacggga ctctatgtcg gcctgctggg ccgtgcagcc ctcgacagcc cccccggggc 2101 cctggcagag ctggccaagc tggcctggga gctgggccgc agacatcaaa gtaccctaca 2161 ggaggaccag ttcccatccg cagacgtgag gacctgggcg atggccaaag gcttagtcca 2221 acacccaccg cgggccgcag agtccgagct ggccttcccc agcttcctcc tgcaatgctt 2281 cctgggggcc ctgtggctgg ctctgagtgg cgaaatcaag gacaaggagc tcccgcagta 2341 cctagcattg accccaagga agaagaggcc ctatgacaac tggctggagg gcgtgccacg 2401 ctttctggct gggctgatct tccagcctcc cgcccgctgc ctgggagccc tactcgggcc 2461 atcggcggct gcctcggtgg acaggaagca gaaggtgctt gcgaggtacc tgaagcggct 2521 gcagccgggg acactgcggg cgcggcagct gctggagctg ctgcactgcg cccacgaggc 2581 cgaggaggct ggaatttggc agcacgtggt acaggagctc cccggccgcc tctcttttct 2641 gggcacccgc ctcacgcctc ctgatgcaca tgtactgggc aaggccttgg aggcggcggg 2701 ccaagacttc tccctggacc tccgcagcac tggcatttgc ccctctggat tggggagcct 2761 cgtgggactc agctgtgtca cccgtttcag ggctgccttg agcgacacgg tggcgctgtg 2821 ggagtccctg cagcagcatg gggagaccaa gctacttcag gcagcagagg agaagttcac 2881 catcgagcct ttcaaagcca agtccctgaa ggatgtggaa gacctgggaa agcttgtgca 2941 gactcagagg acgagaagtt cctcggaaga cacagctggg gagctccctg ctgttcggga 3001 cctaaagaaa ctggagtttg cgctgggccc tgtctcaggc ccccaggctt tccccaaact 3061 ggtgcggatc ctcacggcct tttcctccct gcagcatctg gacctggatg cgctgagtga 3121 gaacaagatc ggggacgagg gtgtctcgca gctctcagcc accttccccc agctgaagtc 3181 cttggaaacc ctcaatctgt cccagaacaa catcactgac ctgggtgcct acaaactcgc 3241 cgaggccctg ccttcgctcg ctgcatccct gctcaggcta agcttgtaca ataactgcat 3301 ctgcgacgtg ggagccgaga gcttggctcg tgtgcttccg gacatggtgt ccctccgggt 3361 gatggacgtc cagtacaaca agttcacggc tgccggggcc cagcagctcg ctgccagcct 3421 tcggaggtgt cctcatgtgg agacgctggc gatgtggacg cccaccatcc cattcagtgt 3481 ccaggaacac ctgcaacaac aggattcacg gatcagcctg agatgatccc agctgtgctc 3541 tggacaggca tgttctctga ggacactaac cacgctggac cttgaactgg gtacttgtgg 3601 acacagctct tctccaggct gtatcccatg agcctcagca tcctggcacc cggcccctgc 3661 tggttcaggg ttggcccctg cccggctgcg gaatgaacca catcttgctc tgctgacaga 3721 cacaggcccg gctccaggct cctttagcgc ccagttgggt ggatgcctgg tggcagctgc 3781 ggtccaccca ggagccccga ggccttctct gaaggacatt gcggacagcc acggccaggc 3841 cagagggagt gacagaggca gccccattct gcctgcccag gcccctgcca ccctggggag 3901 aaagtacttc ttttttttta tttttagaca gagtctcact gttgcccagg ctggcgtgca 3961 gtggtgcgat ctgggttcac tgcaacctcc gcctcttggg ttcaagcgat tcttctgctt 4021 cagcctcccg agtagctggg actacaggca cccaccatca tgtctggcta atttttcatt 4081 tttagtagag acagggtttt gccatgttgg ccaggctggt ctcaaactct tgacctcagg 4141 tgatccaccc acctcagcct cccaaagtgc tgggattaca agcgtgagcc actgcaccgg 4201 gccacagaga aagtacttct ccaccctgct ctccgaccag acaccttgac agggcacacc 4261 gggcactcag aagacactga tgggcaaccc ccagcctgct aattccccag attgcaacag 4321 gctgggcttc agtggcagct gcttttgtct atgggactca atgcactgac attgttggcc 4381 aaagccaaag ctaggcctgg ccagatgcac cagcccttag cagggaaaca gctaatggga 4441 cactaatggg gcggtgagag gggaacagac tggaagcaca gcttcatttc ctgtgtcttt 4501 tttcactaca ttataaatgt ctctttaatg tcacaggcag gtccagggtt tgagttcata 4561 ccctgttacc attttggggt acccactgct ctggttatct aatatgtaac aagcccacccc 4621 aaatcatagt ggcttaaaac aacactcaca ttta

[0125] "Cluster of Differentiation 7 (CD7) polypeptide" means a protein having at least about 85% amino acid sequence identity to NCBI reference sequence: NP_006128.1 or fragments thereof involved in T cell and T cell / B cell interactions. Exemplary CD7 polypeptide sequences are provided below. 1 magpprllll plllalargl pgalaaqevq qsphcttvpv gasvnitcst sgglrgiylr 61 qlgpqpqdii yyedgvvptt drrfrgridf sgsqdnltit mhrlqlsdtg tytcqaitev 121 nvygsgtlvl vteeqsqgwh rcsdappras alpapptgsa lpdpqtasal pdppaasalp 181 aalavisfll glglgvacvl artqikklcs wrdknsaacv vyedmshsrc ntlsspnqyq

[0126] "Cluster of Differentiation 7 (CD7) polynucleotide" refers to a nucleic acid molecule that encodes a CD7 polypeptide. The CD7 gene encodes a transmembrane protein. Exemplary CD7 nucleic acid sequences are provided below. 1 ctctctgagc tctgagcgcc tgcggtctcc tgtgtgctgc tctctgtggg gtcctgtaga 61 cccagagagg ctcagctgca ctcgcccggc tgggagagct gggtgtgggg aacatggccg 121 ggcctccgag gctcctgctg ctgcccctgc ttctggcgct ggctcgcggc ctgcctgggg 181 ccctggctgc ccaagaggtg cagcagtctc cccactgcac gactgtcccc gtgggagcct 241 ccgtcaacat cacctgctcc accagcgggg gcctgcgtgg gatctacctg aggcagctcg 301 ggccacagcc ccaagacatc atttactacg aggacggggt ggtgcccact acggacagac 361 ggttccgggg ccgcatcgac ttctcagggt cccaggacaa cctgactatc accatgcacc 421 gcctgcagct gtcggacact ggcacctaca cctgccaggc catcacggag gtcaatgtct 481 acggctccgg caccctggtc ctggtgacag aggaacagtc ccaaggatgg cacagatgct 541 cggacgcccc accaagggcc tctgccctcc ctgccccacc gacaggctcc gccctccctg 601 acccgcagac agcctctgcc ctccctgacc cgccagcagc ctctgccctc cctgcggccc 661 tggcggtgat ctccttcctc ctcgggctgg gcctgggggt ggcgtgtgtg ctggcgagga 721 cacagataaa gaaactgtgc tcgtggcggg ataagaattc ggcggcatgt gtggtgtacg 781 aggacatgtc gcacagccgc tgcaacacgc tgtcctcccc caaccagtac cagtgaccca 841 gtgggcccct gcacgtcccg cctgtggtcc ccccagcacc ttccctgccc caccatgccc 901 cccaccctgc cacacccctc accctgctgt cctcccacgg ctgcagcaga gtttgaaggg 961 cccagccgtg cccagctcca agcagacaca caggcagtgg ccaggcccca cggtgcttct 1021 cagtggacaa tgatgcctcc tccgggaagc cttccctgcc cagcccacgc cgccaccggg 1081 aggaagcctg actgtccttt ggctgcatct cccgaccatg gccaaggagg gcttttctgt 1141 gggatgggcc tgggcacgcg gccctctcct gtcagtgccg gcccacccac cagcaggccc 1201 ccaaccccca ggcagcccgg cagaggacgg gaggagacca gtcccccacc cagccgtacc 1261 agaaataaag gcttctgtgc ttcc

[0127] "Cluster of Differentiation 5 (CD5) polypeptide" means a protein having at least about 85% amino acid sequence identity to NCBI Reference Sequence: NP_001333385.1 or a fragment thereof expressed on the surface of T cells. Exemplary CD5 polypeptide sequences are provided below. 1 mvcsqswgrs skqwedpsqa skvcqrlncg vplslgpflv tytpqssiic ygqlgsfsnc 61 shsrndmchs lgltclepqk ttppttrppp tttpeptapp rlqlvaqsgg qhcagvvefy 121 sgslggtisy eaqdktqdle nflcnnlqcg sflkhlpete agraqdpgep rehqplpiqw 181 kiqnssctsl ehcfrkikpq ksgrvlallc sgfqpkvqsr lvggssiceg tvevrqgaqw 241 aalcdsssar sslrweevcr eqqcgsvnsy rvldagdpts rglfcphqkl sqchelwern 301 syckkvfvtc qdpnpaglaa gtvasiilal vllvvllvvc gplaykklvk kfrqkkqrqw 361 igptgmnqnm sfhrnhtatv rshaenptas hvdneysqpp rnshlsaypa legalhrssm 421 qpdnssdsdy dlhgaqrl

[0128] "Cluster of Differentiation 5 (CD5) polynucleotide" refers to a nucleic acid molecule that encodes a CD5 polypeptide. The CD5 gene encodes a transmembrane protein. Exemplary CD5 nucleic acid sequences are provided below. 1 gagtcttgct gatgctcccg gctgaataaa ccccttcctt ctttaacttg gtgtctgagg 61 ggttttgtct gtggcttgtc ctgctacatt tcttggttcc ctgaccagga agcaaagtga 121 ttaacggaca gttgaggcag ccccttaggc agcttaggcc tgccttgtgg agcatccccg 181 cggggaactc tggccagctt gagcgacacg gatcctcaga gcgctcccag gtaggcaatt 241 gccccagtgg aatgcctcgt cagagcagtg catggcaggc ccctgtggag gatcaacgca 301 gtggctgaac acagggaagg aactggcact tggagtccgg acaactgaaa cttgtcgctt 361 cctgcctcgg acggctcagc tggtatgacc cagatttcca ggcaaggctc acccgttcca 421 actcgaagtg ccagggccag ctggaggtct acctcaagga cggatggcac atggtttgca 481 gccagagctg gggccggagc tccaagcagt gggaggaccc cagtcaagcg tcaaaagtct 541 gccagcggct gaactgtggg gtgcccttaa gccttggccc cttccttgtc acctacacac 601 ctcagagctc aatcatctgc tacggacaac tgggctcctt ctccaactgc agccacagca 661 gaaatgacat gtgtcactct ctgggcctga cctgcttaga accccagaag acaacacctc 721 caacgacaag gcccccgccc accacaactc cagagcccac agctcctccc aggctgcagc 781 tggtggcaca gtctggcggc cagcactgtg ccggcgtggt ggagttctac agcggcagcc 841 tggggggtac catcagctat gaggcccagg acaagaccca ggacctggag aacttcctct 901 gcaacaacct ccagtgtggc tccttcttga agcatctgcc agagactgag gcaggcagag 961 cccaagaccc aggggagcca cgggaacacc agcccttgcc aatccaatgg aagatccaga 1021 actcaagctg tacctccctg gagcattgct tcaggaaaat caagccccag aaaagtggcc 1081 gagttcttgc cctcctttgc tcaggtttcc agcccaaggt gcagagccgt ctggtggggg 1141 gcagcagcat ctgtgaaggc accgtggagg tgcgccaggg ggctcagtgg gcagccctgt 1201 gtgacagctc ttcagccagg agctcgctgc ggtgggagga ggtgtgccgg gagcagcagt 1261 gtggcagcgt caactcctat cgagtgctgg acgctggtga cccaacatcc cgggggctct 1321 tctgtcccca tcagaagctg tcccagtgcc acgaactttg ggagagaaat tcctactgca 1381 agaaggtgtt tgtcacatgc caggatccaa accccgcagg cctggccgca ggcacggtgg 1441 caagcatcat cctggccctg gtgctcctgg tggtgctgct ggtcgtgtgc ggcccccttg 1501 cctacaagaa gctagtgaag aaattccgcc agaagaagca gcgccagtgg attggcccaa 1561 cgggaatgaa ccaaaacatg tctttccatc gcaaccacac ggcaaccgtc cgatcccatg 1621 ctgagaaccc cacagcctcc cacgtggata acgaatacag ccaacctccc aggaactccc 1681 acctgtcagc ttatccagct ctggaagggg ctctgcatcg ctcctccatg cagcctgaca 1741 actcctccga cagtgactat gatctgcatg gggctcagag gctgtaaaga actgggatcc 1801 atgagcaaaa agccgagagc cagacctgtt tgtcctgaga aaactgtccg ctcttcactt 1861 gaaatcatgt ccctatttct accccggcca gaacatggac agaggccaga agccttccgg 1921 acaggcgctg ctgccccgag tggcaggcca gctcacactc tgctgcacaa cagctcggcc 1981 gcccctccac ttgtggaagc tgtggtgggc agagccccaa aacaagcagc cttccaacta 2041 gagactcggg ggtgtctgaa gggggccccc tttccctgcc cgctggggag cggcgtctca 2101 gtgaaatcgg ctttctcctc agactctgtc cctggtaagg agtgacaagg aagctcacag 2161 ctgggcgagt gcattttgaa tagttttg taagtagtgc tttcctcct tcctgacaaa 2221 tcgagcgctt tgctcttc tgtgcagcat sccacctgc ggatccctt ggggaggaca 2281 ggagggac tcccggagac ctctgcagcc gtggtgtca gaggctgctc acctgagcac 2341 aagacagct ctgcacattc accccagctg ccagccaggg gtctggtgg gcaccattc 2401 gacccacagc gtcacccccctctgtc 2461 aagacacct tccttccac tggctgtca gccacaggg caccagtgcc acccaggcc 2521 cggcacaag gggcgcctag taaaccttaa ccaacttggt ttttgctc acccagcaat 2581 taaaagtccc aagctgaggt agtttcagtc catcacagtt catctctaa cccagagtc 2641 agagatgggg ctgtcatgt tcctttggtt tgaatactc ccttgacgaa aacagactcc 2701 tctagtactt ggagatcttg gacgtacacc taatcccatg gggcctcggc tccttact 2761 gcaagtgaga agaggaggtc taccaggag cctcgggtct gatcaaggga gaggccaggc 2821 gcagctcact gcggcggctc cctaagaggg tgaagcaca tgggacaca tcctagaca 2881 ggtcctttct ccacgccatt tgatgctgta tctcctggga gcacaggcat caatggtcca 2941 agccgcataa taagtctgga agagcaaaag ggagttacta ggatatgggg tgggctgctc 3001 ccagaatctg ctcagctttc tgcccccacc aacaccctcc aaccaggcct tgccttctga 3061 gagcccccgt ggccaagccc aggtcacaga tcttccccg accatgctgg gaatccagaa 3121 acagggaccc catttgtctt cccatatctg gtggaggtga gggggctcct caaaagggaa 3181 ctgagaggct gctcttaggg agggcaaagg ttcgggggca gccagtgtct cccatcagtg 3241 ccttttttaa taaaagctct ttcatctata gtttggccac catacagtgg cctcaaagca 3301 accatggcct acttaaaaac caaaccaaaa ataaagagtt tagttgagga gaaaaaaaa 3361 aaaaaaaaaaaaaaaaaa

[0129] The term "conservative amino acid substitution" or "conservative mutation" refers to the replacement of one amino acid with another amino acid that shares common properties. A practical method for defining common properties between individual amino acids is to analyze the normalized frequency of amino acid changes between corresponding proteins of homologous organisms (Schulz, GE and Schirmer, RH, Principles of Protein Structure, Springer-Verlag, New York (1979)). Such analysis allows for the definition of groups of amino acids in which amino acids within a group preferentially exchange with each other and are therefore most similar to each other in their effect on overall protein structure (Schulz, GE and Schirmer, RH, supra). Non-limiting examples of conservative mutations include amino acid substitutions such as arginine to lysine, which can maintain a positive charge; aspartic acid to glutamic acid, which can maintain a negative charge; threonine to serine, which maintains a free -OH; and asparagine to glutamine, which can maintain a free NH2.

[0130] The terms "coding sequence" or "protein-coding sequence," as used interchangeably herein, refer to a segment of a polynucleotide that encodes a protein. This region or sequence is bounded at its 5'-proximal end by a start codon and at its 3'-proximal end by a stop codon. A coding sequence is also called an open reading frame.

[0131] "Cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) polypeptide" means a protein or fragment thereof having at least about 85% sequence identity with NCBI Accession No. EAW70354.1. Exemplary amino acid sequences are provided below. >EAW70354.1 Cytotoxic T-lymphocyte-associated protein 4 [Homo sapiens] MACLGFQRHKAQLNLATRTWPCTLLFFLLFIPVFCKAMHVAQPAVVLASSRGIASFVCEYASPGKATEVRVTVLRQADSQVTEVCAATYMMGNELTFLDDSICTGTSSGNQ VNLTIQGLRAMDTGLYICKVELMYPPPYYLGIGNGTQIYVIDPEPCPDSDFLLWILAAVSSGLFFYSFLLTAVSLSKMLKKRSPLTTGVYVKMPPTEPECEKQFQPYFIPIN

[0132] "Cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) polynucleotide" refers to a nucleic acid molecule encoding a CTLA-4 polypeptide. The CTLA-4 gene encodes a member of the immunoglobulin superfamily and encodes a protein that transmits inhibitory signals to T cells. Exemplary CTLA-4 nucleic acid sequences are provided below. >BC074842.2 Homo sapiens cytotoxic T-lymphocyte-associated protein 4, mRNA (cDNA clone MGC:104099 IMAGE:30915552), complete CDS GACCTGAACACCGCTCCCATAAAGCCATGGCTTGCCTTGGATTTCAGCGGCACAAGGCTCAGCTGAACCTGGCTACCAGGACCTGGCCCTGCACTCTCCTGTTTTTTCTTCTCTTCATCCCTGTCTTCTGCAAAGCAATGCACGTGGCCCAGCCTGCTGTGGTACTGGCCAGCAGCCGAGGCATCGCCAGCTTTGTGTGTGAGTATGCATCTCCAGGCAAAGCCACTGAGGTCCGGGTGACAGTGCTTCGGCAGGCTGACAGCCAGGTGACTGAAGTCTGTGCGGCAACCTACATGATGGGGAATGAGTTGACCTTCCTAGATGATTCCATCTGCACGGGCACCTCCAGTGGAAATCAAGTGAACCTCACTATCCAAGGACTGAGGGCCATGGACACGGGACTCTACATCTGCAAGGTGGAGCTCATGTACCCACCGCCATACTACCTGGGCATAGGCAACGGAACCCAGATTTATGTAATTGATCCAGAACCGTGCCCAGATTCTGACTTCCTCCTCTGGATCCTTGCAGCAGTTAGTTCGGGGTTGTTTTTTTATAGCTTTCTCCTCACAGCTGTTTCTTTGAGCAAAATGCTAAAGAAAAGAAGCCCTCTTACAACAGGGGTCTATGTGAAAATGCCCCCAACAGAGCCAGAATGTGAAAAGCAATTTCAGCCTTATTTTATTCCCATCAATTGAGAAACCATTATGAAGAAGAGAGTCCATATTTCAATTTCCAAGAGCTGAGG

[0133] As used herein, the term "deaminase" or "deaminase domain" refers to a protein or enzyme that catalyzes a deamination reaction. In some embodiments, the deaminase is an adenosine deaminase, which catalyzes the hydrolytic deamination of adenine to hypoxanthine. In some embodiments, the deaminase is an adenosine deaminase, which catalyzes the hydrolytic deamination of adenosine or adenine (A) to inosine (I). In some embodiments, the deaminase or deaminase domain is an adenosine deaminase, which catalyzes the hydrolytic deamination of adenosine or deoxyadenosine to inosine or deoxyinosine, respectively. In some embodiments, the adenosine deaminase catalyzes the hydrolytic deamination of adenosine in deoxyribonucleic acid (DNA). The adenosine deaminases provided herein (e.g., engineered adenosine deaminases, evolved adenosine deaminases) can be derived from any organism, such as a bacterium. In some embodiments, the adenosine deaminase is derived from a bacterium, such as Escherichia coli, Staphylococcus aureus, Salmonella typhimurium, Shewanella putrefaciens, Haemophilus influenzae, or Caulobacter crescentus.

[0134] In some embodiments, the adenosine deaminase is TadA deaminase. In some embodiments, the TadA deaminase is a TadA variant. In some embodiments, the TadA variant is TadA*8. In some embodiments, the deaminase or deaminase domain is a variant of a naturally occurring deaminase from an organism such as a human, chimpanzee, gorilla, monkey, cow, dog, rat, or mouse. In some embodiments, the deaminase or deaminase domain is non-naturally occurring. For example, in some embodiments, the deaminase or deaminase domain has at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, or at least 99.9% identity to a naturally occurring deaminase. For example, deaminase domains are described in International PCT Applications PCT / 2017 / 045381 (WO 2018 / 027078) and PCT / US2016 / 058344 (WO 2017 / 070632), each of which is incorporated herein by reference in its entirety.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: See also 10.1038 / s41576-018-0059-1, the entire contents of which are incorporated herein by reference.

[0135] "Detection" refers to identifying the presence, absence, or amount of an analyte to be detected. In one embodiment, a sequence variation in a polynucleotide or polypeptide is detected. In another embodiment, the presence of an indel is detected.

[0136] "Detectable label" refers to a composition that, when attached to a molecule of interest, renders the latter detectable via spectroscopic, photochemical, biochemical, immunochemical, or chemical means. For example, useful labels include radioisotopes, magnetic beads, metallic beads, colloidal particles, fluorescent dyes, electron-dense reagents, enzymes (e.g., those commonly used in ELISA), biotin, digoxigenin, or haptens.

[0137] "Disease" means any condition or disorder that damages or interferes with the normal function of a cell, tissue, or organ. In one embodiment, the disease is neoplasia or cancer.

[0138] The term "effective amount" refers to the amount of a bioactive agent sufficient to induce a desired biological response. The effective amount of an active agent used to practice the present invention for the therapeutic treatment of a disease will vary depending on the mode of administration, the age, weight, and general health of the subject. Ultimately, the appropriate amount and dosing regimen will be determined by the attending physician or veterinarian. Such an amount is referred to as an "effective" amount. In one embodiment, an effective amount is the amount of a base editor of the present invention (e.g., a fusion protein comprising a programmable DNA-binding protein, a nucleic acid base editor, and a gRNA) sufficient to introduce a modification into a gene of interest in a cell (e.g., a cell in vitro or in vivo). In one embodiment, an effective amount is the amount of a base editor necessary to achieve a therapeutic effect (e.g., to reduce or control a disease or symptom or condition thereof). Such a therapeutic effect need not be sufficient to alter the gene of interest in all cells of a subject, tissue, or organ; it need only alter the gene of interest in about 1%, 5%, 10%, 25%, 50%, 75%, or more of the cells present in the subject, tissue, or organ.

[0139] "Epitope," as used herein, means an antigenic determinant. An epitope is a portion of an antigen molecule that, by its structure, determines the particular antibody molecule that recognizes and binds to it.

[0140] By "fragment" is meant a portion of a polypeptide or nucleic acid molecule, which portion comprises at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of the entire length of the reference nucleic acid molecule or polypeptide. A fragment can comprise 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100, 200, 300, 400, 500, 600, 700, 800, 900, or 1000 nucleotides or amino acids.

[0141] "Graft versus host disease" (GVHD) refers to a pathological condition in which transplanted donor cells generate an immune response against the host's cells.

[0142] "Guide RNA" or "gRNA" refers to a polynucleotide that is specific for a target sequence and can form a complex with a polynucleotide-programmable nucleotide-binding domain protein (e.g., Cas9 or Cpf1). In one embodiment, the guide polynucleotide is a guide RNA (gRNA). A gRNA can exist as a complex of two or more RNAs or as a single RNA molecule. A gRNA that exists as a single RNA molecule is sometimes referred to as a single guide RNA (sgRNA), although "gRNA" is used interchangeably to refer to a guide RNA that exists as a single molecule or as a complex of two or more molecules. Typically, a gRNA that exists as a single RNA species contains two domains: (1) a domain that shares homology with the target nucleic acid (e.g., directs binding of the Cas9 complex to the target); and (2) a domain that binds to the Cas9 protein. In some embodiments, domain (2) corresponds to a sequence known as tracrRNA and contains a stem-loop structure. For example, in some embodiments, domain (2) is identical to or homologous to a tracrRNA as provided in Jinek et al., Science 337:816-821 (2012), the entire contents of which are incorporated herein by reference. Other examples of gRNAs (e.g., those comprising domain 2) can be found in U.S. Provisional Patent Application No. USSN 61 / 874,682, entitled "Switchable Cas9 Nucleases and Uses Thereof," filed September 6, 2013, and U.S. Provisional Patent Application No. USSN 61 / 874,746, entitled "Delivery System For Functional Nucleases," filed September 6, 2013, the entire contents of which are incorporated herein by reference. In some embodiments, a gRNA comprises two or more of domains (1) and (2) and may be referred to as an "extended gRNA." The extended gRNA binds to two or more Cas9 proteins and binds to the target nucleic acid in two or more different regions, as described herein.The gRNA contains a nucleotide sequence complementary to the target site, which mediates the binding of the nuclease / RNA complex to the target site and provides the sequence specificity of the nuclease:RNA complex. As will be recognized by those skilled in the art, an RNA polynucleotide sequence, such as a gRNA sequence, contains the nucleobase uracil (U), a pyrimidine derivative, rather than the nucleobase thymine (T) contained in a DNA polynucleotide sequence. In RNA, uracil forms a base pair with adenine and replaces thymine during DNA transcription.

[0143] "Heterodimer" refers to a fusion protein containing two domains, such as a wild-type TadA domain and a variant of the TadA domain (e.g., TadA*8), or two variant TadA domains (e.g., TadA*7.10 and TadA*8, or two TadA*8 domains).

[0144] "Host-versus-graft disease" (HVGD) refers to a pathological condition in which the host's immune system generates an immune response against transplanted donor cells.

[0145] "Hybridization" refers to hydrogen bonding between complementary nucleobases, which may be Watson-Crick, Hoogsteen, or reversed Hoogsteen hydrogen bonding. For example, adenine and thymine are complementary nucleobases that form hydrogen bonds to pair.

[0146] "Immune cell" means a cell of the immune system that is capable of producing an immune response.

[0147] "Immune effector cell" means a lymphocyte that, once activated, can bring about an immune response against target cells. T cells are exemplary immune effector cells.

[0148] The term "base repair inhibitor" or "IBR" refers to a protein that can inhibit the activity of nucleic acid repair enzymes, such as base excision repair (BER) enzymes. In one embodiment, IBR is an inhibitor of inosine base excision repair. Examples of base repair inhibitors include inhibitors of APE1, Endo III, Endo IV, Endo V, Endo VIII, Fpg, hOGGl, hNEILl, T7 Endol, T4 PDG, UDG, hSMUGL and hAAG. In one embodiment, IBR is an inhibitor of Endo V or hAAG. In one embodiment, IBR is catalytically inactive EndoV or catalytically inactive hAAG. In some embodiments, the base repair inhibitor is an inhibitor of Endo V or hAAG. In one embodiment, the base repair inhibitor is catalytically inactive EndoV or catalytically inactive hAAG.

[0149] In some embodiments, the base repair inhibitor is a uracil glycosylase inhibitor (UGI). UGI refers to a protein that can inhibit the base excision repair enzyme uracil-DNA glycosylase. In some embodiments, the UGI domain comprises wild-type UGI or a fragment thereof. In some embodiments, the UGI proteins provided herein comprise fragments of UGI and proteins homologous to UGI or UGI fragments. In some embodiments, the base repair inhibitor is an inhibitor of inosine base excision repair. In some embodiments, the base repair inhibitor is a "catalytically inactive inosine-specific nuclease" or "dead inosine-specific nuclease." Without wishing to be bound by any particular theory, catalytically inactive inosine glycosylases (e.g., alkyladenine glycosylases (AAG)) can bind to inosine but cannot create an abasic site or remove the inosine, thereby sterically blocking the newly formed inosine moiety from DNA damage / repair mechanisms. In some embodiments, catalytically inactive inosine-specific nucleases can bind to inosine in nucleic acids but do not cleave nucleic acids.Representative catalytically inactive inosine-specific nucleases include, but are not limited to, catalytically inactive alkyl adenosine glycosylase (AAG nuclease) (e.g., from humans) and catalytically inactive endonuclease V (EndoV nuclease) (e.g., from E. coli).In some embodiments, catalytically inactive AAG nucleases include an E125Q mutation or a corresponding mutation in another AAG nuclease.

[0150] By "increase" is meant a positive change of at least 10%, 25%, 50%, 75%, or 100%.

[0151] An "intein" is a fragment of a protein that can excise itself and link the remaining fragment (extein) with a peptide bond in a process known as protein splicing. Inteins are also called "protein introns." The process by which an intein excises itself and links the remainder of a protein is referred to herein as "protein splicing" or "intein-mediated protein splicing." In some embodiments, the inteins of a precursor protein (the intein-containing protein before intein-mediated protein splicing) are derived from two genes. Such inteins are referred to herein as split inteins (e.g., split intein-N and split intein-C). For example, in cyanobacteria, DnaE, ​​the catalytic subunit a of DNA polymerase III, is encoded by two separate genes, dnaE-n and dnaE-c. The intein encoded by the dnaE-n gene may be referred to herein as "intein N." The intein encoded by the dnaE-c gene may be referred to herein as "intein C."

[0152] Other intein systems can also be used. For example, synthetic inteins based on the dnaE intein, i.e., the intein pair Cfa-N (e.g., split intein-N) and Cfa-C (e.g., split intein-C), have been described (e.g., Stevens et al., J Am Chem Soc. 2016 Feb. 24; 138(7):2162-5, incorporated herein by reference). Non-limiting examples of intein pairs that can be used according to the present disclosure include Cfa DnaE intein, Ssp GyrB intein, Ssp DnaX intein, Ter DnaE3 intein, Ter ThyX intein, Rma DnaB intein, and Cne Prp8 intein (e.g., as described in U.S. Patent No. 8,394,604, incorporated herein by reference).

[0153] Exemplary nucleotide and amino acid sequences of inteins are provided. DnaE Intein-N DNA: TGCCTGTCATACGAAACCGAGATACTGACAGTAGAATATGGCCTTCTGCCAATCGGGAAGATTGTGGAGAAACGGATAGAATGCACAGTTTACTCTGTCGATAACAATGGTAACATTTATACTCAGCCAGTTGCCCAGTGGCACGACCGGGGA GAGCAGGAAGTATTCGAATACTGTCTGGAGGATGGAAGTCTCATTAGGGCCACTAAGGACCACAAATTTATGACAGTCGATGGCCAGATGCTGCCTATAGACGAAATCTTTGAGCGAGAGTTGGACCTCATGCGAGTTGACAACCTTCCTAAT DnaE Intein-N Protein: CLSYETEILTVEYGLLPIGKIVEKRIECTVYSVDNNGNIYTQPVAQWHDR GEQEVFEYCLEDGSLIRATKDHKFMTVDGQMLPIDEIFERELDLMRVDNLPN DnaE Intein-C DNA: ATGATCAAGATAGCTACAAGGAAGTATCTTGGCAAACAAAACGTTTATGA TATTGGAGTCGAAAGAGATCACAACTTTGCTCTGAAGAACGGATTCATAGCTTCTAAT Intein-C: MIKIATRKYLGKQNVYDIGVERDHNFALKNGFIASN Cfa-N DNA: TGCCTGTCTTATGATACCGAGATACTTACCGTTGAATATGGCTTCTTGCCTATTGGAAAGATTGTCGAAGAGGAGAATTGAATGCACAGTATATACTGTAGACAAGAATGGTTTCGTTTACACACAGCCCATTGCTCAATGCACAATCGCGGCGAACAAGAAGTATTTGAGTACTGTCTCGAGGATGGAAGCATCATACGAGCAACTAAAGATCATAAATTCATGACCACTGACGGGCAGATGTTGCCAATAGATGAGATATTCGAGCGGGGCTTGGATCTCAAACAAGTGGATGGATTGCCA Cfa-N protein: CLSYDTEILTVEYGFLPIGKIVEERIECTVYTVDKNGFVYTQPIAQWHNRGEQEVFEYCLEDGSIIRATKDHKFMTTDGQMLPIDEIFERGDLLKQVDGLP Cfa-C DNA: ATGAAGAGGACTGCCGATGGATCAGAGTTTGAATCTCCCAAGAAGAAGAGGAAAGTAAAGATAATATCTCGAAAAGTCTTGGTACCCAAAATGTCTATGATATTGGAGTGGAGAAAGATCACAACTTCCTTCTCAAGAACGGTCTCGTAGCCAGCAAC Cfa-C protein: MKRTADGSEFESPKKKRKVKIISRKSLGTQNVYDIGVEKDHNFLLKNGLVASN

[0154] To join the N-terminal portion of split-Cas9 with the C-terminal portion of split-Cas9, an intein-N and an intein-C can be fused to the N-terminal portion of split-Cas9 and the C-terminal portion of split-Cas9, respectively. For example, in some embodiments, intein-N is fused to the C-terminus of the N-terminal portion of split-Cas9, i.e., forming the structure N--[N-terminal portion of split-Cas9]-[intein-N]--C. In some embodiments, intein-C is fused to the N-terminus of the C-terminal portion of split-Cas9, i.e., forming the structure N--[intein-C]--[C-terminal portion of split-Cas9]-C. The mechanism of intein-mediated protein splicing for linking proteins (e.g., split Cas9) to which an intein is fused is known in the art, for example, as described in Shah et al., Chem Sci. 2014; 5(1):446-461, which is incorporated herein by reference. Methods for designing and using inteins are known in the art and are described, for example, by WO2014004336, WO2017132580, US20150344549, and US20180127780, each of which is incorporated herein by reference in its entirety.

[0155] The terms "isolated," "purified," or "biologically pure" refer to materials that have been removed, to varying degrees, from components that normally accompany them when found in their native state. "Isolated" refers to the degree of separation from the original source or surrounding environment. "Purified" refers to a greater degree of separation than isolation. A "purified" or "biologically pure" protein has been sufficiently free of other substances so that the impurities do not substantially affect the biological properties of the protein or cause other adverse effects. That is, a nucleic acid or peptide of the invention is purified if it is substantially free of cellular material, viral material, or culture medium when produced by recombinant DNA technology, or substantially free of chemical precursors or other chemicals when chemically synthesized. Purity and homogeneity are typically determined using analytical chemistry techniques, such as polyacrylamide gel electrophoresis or high-performance liquid chromatography. The term "purified" can mean that the nucleic acid or protein yields essentially one band in an electrophoretic gel. For proteins that can undergo modifications, such as phosphorylation or glycosylation, different modifications can result in different isolated proteins that can be separately purified.

[0156] By "isolated polynucleotide" is meant a nucleic acid (e.g., DNA) that is free of the genes that flank the nucleic acid molecule of the invention in the naturally occurring genome of the organism from which it is derived. Thus, the term includes recombinant DNA that is present, for example, in a vector; in an autonomously replicating plasmid or virus; in the genomic DNA of a prokaryote or eukaryote; or as a separate molecule independent of other sequences (e.g., cDNA or genomic or cDNA fragments generated by PCR or restriction endonuclease digestion). Furthermore, the term includes RNA molecules transcribed from DNA molecules, as well as recombinant DNA that is part of a hybrid gene that encodes additional polypeptide sequences.

[0157] By "isolated polypeptide" is meant a polypeptide of the invention separated from components that naturally accompany it. Typically, a polypeptide is isolated when it is at least 60% by weight free from the proteins and naturally occurring organic molecules with which it is naturally associated. Preferably, the preparation is at least 75% by weight, more preferably at least 90%, and most preferably at least 99% polypeptide of the invention. Isolated polypeptides of the invention can be obtained, for example, by extraction from a natural source, by expression of a recombinant nucleic acid encoding such a polypeptide; or by chemically synthesizing the protein. Purity can be measured by any appropriate method, for example, column chromatography, polyacrylamide gel electrophoresis, or HPLC analysis.

[0158] The term "linker," as used herein, can refer to a covalent linker (e.g., a covalent bond), a non-covalent linker, a chemical group, or a molecule that connects two molecules or moieties (e.g., two components of a protein complex or ribonucleocomplex, or two domains of a fusion protein, e.g., a polynucleotide programmable DNA-binding domain (e.g., dCas9) and a deaminase domain (e.g., adenosine deaminase, cytidine deaminase, or adenosine deaminase and cytidine deaminase)). A linker can connect different components or different parts of a component of a base editor system. For example, in some embodiments, a linker can connect the guide polynucleotide-binding domain of a polynucleotide programmable nucleotide-binding domain and the catalytic domain of a deaminase. In some embodiments, a linker can connect a CRISPR polypeptide and a deaminase. In some embodiments, a linker can connect Cas9 and a deaminase. In some embodiments, a linker can connect dCas9 and a deaminase. In some embodiments, a linker can connect nCas9 and a deaminase. In some embodiments, a linker can connect a guide polynucleotide and a deaminase. In some embodiments, a linker can connect a deaminating component of a base editor system and a polynucleotide-programmable nucleotide-binding component. In some embodiments, a linker can connect an RNA-binding portion of a deaminating component of a base editor system and a polynucleotide-programmable nucleotide-binding component. In some embodiments, a linker can connect an RNA-binding portion of a deaminating component of a base editor system and an RNA-binding portion of a polynucleotide-programmable nucleotide-binding component. A linker can be positioned between or sandwiched between two groups, molecules, or other moieties, and can be linked to each through covalent or non-covalent interactions, thus connecting the two. In some embodiments, a linker can be an organic molecule, group, polymer, or chemical moiety.In some embodiments, the linker can be a polynucleotide. In some embodiments, the linker can be a DNA linker. In some embodiments, the linker can be an RNA linker. In some embodiments, the linker can comprise an aptamer capable of binding to a ligand. In some embodiments, the ligand can be a carbohydrate, peptide, protein, or nucleic acid. In some embodiments, the linker can comprise an aptamer derived from a riboswitch. The riboswitch from which the aptamer is derived can be selected from a theophylline riboswitch, a thiamine pyrophosphate (TPP) riboswitch, an adenosine cobalamin (AdoCbl) riboswitch, an S-adenosylmethionine (SAM) riboswitch, an SAH riboswitch, a flavin mononucleotide (FMN) riboswitch, a tetrahydrofolate riboswitch, a lysine riboswitch, a glycine riboswitch, a purine riboswitch, a GlmS riboswitch, or a prequeosin 1 (PreQ1) riboswitch. In some embodiments, the linker can comprise an aptamer bound to a protein domain, such as a polypeptide or polypeptide ligand. In some embodiments, the polypeptide ligand can be a K homology (KH) domain, an MS2 coat protein domain, a PP7 coat protein domain, an SfMu Com coat protein domain, a sterile alpha motif, a telomerase Ku binding motif and Ku protein, a telomerase Sm7 binding motif and Sm7 protein, or an RNA recognition motif. In some embodiments, the polypeptide ligand can be part of a base editor system component. For example, a nucleic acid base editing component can include a deaminase domain and an RNA recognition motif.

[0159] In some embodiments, the linker can be an amino acid or multiple amino acids (e.g., a peptide or protein). In some embodiments, the linker can be about 5-100 amino acids in length, e.g., about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 20-30, 30-40, 40-50, 50-60, 60-70, 70-80, 80-90, or 90-100 amino acids in length. In some embodiments, the linker can be about 100-150, 150-200, 200-250, 250-300, 300-350, 350-400, 400-450, or 450-500 amino acids in length. Longer or shorter linkers are also contemplated.

[0160] In some embodiments, a linker connects the gRNA binding domain of an RNA programmable nuclease, including a Cas9 nuclease domain, with the catalytic domain of a nucleic acid editing protein (e.g., cytidine or adenosine deaminase). In some embodiments, a linker connects dCas9 and a nucleic acid editing protein. For example, a linker is placed between two groups, molecules, or other moieties, or is flanked by two groups, molecules, or other moieties, and is linked to each other via a covalent bond, thus connecting the two. In some embodiments, a linker is an amino acid or multiple amino acids (e.g., a peptide or protein). In some embodiments, a linker is an organic molecule, group, polymer, or chemical moiety. In some embodiments, the linker is 5 to 200 amino acids in length, e.g., 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 35, 45, 50, 55, 60, 60, 65, 70, 70, 75, 80, 85, 90, 90, 95, 100, 101, 102, 103, 104, 105, 110, 120, 130, 140, 150, 160, 175, 180, 190, or 200 amino acids in length. Longer and shorter linkers are also contemplated.

[0161] In some embodiments, the nucleobase editor domain has the amino acid sequence SGGSSGSETPGTSESATPESSGGS, SGGSSGGSSGSETPGTSESATPESSGGSSGGS, or GGSGGSPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSTE PSEGSAPGTSTEPSEGSAPGTSESATPESGPGSEPATSGGSGGS In some embodiments, the nucleobase editor domain is fused via a linker comprising the amino acid sequence SGSETPGTSESATPES, also referred to as an XTEN linker. In some embodiments, the linker comprises the amino acid sequence SGGS. In some embodiments, the linker is (SGGS) n , (GGGS) n , (GGGGS) n , (G) n , (EAAAK) n , (GGS) n , SGSETPGTSESATPES, or (XP) n motif, or any combination thereof, where n is independently an integer between 1 and 30 and X is any amino acid. In some embodiments, n is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15.

[0162] In some embodiments, the linker is 24 amino acids in length. In some embodiments, the linker comprises the amino acid sequence SGGSSGGSSGSETPGTSESATPES. In some embodiments, the linker is 40 amino acids in length. In some embodiments, the linker comprises the amino acid sequence SGGSSGGSSGSETPGTSESATPESSGGSSGGSSGGSSGGS. In some embodiments, the linker is 64 amino acids in length. In some embodiments, the linker comprises the amino acid sequence SGGSSGGSSGSETPGTSESATPESSGGSSGGSSGGSSGGSSGSETPGTSESATPESSGGS SGGS. In some embodiments, the linker is 92 amino acids in length. In some embodiments, the linker comprises the amino acid sequence PGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGSEPATS.

[0163] By "marker" is meant any protein or polynucleotide having an altered expression level or activity that is associated with a disease or disorder.

[0164] As used herein, the term "mutation" refers to the substitution of a residue in a sequence, e.g., a nucleic acid or amino acid sequence, with another residue, or the deletion or insertion of one or more residues in a sequence. Mutations are typically described herein by identifying the original residue, then identifying the position of the residue in the sequence, and identifying the newly substituted residue. Various methods for making the amino acid substitutions (mutations) provided herein are well known in the art and are provided, for example, by Green and Sambrook, Molecular Cloning: A Laboratory Manual (4th ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (2012)). In some embodiments, the base editors of the present disclosure can efficiently generate "intended mutations," e.g., point mutations, in a nucleic acid (e.g., a nucleic acid in a subject's genome) without generating a significant number of unintended mutations, e.g., unintended point mutations. In some embodiments, the intended mutation is a mutation caused by a particular base editor (e.g., a cytidine base editor or an adenosine base editor) attached to a guide polynucleotide (e.g., a gRNA) that is specifically designed to produce the intended mutation.

[0165] Generally, mutations made or identified in a sequence (e.g., an amino acid sequence described herein) are numbered relative to a reference (or wild-type) sequence, i.e., a sequence that does not contain the mutation. Those skilled in the art will readily understand how to determine the location of mutations in amino acid and nucleic acid sequences relative to a reference sequence.

[0166] "Neoplasia" refers to cells or tissues that exhibit abnormal growth or proliferation. The term "neoplasia" includes cancer and solid tumors.

[0167] The term "non-conservative mutation" refers to an amino acid substitution between different groups, such as tryptophan to lysine or serine to phenylalanine. In this case, the non-conservative amino acid substitution preferably does not interfere with or inhibit the biological activity of the functional variant. The non-conservative amino acid substitution can enhance the biological activity of the functional variant, such that the biological activity of the functional variant is increased compared to the wild-type protein.

[0168] "Nuclear factor of activated T cells 1 (NFATc1) polypeptide" means a protein or fragment thereof having at least 85% amino acid sequence identity to NCBI Accession No. NM_172390.2, which is a component of the activated T cell DNA-binding transcription complex. Exemplary amino acid sequences are provided below. >NP_765978.1 Nuclear factor of activated T cells, cytoplasmic 1 isoform A [Homo sapiens] MPSTSFPVPSKFPLGPAAAVFGRGETLGPAPRAGGTMKSAEEEHYGYASSNVSPALPLPTAHSTLPAPCHNLQTSTPGIIPPADHPSGYGAALDGGPAGYFLSSGHTRPDGAPALESPRIEITSCLGLYHNNNQFFHDVEVEDVLPSSKRSPSTATLSLPSLEAYRDPSCLSPASSLSS RSCNSEASSYESNYSYPYASPQTSPWQSPCVSPKTTDPEEGFPRGLGACTLLGSPRHSPSTSPRASVTEESWLGARSSRPASPCNKRKYSLNGRQPPYSPHHSPTPSPHGSPRVSVTDDSWLGNTTQYTSSAIVAAINALTTDSSLDLGDGVPVKSRKTTLEQPPSVALKVEPVGEDLG SPPPPADFAPEDYSSFQHIRKGGFCDQYLAVPQHPYQWAKPKPLSPTSYMSPTLPALDWQLPSHSGPYELRIEVQPKSHHRAHYETEGSRGAVKASAGGHPIVQLHGYLENEPLMLQLFIGTADDRLLRPHAFYQVHRITGKTVSTTSHEAILSNTKVLEIPLLPENSMRAVIDCAGIL KLRNSDIELRKGETDIGRKNTRVRLVFRVHVPQPSGRTLSLQVASNPIECSQRSAQELPLVEKQSTDSYPVVGGKKMVLSGHNFLQDSKVIFVEKAPDGHHVWEMEAKTDRDLCKPNSLVVEIPPFRNQRITSPVHVSFYVCNGKRKRSQYQRFTYLPANGNAIFLTVSREHERVGCFF

[0169] "Nuclear factor of activated T cells 1 (NFATc1) polynucleotide" refers to a nucleic acid molecule encoding an NFATc1 polypeptide. The NFATc1 gene encodes a protein involved in the inducible expression of cytokine genes in T cells, particularly IL-2 and IL-4. Exemplary nucleic acid sequences are provided below. >NM_172390.2 Homo sapiens nuclear factor of activated T cells 1 (NFATC1), transcript variant 1, mRNA GGCGGGCGCTCGGCGACTCGTCCCCGGGGCCCCGCGCGGGCCCGGGCAGCAGGGGCGTGATGTCACGGCA GGGAGGGGGCGCGGGAGCCGCCGGGCCGGCGGGGAGGCGGGGGAGGTGTTTTCCAGCTTTAAAAAGGCAG GAGGCAGAGCGCGGCCCTGCGTCAGAGCGAGACTCAGAGGCTCCGAACTCGCCGGCGGAGTCGCCGCGCC AGATCCCAGCAGCAGGGCGCGGGCACCGGGGCGCGGGCAGGGCTCGGAGCCACCGCGCAGGTCCTAGGGC CGCGGCCGGGCCCCGCCACGCGCGCACACGCCCCTCGATGACTTTCCTCCGGGGCGCGCGGCGCTGAGCC CGGGGCGAGGGCTGTCTTCCCGGAGACCCGACCCCGGCAGCGCGGGGCGGCCGCTTCTCCTGTGCCTCCG CCCGCCGCTCCACTCCCCGCCGCCGCCGCGCGGATGCCAAGCACCAGCTTTCCAGTCCCTTCCAAGTTTC CACTTGGCCCTGCGGCTGCGGTCTTCGGGAGAGGAGAAACTTTGGGGCCCGCGCCGCGCGCCGGCGGCAC CATGAAGTCAGCGGAGGAAGAACACTATGGCTATGCATCCTCCAACGTCAGCCCCGCCCTGCCGCTCCCC ACGGCGCACTCCACCCTGCCGGCCCCGTGCCACAACCTTCAGACCTCCACACCGGGCATCATCCCGCCGG CGGATCACCCCTCGGGGTACGGAGCAGCTTTGGACGGTGGGCCCGCGGGCTACTTCCTCTCCTCCGGCCA CACCAGGCCTGATGGGGCCCCTGCCCTGGAGAGTCCTCGCATCGAGATAACCTCGTGCTTGGGCCTGTAC CACAACAATAACCAGTTTTTCCACGATGTGGAGGTGGAAGACGTCCTCCCTAGCTCCAAACGGTCCCCCT CCACGGCCACGCTGAGTCTGCCCAGCCTGGAGGCCTACAGAGACCCCTCGTGCCTGAGCCCGGCCAGCAGCCTGTCCTCCCGGAGCTGCAACTCAGAGGCCTCCTCCTACGAGTCCAACTACTCGTACCCGTACGCGTCC CCCCAGACGTCGCCATGGCAGTCTCCCTGCGTGTCTCCCAAGACCACGGACCCCGAGGAGGGCTTTCCCC GCGGGCTGGGGGCCTGCACACTGCTGGGTTCCCCGCGGCACTCCCCCTCCACCTCGCCCCGCGCCAGCGT CACTGAGGAGAGCTGGCTGGGTGCCCGCTCCTCCAGACCCGCGTCCCCTTGCAACAAGAGGAAGTACAGC CTCAACGGCCGGCAGCCGCCCTACTCACCCCACCACTCGCCCACGCCGTCCCCGCACGGCTCCCCGCGGG TCAGCGTGACCGACGACTCGTGGTTGGGCAACACCACCCAGTACACCAGCTCGGCCATCGTGGCCGCCAT CAACGCGCTGACCACCGACAGCAGCCTGGACCTGGGAGATGGCGTCCCTGTCAAGTCCCGCAAGACCACC CTGGAGCAGCCGCCCTCAGTGGCGCTCAAGGTGGAGCCCGTCGGGGAGGACCTGGGCAGCCCCCCGCCCC CGGCCGACTTCGCGCCCGAAGACTACTCCTCTTTCCAGCACATCAGGAAGGGCGGCTTCTGCGACCAGTA CCTGGCGGTGCCGCAGCACCCCTACCAGTGGGCGAAGCCCAAGCCCCTGTCCCCTACGTCCTACATGAGC CCGACCCTGCCCGCCCTGGACTGGCAGCTGCCGTCCCACTCAGGCCCGTATGAGCTTCGGATTGAGGTGC AGCCCAAGTCCCACCACCGAGCCCACTACGAGACGGAGGGCAGCCGGGGGGCCGTGAAGGCGTCGGCCGG AGGACACCCCATCGTGCAGCTGCATGGCTACTTGGAGAATGAGCCGCTGATGCTGCAGCTTTTCATTGGG ACGGCGGACGACCGCCTGCTGCGCCCGCACGCCTTCTACCAGGTGCACCGCATCACAGGGAAGACCGTGTCCACCACCAGCCACGAGGCCATCCTCTCCAACACCAAAGTCCTGGAGATCCCACTCCTGCCGGAGAACAG CATGCGAGCCGTCATTGACTGTGCCGGAATCCTGAAACTCAGAAACTCCGACATTGAACTTCGGAAAGGA GAGACGGACATCGGGAGGAAGAACACACGGGTACGGCTGGTGTTCCGCGTTCACGTCCCGCAACCCAGCG GCCGCACGCTGTCCCTGCAGGTGGCCTCCAACCCCATCGAATGCTCCCAGCGCTCAGCTCAGGAGCTGCC TCTGGTGGAGAAGCAGAGCACGGACAGCTATCCGGTCGTGGGCGGGAAGAAGATGGTCCTGTCTGGCCAC AACTTCCTGCAGGACTCCAAGGTCATTTTCGTGGAGAAAGCCCCAGATGGCCACCATGTCTGGGAGATGG AAGCGAAAACTGACCGGGACCTGTGCAAGCCGAATTCTCTGGTGGTTGAGATCCCGCCATTTCGGAATCA GAGGATAACCAGCCCCGTTCACGTCAGTTTCTACGTCTGCAACGGGAAGAGAAAGCGAAGCCAGTACCAG CGTTTCACCTACCTTCCCGCCAACGGTAACGCCATCTTTCTAACCGTAAGCCGTGAACATGAGCGCGTGG GGTGCTTTTTCTAAAGACGCAGAAACGACGTCGCCGTAAAGCAGCGTGGCGTGTTGCACATTTAACTGTG TGATGTCCCGTTAGTGAGACCGAGCCATCGATGCCCTGAAAAGGAAAGGAAAAGGGAAGCTTCGGATGCA TTTTCCTTGATCCCTGTTGGGGGTGGGGGGCGGGGGTTGCATACTCAGATAGTCACGGTTATTTTGCTTC TTGCGAATGTATAACAGCCAAGGGGAAAACATGGCTCTTCTGCTCCAAAAAACTGAGGGGGTCCTGGTGT GCATTTGCACCCTAAAGCTGCTTACGGTGAAAAGGCAAATAGGTATAGCTATTTTGCAGGCACCTTTAGGAATAAACTTTGCTTTTAAGCCTGTAAAAAAAAAAAAAAA

[0170] The term "nuclear localization sequence," "nuclear localization signal," or "NLS" refers to an amino acid sequence that promotes the import of a protein into the cell nucleus. Nuclear localization sequences are known in the art and are described, for example, in International PCT Application PCT / EP 2000 / 011690, filed November 23, 2000, and published May 31, 2001 as WO / 2001 / 038547, by Plank et al., the contents of which are incorporated herein by reference for their disclosure of exemplary nuclear localization sequences. In other embodiments, the NLS is an optimized NLS, for example, as described by Koblan et al., Nature Biotech. 2018 doi:10.1038 / nbt.4172. In some embodiments, the NLS comprises the amino acid sequence KRTADGSEFESPKKKRKV, KRPAATKKAGQAKKKK, KKTELQTTNAENKTKKL, KRGINDRNFWRGENGRKTR, RKSGKIAAIVVKRPRK, PKKKRKV, or MDSLLMNRRKFLYQFKNVRWAKGRRETYLC.

[0171] As used herein, the terms "nucleic acid" and "nucleic acid molecule" refer to a compound containing a nucleobase and an acidic moiety, such as a nucleoside, a nucleotide, or a polymer of nucleotides. Typically, polymeric nucleic acids, e.g., nucleic acid molecules containing three or more nucleotides, are linear molecules in which adjacent nucleotides are linked to each other via phosphodiester bonds. In certain embodiments, "nucleic acid" refers to individual nucleic acid residues (e.g., nucleotides and / or nucleosides). In certain embodiments, "nucleic acid" refers to an oligonucleotide chain containing three or more individual nucleotide residues. As used herein, the terms "oligonucleotide" and "polynucleotide" can be used interchangeably to refer to a polymer of nucleotides (e.g., a chain of at least three nucleotides). In certain embodiments, "nucleic acid" encompasses RNA and single- and / or double-stranded DNA. Nucleic acids can naturally occur, for example, in the context of a genome, transcript, mRNA, tRNA, rRNA, siRNA, snRNA, plasmid, cosmid, chromosome, chromatid, or other naturally occurring nucleic acid molecule. Alternatively, a nucleic acid molecule may be, for example, a non-naturally occurring molecule, recombinant DNA or RNA, an artificial chromosome, an engineered genome, or a fragment thereof, or synthetic DNA, RNA, DNA / RNA hybrid, or a non-naturally occurring molecule containing non-naturally occurring nucleotides or nucleosides. Furthermore, the terms "nucleic acid," "DNA," "RNA," and / or similar terms include nucleic acid analogs, e.g., analogs having other than a phosphodiester backbone. Nucleic acids can be purified from natural sources, produced using recombinant expression systems and optionally purified, chemically synthesized, etc. In the case of chemically synthesized molecules, the nucleic acid may contain nucleoside analogs, such as, for example, chemically modified bases or sugars, and analogs with backbone modifications, where appropriate. Nucleic acid sequences are shown in the 5' to 3' direction unless otherwise indicated.In some embodiments, nucleic acids are selected from natural nucleosides (e.g., adenosine, thymidine, guanosine, cytidine, uridine, deoxyadenosine, deoxythymidine, deoxyguanosine, and deoxycytidine); nucleoside analogs (e.g., 2-aminoadenosine, 2-thiothymidine, inosine, pyrrolopyrimidine, 3-methyladenosine, 5-methylcytidine, 2-aminoadenosine, C5-bromouridine, C5-fluorouridine, C5-iodouridine, C5-propynyl-uridine, C5-propynyl-cytidine, C5-amino-3-methyl-4-methyl-5 ... The bases may be or contain: 5-methylcytidine, 2-aminoadenosine, 7-deazaadenosine, 7-deazaguanosine, 8-oxoadenosine, 8-oxoguanine, O6-methylguanine, and 2-thiocytidine; chemically modified bases; biologically modified bases (e.g., methylated bases); inserted bases; modified sugars (e.g., 2'-fluororibose, ribose, 2'-deoxyribose, arabinose, and hexose); and / or modified phosphate groups (e.g., phosphorothioate and 5'-N-phosphoramidite linkages).

[0172] The term "nucleic acid programmable DNA binding protein" or "napDNAbp" may be used interchangeably with "polynucleotide programmable nucleotide binding domain" and refers to a protein that associates with a nucleic acid (e.g., DNA or RNA), such as a guide nucleic acid or guide polynucleotide (e.g., gRNA), that guides the napDNAbp to a specific nucleic acid sequence. In some embodiments, the polynucleotide-programmable nucleotide binding domain is a polynucleotide-programmable DNA binding domain. In some embodiments, the polynucleotide-programmable nucleotide binding domain is a polynucleotide-programmable RNA binding domain. In some embodiments, the polynucleotide-programmable nucleotide binding domain is a Cas9 protein. The Cas9 protein can bind to a guide RNA that guides the Cas9 protein to a specific DNA sequence complementary to the guide RNA. In some embodiments, the napDNAbp is a Cas9 domain, such as a nuclease-active Cas9, Cas9 nickase (nCas9), or nuclease-inactive Cas9 (dCas9). Non-limiting examples of nucleic acid programmable DNA binding proteins include Cas9 (e.g., dCas9 and nCas9), Cas12a / Cpfl, Cas12b / C2cl, Cas12c / C2c3, Cas12d / CasY, Cas12e / CasX, Cas12g, Cas12h, and Cas12i.Non-limiting examples of Cas enzymes include Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas5d, Cas5t, Cas5h, Cas5a, Cas6, Cas7, Cas8, Cas8a, Cas8b, Cas8c, Cas9 (also called Csn1 or Csx12), Cas10, Cas10d, Cas12a / Cpfl, Cas12b / C2cl, Cas12c / C2c3, Cas12d / CasY, Cas12e / CasX, Cas12g, Cas12h, Cas12i, Csy1, Csy2, Csy3, Csy4, and Cse 1, Cse2, Cse3, Cse4, Cse5e, Csc1, Csc2, Csa5, Csn1, Csn2, Csm1, Csm2, Csm3 , Csm4, Csm5, Csm6, Cmr1, Cmr3, Cmr4, Cmr5, Cmr6, Csb1, Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, Csx1, Csx1S, Csx11, Csf1, Csf2, CsO, Csf4, Csd1, Csd2, Cst1, Cst2, Csh1, Csh2, Csa1, Csa2, Csa3, Csa4, Csa5, Type II Examples of such proteins include Cas effector proteins, type V Cas effector proteins, type VI Cas effector proteins, CARF, DinG, homologs thereof, or modified or engineered versions thereof. Other nucleic acid programmable DNA-binding proteins are also within the scope of this disclosure, even if they may not be specifically listed herein. See, for example, Makarova et al. "Classification and Nomenclature of CRISPR-Cas Systems: Where from Here?" CRISPR J. 2018 Oct;1:325-336. doi: 10.1089 / crispr.2018.0033; Yan et al., "Functionally diverse type V CRISPR-Cas systems" Science. 2019 Jan 4;363(6422):88-91. doi: 10.1126 / science.aav7271 (the entire contents of each are incorporated herein by reference).

[0173] The terms "nucleobase," "nitrogenous base," or "base" are used interchangeably herein and refer to nitrogen-containing biological compounds that form nucleosides, which are the building blocks of nucleotides. The ability of nucleobases to base pair and stack with each other directly leads to long-chain helical structures such as ribonucleic acid (RNA) and deoxyribonucleic acid (DNA). The five nucleobases, adenine (A), cytosine (C), guanine (G), thymine (T), and uracil (U), are referred to as primary or canonical. Adenine and guanine are derived from purines, while cytosine, uracil, and thymine are derived from pyrimidines. DNA and RNA may also contain other (non-primary) modified bases. Non-limiting exemplary modified nucleobases include hypoxanthine, xanthine, 7-methylguanine, 5,6-dihydrouracil, 5-methylcytosine (m5C), and 5-hydromethylcytosine. Hypoxanthine and xanthine can be produced in the presence of mutagens, and both are produced by deamination (replacement of an amine group with a carbonyl group). Hypoxanthine can be modified from adenine. Xanthine can be modified from guanine. Uracil can be produced by deamination of cytosine. A "nucleoside" consists of a nucleobase and a five-carbon sugar (ribose or deoxyribose). Examples of nucleosides include adenosine, guanosine, uridine, cytidine, 5-methyluridine (m5U), deoxyadenosine, deoxyguanosine, thymidine, deoxyuridine, and deoxycytidine. Nucleosides having modified nucleobases include inosine (I), xanthosine (X), 7-methylguanosine (m7G), dihydrouridine (D), 5-methylcytidine (m5C), and pseudouridine (Ψ). A "nucleotide" consists of a nucleobase, a five-carbon sugar (ribose or deoxyribose), and at least one phosphate group. The term "nucleobase editing domain" or "nucleobase editing protein," as used herein, refers to a protein or enzyme that can catalyze nucleobase modifications in RNA or DNA, such as the deamination of cytosine (or cytidine) to uracil (or uridine) or thymine (or thymidine), and adenine (or adenosine) to hypoxanthine (or inosine), as well as non-templated nucleotide addition and insertion. In some embodiments, the nucleobase editing domain is a deaminase domain (e.g., adenine deaminase or adenosine deaminase; or cytidine deaminase or cytosine deaminase). In some embodiments, the nucleobase editing domain is a multiple deaminase domain (e.g., adenine deaminase or adenosine deaminase and cytidine or cytosine deaminase). In some embodiments, the nucleobase editing domain can be a naturally occurring nucleobase editing domain. In some embodiments, the nucleobase-editing domain can be a nucleobase-editing domain engineered or evolved from a naturally occurring nucleobase-editing domain. The nucleobase-editing domain can be from any organism, such as a bacterium, human, chimpanzee, gorilla, monkey, cow, dog, rat, or mouse.

[0174] As used herein, "obtaining," as in "obtaining a drug," includes synthesizing, purchasing, or otherwise acquiring the drug.

[0175] As used herein, "patient" or "subject" refers to a mammalian subject or individual who has been diagnosed with, is at risk of developing, or is suspected of having or developing a disease or disorder. In some embodiments, the term "patient" refers to a mammalian subject who has a higher than average likelihood of developing a disease or disorder. Exemplary patients may be humans, non-human primates, cats, dogs, pigs, cows, cats, horses, camels, llamas, goats, sheep, rodents (e.g., mice, rabbits, rats, guinea pigs), and other mammals that may benefit from the treatments disclosed herein. Exemplary human patients may be male and / or female.

[0176] A "patient in need thereof" or "subject in need thereof" is referred to herein as a patient who has been diagnosed with, is at risk of having, has been predetermined to have, or is suspected of having a disease or disorder.

[0177] The terms "pathogenic mutation," "pathogenic variant," "disease-causing mutation," "disease-causing variant," "deleterious mutation," or "predisposing mutation" refer to a genetic change or mutation that increases an individual's susceptibility or predisposition to a particular disease or disorder. In some embodiments, a pathogenic mutation comprises the substitution of at least one wild-type amino acid in a protein encoded by a gene with at least one pathogenic amino acid.

[0178] The terms "protein," "peptide," "polypeptide," and their grammatical equivalents are used interchangeably herein and refer to a polymer of amino acid residues linked together by peptide (amide) bonds. The term refers to a protein, peptide, or polypeptide of any size, structure, or function. Typically, a protein, peptide, or polypeptide is at least three amino acids in length. A protein, peptide, or polypeptide can refer to an individual protein or a group of proteins. One or more amino acids in a protein, peptide, or polypeptide can be modified by the addition of chemical entities such as carbohydrate groups, hydroxyl groups, phosphate groups, farnesyl groups, isofarnesyl groups, fatty acid groups, linkers for conjugation, functionalization, or other modifications. A protein, peptide, or polypeptide can also be a single molecule or a multimolecular complex. A protein, peptide, or polypeptide can be simply a fragment of a naturally occurring protein or peptide. A protein, peptide, or polypeptide can be naturally occurring, recombinant, synthetic, or any combination thereof. As used herein, the term "fusion protein" refers to a hybrid polypeptide containing protein domains from at least two different proteins. One protein can be located at the amino-terminal (N-terminal) or carboxy-terminal (C-terminal) portion of the fusion protein, thus forming an amino-terminal fusion protein or a carboxy-terminal fusion protein, respectively. The protein can contain different domains, such as a nucleic acid binding domain (e.g., the gRNA binding domain of Cas9, which guides the protein to bind to the target site) and a nucleic acid cleavage domain, or the catalytic domain of a nucleic acid editing protein. In some embodiments, the protein includes a proteinaceous portion, such as an amino acid sequence constituting the nucleic acid binding domain, and an organic compound, such as a compound that can act as a nucleic acid cleavage agent. In some embodiments, the protein is complexed with or associated with a nucleic acid (e.g., RNA or DNA).Any protein provided herein can be produced by any method known in the art.For example, the protein provided herein can be produced through recombinant protein expression and purification, which is particularly suitable for fusion proteins containing peptide linkers.The method for recombinant protein expression and purification is well known, including that described by Green and Sambrook, Molecular Cloning: A Laboratory Manual (4th ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (2012)), the entire contents of which are incorporated herein by reference.

[0179] The polypeptides and proteins (including functional portions and functional variants thereof) disclosed herein can contain synthetic amino acids in place of one or more naturally occurring amino acids. Such synthetic amino acids are known in the art and include, for example, aminocyclohexanecarboxylic acid, norleucine, α-amino n-decanoic acid, homoserine, S-acetylaminomethyl-cysteine, trans-3- and trans-4-hydroxyproline, 4-aminophenylalanine, 4-nitrophenylalanine, 4-chlorophenylalanine, 4-carboxyphenylalanine, β-phenylserine, β-hydroxyphenylalanine, phenylglycine, α-naphthylalanine, cyclohexylalanine, cyclohexylglycine, and indoline-2-carboxylic acid. , 1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid, aminomalonic acid, aminomalonic acid monoamide, N'-benzyl-N'-methyllysine, N',N'-dibenzyl-lysine, 6-hydroxylysine, ornithine, α-aminocyclopentanecarboxylic acid, aminocyclohexanecarboxylic acid, aminocyclohexanecarboxylic acid, α-aminocycloheptanecarboxylic acid, α-(2-amino-2-norbornane)-carboxylic acid, α,γ-diaminobutyric acid, α,β-diaminopropionic acid, homophenylalanine, and α-tert-butylglycine. Polypeptides and proteins can be associated with post-translational modifications of one or more amino acids of the polypeptide construct. Non-limiting examples of post-translational modifications include phosphorylation, acylation, including acetylation and formylation, glycosylation (including N-linked and O-linked), amidation, hydroxylation, alkylation, including methylation and ethylation, ubiquitination, addition of pyrrolidone carboxylic acid, formation of disulfide bridges, sulfation, myristoylation, palmitoylation, isoprenylation, farnesylation, geranylation, glypiation, lipoylation, and iodination.

[0180] "Programmed cell death 1 (PDCD1 or PD-1) polypeptide" means a protein or fragment thereof having at least 85% amino acid sequence identity with NCBI Accession No. AJS10360.1. The PD-1 protein is thought to be involved in regulating T cell function during immune responses and in tolerant states. Exemplary B2M polypeptide sequences are provided below. >AJS10360.1 Programmed cell death 1 protein [Homo sapiens] MQIPQAPWPVVWAVLQLGWRPGWFLDSPDRPWNPPTFSPALLVVTEGDNATFTCSFSNTSESFVLNWYRMSPSNQTDKLAAFPEDRSQPGQDCRFRVTQLPNGRDFHMSVVRARRNDSGTYLCGAISLAPKAQIKESLRAELRV TERRAEVPTAHPSPSPRPAGQFQTLVVGVVGGLLGSLVLLVWVLAVICSRAARGTIGARRTGQPLKEDPSAVPVFSVDYGELDFQWREKTPEPPVPCVPEQTEYATIVFPSGMGTSPARRGSADGPRSAQPLRPEDGHCSWPL

[0181] "Programmed cell death 1 (PDCD1 or PD-1) polynucleotide" refers to a nucleic acid molecule that encodes a PD-1 polypeptide. The PDCD1 gene encodes an inhibitory cell surface receptor that inhibits T cell effector function in an antigen-specific manner. Exemplary PDCD1 nucleic acid sequences are provided below. >AY238517.1 Homo sapiens programmed cell death 1 (PDCD1) mRNA, complete cds ATGCAGATCCCACAGGCGCCCTGGCCAGTCGTCTGGGCGGTGCTACAACTGGGCTGGCGGCCAGGATGGTTCTTAGACTCCCCAGACAGGCCCTGGAACCCCCCCACCTTCTCCCCAGCCCTGCTCGTGGTGACCGAAGGGGACAACGCCACCTTCACCTGCAGCTTCTCCAACACATCGGAGAGCTTCGTGCTAAACTGGTACCGCATGAGCCCCAGCAACCAGACGGACAAGCTGGCCGCCTTCCCCGAGGACCGCAGCCAGCCCGGCCAGGACTGCCGCTTCCGTGTCACACAACTGCCCAACGGGCGTGACTTCCACATGAGCGTGGTCAGGGCCCGGCGCAATGACAGCGGCACCTACCTCTGTGGGGCCATCTCCCTGGCCCCCAAGGCGCAGATCAAAGAGAGCCTGCGGGCAGAGCTCAGGGTGACAGAGAGAAGGGCAGAAGTGCCCACAGCCCACCCCAGCCCCTCACCCAGGCCAGCCGGCCAGTTCCAAACCCTGGTGGTTGGTGTCGTGGGCGGCCTGCTGGGCAGCCTGGTGCTGCTAGTCTGGGTCCTGGCCGTCATCTGCTCCCGGGCCGCACGAGGGACAATAGGAGCCAGGCGCACCGGCCAGCCCCTGAAGGAGGACCCCTCAGCCGTGCCTGTGTTCTCTGTGGACTATGGGGAGCTGGATTTCCAGTGGCGAGAGAAGACCCCGGAGCCCCCCGTGCCCTGTGTCCCTGAGCAGACGGAGTATGCCACCATTGTCTTTCCTAGCGGAATGGGCACCTCATCCCCCGCCCGCAGGGGCTCAGCTGACGGCCCTCGGAGTGCCCAGCCACTGAGGCCTGAGGATGGACACTGCTCTTGGCCCCTCTGA

[0182] The term "recombinant," as used herein with respect to a protein or nucleic acid, refers to a protein or nucleic acid that does not occur in nature but is the product of human engineering. For example, in some embodiments, a recombinant protein or nucleic acid molecule comprises an amino acid or nucleotide sequence that contains at least one, at least two, at least three, at least four, at least five, at least six, or at least seven mutations compared to any naturally occurring sequence.

[0183] By "decreasing" is meant a negative change of at least 10%, 25%, 50%, 75%, or 100%.

[0184] "Reference" refers to a standard or control condition. In one embodiment, the reference is a wild-type or healthy cell. In another embodiment, without limitation, the reference is an untreated cell that is not exposed to the test condition or is exposed to a placebo or normal saline, medium, buffer, and / or a control vector that does not carry the polynucleotide of interest.

[0185] A "reference sequence" is a defined sequence used as the basis for sequence comparison. A reference sequence can be a subset or the entirety of a specific sequence; for example, a segment of a full-length cDNA or gene sequence, or the entire cDNA or gene sequence. For polypeptides, the length of a reference polypeptide sequence is generally at least about 16 amino acids, at least about 20 amino acids, at least about 25 amino acids, about 35 amino acids, about 50 amino acids, or about 100 amino acids. For nucleic acids, the length of a reference nucleic acid sequence is generally at least about 50 nucleotides, at least about 60 nucleotides, at least about 75 nucleotides, about 100 nucleotides, or about 300 nucleotides, or any integer therebetween or thereabout. In some embodiments, the reference sequence is the wild-type sequence of a target protein. In other embodiments, the reference sequence is a polynucleotide sequence encoding a wild-type protein.

[0186] The terms "RNA-programmable nuclease" and "RNA-guided nuclease" are used in conjunction with (e.g., bound to or associated with) one or more RNAs that are not targets for cleavage. In certain embodiments, when an RNA-programmable nuclease is complexed with an RNA, it can be referred to as a nuclease:RNA complex. Typically, the bound RNA is referred to as a guide RNA (gRNA). A gRNA can exist as a complex of two or more RNAs or as a single RNA molecule. A gRNA that exists as a single RNA molecule is sometimes referred to as a single guide RNA (sgRNA), although "gRNA" is used interchangeably to refer to a guide RNA that exists as a single molecule or as a complex of two or more molecules. Typically, a gRNA that exists as a single RNA species contains two domains: (1) a domain that shares homology with the target nucleic acid (e.g., directs binding of the Cas9 complex to the target); and (2) a domain that binds to the Cas9 protein. In some embodiments, domain (2) corresponds to a sequence known as tracrRNA and contains a stem-loop structure. For example, in some embodiments, domain (2) is identical to or homologous to the tracrRNA as provided in Jinek et al., Science 337:816-821 (2012), the entire contents of which are incorporated herein by reference. Other examples of gRNAs (e.g., those comprising domain 2) can be found in U.S. Provisional Patent Application No. USSN 61 / 874,682, entitled "Switchable Cas9 Nucleases and Uses Thereof," filed September 6, 2013, and U.S. Provisional Patent Application No. USSN 61 / 874,746, entitled "Delivery System For Functional Nucleases," filed September 6, 2013, the entire contents of each of which are incorporated herein by reference. In some embodiments, a gRNA comprises two or more of domains (1) and (2), and may be referred to as an "extended gRNA." By way of example, the extended gRNA may bind, e.g., to two or more Cas9 proteins and bind to the target nucleic acid in two or more different regions, as described herein.The gRNA contains a nucleotide sequence complementary to a target site, which mediates binding of the nuclease / RNA complex to said target site and provides sequence specificity for the nuclease:RNA complex.

[0187] In certain embodiments, the RNA programmable nuclease is a (CRISPR-associated system) Cas9 endonuclease, e.g., Cas9 (Casnl) from Streptococcus pyogenes (e.g., "Complete genome sequence of an Ml strain of Streptococcus pyogenes." Ferretti JJ, McShan WM, Ajdic DJ, Savic DJ, Savic G., Lyon K., Primeaux C, Sezate S., Suvorov AN, Kenton S., Lai HS, Lin SP, Qian Y., Jia HG, Najar FZ, Ren Q., Zhu H., Song L., White J., Yuan X., Clifton SW, Roe BA, McLaughlin RE, Proc. Natl. Acad. Sci. USA 98:4658-4663(2001); trans-encoded small RNA and host factor RNase III." Deltcheva E., Chylinski K., Sharma CM., Gonzales K., Chao Y., Pirzada ZA, Eckert MR, Vogel J., Charpentier E., Nature 471:602-607 (2011)).

[0188] Because RNA-programmable nucleases (e.g., Cas9) use RNA:DNA hybridization to target DNA cleavage sites, these proteins can, in principle, target any sequence specified by a guide RNA. Methods of using RNA-programmable nucleases such as Cas9 for site-specific cleavage (e.g., to modify genomes) are known in the art (e.g., Cong, L. et al., Multiplex genome engineering using CRISPR / Cas systems. Science 339, 819-823 (2013); Mali, P. et al., RNA-guided human genome engineering via Cas9. Science 339, 823-826 (2013); Hwang, WY et al., Efficient genome editing in zebrafish using a CRISPR-Cas system. Nature biotechnology 31, 227-229 (2013); Jinek, M. et al., RNA-programmed genome editing in human cells. eLife 2, e00471 (2013); Dicarlo, JE et al., Genome engineering in Saccharomyces cerevisiae using CRISPR-Cas systems. Nucleic acids research (2013); Jiang, W. et al., RNA-guided editing of bacterial genomes using CRISPR-Cas systems. Nature biotechnology 31, 233-239 (2013), the entire contents of each of which are incorporated herein by reference.

[0189] The term "single nucleotide polymorphism (SNP)" refers to a single nucleotide variation occurring at a specific position in the genome, where each variation is present to a noticeable degree in a population (e.g., >1%). For example, at a particular base position in the human genome, a C nucleotide can occur in most individuals, but in a minority of individuals, that position is occupied by an A. This means that there is an SNP at this specific position, and the two nucleotide variations, C or A, are alleles at this position. SNPs underlie differences in susceptibility to disease. Disease severity and the body's response to treatment are also manifestations of genetic variation. SNPs can occur in the coding region of a gene, the noncoding region of a gene, or intergenic regions (regions between genes). In some embodiments, SNPs within a coding sequence do not necessarily change the amino acid sequence of the resulting protein due to the degeneracy of the genetic code. SNPs in coding regions are of two types: synonymous and nonsynonymous SNPs. Synonymous SNPs do not affect the protein sequence, while nonsynonymous SNPs do alter the amino acid sequence of a protein. There are two types of nonsynonymous SNPs: missense and nonsense. SNPs that are not located in protein-coding regions can affect gene splicing, transcription factor binding, messenger RNA degradation, or the sequence of non-coding RNA. Gene expression affected by this type of SNP is called an eSNP (expressed SNP) and can be upstream or downstream of the gene. Single-nucleotide variants (SNVs) are single-nucleotide variations with unlimited frequency that can occur somatically. Somatic single-nucleotide variations can also be called single-nucleotide modifications.

[0190] By "specifically binds" is meant a nucleic acid molecule, polypeptide, or complex thereof (e.g., a nucleic acid programmable DNA binding domain and a guide nucleic acid), compound, or molecule that recognizes and binds to a polypeptide and / or nucleic acid molecule of the invention, but does not substantially recognize or bind to other molecules in a sample (e.g., a biological sample).

[0191] Nucleic acid molecules useful in the methods of the present invention include any nucleic acid molecule encoding a polypeptide of the present invention or a fragment thereof. Such nucleic acid molecules need not be 100% identical to an endogenous nucleic acid sequence, but typically exhibit substantial identity. A polynucleotide having "substantial identity" to an endogenous sequence is typically capable of hybridizing with at least one strand of a double-stranded nucleic acid molecule. Nucleic acid molecules useful in the methods of the present invention include any nucleic acid molecule encoding a polypeptide of the present invention or a fragment thereof. Such nucleic acid molecules need not be 100% identical to an endogenous nucleic acid sequence, but typically exhibit substantial identity. A polynucleotide having "substantial identity" to an endogenous sequence is typically capable of hybridizing with at least one strand of a double-stranded nucleic acid molecule. "Hybridizing" refers to pairing to form a double-stranded molecule between complementary polynucleotide sequences (e.g., genes described herein) or portions thereof under various stringency conditions. (See, e.g., Wahl, GM and SL Berger (1987) Methods Enzymol. 152:399; Kimmel, AR (1987) Methods Enzymol. 152:507).

[0192] For example, stringent salt concentrations are typically less than about 750 mM NaCl and 75 mM trisodium citrate, preferably less than about 500 mM NaCl and 50 mM trisodium citrate, and more preferably less than about 250 mM NaCl and 25 mM trisodium citrate. Low stringency hybridization can be achieved in the absence of organic solvents, such as formamide, while high stringency hybridization can be achieved in the presence of at least about 35% formamide, more preferably at least about 50% formamide. Stringent temperature conditions will typically include a temperature of at least about 30°C, more preferably at least about 37°C, and most preferably at least about 42°C. Various additional parameters, such as hybridization time, detergent (e.g., sodium dodecyl sulfate (SDS)) concentration, and the inclusion or exclusion of carrier DNA, are well known to those skilled in the art. Various levels of stringency can be achieved by combining these various conditions as needed. In one embodiment, hybridization occurs at 30° C. in 750 mM NaCl, 75 mM trisodium citrate, and 1% SDS. In another embodiment, hybridization occurs at 37° C. in 500 mM NaCl, 50 mM trisodium citrate, 1% SDS, 35% formamide, and 100 μg / ml denatured salmon sperm DNA (ssDNA). In another embodiment, hybridization occurs at 42° C. in 250 mM NaCl, 25 mM trisodium citrate, 1% SDS, 50% formamide, and 200 μg / ml ssDNA. Useful variations of these conditions will be readily apparent to those of skill in the art.

[0193] In most applications, the washing steps following hybridization also vary in stringency. Wash stringency conditions can be defined by salt concentration and temperature. As described above, washing stringency can be increased by decreasing salt concentration or increasing temperature. For example, stringent salt concentrations for washing steps are preferably less than about 30 mM NaCl and 3 mM trisodium citrate, and most preferably less than about 15 mM NaCl and 1.5 mM trisodium citrate. Stringent temperature conditions for washing steps typically include temperatures of at least about 25°C, more preferably at least about 42°C, and even more preferably at least about 68°C. In one embodiment, washing steps are performed at 25°C in 30 mM NaCl, 3 mM trisodium citrate, and 0.1% SDS. In a more preferred embodiment, washing steps are performed at 42°C in 15 mM NaCl, 1.5 mM trisodium citrate, and 0.1% SDS. In a more preferred embodiment, wash steps are performed in 15 mM NaCl, 1.5 mM trisodium citrate, and 0.1% SDS at 68° C. Further variations of these conditions will be readily apparent to those of skill in the art.Hybridization techniques are well known to those skilled in the art and are described, for example, in Benton and Davis (Science 196:180, 1977); Grunstein and Hogness (Proc. Natl. Acad. Sci., USA 72:3961, 1975); Ausubel et al. (Current Protocols in Molecular Biology, Wiley Interscience, New York, 2001); Berger and Kimmel (Guide to Molecular Cloning Techniques, 1987, Academic Press, New York); and Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, New York.

[0194] "Split" means divided into two or more pieces.

[0195] "Split Cas9 protein" or "split-Cas9" refers to a Cas9 protein that is provided as an N-terminal fragment and a C-terminal fragment encoded by two separate nucleotide sequences. Polypeptides corresponding to the N-terminal and C-terminal portions of the Cas9 protein can be spliced ​​to form a "reconstituted" Cas9 protein. In certain embodiments, the Cas9 protein is split into two fragments within a disordered region of the protein, e.g., as described in Nishimasu et al., Cell, Volume 156, Issue 5, pp. 935-949, 2014, or as described in Jiang et al. (2016) Science 351: 867-871. PDB file: 5F9R (each incorporated herein by reference). In some embodiments, the protein is split into two fragments at any C, T, A, or S within the region between approximately amino acids A292-G364, F445-K483, or E565-T637 of SpCas9, or at the corresponding position in any other Cas9, Cas9 variant (e.g., nCas9, dCas9), or other napDNAbp. In certain embodiments, the protein is split into two fragments at SpCas9 T310, T313, A456, S469, or C574. In some embodiments, the process of splitting a protein into two fragments is referred to as "splitting" the protein.

[0196] In other embodiments, the N-terminal portion of the Cas9 protein comprises amino acids 1-573 or 1-637 of wild-type S. pyogenes Cas9 (SpCas9) (NCBI Reference Sequence NC_002737.2, Uniprot Reference Sequence: Q99ZW2), and the C-terminal portion of the Cas9 protein comprises amino acids 574-1368 or 638-1368 of wild-type SpCas9, or their corresponding positions.

[0197] The C-terminal portion of a split Cas9 can be joined with the N-terminal portion of a split Cas9 to form a complete Cas9 protein. In some embodiments, the C-terminal portion of the Cas9 protein begins where the N-terminal portion of the Cas9 protein ends. Thus, in some embodiments, the C-terminal portion of the split Cas9 comprises a portion of amino acids (551-651)-1368 of spCas9. "(551-651)-1368" means beginning with an amino acid between amino acids 551 and 651 (inclusive) and ending at amino acid 1368.For example, the C-terminal portion of a split-Cas9 consists of amino acids 551-1368, 552-1368, 553-1368, 554-1368, 555-1368, 556-1368, 557-1368, 558-1368, 559-1368, 560-1368, 561-1368, 562-1368, 563-1368, 564-1368, 565-1368, 566-1368, 567-1368, 568-1368, 569-1368, 570-1368, 571-1368, 572-1368, 573-1368, 574 -1368, 575-1368, 576-1368, 577-1368, 578-1368, 579-1368, 580-1368, 581-1368, 582-1368, 583-1368, 584-1368, 585-1368, 586-1368, 587-1368, 588-1368, 589-1368, 590-1368, 591-1368, 592-1368, 593-1368, 594-1368, 595-1368, 596-1368, 597-1368, 598-1368, 599-1368, 600-1368 , 601-1368, 602-1368, 603-1368, 604-1368, 605-1368, 606-1368, 607-1368, 608-1368, 609-1368, 610-1368, 611-1368, 612-1368, 613-1368, 614-1368, 615-1368, 616-1368, 617-1368, 618-1368, 619-1368, 620-1368, 621-1368, 622-1368, 623-1368, 624-1368, 625-1368, 626-1368, 627- 1368, 628-1368, 629-1368, 630-1368, 631-1368, 632-1368, 633-1368, 634-1368, 635-1368, 636-1368, 637-1368, 638-1368, 639-1368, 640-1368, 641-1368, 642-1368, 643-1368, 644-1368, 645-1368, 646-1368, 647-1368, 648-1368, 649-1368, 650-1368, or 651-1368.In some embodiments, the C-terminal portion of the split Cas9 protein comprises a portion of amino acids 574-1368 or 638-1368 of SpCas9.

[0198] "Subject" means a mammal, including, but not limited to, a human or a non-human mammal such as a cow, horse, dog, sheep, or cat. Subjects also include livestock, domestic animals raised to produce labor and provide commodities such as food, including, but not limited to, cows, goats, chickens, horses, pigs, rabbits, and sheep.

[0199] By "substantially identical" is meant a polypeptide or nucleic acid molecule that exhibits at least 50% identity to a reference amino acid sequence (e.g., any one of the amino acid sequences described herein) or nucleic acid sequence (e.g., any one of the nucleic acid sequences described herein). In one embodiment, such a sequence has at least 60%, 80%, or 85%, 90%, 95%, or even 99% identity at the amino acid level or nucleic acid to the sequence used for comparison.

[0200] Sequence identity is typically measured using sequence analysis software (e.g., the Sequence Analysis Software Package, BLAST, BESTFIT, GAP, or PILEUP / PRETTYBOX programs of the Genetics Computer Group, University of Wisconsin Biotechnology Center, 1710 University Avenue, Madison, Wis. 53705). Such software matches identical or similar sequences by assigning degrees of homology to various substitutions, deletions, and / or other modifications. Conservative substitutions typically include substitutions within the following groups: glycine, alanine; valine, isoleucine, leucine; aspartic acid, glutamic acid, asparagine, glutamine; serine, threonine; lysine, arginine; phenylalanine, tyrosine. In an exemplary approach to determining the degree of identity, the BLAST program can be used, e.g., -3 and e -100 A probability score between indicates closely related sequences. COBALT can be used, for example, with the following parameters: a) Alignment parameters: Gap penalties -11, -1 and End-Gap penalties -5, -1 b) CDD parameters: Use RPS BLAST on; Blast E-value 0.003; Find Conserved columns and Recompute on c) Query clustering parameters: Use query clusters on; Word Size 4; Max cluster distance 0.8; Alphabet Regular. The EMBOSS Needle is used, for example, with the following parameters: a) Matrix: BLOSUM62; b) GAP OPEN: 10; c) GAP EXTEND: 0.5; d) OUTPUT FORMAT: pair; e) END GAP PENALTY: false; f) END GAP OPEN: 10; and g) END GAP EXTEND: 0.5.

[0201] The term "target site" refers to a sequence within a nucleic acid molecule that is modified by a nucleobase editor. In one embodiment, the target site is deaminated by a deaminase or a fusion protein comprising a deaminase (e.g., a cytidine or adenine deaminase). "Tet methylcytosine dioxygenase 2 (TET2) polypeptide" refers to a protein having at least about 85% amino acid sequence identity to NCBI Accession No. FM992369.1, or a fragment thereof, and having catalytic activity to convert methylcytosine to 5-hydroxymethylcytosine. Genetic defects are associated with myeloproliferative disorders, and the enzyme's ability to methylate cytosine contributes to transcriptional regulation. An exemplary TET2 amino acid sequence is shown below.

[0202] >CAX30492.1 tet oncogene family member 2[Homo sapiens]

[0203] "Tet methylcytosine dioxygenase 2 (TET2) polynucleotide" refers to a nucleic acid molecule encoding a TET2 polypeptide. A TET polypeptide encodes a methylcytosine dioxygenase and has transcriptional regulatory activity. Exemplary TET2 nucleic acids are provided below. >FM992369.1 Homo sapiens mRNA for tet oncogene family member 2(TET2 gene)

[0204] By "transforming growth factor receptor 2 (TGFBRII) polypeptide" is meant a protein having at least about 85% sequence identity to NCBI Accession No. ABG65632.1, or a fragment thereof, and having immunosuppressive activity. An exemplary amino acid sequence is shown below. >ABG65632.1 transforming growth factor beta receptor II[Homo sapiens] MGRGLLRGLWPLHIVLWTRIASTIPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSD ECNDNIIFSEEYNTSNPDLLLVIFQVTGISLLPPLGVAISVIIIFYCYRVNRQQKLSSTWETGKTRKLMEFSEHCAIILEDDRSDISSTCANNINHNTELLPIELDTLVGKGRFAEVYKAKLKQNTSEQFETVAVKIFPYEE YASWKTEKDIFSDINLKHENILQFLTAEERKTELGKQYWLITAFHAKGNLQEYLTRHVISWEDLRKLGSSLARGIAHLHSDHTPCGRPKMPIVHRDLKSSNILVKNDLTCCLCDFGLSLRLDPTLSVDDLANSGQVGTARYM APEVLESRMNLENVESFKQTDVYSMALVLWEMTSRCNAVGEVKDYEPPFGSKVREHPCVESMKDNVLRDRGRPEIPSFWLNHQGIQMVCETLTECWDHDPEARLTAQCVAERFSELEHLDRLSGRSCSEEKIPEDGSLNTTK

[0205] "Transforming growth factor receptor 2 (TGFBRII) polynucleotide" refers to a nucleic acid encoding a TGFBRII polypeptide. The TGFBRII gene encodes a transmembrane protein with serine / threonine kinase activity. Exemplary TGFBRII nucleic acids are shown below. >M85079.1 Human TGF-beta type II receptor mRNA, complete CDs

[0206] By "T cell immunoreceptor (TIGIT) polypeptide with Ig and ITIM domains" is meant a protein having at least about 85% sequence identity to NCBI Accession No. ACD74757.1, or a fragment thereof, and having immunomodulatory activity. An exemplary TIGIT amino acid sequence is shown below. >ACD74757.1 T Cell immunoreceptor with Ig and ITIM domains[Homo sapiens] MRWCLLLIWAQGLRQAPLASGMMTGTIETTGNISAEKGGSIILQCHLSSTTAQVTQVNWEQQDQLLAICNADLGWHISPSFKDRVAPGPGLGLTLQSLTVNDTGEYFCIYHTYPDGTYTGRI FLEVLESSVAEHGARFQIPLLGAMAATLVVICTAVIVVVALTRKKKALRIHSVEGDLRRKSAGQEEWSPSAPSPPGSCVQAEAAPAGLCGEQRGEDCAELHDYFNVLSYRSLGNCSFFTETG

[0207] "T cell immunoreceptor with Ig and ITIM domains (TIGIT) polynucleotide" refers to a nucleic acid encoding a TIGIT polypeptide. The TIGIT gene encodes an inhibitory immunoreceptor associated with neoplasia and T cell depletion. Exemplary nucleic acid sequences are shown below. >EU675310.1 Homo sapiens T Cell immunoreceptor with Ig and ITIM domains(TIGIT)mRNA,complete cds CGTCCTATCTGCAGTCGGCTACTTTCAGTGGCAGAAGAGGCCACATCTGCTTCCTGTAGGCCCTCTGGGCAGAAGCATGCGCTGGTGTCTCCTCCTGATCTGGGCCCAGGGGCTGAGGCAGGCTCCCCTCGCCTCAGGAATGATGACAGGCACAATAGAAACAACGGGGAACATTTCTGCAGAGAAAGGTGGCTCTATCATCTTACA ATGTCACCTCTCCTCCACCACGGCACAAGTGACCCAGGTCAACTGGGAGCAGCAGGACCAGCTTCTGGCCATTTGTAATGCTGACTTGGGTGGCACATCTCCCCATCCTTCAAGGATCGAGTGGCCCCAGGTCCCGGCCTGGGCCTCACCCTCCAGTCGCTGACCGTGAACGATACAGGGGAGTACTTCTGCATCTATCACACCTAC CCTGATGGGACGTACACTGGGAGAATCTTCCTGGAGGTCCTAGAAAGCTCAGTGGCTGAGCACGGTGCCAGGTTCCAGATTCCATTGCTTGGAGCCATGGCCGCGACGCTGGTGGTCATCTGCACAGCAGTCATCGTGGTGGTCGCGTTGACTAGAAAGAAGAAAGCCCTCAGAATCCATTCTGTGGAAGGTGACCTCAGGAGAAAAT CAGCTGGACAGGAGGAATGGAGCCCCAGTGCTCCCTCACCCCAGGAAGCTTGTGTCCAGGCAGAAGCTGCACCTGCTGGGCTCTGTGGAGAGCAGCGGGGAGAGGACTGTGCCGAGCTGCATGACTACTTCAATGTCCTGAGTTACAGAAGCCTGGGTAACTGCAGCTTCTTCACAGAGACTGGTTAGCAACCAGAGGCATCTTCTGG

[0208] By "T-cell receptor alpha constant (TRAC) polypeptide" is meant a protein having at least about 85% amino acid sequence identity to NCBI Accession No. P01848.2, or a fragment thereof, and having immunomodulatory activity. An exemplary amino acid sequence is shown below. >sp|P01848.2|TRAC_HUMAN RecName:Full=T Cell receptor alpha constant IQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKTVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRILLLKVAGFNLLMTLRLWSS

[0209] "T-cell receptor alpha constant (TRAC) polynucleotide" means a nucleic acid that encodes a TRAC polypeptide. An exemplary TRAC nucleic acid sequence is shown below. >X02592.1 Human mRNA for T-Cell receptor alpha chain(TCR-alpha)

[0210] As used herein, "transduction" means the transfer of a gene or genetic material into a cell via a viral vector.

[0211] As used herein, "transformation" refers to the process of introducing a genetic alteration into a cell produced by the introduction of an exogenous nucleic acid.

[0212] "Transfection" refers to the transfer of a gene or genetic material into a cell via chemical or physical means.

[0213] By "translocation" is meant a rearrangement of nucleic acid segments between non-homologous chromosomes.

[0214] As used herein, the terms "treat," "treating," "treatment," and the like refer to alleviating or ameliorating a disorder and / or its associated symptoms, or achieving a desired pharmacological and / or physiological effect. It will be understood that treating a disorder or condition does not require (nor does complete elimination preclude) the complete elimination of the associated disorder, condition, or symptoms. In some embodiments, the effect is therapeutic, i.e., without limitation, the effect partially or completely reduces, diminishes, eliminates, alleviates, alleviates, reduces the intensity of, or cures, the disease and / or adverse symptoms resulting therefrom. In certain embodiments, the effect is prophylactic, i.e., the effect protects against or prevents the occurrence or recurrence of the disease or condition. To this end, the methods of the present disclosure comprise administering a therapeutically effective amount of a composition as described herein.

[0215] "Uracil glycosylase inhibitor," or alternatively "UGI," refers to an agent that inhibits the uracil excision repair system. In one embodiment, the agent is a protein or fragment thereof that binds to host uracil-DNA glycosylase and prevents the removal of uracil residues from DNA. In one embodiment, UGI is a protein, fragment, or domain thereof that can inhibit the uracil-DNA glycosylase base excision repair enzyme. In some embodiments, the UGI domain comprises wild-type UGI or a modified version thereof. In some embodiments, the UGI domain comprises a fragment of an exemplary amino acid sequence provided below. In some embodiments, the UGI fragment comprises an amino acid sequence that comprises at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% of the exemplary UGI sequence provided below. In some embodiments, the UGI comprises an amino acid sequence homologous to an exemplary UGI amino acid sequence or fragment thereof, as described below. In some embodiments, UGI or a portion thereof has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.9%, or 100% identity to wild-type UGI or a UGI sequence or a portion thereof, as described below. Exemplary UGIs include the following amino acid sequences: >splP14739IUNGI_BPPB2 Uracil-DNA glycosylase inhibitor MTNLSDIIEKETGKQLVIQESILMLPEEVEEVIGNKPESDILVHTAYDESTDENVMLLTSD APEYKPWALVIQDSNGENKIKML.

[0216] The term "vector" refers to a means for introducing a nucleic acid sequence into a cell, resulting in a transformed cell. Vectors include plasmids, transposons, phages, viruses, liposomes, and episomes. An "expression vector" is a nucleic acid sequence that contains a nucleotide sequence that is expressed in a recipient cell. Expression vectors may contain additional nucleic acid sequences to promote and / or facilitate expression of the introduced sequence, such as initiation, termination, enhancer, promoter, and secretion sequences.

[0217] By "zeta chain of T-cell receptor-associated protein kinase 70 (ZAP70) polypeptide" is meant a protein having at least about 85% amino acid sequence identity to NCBI Accession No. AAH53878.1 and having kinase activity. An exemplary amino acid sequence is shown below. >AAH53878.1 Zeta-chain(TCR) associated protein kinase 70kDa[Homo sapiens] MPDPAAHLPFFYGSISRAEAEEHLKLAGMADGLFLLRQCLRSLGYVLSLVHDVRFHHFPIERQLNGTYAIAGGKAHCGPAELCEFYSRDPDGLPCNLRKPCNRPSGLEPQPGVFDCLRDAMVRDYVRQTWKLEGEALEQAIISQAPQVEKLIA TTAHERMPWYHSSLTREEAERKLYSGAQTDGKFLLRPRKEQGTYALSLIYGKTVYHYLISQDKAGKYCIPEGTKFDTLWQLVEYLKLKADGLIYCLKEACPNSSASNASGAAAPTLPAHPSTLTHPQRRIDTLNSDGYTPEPARITSPDKPRPMP MDTSVYESPYSDPEELKDKKLFLKRDNLLIADIELGCGNFGSVRQGVYRMRKKQIDVAIKVLKQGTEKADTEEMMREAQIMHQLDNPYIVRLIGVCQAEALMLVMEMAGGGPLHKFLVGKREEIPVSNVAELLHQVSMGMKYLEEKNFVHRDLAA RNVLLVNRHYAKISDFGLSKALGADDSYYTARSAGKWPLKWYAPECINFRKFSSRSDVWSYGVTMWEALSYGQKPYKKMKGPEVMAFIEQGKRMECPECPPELYALMSDCWIYKWEDRPDFLTVEQRMRACYYSLASKVEGPPGSTQKAEAACA

[0218] "Zeta chain of T cell receptor-associated protein kinase 70 (ZAP70) polynucleotide" refers to a nucleic acid encoding a ZAP70 polypeptide. The ZAP70 gene encodes a tyrosine kinase involved in T cell development and lymphocyte activation. Lack of functional ZAP10 can lead to severe combined immunodeficiency, characterized by a lack of CD8+ T cells. An exemplary ZAP70 nucleic acid sequence is shown below. >BC053878.1 Homo sapiens zeta-chain(TCR) associated protein kinase 70kDa,mRNA(cDNA clone MGC:61743 IMAGE:5757161),complete cds

[0219] Any composition or method provided herein may be combined with one or more of any other compositions and methods provided herein.

[0220] DNA editing has emerged as a viable means to modify disease states by correcting pathogenic mutations at the gene level. Until recently, all DNA editing platforms functioned by inducing DNA double-strand breaks (DSBs) at specific genomic sites and relying on endogenous DNA repair pathways to semi-stochastically determine the resulting product, resulting in a complex population of gene products. Accurate, user-defined repair outcomes can be achieved via the homology-directed repair (HDR) pathway; however, numerous challenges have prevented highly efficient repair using HDR in therapeutically relevant cell types. In practice, this pathway is less efficient than the competing, error-prone non-homologous end-joining pathway. Furthermore, HDR is strictly restricted to the G1 and S phases of the cell cycle, preventing accurate repair of DSBs in post-mitotic cells. As a result, it has proven difficult or impossible to modify genome sequences in a user-defined, programmable manner with high efficiency in these populations. [Brief explanation of the drawings]

[0221] [Figure 1] Figures 1A and 1B are diagrams of three proteins that influence T cell function. Figure 1A is a diagram of the TRAC protein, a key component of graft-versus-host disease. Figure 1B is a diagram of the B2M protein, a component of the MHC class 1 antigen-presenting complex present on nucleated cells that can be recognized by host CD8+ T cells. Figure 1C is a diagram of T cell signaling that leads to expression of the PDCD1 gene, and the resulting PD-1 protein acts to inhibit T cell signaling. [Figure 2]Figures 2A-2D show A·T to G·C conversions and phenotypic consequences in primary cells. Figure 2A is a diagram showing the reduction in protein expression measured by flow cytometry after primary human T cells were electroporated with 41 individual sgRNAs targeting the indicated mRNAs and six genes. Individual values ​​shown represent the average percentage of cells with reduced protein expression from two replicates of cells edited with the indicated mRNA and one of the 41 sgRNAs tested. Figure 2B is a heatmap showing NGS analysis of A·T to G·C conversions at six target sites by eight ABE8 mRNAs and ABE7.10-m / d. Values ​​shown reflect the average of three independent biological replicates. The position of the edited nucleotide at each target site is indicated above the heatmap. Figure 2C is a graph showing NGS analysis of A·T to G·C conversions in multiply edited T cells at site 21 (B2M), site 25 (TRAC), and site 24 (CIITA) after electroporation of primary human T cells with the indicated mRNAs and three sgRNAs in a multiply edited format. Figure 2D (top panel) is a graph of protein expression of B2M, CIITA, and TRAC proteins measured by flow cytometry for the cell population in Figure 2C 5 days after electroporation. Values ​​shown are from a representative donor. Figure 2D (bottom panel) is a table showing the percentage of cellular expression measured by flow cytometry after editing with the indicated ABEs. [Figure 3] Figure 3 is a heatmap showing protein knockdown measured by flow cytometry with the ABE editor in primary T cells. Eight mRNAs encoding the ABE8 editor and two mRNAs encoding ABE7.10-m / d were individually transfected into T cells along with 41 sgRNAs targeting six genes, and their effect on protein expression was measured using flow cytometry. Values ​​shown are the average of n=2 independent replicates. [Figure 4]Figure 4 is a graph showing ABE-edited CAR-T cells with potent cytotoxic activity in response to antigen-positive tumor cells. Fluorescently tagged RPMI-8226 cells were seeded at time = 0 h, and their proliferation was monitored using an IncuCyte live cell imaging system for 28 h before introducing CAR-T cells. Multiply edited T cells using the indicated ABE (Figure 1C) were transduced with lentivirus encoding an anti-BCMA CAR molecule and introduced into RPMI-8226 cells at time = 28 h. RPMI-8226 cell proliferation was monitored for an additional 68 h. Values ​​shown are the average of n = 3 independent biological replicates. [Figure 5] Figures 5A and 5B show RNA amplicon sequencing to detect cellular A to I editing in RNA associated with ABE treatment. Individual data points are shown, and error bars represent the standard deviation (s.d.) for n=3 independent biological replicates performed on different days. Figure 5A is a graph showing the frequency of A to I editing in target RNA amplicons of the core ABE8 construct compared to ABE7 and Cas9(D10A) nickase controls. Figure 5B is a graph showing the frequency of A to I editing in target RNA amplicons of ABE8 with mutations reported to improve RNA off-target editing. [Figure 6] Figures 6A and 6B are graphs showing examples of gates used to assess protein knockdown in T cells. A representative gating strategy for population analysis of live single lymphocytes to determine surface protein reduction via flow cytometry. [Figure 7] 1 is a graph showing alleles generated by ABE across eight different genomic sites in HEK293T cells. [Figure 8]Figures 8A and 8B show whole-transcriptome and whole-genome sequencing data from cells treated with base editor mRNAs. Figure 8A is a strip plot showing whole-transcriptome sequencing in HEK293T cells treated with the indicated mRNAs. The variant allele frequencies of transcriptome-wide A-to-G mutations in the RNA were observed in replicate HEK293T cell experiments. The total A-to-G mutations are shown above each sample. Figure 8B is a strip plot showing whole-transcriptome sequencing in T cells treated with the indicated mRNAs. The variant allele frequencies of transcriptome-wide A-to-G mutations in the RNA were observed in three different T cell donors. The total A-to-G mutations are shown above each sample. [Figure 9] Figures 9A and 9B show representative examples of gates used to flow-sort B2M-positive and B2M-negative cells prior to whole genome sequencing. Figure 9A shows a representative plot and gate of live B2M-positive HEK293T cells sorted into single-cell clones for the untreated condition. Figure 9B shows a representative plot and gate of live B2M-negative HEK293T cells sorted for all treated conditions (cells treated with ABE, CBE, or Cas9). [Figure 10] This table shows Cas9 variants that access all possible PAMs within the NRNN PAM space. Only Cas9 variants that require recognition of three or fewer defined nucleotides at the PAM are listed. Non-GPAM variants include SpCas9-NRRH, SpCas9-NRTH, and SpCas9-NRCH. DETAILED DESCRIPTION OF THE INVENTION

[0222] The present invention features genetically modified immune cells containing novel adenosine base editors (e.g., ABE8) that have enhanced anti-neoplastic activity, resistance to immunosuppression, and a reduced risk of eliciting a graft-versus-host response or a host-versus-graft response, or a combination thereof. The present invention also features methods for producing and using these modified immune effector cells (e.g., immune effector cells such as T cells). The present invention also features methods for treating a subject with or prone to developing a neoplasm, graft-versus-host disease (GVHD), or host-versus-graft disease (HVGD) using an effective amount of the modified immune effector cells (e.g., CAR-T cells).

[0223] Modification of immune effector cells to express chimeric antigen receptors (CARs) and knock out or knock down specific genes to mitigate the adverse effects their expression may have on immune cell function is achieved using the adenosine deaminase-containing base editor system described herein.

[0224] Autologous, patient-derived chimeric antigen receptor T-cell (CAR-T) therapies have demonstrated remarkable efficacy in the treatment of several hematologic cancers. While these products have provided significant clinical benefit to patients, the need to create individualized therapies creates significant manufacturing challenges and economic burdens. Allogeneic CAR-T therapies were developed as a potential solution to these challenges, treating many patients with cells derived from a single healthy donor while possessing a clinical efficacy profile similar to that of autologous products, thereby substantially reducing the cost of goods and lot-to-lot variability.

[0225] Most first-generation allogeneic CAR-Ts use nucleases to introduce two or more targeted genomic DNA double-strand breaks (DSBs) into a target T cell population and rely on error-prone DNA repair to generate mutations that knock out the target gene in a semi-stochastic manner. Such nuclease-based gene knockout strategies aim to reduce the risk of graft-versus-host disease (GVHD) and host rejection in CAR-Ts. However, the simultaneous induction of multiple DSBs results in a final cell product containing large-scale genomic rearrangements, such as balanced and unbalanced translocations, as well as relatively large amounts of local rearrangements, such as inversions and large deletions. Furthermore, as the number of simultaneously induced DSBs increases, considerable genotoxicity is observed in the treated cell population. This can significantly reduce the potential for cell expansion from each manufacturing trial, thereby reducing the number of treatable patients per healthy donor.

[0226] Base editors (BEs) are an emerging class of gene editing reagents that enable highly efficient, user-defined modifications of target genomic DNA without creating DSBs. Here, we propose an alternative approach to generating allogeneic CAR-T cells by using base editing technology to reduce or eliminate detectable genomic rearrangements while simultaneously improving cell proliferation. As shown herein, in contrast to nuclease-only editing strategies, simultaneous modification of three gene loci by base editing generates highly efficient gene knockouts without detectable translocation events. In one embodiment, a base editor (e.g., ABE8) is used for multiplexed base editing of at least one cell surface target in T cells (e.g., including, but not limited to, TRAC, B2M, CD7, PDCD1, CBLB, and / or CIITA). In one embodiment, ABE8 is used for multiplexed base editing of TRAC, B2M, and CIITA in T cells. Multiplexed gene editing may be useful for creating CAR-T cell therapies with improved therapeutic properties. This method addresses known limitations of multiply edited T cell products and is a promising development toward the next generation of precision cell-based therapies.

[0227] [Chimeric antigen receptor and CAR-T cells] The present invention provides immune cells modified using the nucleobase editors described herein to express chimeric antigen receptors (CARs). Modification of immune cells to express chimeric antigen receptors can enhance the immune cell's immune response activity, where the chimeric antigen receptor has affinity for an epitope on an antigen, and the antigen is associated with altered organismal fitness. For example, the chimeric antigen receptor can have affinity for an epitope on a protein expressed on a neoplastic cell. Because CAR-T cells can act independently of major histocompatibility complex (MHC), activated CAR-T cells can kill neoplastic cells expressing the antigen. The direct action of CAR-T cells circumvents neoplastic cell defense mechanisms that evolved in response to MHC presentation of the antigen to immune cells.

[0228] In some embodiments, the present invention provides immune effector cells expressing chimeric antigen receptors that target B cells involved in autoimmune responses (e.g., B cells of a subject that express antibodies directed against the subject's own tissues).

[0229] Some embodiments include autologous immune cell immunotherapy, in which immune cells are obtained from a subject with a disease or altered fitness characterized by cancerous or otherwise altered cells expressing a surface marker. The obtained immune cells are genetically modified to express a chimeric antigen receptor, effectively redirecting them against a specific antigen. Thus, in some embodiments, immune cells are obtained from a subject in need of CAR-T immunotherapy. In some embodiments, these autologous immune cells are cultured and modified immediately after they are obtained from the subject. In other embodiments, autologous cells are obtained and then stored for future use. This practice may be recommended for individuals who may be undergoing concurrent treatments that will reduce immune cell numbers in the future. In allogeneic immune cell immunotherapy, immune cells may be obtained from a donor other than the subject receiving treatment. The immune cells are modified to express a chimeric antigen receptor and then administered to a subject to treat a neoplasm. In some embodiments, the immune cells modified to express a chimeric antigen receptor may be obtained from an existing stock culture of immune cells.

[0230] Immune cells and / or immune effector cells may be isolated or purified from samples collected from subjects or donors using standard techniques known in the art. For example, immune effector cells can be isolated or purified from whole blood samples by lysing red blood cells and removing peripheral blood mononuclear cells by centrifugation. Immune effector cells can be further isolated or purified using selective purification methods that isolate immune effector cells based on cell-specific markers such as CD25, CD3, CD4, CD8, CD28, CD45RA, or CD45RO. In one embodiment, CD25+ is used as a marker to select regulatory T cells. In another embodiment, the present invention provides T cells with a targeted gene knockout in the TCR constant region (TRAC) involved in TCRαβ surface expression. TCR alpha beta-deficient CART cells are compatible with allogeneic immunotherapy (Qasim et al., Sci. Transl. Med. 9, eaaj2013(2017); Valton et al., Mol Ther. 2015 Sep;23(9):1507-1518). If necessary, residual TCR alpha beta T cells are removed using CliniMACS magnetic bead depletion to minimize the risk of GVHD. In another embodiment, the present invention provides donor T cells selected ex vivo to recognize minor histocompatibility antigens expressed on recipient hematopoietic cells, thereby minimizing the risk of graft-versus-host disease (GVHD), a major cause of post-transplant morbidity and mortality (Warren et al., Blood 2010;115(19):3869-3878). Another technique for isolating or purifying immune effector cells is flow cytometry. In fluorescence-activated cell sorting, immune effector cells in a sample are labeled using fluorescently labeled antibodies with affinity to immune effector cell markers. Cells are then separated using a gating strategy appropriate for cells expressing the marker. For example, T lymphocytes can be separated from other cells in a sample by using fluorescently labeled antibodies specific to immune effector cell markers (e.g., CD4, CD8, CD28, CD45) and the corresponding gating strategy.In one embodiment, a CD45 gating strategy is employed. In some embodiments, gating strategies for other markers specific to immune effector cells are used instead of or in combination with the CD45 gating strategy.

[0231] Immune effector cells contemplated by the present invention are effector T cells. In some embodiments, effector T cells are naive CD8 + In some embodiments, the effector T cells are thymocytes, immature T lymphocytes, mature T lymphocytes, resting T lymphocytes, or activated T lymphocytes. In some embodiments, the immune effector cells are CD4 + CD8 + T cells or CD4 - CD8 - In some embodiments, the immune effector cells are T cells. In some embodiments, the T helper cells are T helper cells. In some embodiments, the T helper cells are T helper 1 (Th1), T helper 2 (Th2) cells, or CD4-expressing helper T cells (CD4+ T cells). In some embodiments, the immune effector cells are any other subset of T cells. In addition to the chimeric antigen receptor, the engineered immune effector cells may express exogenous cytokines, different chimeric receptors, or any other agents that enhance the signaling or function of immune effector cells. For example, coexpression of a chimeric antigen receptor and a cytokine may enhance the ability of CAR-T cells to lyse target cells.

[0232] The chimeric antigen receptors contemplated by the present invention comprise an extracellular binding domain, a transmembrane domain, and an intracellular domain. Binding of an antigen to the extracellular binding domain can activate CAR-T cells and generate an effector response, including CAR-T cell proliferation, cytokine production, and other processes leading to the death of antigen-expressing cells. In some embodiments of the present invention, the chimeric antigen receptor further comprises a linker.

[0233] The extracellular binding domain of the chimeric antigen receptor contemplated herein comprises the amino acid sequence of an antibody or its antigen-binding fragment that has affinity for a specific antigen. In various embodiments, the CAR specifically binds to 5T4. Exemplary anti-5T4 CARs include, but are not limited to, CART-5T4 (Oxford BioMedica plc) and UCART-5T4 (Cellectis SA).

[0234] In various embodiments, the CAR specifically binds to alphafetoprotein. Exemplary anti-alphafetoprotein CARs include, but are not limited to, ET-1402 (Eureka Therapeutics Inc). In various embodiments, the CAR specifically binds to Axl. Exemplary anti-Axl CARs include, but are not limited to, CCT-301-38 (F1 Oncology Inc). In various embodiments, the CAR specifically binds to B7H6. Exemplary anti-B7H6 CARs include, but are not limited to, CYAD-04 (Celyad SA).

[0235] In various embodiments, the CAR specifically binds to BCMA. Exemplary anti-BCMA CARs include, but are not limited to, ACTR-087 + SEA-BCMA (Seattle Genetics Inc), ALLO-715 (Cellectis SA), ARI-0002 (Institut d'Investigacions Biomediques August Pi I Sunyer), bb-2121 (bluebird bio Inc), bb-21217 (birdblue bio Inc), CART-BCMA(University of Pennsylvania), CT-053(Carsgen Therapeutics Ltd), Descartes-08(Cartesian Therapeutics), FCARH-143(Juno Therapeutics Inc), ICTCAR-032(Innovative Cellular Therapeutics Co Ltd), IM21 CART(Beijing Immunochina Medical Science & Technology Co Ltd), JCARH-125(Memorial Sloan-Kettering Cancer Center), KITE-585 (Kite Pharma Inc), LCAR-B38M (Nanjing Legend Biotech Co Ltd), LCAR-B4822M (Nanjing Legend Biotech Co Ltd), MCARH-171 (Memorial Sloan-Kettering Cancer Center), P-BCMA-101 (Poseida Therapeutics Inc), P-BCMA-ALLO1 (Poseida Therapeutics Inc), spCART-269 (Shanghai Unicar-Therapy Bio-medicine Technology Co Ltd), and BCMA02 / bb2121 (bluebird bio Inc). The polypeptide sequence of BCMA02 / bb2121 CAR is shown below: MALPVTALLLPLALLLHAARPDIVLTQSPPSLAMSLGKRATISCRASESVTILGSHLIHW YQQKPGQPPTLLIQLASNVQTGVPARFSGSGSRTDFTLTIDPVEEDDVAVYYCLQSRTIP RTFGGTKLEIKGSTSGSGKPGSGEGSTKGQIQLVQSGPELKKPGETVKISCKASGYTFT DYSINWVKRAPGKGLKWMGWINTETREPAYAYDFRGRFAFSLETSASTAYLQINNLKYED TATYFCALDYSYAMDYWGQGTSVTVSSAAATTTPAPRPPTPAPTIASQPLSLRPEACRPA AGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQT TQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRR GRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKD TYDALHMQALPPR

[0236] In various embodiments, the CAR specifically binds to CCK2R. Exemplary anti-CCK2R CARs include, but are not limited to, anti-CCK2R CAR-T adapter molecule (CAM) + anti-FITC CAR T cell therapy (cancer), Endocyte / Purdue (Purdue University).

[0237] In various embodiments, the CAR specifically binds to a CD antigen. Exemplary anti-CD antigen CARs include, but are not limited to, VM-802 (ViroMed Co Ltd). In various embodiments, the CAR specifically binds to CD123. Exemplary anti-CD123 CARs include, but are not limited to, MB-102 (Fortress Biotech Inc), RNA CART123 (University of Pennsylvania), SFG-iMC-CD123.zeta (Bellicum Pharmaceuticals Inc), and UCART-123 (Cellectis SA). In various embodiments, the CAR specifically binds to CD133. Exemplary anti-CD133 CARs include, but are not limited to, KD-030 (Nanjing Kaedi Biotech Inc). In various embodiments, the CAR specifically binds to CD138. Exemplary anti-CD138 CARs include, but are not limited to, ATLCAR.CD138 (UNC Lineberger Comprehensive Cancer Center) and CART-138 (Chinese PLA General Hospital). In various embodiments, the CAR specifically binds to CD171. Exemplary anti-CD171 CARs include, but are not limited to, JCAR-023 (Juno Therapeutics Inc). In various embodiments, the CAR specifically binds to CD19. Exemplary anti-CD19 CARs include, but are not limited to, 1928z-41BBL (Memorial Sloan-Kettering Cancer Center), 1928z-E27 (Memorial Sloan-Kettering Cancer Center), 19-28z-T2 (Guangzhou Institutes of Biomedicine and Health), 4G7-CARD (University College London), 4SCAR19 (Shenzhen Geno-Immune Medical Institute), ALLO-501 (PfizerInc)、ATA-190(QIMR Berghofer Medical Research Institute)、AUTO-1(University College London)、AVA-008(Avacta Ltd)、アキシカブタゲンシロルユーセル(axicabtagene ciloleucel)(Kite Pharma Inc)、BG-T19(Guangzhou Bio-gene Technology Co Ltd)、BinD-19(Shenzhen BinDeBio Ltd.)、BPX-401(Bellicum Pharmaceuticals Inc)、CAR19h28TM41BBz(Westmead Institute for Medical Research)、C-CAR-011(Chinese PLA General Hospital)、CD19CART(Innovative Cellular Therapeutics Co Ltd)、CIK-CAR.CD19(Formula Pharmaceuticals Inc)、CLIC-1901(Ottawa Hospital Research Institute)、CSG-CD19(Carsgen Therapeutics Ltd)、CTL-119(University of Pennsylvania)、CTX-101(CRISPR Therapeutics AG)、DSCAR-01(Shanghai Hrain Biotechnology)、ET-190(Eureka Therapeutics Inc)、FT-819(Memorial Sloan-Kettering Cancer Center)、ICAR-19(Immune Cell Therapy Inc)、IM19 CAR-T(Beijing Immunochina Medical Science & Technology Co Ltd)、JCAR-014(Juno Therapeutics Inc)、JWCAR-029(MingJu Therapeutics(Shanghai) Co., Ltd)、KD-C-19(Nanjing Kaedi Biotech Inc)、LinCART19(iCell GeneTherapeutics), lisocabtagene maraleucel (Juno Therapeutics Inc), MatchCART (Shanghai Harain Biotechnology), MB-CART19.1 (Shanghai Children's Medical Center), PBCAR-0191 (Precision BioSciences Inc), PCAR-019 (PersonGen Biomedicine (Suzhou) Co Ltd), pCAR-19B (Chongqing Precision Biotech Co Ltd), PZ-01 (Pinze Lifetechnology Co Ltd), RB-1916 (Refuge Biotechnologies Inc), SKLB-083019 (Chengdu Yinhe Biomedical Co Ltd), spCART-19 (Shanghai Unicar-Therapy Bio-medicine Technology Co Ltd), TBI-1501 (Takara Bio Inc), TC-110(TCR2 Therapeutics Inc), TI-1007 (Timmune Biotech Inc), tisagenlecleucel (Abramson Cancer Center of the University of Pennsylvania), U-CART (Shanghai Bioray Laboratory Inc), UCART-19 (Wugen Inc), UCART-19 (Cellectis SA), vadacabtagene leraleucel (Memorial Sloan-Kettering Cancer Center), XLCART-001 (Nanjing Medical University), and yinnuokati-19 (Shenzhen Innovation Immunotechnology Co Ltd). In various embodiments, the CAR specifically binds to CD2. Exemplary anti-CD2 CARs include, but are not limited to, UCART-2 (WugenIn various embodiments, the CAR specifically binds to CD20. Exemplary anti-CD20 CARs include, but are not limited to, ACTR-087 (National University of Singapore), ACTR-707 (Unum Therapeutics Inc), CBM-C20.1 (Chinese PLA General Hospital), MB-106 (Fred Hutchinson Cancer Research Center), and MB-CART20.1 (Miltenyi Biotec GmbH).

[0238] In various embodiments, the CAR specifically binds to CD22. Exemplary anti-CD22 CARs include, but are not limited to, anti-CD22 CAR T-cell therapy (B-cell acute lymphoblastic leukemia), University of Pennsylvania, CD22-CART (Shanghai Unicar-Therapy Bio-medicine Technology Co Ltd), JCAR-018 (Opus Bio Inc), MendCART (Shanghai Hrain Biotechnology), and UCART-22 (Cellectis SA). In various embodiments, the CAR specifically binds to CD30. Exemplary anti-CD30 CARs include, but are not limited to, ATLCAR.CD30 (UNC Lineberger Comprehensive Cancer Center), CBM-C30.1 (Chinese PLA General Hospital), and Hu30-CD28zeta (National Cancer Institute). In various embodiments, the CAR specifically binds to CD33. Exemplary anti-CD33 CARs include, but are not limited to, anti-CD33 CAR gamma delta T cell therapy (acute myeloid leukemia), TC BioPharm / University College London (University College London), CAR33VH (Opus Bio Inc), CART-33 (Chinese PLA General Hospital), CIK-CAR.CD33 (Formula Pharmaceuticals Inc), UCART-33 (Cellectis SA), and VOR-33 (Columbia University).

[0239] In various embodiments, the CAR specifically binds to CD38. Exemplary anti-CD38 CARs include, but are not limited to, UCART-38 (Cellectis SA). In various embodiments, the CAR specifically binds to CD38 A2. Exemplary anti-CD38 A2 CARs include, but are not limited to, T-007 (TNK Therapeutics Inc). In various embodiments, the CAR specifically binds to CD4. Exemplary anti-CD4 CARs include, but are not limited to, CD4CAR (iCell Gene Therapeutics). In various embodiments, the CAR specifically binds to CD44. Exemplary anti-CD44 CARs include, but are not limited to, CAR-CD44v6 (Istituto Scientifico H San Raffaele). In various embodiments, the CAR specifically binds to CD5. Exemplary anti-CD5 CARs include, but are not limited to, CD5CAR (iCell Gene Therapeutics). In various embodiments, the CAR specifically binds to CD7. Exemplary anti-CD7 CARs include, but are not limited to, CAR-pNK (PersonGen Biomedicine (Suzhou) Co Ltd), and CD7.CAR / 28zeta CAR T cells (Baylor College of Medicine), UCART7 (Washington University in St Louis).

[0240] In various embodiments, the CAR specifically binds to CDH17. Exemplary anti-CDH17 CARs include, but are not limited to, ARB-001.T (Arbele Ltd). In various embodiments, the CAR specifically binds to CEA. Exemplary anti-CEA CARs include, but are not limited to, HORC-020 (HumOrigin Inc). In various embodiments, the CAR specifically binds to chimeric TGF-beta receptor (CTBR). Exemplary anti-chimeric TGF-beta receptor (CTBR) CARs include, but are not limited to, CAR-CTBR T cells (bluebird bio Inc). In various embodiments, the CAR specifically binds to claudin 18.2. Exemplary anti-claudin 18.2 CARs include, but are not limited to, CAR-CLD18 T cells (Carsgen Therapeutics Ltd) and KD-022 (Nanjing Kaedi Biotech Inc).

[0241] In various embodiments, the CAR specifically binds to CLL1. Exemplary anti-CLL1 CARs include, but are not limited to, KITE-796 (Kite Pharma Inc). In various embodiments, the CAR specifically binds to DLL3. Exemplary anti-DLL3 CARs include, but are not limited to, AMG-119 (Amgen Inc). In various embodiments, the CAR specifically binds to Dual BCMA / TACI (APRIL). Exemplary anti-Dual BCMA / TACI (APRIL) CARs include, but are not limited to, AUTO-2 (Autolus Therapeutics Limited). In various embodiments, the CAR specifically binds to Dual CD19 / CD22. Exemplary anti-Dual CD19 / CD22 CARs include, but are not limited to, AUTO-3 (Autolus Therapeutics Limited) and LCAR-L10D (Nanjing Legend Biotech Co Ltd). In various embodiments, the CAR specifically binds to CD19. In various embodiments, the CAR specifically binds to dual CLL1 / CD33. An exemplary anti-dual CLL1 / CD33 CAR includes, but is not limited to, ICG-136 (iCell Gene Therapeutics). In various embodiments, the CAR specifically binds to dual EpCAM / CD3. An exemplary anti-dual EpCAM / CD3 CAR includes, but is not limited to, IKT-701 (Icell Kealex Therapeutics). In various embodiments, the CAR specifically binds to dual ErbB / 4ab. An exemplary anti-dual ErbB / 4ab CAR includes, but is not limited to, LEU-001 (King's College London). In various embodiments, the CAR specifically binds to dual FAP / CD3.Exemplary anti-dual FAP / CD3 CARs include, but are not limited to, IKT-702 (Icell Kealex Therapeutics). In various embodiments, the CAR specifically binds to EBV. Exemplary anti-EBV CARs include, but are not limited to, TT-18 (Tessa Therapeutics Pte Ltd).

[0242] In various embodiments, the CAR specifically binds to EGFR. Exemplary anti-EGFR CARs include, but are not limited to, anti-EGFR CAR T-cell therapy (CBLB MegaTAL, cancer), bluebird bio (bluebird bio Inc), anti-EGFR CAR T-cell therapy expressing CTLA-4 checkpoint inhibitor + PD-1 checkpoint inhibitor mAbs (EGFR-positive advanced solid tumors), Shanghai Cell Therapy Research Institute (Shanghai Cell Therapy Research Institute), CSG-EGFR (Carsgen Therapeutics Ltd), and EGFR-IL12-CART (Pregene (Shenzhen) Biotechnology Co Ltd).

[0243] In various embodiments, the CAR specifically binds to EGFRvIII. Exemplary anti-EGFRvIII CARs include, but are not limited to, KD-035 (Nanjing Kaedi Biotech Inc) and UCART-EgfrVIII (Cellectis SA). In various embodiments, the CAR specifically binds to Flt3. Exemplary anti-Flt3 CARs include, but are not limited to, ALLO-819 (Pfizer Inc) and AMG-553 (Amgen Inc). In various embodiments, the CAR specifically binds to the folate receptor. Exemplary anti-folate receptor CARs include, but are not limited to, EC17 / CAR T (Endocyte Inc). In various embodiments, the CAR specifically binds to G250. Exemplary anti-G250 CARs include, but are not limited to, autologous T-lymphocyte cell therapy (G250-scFV-transduced, renal cell carcinoma), Erasmus Medical Center (Daniel den Hoed Cancer Center).

[0244] In various embodiments, the CAR specifically binds to GD2. Exemplary anti-GD2 CARs include, but are not limited to, 1RG-CART (University College London), 4SCAR-GD2 (Shenzhen Geno-Immune Medical Institute), C7R-GD2.CART cells (Baylor College of Medicine), CMD-501 (Baylor College of Medicine), CSG-GD2 (Carsgen Therapeutics Ltd), GD2-CART01 (Bambino Gesu Hospital and Research Institute), GINAKIT cells (Baylor College of Medicine), iC9-GD2-CAR-IL-15 T cells (UNC Lineberger Comprehensive Cancer Center), and IKT-703 (Icell Kealex Therapeutics). In various embodiments, the CAR specifically binds to GD2 and MUC1. Exemplary anti-GD2 / MUC1 CARs include, but are not limited to, PSMA CAR-T (University of Pennsylvania).

[0245] In various embodiments, the CAR specifically binds to GPC3. Exemplary anti-GPC3 CARs include, but are not limited to, ARB-002.T (Arbele Ltd), CSG-GPC3 (Carsgen Therapeutics Ltd), GLYCAR (Baylor College of Medicine), and TT-14 (Tessa Therapeutics Pte Ltd). In various embodiments, the CAR specifically binds to Her2. Exemplary anti-Her2 CARs include, but are not limited to, ACTR-087 + trastuzumab (Unum Therapeutics Inc), ACTR-707 + trastuzumab (Unum Therapeutics Inc), CIDeCAR (Bellicum Pharmaceuticals Inc), MB-103 (Mustang Bio Inc), RB-H21 (Refuge Biotechnologies Inc), and TT-16 (Baylor College of Medicine). In various embodiments, the CAR specifically binds to IL13R. Exemplary anti-IL13R CARs include, but are not limited to, MB-101 (City of Hope) and YYB-103 (YooYoung Pharmaceuticals Co Ltd). In various embodiments, the CAR specifically binds to integrin beta-7. Exemplary anti-integrin beta-7 CARs include, but are not limited to, MMG49 CAR T cell therapy (Osaka University). In various embodiments, the CAR specifically binds to LC antigen. Exemplary anti-LC antigen CARs include, but are not limited to, VM-803 (ViroMed Co Ltd) and VM-804 (ViroMed Co Ltd).

[0246] In various embodiments, the CAR specifically binds to mesothelin. Exemplary anti-mesothelin CARs include, but are not limited to, CARMA-hMeso (Johns Hopkins University), CSG-MESO (Carsgen Therapeutics Ltd), iCasp9M28z (Memorial Sloan-Kettering Cancer Center), KD-021 (Nanjing Kaedi Biotech Inc), m-28z-T2 (Guangzhou Institutes of Biomedicine and Health), MesoCART (University of Pennsylvania), meso-CAR-T+PD-78 (MirImmune LLC), RB-M1 (Refuge Biotechnologies Inc), and TC-210 (TCR2 Therapeutics Inc).

[0247] In various embodiments, the CAR specifically binds to MUC1. Exemplary anti-MUC1 CARs include, but are not limited to, anti-MUC1 CAR T cell therapy + PD-1 knockout T cell therapy (esophageal cancer / NSCLC), Guangzhou Anjie Biomedical Technology / University of Technology Sydney (Guangzhou Anjie Biomedical Technology Co LTD), ICTCAR-043 (Innovative Cellular Therapeutics Co Ltd), ICTCAR-046 (Innovative Cellular Therapeutics Co Ltd), P-MUC1C-101 (Poseida Therapeutics Inc), and TAB-28z (OncoTab Inc). In various embodiments, the CAR specifically binds to MUC16. Exemplary anti-MUC16 CARs include, but are not limited to, 4H1128Z-E27 (Eureka Therapeutics Inc) and JCAR-020 (Memorial Sloan-Kettering Cancer Center).

[0248] In various embodiments, the CAR specifically binds to nfP2X7. Exemplary anti-nfP2X7 CARs include, but are not limited to, BIL-022c (Biosceptre International Ltd). In various embodiments, the CAR specifically binds to PSCA. Exemplary anti-PSCA CARs include, but are not limited to, BPX-601 (Bellicum Pharmaceuticals Inc). In various embodiments, the CAR specifically binds to PSMA.CIK-CAR.PSMA (Formula Pharmaceuticals Inc), and P-PSMA-101 (Poseida Therapeutics Inc). In various embodiments, the CAR specifically binds to ROR1. Exemplary anti-ROR1 CARs include, but are not limited to, JCAR-024 (Fred Hutchinson Cancer Research Center). In various embodiments, the CAR specifically binds to ROR2. Exemplary anti-ROR2 CARs include, but are not limited to, CCT-301-59 (F1 Oncology Inc). In various embodiments, the CAR specifically binds to SLAMF7. Exemplary anti-SLAMF7 CARs include, but are not limited to, UCART-CS1 (Cellectis SA). In various embodiments, the CAR specifically binds to TRBC1. Exemplary anti-TRBC1 CARs include, but are not limited to, AUTO-4 (Autolus Therapeutics Limited). In various embodiments, the CAR specifically binds to TRBC2. Exemplary anti-TRBC2 CARs include, but are not limited to, AUTO-5 (Autolus Therapeutics Limited). In various embodiments, the CAR specifically binds to TSHR. Exemplary anti-TSHR CARs include, but are not limited to, ICTCAT-023 (Innovative Cellular Therapeutics Co Ltd).In various embodiments, the CAR specifically binds to VEGFR-1. Exemplary anti-VEGFR-1 CARs include, but are not limited to, SKLB-083017 (Sichuan University).

[0249] In various embodiments, the CAR is selected from the group consisting of AT-101 (AbClon Inc); AU-101, AU-105, and AU-180 (Aurora Biopharma Inc); CARMA-0508 (Carisma Therapeutics); CAR-T (Fate Therapeutics Inc); CAR-T (Cell Design Labs Inc); CM-CX1 (Celdara Medical LLC); CMD-502, CMD-503, and CMD-504 (Baylor College of Medicine); CSG-002 and CSG-005 (Carsgen Therapeutics Ltd); ET-1501, ET-1502, and ET-1504 (Eureka Therapeutics Inc); FT-61314 (Fate Therapeutics Inc); GB-7001 (Shanghai GeneChem Co Ltd); IMA-201 (Immatics Biotechnologies GmbH); IMM-005 and IMM-039 (Immunome Inc); ImmuniCAR (TC BioPharm Ltd); NT-0004 and NT-0009 (BioNTech Cell and Gene Therapies GmbH), OGD-203 (OGD2 Pharma SAS), PMC-005B (PharmAbcine), and TI-7007 (Timmune Biotech Inc).

[0250] In some embodiments, the chimeric antigen receptor comprises the amino acid sequence of an antibody. In some embodiments, the chimeric antigen receptor comprises the amino acid sequence of an antigen-binding fragment of an antibody. The antibody (or fragment thereof) portion of the extracellular binding domain recognizes and binds to an epitope of an antigen. In some embodiments, the antibody fragment portion of the chimeric antigen receptor is a single-chain variable fragment (scFv). An scFv comprises the light and variable fragments of a monoclonal antibody. In other embodiments, the antibody fragment portion of the chimeric antigen receptor is a multi-chain variable fragment, which may comprise two or more extracellular binding domains and thus may simultaneously bind to two or more antigens. In multi-chain variable fragment embodiments, a hinge region may separate different variable fragments to provide the necessary spatial arrangement and flexibility.

[0251] In other embodiments, the antibody portion of the chimeric antigen receptor comprises at least one heavy chain and at least one light chain. In some embodiments, the antibody portion of the chimeric antigen receptor comprises two heavy chains and two light chains connected by disulfide bridges, with each light chain connected to one of the heavy chains by a disulfide bridge. In some embodiments, the light chain comprises a constant region and a variable region. The complementarity-determining regions present in the variable region of an antibody are responsible for the affinity of the antibody for a specific antigen. Thus, antibodies that recognize different antigens contain different complementarity-determining regions. The complementarity-determining regions are present in the variable domain of the extracellular binding domain, and the variable domains (i.e., the variable heavy and variable light) may be linked by a linker or, in some embodiments, by a disulfide bridge.

[0252] In some embodiments, the antigen recognized and bound by the extracellular domain is a protein or peptide, a nucleic acid, a lipid, or a polysaccharide. The antigen may be heterologous, such as one expressed by a pathogenic bacterium or virus. The antigen may be synthetic; for example, some individuals are extremely allergic to synthetic latex, and exposure to this antigen may result in an extreme immune response. In some embodiments, the antigen is autologous and expressed on diseased or otherwise modified cells. For example, in some embodiments, the antigen is expressed on a neoplastic cell. In some embodiments, the neoplastic cell is a solid tumor cell. In other embodiments, the neoplastic cell is a blood cancer, such as a B-cell cancer. In some embodiments, the B-cell cancer is a lymphoma (e.g., Hodgkin's lymphoma or non-Hodgkin's lymphoma) or a leukemia (e.g., B-cell acute lymphoblastic leukemia). Exemplary B-cell lymphomas include diffuse large B-cell lymphoma (DLBCL), primary mediastinal B-cell lymphoma, follicular lymphoma, chronic lymphocytic leukemia (CLL), small lymphocytic lymphoma (SLL), mantle cell lymphoma, marginal zone lymphoma, Burkitt lymphoma, Burkitt-like lymphoma, lymphoplasmacytic lymphoma (Waldenstrom macroglobulinemia), and hairy cell leukemia. In some embodiments, the B-cell cancer is multiple myeloma.

[0253] Antibody-antigen interactions are non-covalent interactions resulting from hydrogen bonds, electrostatic or hydrophobic interactions, or van der Waals forces. The affinity of the extracellular binding domain of a chimeric antigen receptor for an antigen can be calculated using the following formula: K A = [antibody-antigen] / [antibody][antigen], where [Ab] = molar concentration of unoccupied binding sites on the antibody; [Ag] = molar concentration of unoccupied binding sites on the antigen; and [Ab-Ag] = molar concentration of antibody-antigen complex.

[0254] Antibody-antigen interactions can also be characterized based on the dissociation of the antigen from the antibody. The dissociation constant (K D ) is the ratio of the association rate to the dissociation rate and is inversely proportional to the affinity constant. Therefore, K D =1 / K A Those skilled in the art are familiar with these concepts and know that they can calculate these constants using conventional methods such as ELISA assays.

[0255] The transmembrane domain of the chimeric antigen receptor described herein spans the CAR-T cell lipid bilayer membrane, separating the extracellular binding domain from the intracellular signaling domain. In some embodiments, this domain is derived from another receptor with a transmembrane domain, while in other embodiments, this domain is synthetic. In some embodiments, the transmembrane domain may be derived from a non-human transmembrane domain and, in some embodiments, may be humanized. "Humanized" means that the nucleic acid sequence encoding the transmembrane domain has been optimized for more reliable or efficient expression in human subjects. In some embodiments, the transmembrane domain is derived from another transmembrane protein expressed in human immune effector cells. Examples of such proteins include, but are not limited to, a subunit of the T cell receptor (TCR) complex, PD1, or any cluster of differentiation protein, or other protein with a transmembrane domain expressed in immune effector cells. In some embodiments, the transmembrane domain is synthetic, and such a sequence will contain many hydrophobic residues.

[0256] In some embodiments, chimeric antigen receptors are designed to include a spacer between the transmembrane domain and the extracellular domain, the intracellular domain, or both. Such spacers can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acids in length. In some embodiments, spacers can be 20, 30, 40, 50, 60, 70, 80, 90, or 100 amino acids in length. In yet other embodiments, spacers can be 100 to 500 amino acids in length. A spacer can be any polypeptide used to link one domain to another and position such linked domains to enhance or optimize chimeric antigen receptor function.

[0257] The intracellular signaling domain of a chimeric antigen receptor contemplated herein comprises a primary signaling domain. In some embodiments, the chimeric antigen receptor comprises a primary signaling domain and a secondary or costimulatory signaling domain. In some embodiments, the primary signaling domain comprises one or more immunoreceptor tyrosine-based activation motifs (ITAMs). In some embodiments, the primary signaling domain comprises two or more ITAMs. The ITAMs incorporated into the chimeric antigen receptor may be derived from ITAMs from other cellular receptors. In some embodiments, the ITAM-containing primary signaling domain may be derived from a subunit of the TCR complex, such as CD3γ, CD3ε, CD3ζ, or CD3δ (see Figure 1A). In some embodiments, the ITAM-containing primary signaling domain may be derived from FcRγ, FcRβ, CD5, CD22, CD79a, CD79b, or CD66d. The secondary signaling domain, in some embodiments, is derived from CD28. In other embodiments, the secondary signaling domain is derived from CD2, CD4, CDS, CD8α, CD83, CD134, CD137, ICOS, or CD154.

[0258] Also provided herein is a nucleic acid encoding the chimeric antigen receptor described herein. In some embodiments, the nucleic acid is isolated or purified. Ex vivo nucleic acid delivery can be achieved using methods known in the art. For example, immune cells obtained from a subject can be transformed with a nucleic acid vector encoding the chimeric antigen receptor. The vector can then be used to transform recipient immune cells so that these cells express the chimeric antigen receptor. Efficient means of transforming immune cells include transfection and transduction. Such methods are well known in the art. For example, applicable methods for delivering nucleic acid molecules encoding chimeric antigen receptors (and nucleic acid(s) encoding base editors) can be found in International Application No. PCT / US2009 / 040040 and U.S. Patent Nos. 8,450,112; 9,132,153; and 9,669,058 (each of which is incorporated herein in its entirety). Furthermore, these methods and vectors described herein for delivering nucleic acids encoding base editors (e.g., ABE8) are applicable for delivering nucleic acids encoding chimeric antigen receptors.

[0259] Some aspects of the present invention provide immune cells comprising a chimeric antigen and a modified endogenous gene that enhances immune cell function, resistance to immune suppression or inhibition, or a combination thereof. Allogeneic immune cells expressing endogenous immune cell receptors and chimeric antigen receptors can recognize and attack host cells, a condition known as graft-versus-host disease (GVHD). The alpha component of the immune cell receptor complex is encoded by the TRAC gene, and in some embodiments, this gene is edited to render the alpha subunit of the TCR complex nonfunctional or absent. Because this subunit is required for endogenous immune cell signaling, editing this gene can reduce the risk of graft-versus-host disease (GVHD) caused by allogeneic immune cells.

[0260] Host immune cells may recognize allogeneic CAR-T cells as non-self and elicit an immune response to eliminate the non-self cells. B2M is expressed on nearly all nucleated cells and is associated with the MHC class I complex (Figure 1B). Circulating host CD8 + T cells can recognize this B2M protein as non-self and kill allogeneic cells. To overcome this graft rejection, in some embodiments, the B2M gene is edited to either knock out or knock down expression.

[0261] In some embodiments of the present invention, the PDCD1 gene is edited in CAR-T cells to knock out or knock down its expression. The PDCD1 gene encodes the cell surface receptor PD-1, an immune system checkpoint expressed on immune cells, and is involved in reducing autoimmunity by promoting apoptosis of antigen-specific immune cells. By knocking out or knocking down the expression of the PDCD1 gene, the modified CAR-T cells are less likely to undergo apoptosis, more likely to proliferate, and can escape programmed cell death immune checkpoints.

[0262] The CBLB gene encodes an E3 ubiquitin ligase that plays a key role in inhibiting immune effector cell activation. Referring to Figure 1C, the CBLB protein actively inhibits signaling pathways that lead to immune effector cell tolerance, favoring immune effector cell tolerance. Because immune effector cell activation is required for CAR-T cell proliferation in vivo after transplantation, in some embodiments of the present invention, CBLB is edited to knock out or down expression.

[0263] In some embodiments, gene editing to enhance immune cell function or reduce immunosuppression or inhibition may occur in immune cells before the cells are transformed to express the chimeric antigen receptor. In other aspects, gene editing to enhance immune cell function or reduce immunosuppression or inhibition may occur in CAR-T cells, i.e., after the immune cells are transformed to express the chimeric antigen receptor. In some embodiments, the immune cells comprise a chimeric antigen receptor and an edited TRAC, B2M, PDCD1, CD7, CIITA, CBLB gene, or a combination thereof, and the expression of the edited gene is knocked out or knocked down.

[0264] In some embodiments, the immune cells comprise a chimeric antigen receptor and an edited TRAC gene, and expression of the edited gene is knocked out or knocked down. In some embodiments, the immune cells comprise a chimeric antigen receptor and an edited TRAC gene, and one or more of B2M, PDCD1, CD7, CIITA, and / or CBLB genes, and expression of the edited gene is knocked out or knocked down. In some embodiments, the immune cells comprise a chimeric antigen receptor and edited TRAC and B2M genes, and expression of the edited gene is knocked out or knocked down. In some embodiments, the immune cells comprise a chimeric antigen receptor and edited TRAC and PDCD1 genes, and expression of the edited gene is knocked out or knocked down. In some embodiments, the immune cells comprise a chimeric antigen receptor and edited TRAC and CBLB genes, and expression of the edited gene is knocked out or knocked down. In some embodiments, the immune cells comprise a chimeric antigen receptor and edited TRAC and CD7 genes, and expression of the edited gene is knocked out or knocked down. In some embodiments, the immune cells comprise a chimeric antigen receptor and edited TRAC and CIITA genes, and expression of the edited genes is knocked out or knocked down. In some embodiments, the immune cells comprise a chimeric antigen receptor and edited TRAC, B2M, and PDCD1 genes, and expression of the edited genes is knocked out or knocked down. In some embodiments, the immune cells comprise a chimeric antigen receptor and edited TRAC, B2M, and CBLB genes, and expression of the edited genes is knocked out or knocked down. In some embodiments, the immune cells or immune effector cells comprise a chimeric antigen receptor and edited TRAC, PDCD1, and CBLB genes, and expression of the edited genes is knocked out or knocked down.In some embodiments, the immune cells comprise a chimeric antigen receptor and edited TRAC, B2M, and CIITA genes, and expression of the edited genes is knocked out or knocked down. In some embodiments, the immune cells comprise a chimeric antigen receptor and edited TRAC, B2M, and CD7 genes, and expression of the edited genes is knocked out or knocked down. In some embodiments, the immune cells comprise a chimeric antigen receptor and edited TRAC, PDCD1, and CD7 genes, and expression of the edited genes is knocked out or knocked down. In some embodiments, the immune cells comprise a chimeric antigen receptor and edited TRAC, PDCD1, and CIITA genes, and expression of the edited genes is knocked out or knocked down. In some embodiments, the immune cells comprise a chimeric antigen receptor and edited TRAC, PDCD1, and CBLB genes, and expression of the edited genes is knocked out or knocked down. In some embodiments, the immune cells comprise a chimeric antigen receptor and edited TRAC, CD7, and CIITA genes, and expression of the edited genes is knocked out or knocked down. In some embodiments, the immune cells comprise a chimeric antigen receptor and edited TRAC, CD7, and CBLB genes, and expression of the edited genes is knocked out or knocked down. In some embodiments, the immune cells comprise a chimeric antigen receptor and edited TRAC, CIITA, and CBLB genes, and expression of the edited genes is knocked out or knocked down.

[0265] In some embodiments, the immune cells comprise a chimeric antigen and edited TRAC, B2M, PDCD1, and CBLB genes, and expression of the edited genes is knocked out or knocked down. In some embodiments, the immune cells comprise a chimeric antigen and edited TRAC, B2M, PDCD1, and CD7 genes, and expression of the edited genes is knocked out or knocked down. In some embodiments, the immune cells comprise a chimeric antigen and edited TRAC, B2M, CD7, and CIITA genes, and expression of the edited genes is knocked out or knocked down. In some embodiments, the immune cells comprise a chimeric antigen and edited TRAC, B2M, CD7, and CBLB genes, and expression of the edited genes is knocked out or knocked down. In some embodiments, the immune cells comprise a chimeric antigen and edited TRAC, B2M, PDCD1, and CIITA genes, and expression of the edited genes is knocked out or knocked down. In some embodiments, the immune cells comprise a chimeric antigen and edited TRAC, B2M, CBLB, and CIITA genes, and expression of the edited genes is knocked out or knocked down. In some embodiments, the immune cells comprise a chimeric antigen and edited TRAC, PDCD1, CD7, and CIITA genes, and expression of the edited genes is knocked out or knocked down. In some embodiments, the immune cells comprise a chimeric antigen and edited TRAC, PDCD1, CD7, and CBLB genes, and expression of the edited genes is knocked out or knocked down. In some embodiments, the immune cells comprise a chimeric antigen and edited TRAC, PDCD1, CIITA, and CBLB genes, and expression of the edited genes is knocked out or knocked down. In some embodiments, the immune cells comprise a chimeric antigen and edited TRAC, CIITA, CD7, and CBLB genes, and expression of the edited genes is knocked out or knocked down.

[0266] In some embodiments, the immune cells comprise a chimeric antigen and edited TRAC, B2M, PDCD1, CD7, and CIITA genes, and expression of the edited genes is knocked out or knocked down. In some embodiments, the immune cells comprise a chimeric antigen and edited TRAC, B2M, PDCD1, CD7, and CBLB genes, and expression of the edited genes is knocked out or knocked down. In some embodiments, the immune cells comprise a chimeric antigen and edited TRAC, B2M, CD7, CIITA, and CBLB genes, and expression of the edited genes is knocked out or knocked down. In some embodiments, the immune cells comprise a chimeric antigen and edited TRAC, B2M, PDCD1, CIITA, and CBLB genes, and expression of the edited genes is knocked out or knocked down. In some embodiments, the immune cells comprise a chimeric antigen and edited TRAC, B2M, PDCD1, CD7, CIITA, and CBLB genes, and expression of the edited genes is knocked out or knocked down. In some embodiments, the immune cells comprise a chimeric antigen and edited TRAC, B2M, PDCD1, CD7, CIITA, and CBLB genes, and expression of the edited genes is knocked out or knocked down.

[0267] In some embodiments, the immune cells comprise a chimeric antigen receptor and an edited B2M gene, and expression of the edited gene is knocked out or knocked down. In some embodiments, the immune cells comprise a chimeric antigen receptor and an edited B2M gene, and one or more of the CBLB, PDCD1, CD7, CIITA, and / or TRAC genes, and expression of the edited gene is knocked out or knocked down. In some embodiments, the immune cells comprise a chimeric antigen receptor and edited B2M and PDCD1 genes, and expression of the edited gene is knocked out or knocked down. In some embodiments, the immune cells comprise a chimeric antigen receptor and edited B2M and CBLB genes, and expression of the edited gene is knocked out or knocked down. In some embodiments, the immune cells comprise a chimeric antigen receptor and edited B2M and CIITA genes, and expression of the edited gene is knocked out or knocked down. In some embodiments, the immune cells comprise a chimeric antigen receptor and edited B2M and CD7 genes, and expression of the edited gene is knocked out or knocked down. In some embodiments, the immune cells comprise a chimeric antigen receptor and edited B2M, CIITA, and CBLB genes, and expression of the edited genes is knocked out or knocked down. In some embodiments, the immune cells comprise a chimeric antigen receptor and edited B2M, PDCD1, and CBLB genes, and expression of the edited genes is knocked out or knocked down. In some embodiments, the immune cells comprise a chimeric antigen receptor and edited B2M, PDCD1, and CIITA genes, and expression of the edited genes is knocked out or knocked down. In some embodiments, the immune cells comprise a chimeric antigen receptor and edited B2M, CD7, and CBLB genes, and expression of the edited genes is knocked out or knocked down.In some embodiments, the immune cells comprise a chimeric antigen receptor and edited B2M, CD7, and PDCD1 genes, and expression of the edited genes is knocked out or knocked down. In some embodiments, the immune cells comprise a chimeric antigen receptor and edited B2M, CD7, and CIITA genes, and expression of the edited genes is knocked out or knocked down. In some embodiments, the immune cells comprise a chimeric antigen receptor and edited B2M, PDCD1, CIITA, and CBLB genes, and expression of the edited genes is knocked out or knocked down. In some embodiments, the immune cells comprise a chimeric antigen receptor and edited B2M, PDCD1, CIITA, and CD7 genes, and expression of the edited genes is knocked out or knocked down. In some embodiments, the immune cells comprise a chimeric antigen receptor and edited B2M, PDCD1, CD7, and CBLB genes, and expression of the edited genes is knocked out or knocked down. In some embodiments, the immune cells comprise a chimeric antigen receptor and edited B2M, PDCD1, CD7, CIITA, and CBLB genes, and expression of the edited genes is knocked out or knocked down.

[0268] In some embodiments, the immune cells comprise a chimeric antigen receptor and an edited PDCD1 gene, and expression of the edited gene is knocked out or knocked down. In some embodiments, the immune cells comprise a chimeric antigen receptor and an edited PDCD1 gene and one or more of B2M, CBLB, CD7, CIITA, and / or TRAC genes, and expression of the edited genes is knocked out or knocked down. In some embodiments, the immune cells comprise a chimeric antigen receptor and edited PDCD1 and CBLB genes, and expression of the edited genes is knocked out or knocked down. In some embodiments, the immune cells comprise a chimeric antigen receptor and edited PDCD1 and CD7 genes, and expression of the edited genes is knocked out or knocked down. In some embodiments, the immune cells comprise a chimeric antigen receptor and edited PDCD1 and CIITA genes, and expression of the edited genes is knocked out or knocked down. In some embodiments, the immune cells comprise a chimeric antigen receptor and edited PDCD1, CIITA and CBLB genes, and expression of the edited genes is knocked out or knocked down.

[0269] In some embodiments, the immune cells comprise a chimeric antigen receptor and an edited CD7, and expression of the edited gene is knocked out or knocked down. In some embodiments, the immune cells comprise a chimeric antigen receptor and an edited CBLB, and expression of the edited gene is knocked out or knocked down. In some embodiments, the immune cells comprise a chimeric antigen receptor and edited CD7 and CIITA genes, and expression of the edited gene is knocked out or knocked down. In some embodiments, the immune cells comprise a chimeric antigen receptor and edited CD7 and CBLB genes, and expression of the edited gene is knocked out or knocked down. In some embodiments, the immune cells comprise a chimeric antigen receptor and edited CD7, PDCD1, and CIITA genes, and expression of the edited gene is knocked out or knocked down. In some embodiments, the immune cells comprise a chimeric antigen receptor and edited CD7, PDCD1, CIITA, and CBLB genes, and expression of the edited gene is knocked out or knocked down.

[0270] In some embodiments, the immune cells comprise a chimeric antigen receptor and an edited CBLB gene, and expression of the edited gene is knocked out or knocked down. In some embodiments, the immune cells comprise a chimeric antigen receptor and an edited CBLB gene, and one or more of B2M, PDCD1, CD7, CIITA, and / or TRAC genes, and expression of the edited genes is knocked out or knocked down. In some embodiments, the immune cells comprise a chimeric antigen receptor and edited CBLB and CIITA genes, and expression of the edited genes is knocked out or knocked down.

[0271] In some embodiments, the immune cells comprise a chimeric antigen receptor and an edited CIITA, and expression of the edited gene is knocked out or knocked down. In some embodiments, the immune cells comprise a chimeric antigen receptor and an edited CBLB gene, and one or more of B2M, PDCD1, CD7, CBLB, and / or TRAC genes, and expression of the edited genes is knocked out or knocked down.

[0272] In some embodiments, immune cells, including but not limited to any immune cells containing any of the aforementioned gene edits, can be edited to generate mutations in other genes that enhance CAR-T function or reduce immune suppression or inhibition of the cells. For example, in some embodiments, the immune cells contain a chimeric antigen receptor and an edited TGFBR2, ZAP70, NFATc1, TET2 gene, or a combination thereof, and the expression of the edited gene is knocked out or knocked down. In some embodiments, the immune cells contain a chimeric antigen receptor and an edited TGFBR2 gene, and the expression of the edited gene is knocked out or knocked down. In some embodiments, the immune cells contain a chimeric antigen receptor and edited TGFBR2 and ZAP70 genes, and the expression of the edited gene is knocked out or knocked down. In some embodiments, the immune cells contain a chimeric antigen receptor and edited TGFBR2 and ZAP70 genes, and the expression of the edited gene is knocked out or knocked down. In some embodiments, the immune cells comprise a chimeric antigen receptor and edited TGFBR2 and NFATC1 genes, and expression of the edited genes is knocked out or knocked down. In some embodiments, the immune cells comprise a chimeric antigen receptor and edited TGFBR2 and TET2 genes, and expression of the edited genes is knocked out or knocked down. In some embodiments, the immune cells comprise a chimeric antigen receptor and edited TGFBR2, ZAP70, and NFATC1 genes, and expression of the edited genes is knocked out or knocked down. In some embodiments, the immune cells comprise a chimeric antigen receptor and edited TGFBR2, ZAP70, and TET2 genes, and expression of the edited genes is knocked out or knocked down. In some embodiments, the immune cells comprise a chimeric antigen receptor and edited TGFBR2, NFATC1, and TET2 genes, and expression of the edited genes is knocked out or knocked down.In some embodiments, the immune cells comprise a chimeric antigen and edited TGFBR2, ZAP70, NFATC1, and TET2 genes, and expression of the edited gene is knocked out or knocked down. In some embodiments, the immune cells comprise a chimeric antigen receptor and edited ZAP70 gene, and expression of the edited gene is knocked out or knocked down. In some embodiments, the immune cells comprise a chimeric antigen receptor and edited ZAP70 and NFATC1 genes, and expression of the edited gene is knocked out or knocked down. In some embodiments, the immune cells comprise a chimeric antigen receptor and edited ZAP70 and TET2 genes, and expression of the edited gene is knocked out or knocked down. In some embodiments, the immune cells comprise a chimeric antigen receptor and edited ZAP70, PDCD1, and TET2 genes, and expression of the edited gene is knocked out or knocked down. In some embodiments, the immune cells comprise a chimeric antigen receptor and edited PDCD1 gene, and expression of the edited gene is knocked out or knocked down. In some embodiments, the immune cells comprise a chimeric antigen receptor and edited PDCD1 and TET2 genes, and expression of the edited genes is knocked out or knocked down, and in some embodiments, the immune cells comprise a chimeric antigen receptor and edited TET2, and expression of the edited genes is knocked out or knocked down.

[0273] In some embodiments, the chimeric antigen receptor is inserted into the TRAC gene. This has advantages. First, because TRAC is highly expressed in immune cells, if the construct is designed to insert the chimeric antigen receptor into the TRAC gene so that its expression is driven by the TRAC promoter, the chimeric antigen receptor will be similarly expressed. Second, inserting the chimeric antigen receptor into the TRAC gene knocks out TRAC expression. In some embodiments, the gene editing system described herein can be used to insert the chimeric antigen receptor into the TRAC locus. A gRNA specific to the TRAC locus can guide the gene editing system to the locus and initiate double-stranded DNA cleavage. In certain embodiments, the gRNA is used in combination with Cas12b. In various embodiments, this gene editing system is used in combination with a nucleic acid having a sequence encoding a CAR receptor. Exemplary guide RNAs are shown in Table 1A below.

[0274] Table 1A: TRAC guide RNAs [Table 1-1]

[0275] A DNA construct encoding a chimeric antigen receptor and a nucleic acid containing an extended stretch of TRAC DNA flanked by gRNA target sequences. Without being bound by theory, this construct binds to the complementary TRAC sequence, and then the chimeric antigen receptor DNA near the TRAC sequence on the construct is inserted into the lesion site, effectively knocking out the TRAC gene and knocking in the chimeric antigen receptor nucleic acid. Table 1B provides guide RNAs for the TRAC gene that can direct base editing machinery to the TRAC locus, thereby enabling insertion of the chimeric antigen receptor nucleic acid. The first 11 gRNAs are for the BhCas12b nuclease. The second set of 11 are for the BvCas12b nuclease. In the first example, the scaffold sequence is in bold. All of these are intended to insert a CAR into TRAC by creating a double-stranded break, not for base editing.

[0276] Table 1-B: TRAC guide RNAs [Table 1-2] TIFF0007672982000013.tif110167

[0277] In some embodiments, nucleic acids encoding the chimeric antigen receptor of the present invention can be targeted to the TRAC locus using ABE8. In some embodiments, the chimeric antigen receptor is targeted to the TRAC locus using the CRISPR / Cas9 base editing system. To perform the above gene editing, immune cells are collected from a subject and contacted with two or more guide RNAs and a nucleobase editor polypeptide comprising a nucleic acid-programmable DNA-binding protein (napDNAbp) and an adenosine deaminase (e.g., TadA*8). In some embodiments, the collected immune cells are contacted with at least one nucleic acid, where the at least one nucleic acid encodes two or more guide RNAs and a nucleobase editor polypeptide comprising a nucleic acid-programmable DNA-binding protein (napDNAbp) and an adenosine deaminase (e.g., TadA*8). In some embodiments, the gRNA contains nucleotide analogs. These nucleotide analogs can inhibit degradation of the gRNA from cellular processes. Table 2 lists target sequences used for the gRNA.

[0278] Table 2: Exemplary target sequences [Table 2] TIFF0007672982000015.tif244166TIFF0007672982000016.tif155169

[0279] The adenosine deaminase nucleobase editors used in the present invention (e.g., ABE8) can act on DNA, including single-stranded DNA. Methods for using them to generate modifications in target nucleobase sequences in immune cells are presented. In certain embodiments, the fusion proteins provided herein include one or more features that improve the base editing activity of the fusion protein. For example, any of the fusion proteins provided herein can include a Cas9 domain with reduced nuclease activity. In some embodiments, any fusion protein provided herein can have a Cas9 domain with no nuclease activity (dCas9) or a Cas9 domain that cleaves one strand of a double-stranded DNA molecule, called a Cas9 nickase (nCas9). Without wishing to be bound by theory, the presence of a catalytic residue (e.g., H840) maintains the activity of Cas9 to cleave the unedited (e.g., unmethylated) strand opposite the target nucleobase. Mutation of the catalytic residue (e.g., D10 to A10) prevents cleavage of the edited strand containing the target A residue. Such Cas9 variants can generate single-stranded DNA breaks (nicks) at specific locations based on the target sequence defined by the gRNA, leading to repair of the unedited strand and ultimately to alteration of the nucleobases of the unedited strand.

[0280] [Nucleobase Editor] Disclosed herein are base editors or nucleobase editors for editing, modifying, or altering a target nucleotide sequence of a polynucleotide. Described herein are nucleobase editors or base editors comprising a polynucleotide-programmable nucleotide-binding domain and a nucleobase-editing domain (e.g., adenosine deaminase). The polynucleotide-programmable nucleotide-binding domain, when combined with a bound guide polynucleotide (e.g., gRNA), can specifically bind to the target polynucleotide sequence (via complementary base pairing between the bases of the bound guide nucleic acid and the bases of the target polynucleotide sequence), thereby localizing the base editor to the target nucleic acid sequence desired to be edited. In some embodiments, the target polynucleotide sequence comprises single-stranded DNA or double-stranded DNA. In some embodiments, the target polynucleotide sequence comprises RNA. In some embodiments, the target polynucleotide sequence comprises a DNA-RNA hybrid.

[0281] Polynucleotide-programmable nucleotide-binding domains It should be understood that polynucleotide programmable nucleotide binding domain can also comprise nucleic acid programmable protein that binds to RNA.For example, polynucleotide programmable nucleotide binding domain can be bound to nucleic acid that guides polynucleotide programmable nucleotide binding domain to RNA.Other nucleic acid programmable DNA binding proteins are also within the scope of the present disclosure, but they are not specifically listed in the present disclosure.

[0282] The polynucleotide-programmable nucleotide-binding domain of a base editor can itself comprise one or more domains. For example, a polynucleotide-programmable nucleotide-binding domain can comprise one or more nuclease domains. In certain embodiments, the nuclease domain of a polynucleotide-programmable nucleotide-binding domain can comprise an endonuclease or exonuclease. As used herein, the term "exonuclease" refers to a protein or polypeptide capable of digesting nucleic acids (e.g., RNA or DNA) from free ends, and the term "endonuclease" refers to a protein or polypeptide capable of catalyzing (e.g., cleaving) an internal region of a nucleic acid (e.g., DNA or RNA). In certain embodiments, an endonuclease can cleave one strand of a double-stranded nucleic acid. In certain embodiments, an endonuclease can cleave both strands of a double-stranded nucleic acid molecule. In certain embodiments, a polynucleotide-programmable nucleotide-binding domain can be a deoxyribonuclease. In certain embodiments, a polynucleotide-programmable nucleotide-binding domain can be a ribonuclease.

[0283] In some embodiments, the nuclease domain of a polynucleotide-programmable nucleotide-binding domain can cleave zero, one, or two strands of a target polynucleotide. In some embodiments, the polynucleotide-programmable nucleotide-binding domain can comprise a nickase domain. As used herein, the term "nickase" refers to a polynucleotide-programmable nucleotide-binding domain that comprises a nuclease domain that can cleave only one strand of a double-stranded nucleic acid molecule (e.g., DNA). In some embodiments, a nickase can be derived from a fully catalytically active (e.g., native) form of a polynucleotide-programmable nucleotide-binding domain by introducing one or more mutations into the active polynucleotide-programmable nucleotide-binding domain. For example, if the polynucleotide-programmable nucleotide-binding domain comprises a nickase domain derived from Cas9, the nickase domain derived from Cas9 can comprise a D10A mutation and a histidine at position 840. In such embodiments, residue H840 retains catalytic activity, thereby enabling single-strand cleavage of a nucleic acid duplex. In another example, a Cas9-derived nickase domain can include an H840A mutation, while the amino acid residue at position 10 remains D. In some embodiments, a nickase can be derived from a fully catalytically active (e.g., native) form of a polynucleotide-programmable nucleotide-binding domain by removing all or a portion of a nuclease domain that is not required for nickase activity. For example, if a polynucleotide-programmable nucleotide-binding domain includes a nickase domain from Cas9, the nickase domain from Cas9 can include a deletion of all or a portion of the RuvC domain or the HNH domain.

[0284] The amino acid sequence of an exemplary catalytically active Cas9 is as follows:

[0285] Thus, a base editor comprising a polynucleotide-programmable nucleotide-binding domain comprising a nickase domain can generate a single-stranded DNA break (nick) in a specific polynucleotide target sequence (e.g., as determined by the complementary sequence of a bound guide nucleic acid). In some embodiments, the strand of a nucleic acid double-stranded target polynucleotide sequence cleaved by a base editor comprising a nickase domain (e.g., a nickase domain derived from Cas9) is the strand not edited by the base editor (i.e., the strand cleaved by the base editor is the opposite strand to the strand containing the base to be edited). In other embodiments, a base editor comprising a nickase domain (e.g., a nickase domain derived from Cas9) can cleave the strand of a DNA molecule targeted for editing. In such embodiments, the non-target strand is not cleaved.

[0286] Also provided herein are base editors comprising a catalytically dead (i.e., incapable of cleaving a target polynucleotide sequence) polynucleotide-programmable nucleotide-binding domain. As used herein, the terms "catalytically dead" and "nuclease-inactive" are used interchangeably to refer to a polynucleotide-programmable nucleotide-binding domain having one or more mutations and / or deletions that result in an inability to cleave a strand of nucleic acid. In some embodiments, a catalytically dead polynucleotide-programmable nucleotide-binding domain base editor can lack nuclease activity as a result of specific point mutations in one or more nuclease domains. For example, in the case of a base editor comprising a Cas9 domain, Cas9 can contain both the D10A and H840A mutations. Such mutations inactivate both nuclease domains, resulting in the loss of nuclease activity. In other embodiments, a catalytically dead polynucleotide-programmable nucleotide-binding domain can contain one or more deletions of all or part of a catalytic domain (e.g., the RuvC1 and / or HNH domain). In further embodiments, the catalytically dead polynucleotide programmable nucleotide binding domain comprises a point mutation (e.g., D10A or H840A) as well as a deletion of all or part of the nuclease domain.

[0287] Also contemplated herein are mutations that can generate catalytically dead polynucleotide-programmable nucleotide-binding domains from previously functional versions of the polynucleotide-programmable nucleotide-binding domain. For example, in the case of catalytically dead Cas9 ("dCas9"), variants are provided that have mutations other than D10A and H840A, resulting in nuclease-inactivated Cas9. Such mutations include, for example, other amino acid substitutions at D10 and H840, or other substitutions within the nuclease domain of Cas9 (e.g., substitutions in the HNH nuclease subdomain and / or RuvC1 subdomain). Additional suitable nuclease-inactive dCas9 domains will be apparent to those skilled in the art based on this disclosure and knowledge in the art, and are within the scope of this disclosure. Such additional exemplary suitable nuclease-inactive Cas9 domains include, but are not limited to, D10A / H840A, D10A / D839A / H840A, and D10A / D839A / H840A / N863A mutant domains (see, e.g., Prashant et al., CAS9 transcriptional activators for target specificity screening and paired nickases for cooperative genome engineering. Nature Biotechnology. 2013; 31(9): 833-838, the entire contents of which are incorporated herein by reference).

[0288] Non-limiting examples of polynucleotide-programmable nucleotide-binding domains that can be incorporated into base editors include domains derived from CRISPR proteins, restriction nucleases, meganucleases, TAL nucleases (TALENs), and zinc finger nucleases (ZFNs). In some embodiments, the base editor comprises a polynucleotide-programmable nucleotide-binding domain comprising a natural or modified protein, or a portion thereof, that can bind to a nucleic acid sequence via a binding guide nucleic acid during CRISPR (i.e., Clustered Regularly Interspaced Short Palindromic Repeats)-mediated modification of the nucleic acid. Such proteins are referred to herein as "CRISPR proteins." Accordingly, disclosed herein are base editors that comprise a polynucleotide-programmable nucleotide-binding domain comprising all or a portion of a CRISPR protein (i.e., a base editor that comprises all or a portion of a CRISPR protein as a domain (also referred to as the "CRISPR protein-derived domain" of the base editor). The CRISPR protein-derived domain incorporated into the base editor can be modified compared to a wild-type or natural CRISPR protein. For example, as described below, a domain derived from a CRISPR protein can contain one or more mutations, insertions, deletions, rearrangements, and / or recombinations compared to a wild-type or naturally occurring CRISPR protein.

[0289] CRISPR is an adaptive immune system that provides defense against mobile genetic elements (viruses, transposable elements, and conjugative plasmids). CRISPR clusters contain a spacer, a sequence complementary to the preceding mobile element, and a target invading nucleic acid. CRISPR clusters are transcribed and processed into CRISPR RNA (crRNA). In type II CRISPR systems, correct processing of the pre-crRNA requires a transcoding small RNA (tracrRNA), endogenous ribonuclease 3 (rnc), and the Cas9 protein. tracrRNA guides ribonuclease 3 processing of the pre-crRNA. Cas9 / crRNA / tracrRNA then endonucleolytically cleaves linear or circular dsDNA targets complementary to the spacer. Target strands not complementary to the crRNA are first endonucleolytically cleaved and then exonucleolytically trimmed 3'-5'. In nature, both proteins and RNAs are required for DNA binding and cleavage. However, single guide RNAs ("sgRNAs," or simply "gRNAs") can be engineered to incorporate aspects of both the crRNA and tracrRNA into a single RNA species. See, e.g., Jinek M., Chylinski K., Fonfara I., Hauer M., Doudna JA, Charpentier E. Science 337:816-821 (2012), the entire contents of which are incorporated herein by reference. Cas9 recognizes a short motif (the PAM or protospacer adjacent motif) in the CRISPR repeat sequence to help distinguish between "self" and "non-self."

[0290] In some embodiments, the methods described herein can utilize engineered Cas proteins. Guide RNAs (gRNAs) are short synthetic RNAs consisting of a scaffold sequence required for Cas binding and a user-defined approximately 20-base spacer that defines the genomic target to be modified. Thus, one skilled in the art can vary the genomic target of Cas protein specificity, which is determined in part by how specific the gRNA targeting sequence is for the genomic target relative to other parts of the genome.

[0291] In some embodiments, the gRNA scaffold sequence is: GUUUUAGAGC UAGAAAUAGC AAGUUAAAAU AAGGCUAGUC CGUUAUCAAC UUGAAAAAGU GGCACCGAGU CGGUGCUUUU.

[0292] In some embodiments, the domain derived from a CRISPR protein incorporated into the base editor is an endonuclease (e.g., a deoxyribonuclease or ribonuclease) capable of binding to a target polynucleotide when combined with a bound guide nucleic acid. In some embodiments, the domain derived from a CRISPR protein incorporated into the base editor is a nickase capable of binding to a target polynucleotide when combined with a bound guide nucleic acid. In some embodiments, the domain derived from a CRISPR protein incorporated into the base editor is a catalytically dead domain capable of binding to a target polynucleotide when combined with a bound guide nucleic acid. In some embodiments, the target polynucleotide that binds to the CRISPR protein-derived domain of the base editor is DNA, and in some embodiments, the target polynucleotide that binds to the CRISPR protein-derived domain of the base editor is RNA.

[0293] CAs proteins that can be used herein include class 1 and class 2. Non-limiting examples of Cas proteins include Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5d, Cas5t, Cas5h, Cas5a, Cas6, Cas7, Cas8, Cas9 (also called Csn1 or Csx12), Cas10, Csy1, Csy2, Csy3, Csy4, Cse1, Cse2, Cse3, Cse4, Cse5, Csn1, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmr1, Cmr3, Cmr4, Cmr5, Csb1, Csb2, Csb3, Csx17, Csx14, Csx10, Cssx16, Cx16, Cx, Csx3, Csx1, Csx1S, Csf1, Csf2, CsO, Csf4, Csd1, Csd2, Cst1, Cst2, Csh1, Csh2, Csa1, Csa2, Csa3, Csa4, Csa5, Cas12a / Cpf1, Cas12b / C2c1, Cas12c / C2c3, Cas12d / CasY, Cas12e / CasX, Cas12g, Cas12h, and Cas12i, CARF, DinG, their homologs, or their variants.Unmodified CRISPR enzyme, like Cas9, can have the DNA cleavage activity with two functional endonuclease regions, RuvC and HNH. CRISPR enzymes can induce cleavage of one or both strands of a target sequence, such as within the target sequence and / or within the complementary strand of the target sequence. For example, CRISPR enzymes can induce cleavage of one or both strands at about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 50, 100, 200, 500 or more base pairs from the first or last nucleotide of the target sequence.

[0294] Vectors can be used that encode CRISPR enzymes that are mutated relative to the corresponding wild-type enzyme so that they lack the ability to cleave one or both strands of a target polynucleotide containing the target sequence. Cas9 can refer to a polypeptide that has at least, or at least about, 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity and / or sequence homology to a wild-type exemplary Cas9 polypeptide (e.g., Cas9 from S. pyogenes). Cas9 can refer to a polypeptide having at most, or at most, about 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity and / or sequence homology to a wild-type exemplary Cas9 polypeptide (e.g., from S. pyogenes). Cas9 can refer to wild-type or modified forms of the Cas9 protein, which can include amino acid changes such as deletions, insertions, substitutions, variants, mutations, fusions, chimeras, or any combination thereof.

[0295] In some embodiments, the base editor CRISPR protein-derived domain is derived from Corynebacterium ulcerans (NCBI Refs: NC_015683.1, NC_017317.1); Corynebacterium diphtheria (NCBI Refs: NC_016782.1, NC_016786.1); Spiroplasma syrphidicola (NCBI Ref: NC_021284.1); Prevotella intermedia (NCBI Ref: NC_017861.1); Spiroplasma taiwanense (NCBI Ref: NC_021846.1); Streptococcus iniae (NCBI Ref: NC_021314.1); Belliella baltica (NCBI Ref: NC_018010.1); Psychroflexus torquis (NCBI Ref: NC_018721.1); Streptococcus thermophilus (NCBI Ref: YP_820832.1); Listeria innocua (NCBI Ref: NP_472073.1); Campylobacter jejuni (NCBI Ref: YP_002344900.1); Neisseria meningitidis (NCBI Ref: YP_002342100.1), Streptococcus pyogenes, or Staphylococcus aureus.

[0296] [Cas9 domain, a nucleobase editor] The sequence and structure of Cas9 nuclease are well known to those of skill in the art (see, e.g., "Complete genome sequence of an M1 strain of Streptococcus pyogenes," Ferretti et al., Proc. Natl. Acad. Sci. USA 98:4658-4663(2001); "CRISPR RNA maturation by trans-encoded small RNA and host factor RNase III," Deltcheva E. et al., Nature 471:602-607(2011); and "A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity," Jinek M et al., Science 337:816-821(2012), the entire contents of which are incorporated herein by reference). Cas9 orthologs have been described in various species, including, but not limited to, S. pyogenes and S. thermophilus. Additional suitable Cas9 nucleases and sequences will be apparent to those of skill in the art based on this disclosure, including Cas9 sequences from the organisms and loci disclosed in Chylinski, Rhun, and Charpentier, "The tracrRNA and Cas9 families of type II CRISPR-Cas immunity systems" (2013) RNA Biology 10:5, 726-737, the entire contents of which are incorporated herein by reference.

[0297] In some embodiments, the nucleic acid programmable DNA binding protein (napDNAbp) is a Cas9 domain. Non-limiting exemplary Cas9 domains are provided herein. The Cas9 domain can be a nuclease-active Cas9 domain, a nuclease-inactive Cas9 domain (dCas9), or a Cas9 nickase (nCas9). In some embodiments, the Cas9 domain is a nuclease-active domain. For example, the Cas9 domain can be a Cas9 domain that cleaves both strands of a double-stranded nucleic acid (e.g., both strands of a double-stranded DNA molecule). In some embodiments, the Cas9 domain comprises any one of the amino acid sequences described herein. In some embodiments, the Cas9 domain comprises an amino acid sequence that is at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identical to any one of the amino acid sequences described herein. In some embodiments, the Cas9 domain comprises an amino acid sequence having 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 21, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, or more mutations compared to any one of the amino acid sequences described herein.In some embodiments, the Cas9 domain comprises an amino acid sequence having at least 10, at least 15, at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 150, at least 200, at least 250, at least 300, at least 350, at least 400, at least 500, at least 600, at least 700, at least 800, at least 900, at least 1000, at least 1100, or at least 1200 identical stretches of amino acid residues compared to any one of the amino acid sequences described herein.

[0298] In some embodiments, proteins comprising a fragment of Cas9 are provided. For example, in some embodiments, the protein comprises one of the following two Cas9 domains: (1) the gRNA binding domain of Cas9; (2) the DNA cleavage domain of Cas9. In some embodiments, proteins comprising Cas9 or a fragment thereof are referred to as "Cas9 variants." Cas9 variants share homology with Cas9 or a fragment thereof. For example, Cas9 variants are at least about 70% identical, at least about 80% identical, at least about 90% identical, at least about 95% identical, at least about 96% identical, at least about 97% identical, at least about 98% identical, at least about 99% identical, at least about 99.5% identical, or at least about 99.9% identical to wild-type Cas9. In some embodiments, the Cas9 mutant may have 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 21, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50 or more amino acid changes compared to wild-type Cas9. In some embodiments, the Cas9 variant comprises a fragment of Cas9 (e.g., a gRNA binding domain or a DNA cleavage domain) that is at least about 70% identical, at least about 80% identical, at least about 90% identical, at least about 95% identical, at least about 96% identical, at least about 97% identical, at least about 98% identical, at least about 99% identical, at least about 99.5% identical, or at least about 99.9% identical to the corresponding fragment of wild-type Cas9.In some embodiments, a fragment is at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% identical, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% of the amino acid length of a corresponding wild-type Cas9. In some embodiments, a fragment is at least 100 amino acids in length. In certain embodiments, fragments are at least 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1050, 1100, 1150, 1200, 1250, or at least 1300 amino acids in length.

[0299] In some embodiments, the Cas9 fusion proteins provided herein comprise the full-length amino acid sequence of a Cas9 protein, e.g., one of the Cas9 sequences provided herein. However, in other embodiments, the fusion proteins provided herein do not comprise the full-length Cas9 sequence, but rather comprise only one or more fragments thereof. Exemplary amino acid sequences of suitable Cas9 domains and Cas9 fragments are provided herein, and additional suitable sequences of Cas9 domains and fragments will be apparent to those skilled in the art.

[0300] The Cas9 protein can bind to a guide RNA, which guides the Cas9 protein to a specific DNA sequence complementary to the guide RNA. In some embodiments, the polynucleotide-programmable nucleotide-binding domain is a Cas9 domain, such as a nuclease-active Cas9, a Cas9 nickase (nCas9), or a nuclease-inactive Cas9 (dCas9). Examples of nucleic acid-programmable DNA-binding proteins include, but are not limited to, Cas9 (e.g., dCas9 and nCas9), CasX, CasY, Cpf1, Cas12b / C2C1, and Cas12c / C2C3.

[0301] In some embodiments, the wild-type Cas9 corresponds to Cas9 from Streptococcus pyogenes (NCBI Reference Sequence: NC_017053.1, nucleotide and amino acid sequences are as follows): TIFF0007672982000017.tif170165 (single underline: HNH domain, double underline: RuvC domain)

[0302] In some embodiments, the wild-type Cas9 corresponds to or comprises the following nucleotide and / or amino acid sequence: TIFF0007672982000018.tif167163 (single underline: HNH domain, double underline: RuvC domain)

[0303] In some embodiments, the wild-type Cas9 corresponds to Cas9 from Streptococcus pyogenes (NCBI Reference Sequence: NC_002737.2 (nucleotide sequence as follows), and Uniprot Reference Sequence: Q99ZW2 (amino acid sequence as follows). TIFF0007672982000019.tif170163 (single underline: HNH domain, double underline: RuvC domain)

[0304] In some embodiments, Cas9 is Corynebacterium ulcerans (NCBI Refs: NC_015683.1, NC_017317.1); Corynebacterium diphtheria (NCBI Refs: NC_016782.1, NC_016786.1); Spiroplasma syrphidicola (NCBI Ref: NC_021284.1); Prevotella intermedia (NCBI Ref: NC_017861.1); Spiroplasma taiwanense (NCBI Ref: NC_021846.1); Streptococcus iniae (NCBI Ref: NC_021314.1); Belliella baltica (NCBI Ref: NC_018010.1); Psychroflexus torquisI (NCBI Ref: NC_018721.1); Streptococcus thermophilus (NCBI Ref: YP_820832.1), Listeria innocua (NCBI Ref: NP_472073.1), Campylobacter jejuni (NCBI Ref: YP_002344900.1) or Neisseria meningitidis (NCBI Ref: YP_002342100.1), or Cas9 from any other organism.

[0305] It should be appreciated that additional Cas9 proteins, including variants and homologs thereof (e.g., nuclease-inactive Cas9 (dCas9), Cas9 nickase (nCas9), or nuclease-active Cas9), are within the scope of this disclosure. Exemplary Cas9 proteins include, without limitation, those provided below. In some embodiments, the Cas9 protein is a nuclease-inactive Cas9 (dCas9). In some embodiments, the Cas9 protein is a Cas9 nickase (nCas9). In some embodiments, the Cas9 protein is a nuclease-active Cas9.

[0306] In some embodiments, the Cas9 domain is a nuclease-inactive Cas9 domain (dCas9). For example, the dCas9 domain can bind to a double-stranded nucleic acid molecule (e.g., via a gRNA molecule) without cleaving either strand of the double-stranded nucleic acid molecule. In some embodiments, the nuclease-inactive dCas9 domain comprises a D10X mutation and a H840X mutation in the amino acid sequence described herein, or a corresponding mutation in any of the amino acid sequences provided herein, where X is any amino acid change. In some embodiments, the nuclease-inactive dCas9 domain comprises a D10A mutation and a H840A mutation in the amino acid sequence described herein, or a corresponding mutation in any of the amino acid sequences provided herein. As an example, the nuclease-inactive Cas9 domain comprises the following amino acid sequence provided in the cloning vector pPlatTET-gRNA 2 (Accession No. BAV54124):

[0307] The amino acid sequence of an exemplary catalytically inactive Cas9 (dCas9) is as follows: (See, e.g., Qi et al., "Repurposing CRISPR as an RNA-guided platform for sequence-specific control of gene expression." Cell. 2013; 152(5):1173-83, the entire contents of which are incorporated herein by reference.)

[0308] Additional suitable nuclease-inactive dCas9 domains will be apparent to those skilled in the art based on this disclosure and knowledge in the art and are within the scope of this disclosure. Such additional exemplary suitable nuclease-inactive Cas9 domains include, but are not limited to, D10A / H840A, D10A / D839A / H840A, and D10A / D839A / H840A / N863A mutant domains (see, e.g., Prashant et al., CAS9 transcriptional activators for target specificity screening and paired nickases for cooperative genome engineering. Nature Biotechnology. 2013; 31(9): 833-838, the entire contents of which are incorporated herein by reference).

[0309] In some embodiments, the Cas9 nuclease has an inactive (e.g., inactivated) DNA cleavage domain, i.e., Cas9 is a nickase, referred to as a "nCas9" protein (for "nickase" Cas9). A nuclease-inactivated Cas9 protein may also be interchangeably referred to as a "dCas9" protein (for nuclease-"dead" Cas9) or catalytically inactive Cas9. Methods for generating a Cas9 protein (or a fragment thereof) with an inactive DNA cleavage domain are known (see, e.g., Jinek et al., Science. 337:816-821 (2012); Qi et al., "Repurposing CRISPR as an RNA-Guided Platform for Sequence-Specific Control of Gene Expression" (2013) Cell. 28; 152(5): 1173-83, the contents of each of which are incorporated herein by reference). For example, the DNA cleavage domain of Cas9 is known to contain two subdomains: the HNH nuclease subdomain and the RuvC1 subdomain. The HNH subdomain cleaves the strand complementary to the gRNA, while the RuvC1 subdomain cleaves the non-complementary strand. Mutations within these subdomains can suppress the nuclease activity of Cas9. For example, mutations D10A and H840A completely inactivate the nuclease activity of S. pyogenes Cas9 (Jinek et al., Science. 337:816-821(2012); Qi et al., Cell. 28;152(5):1173-83 (2013)).

[0310] In some embodiments, the dCas9 domain comprises an amino acid sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identity to any of the dCas9 domains provided herein. In some embodiments, the Cas9 domain comprises an amino acid sequence having 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 21, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50 or more mutations compared to any of the amino acid sequences described herein. In some embodiments, the Cas9 domain comprises an amino acid sequence having at least 10, at least 15, at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 150, at least 200, at least 250, at least 300, at least 350, at least 400, at least 500, at least 600, at least 700, at least 800, at least 900, at least 1000, at least 1100, or at least 1200 identical contiguous amino acid residues compared to any of the amino acid sequences described herein.

[0311] In some embodiments, the dCas9 corresponds to, or comprises part or all of, a Cas9 amino acid sequence with one or more mutations that inactivate Cas9 nuclease activity. For example, in some embodiments, the dCas9 domain comprises D10A and H840A mutations or corresponding mutations in another Cas9.

[0312] In some embodiments, the dCas9 comprises the amino acid sequence of dCas9 (D10A and H840A): TIFF0007672982000020.tif169164 (single underline: HNH domain; double underline: RuvC domain)

[0313] In some embodiments, the Cas9 domain comprises a D10A mutation, while the residue at position 840 in the amino acid sequence provided above, or the residue at the corresponding position in any of the amino acid sequences provided herein, remains a histidine.

[0314] In other embodiments, dCas9 variants are provided that have mutations other than D10A and H840A, e.g., that result in nuclease-inactivated Cas9 (dCas9). Such mutations include, for example, other amino acid substitutions at D10 and H840, or other substitutions within the nuclease domain of Cas9 (e.g., substitutions in the HNH nuclease subdomain and / or the RuvC1 subdomain). In some embodiments, dCas9 variants or homologs are provided that are at least about 70% identical, at least about 80% identical, at least about 90% identical, at least about 95% identical, at least about 98% identical, at least about 99% identical, at least about 99.5% identical, or at least about 99.9% identical. In some embodiments, variants of dCas9 are provided that have amino acid sequences that are shorter or longer by about 5 amino acids, about 10 amino acids, about 15 amino acids, about 20 amino acids, about 25 amino acids, about 30 amino acids, about 40 amino acids, about 50 amino acids, about 75 amino acids, about 100 amino acids or more.

[0315] In some embodiments, the Cas9 domain is a Cas9 nickase. The Cas9 nickase can be a Cas9 protein that can cleave only one strand of a double-stranded nucleic acid molecule (e.g., a double-stranded DNA molecule). In some embodiments, the Cas9 nickase cleaves the target strand of a double-stranded nucleic acid molecule, meaning that the Cas9 nickase cleaves the strand that is base-paired (complementary) to a gRNA (e.g., an sgRNA) bound to the Cas9. In some embodiments, the Cas9 nickase comprises a D10A mutation and has a histidine at position 840. In some embodiments, the Cas9 nickase cleaves the non-target, non-base-edited strand of a double-stranded nucleic acid molecule, meaning that the Cas9 nickase cleaves the strand that is not base-paired to a gRNA (e.g., an sgRNA) bound to the Cas9. In some embodiments, the Cas9 nickase comprises a H840A mutation and has an aspartic acid residue at position 10, or a corresponding mutation. In some embodiments, the Cas9 nickase comprises an amino acid sequence that is at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identical to any of the Cas9 nickases provided herein. Additional suitable Cas9 nickases will be apparent to those of skill in the art based on this disclosure and knowledge in the art, and are within the scope of this disclosure.

[0316] The amino acid sequence of an exemplary catalytic Cas9 nickase (nCas9) is as follows:

[0317] In some embodiments, Cas9 refers to Cas9 from archaea (e.g., nanoarchaea), which constitute the domain and kingdom of unicellular prokaryotic microorganisms. In some embodiments, the programmable nucleotide-binding protein can be a CasX or CasY protein, as described, for example, in Burstein et al., "New CRISPR-Cas systems from uncultivated microbes." Cell Res. 2017 Feb 21. doi: 10.1038 / cr.2017.21, the entire contents of which are incorporated herein by reference. Using genome-resolved metagenomics, many CRISPR-Cas systems have been identified, including the first reported Cas9 in the archaeal domain of life. This divergent Cas9 protein was discovered as part of an active CRISPR-Cas system in the little-studied nanoarchaea. In bacteria, two previously unknown systems, CRISPR-CasX and CRISPR-CasY, have been discovered, which are among the most compact systems discovered to date. In some embodiments, in the base editor systems described herein, Cas9 is replaced by CasX or a variant of CasX. In some embodiments, in the base editor systems described herein, Cas9 is replaced by CasY or a variant of CasY. It should be understood that other RNA-guided DNA-binding proteins can also be used as nucleic acid programmable DNA-binding proteins (napDNAbp) and are within the scope of the present disclosure.

[0318] In some embodiments, the nucleic acid programmable DNA-binding protein (napDNAbp) of any of the fusion proteins provided herein can be a CasX or CasY protein. In some embodiments, the napDNAbp is a CasX protein. In some embodiments, the napDNAbp is a CasY protein. In some embodiments, the napDNAbp comprises an amino acid sequence having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identity to a naturally occurring CasX or CasY protein. In some embodiments, the programmable nucleotide-binding protein is a naturally occurring CasX or CasY protein. In some embodiments, the programmable nucleotide-binding protein comprises an amino acid sequence having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identity to any CasX or CasY protein described herein. It should be understood that CasX and CasY from other bacterial species may also be used in accordance with the present disclosure.

[0319] The amino acid sequence of an exemplary CasX ((uniprot.org / uniprot / F0NN87; uniprot.org / uniprot / F0NH53) tr|F0NN87|F0NN87_SULIH CRISPR-associated Casx protein OS = Sulfolobus islandicus (strain HVE10 / 4) GN = SiH_0402 PE=4 SV=1) is as follows: MEVPLYNIFGDNYIIQVATEAENSTIYNNKVEIDDEELRNVLNLAYKIAKNNEDAAAERRGKAKKKKGEEGETTSNIILPLSGNDKNPWTETLKCYNFPTTTVALSEVFKNFSQVKECEEVSAPSFVKPEFYEFGRSPGMVERTRRVKLEVEPHYLIIAAGWVLTRLGKAKVSEGDYVGVNVFTPTRGILYSLIQNVNGIVPGIKPETAFGLWIARKVVSSVTNPNVSVVRIYTISDAVGQNPTTINGGFSIDLTKLLEKRYLLSERLEAIARNALSISSNMRERYIVLANYIYEYLTG SKRLEDLLYFANRDLIMNLNSDDGKVRDLKLISAYVNGELIRGEG.

[0320] Exemplary CasX (>tr|F0NH53|F0NH53_SULIR CRISPR associated protein, Casx OS = Sulfolobus islandicus (strain REY15A) GN=SiRe_0771 PE=4 SV=1) の amino acid sequence is as follows: MEVPLYNIFGDNYIIQVATEAENSTIYNNKVEIDDEELRNVLNLAYKIAKNNEDAAAERRGKAKKKKGEEGETTSNIILPLSGNDKNPWTETLKCYNFPTTTVALSEVFKNFSQVKECEEVSAPSFVKPFEYKFGRSPGMVERTRRVKLEVEPHYLIMAAAGWVLTRLGKAKVSEGDYVGVNVFTPTRGILYSLIQNVNGIVPGIKPETAFGLWIARKVVSSVTNPNVSVVSIYTISDAVGQNPTTINGGFSIDLTKLLEKRDLLSERLEAIAR...

Claims

1. 1. An in vitro or ex vivo method for producing an engineered immune cell, the method comprising expressing in or introducing into an immune cell a nucleobase editor polypeptide and contacting the cell with two or more guide RNAs that target the nucleobase editor polypeptide to effect an alteration in a nucleic acid molecule encoding at least one polypeptide selected from the group consisting of T cell receptor alpha constant (TRAC), beta-2 microglobulin (B2M), programmed cell death 1 (PD1), cluster of differentiation 7 (CD7), cluster of differentiation 5 (CD5), cluster of differentiation 33 (CD33), cluster of differentiation 123 (CD123), Cbl proto-oncogene B (CBLB), and class II major histocompatibility complex transactivator (CIITA) polypeptides, thereby reducing expression of the at least one polypeptide compared to a wild-type immune cell, wherein the nucleobase editor polypeptide is selected from the group consisting of a nucleic acid programmable DNA binding protein (napDNAbp), The amino acid sequence MSEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWNRAIGLHDPTAHAEIMALRQGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIGRVVFGVRNAKTGAAGSLMDVLHYPGMNHRVEITEGILADECAALLCYFFRMPRQVFNAQKKAQSSTD (SEQ ID NO: 3) or a fragment thereof lacking only the N-terminal methionine, and having a total of up to 10 modifications. wherein said up to a total of 10 modifications include: i) a combination of modifications selected from any of the following with reference to SEQ ID NO:3: a) Y147R, Q154R, and Y123H; b) I76Y, Y123H, Y147R, and Q154R; c) V82S and Q154R; d) I76Y, V82S, Y123H, Y147R, and Q154R; e) Y147R, Q154R, Y123H, and V106W; f) I76Y, Y123H, Y147R, Q154R, I76Y, and V106W; g) V82S, Q154R, and V106W; h) I76Y, V82S, Y123H, Y147R, Q154R, and V106W; and ii) one or more amino acid modifications selected from the group consisting of I76Y, V82S, Y123H, Y147R, Y147T, Q154S, Q154R, and T166R with reference to SEQ ID NO:3, and / or S2A, H8Y, T17S, L18E, W23R, W23L, W23G, D24G, E25M, E25D, E25A, E25R, E25V, E25S, E25Y, E25G, R26W, R26G, R26N, R26Q, R26C, R26L, R26K, L34S, H3 6L, N37T, N37S, W45L, P48A, P48S, P48L, P48T, I49F, I49V, R51L, R51H, R52H, A56E, A56S, E59A, E59G, M61I, G67V, L68Q, M70V, M70L, Q71 R, Q71L, N72S, N72D, R74A, R74Q, D77G, L84F, E85K, E85G, A91T, M94L, I95L, H96L, S97C, R98Q, V102A, F104I, F104L, A106V, A106T, R107C , R107H, R107N, R107K, R107P, R107A, R107W, R107S, D108Y, D108N, D108G, D108R, D108Q, D108M, D108L, D108K, D108I, D108F, D108A, D1 08V, A109T, K110I, M118K, H123Y, G125A, N127S, R129Q, E134G, L137M, A138V, A142N, A142G, A142D, A143D, A143G, A143E, A143L, A143W, one or more amino acid modifications selected from the group consisting of A143M, A143S, A143Q, A143R, S146C, S146T, S146R, D147Y, F149Y, M151V, R152P, R152H, R152C, R153C, Q154H, Q154L, Q154R, E155V, E155G, E155D, I156F, I156Y, I156D, K157N, K157R, L157N, Q159L, K160S, K160E, K161Q, K161T, Q163H, and T166P A method comprising:

2. The napDNAbp has the following sequence: wherein the bolded sequence represents a sequence derived from Cas9, the italicized sequence represents a linker sequence, and the underlined sequence represents a bipartite nuclear localization sequence.

3. 3. The method of claim 1 or 2, wherein the napDNAbp is Staphylococcus aureus Cas9 (SaCas9), Streptococcus thermophilus 1 Cas9 (St1Cas9), Streptococcus pyogenes Cas9 (SpCas9), or a variant thereof.

4. The method of any one of claims 1 to 3, wherein the napDNAbp comprises a nuclease-inactive Cas9 (dCas9), a Cas9 nickase (nCas9), or a nuclease-active Cas9.

5. 2. The method of claim 1, wherein the nucleobase editor polypeptide further comprises a linker between the napDNAbp and the adenosine deaminase variant domain.

6. 2. The method of claim 1, wherein the nucleobase editor polypeptide further comprises one or more nuclear localization signals (NLS).

7. The method of claim 1 , wherein the immune cell is a T cell.

8. 2. The method of claim 1, wherein the guide RNA targets B2M, TRAC, and CIITA polynucleotides, respectively, thereby reducing expression of B2M, TRAC, and CIITA compared to wild-type immune cells.

9. 2. The method of claim 1, wherein the two or more guide RNAs target a splice acceptor site or a splice donor site in the target polynucleotide.

10. 2. The method of claim 1, wherein the nucleobase editor polypeptide generates a stop codon in the target polynucleotide.

11. 2. The method of claim 1, wherein the nucleobase editor polypeptide further comprises one or more uracil glycosylase inhibitors.

12. 13. The method of claim 1, further comprising expressing a chimeric antigen receptor (CAR) in the modified immune cell.

13. The method of claim 1 , wherein the immune cell is a cytotoxic T cell, a regulatory T cell, or a T helper cell.

14. 10. The method of claim 1, wherein the modified immune cells produced by said method have reduced immunogenicity and increased anti-neoplastic activity.

15. The method of claim 14, wherein the immune cell is a T cell.

16. 16. The method of claim 14 or 15, wherein the method comprises introducing a mutation into a polynucleotide encoding a polypeptide selected from the group consisting of B2M, CD7, CIITA, PD1, CBLB, and TRAC, thereby reducing expression of the polypeptide compared to a wild-type immune cell.

17. The method of claim 1, wherein the method comprises introducing a mutation into a polynucleotide encoding a polypeptide selected from the group consisting of TIGIT, TGFBR2, ZAP70, NFATc1, and TET2.

18. The method includes the step of detecting a specific marker for V-Set immunoregulatory receptor (VISTA), T cell immunoglobulin mucin 3 (Tim-3), T cell immunoreceptor with Ig and ITIM domains (TIGIT), transforming growth factor beta receptor II (TGFbRII), regulatory factor X-related ankyrin-containing protein (RFXANK), PVR-related immunoglobulin domain containing (PVRIG), lymphocyte activation gene 3 (Lag3), cytotoxic T lymphocyte-associated protein 4 (CTLA-4), chitinase 3-like 1 (Chi3l1), cluster of differentiation 96 (CD96), B and T lymphocyte-associated (BTLA), Tet methylcytosine dioxygenase 2 (TET2), Sprouty RTK signaling antagonist 1 (Spry1), Sprouty RTK signaling antagonist 2 (Spry2), class II major histocompatibility complex transactivator (CIITA), cluster of differentiation 7 (CD7), cluster of differentiation 33 (CD33), differentiation 2. The method of claim 1, comprising introducing a mutation into a polynucleotide encoding a polypeptide selected from the group consisting of cluster of differentiation 52 (CD52), cluster of differentiation 123 (CD123), T cell receptor beta constant 1 (TRBC1), T cell receptor beta constant 2 (TRBC2), cytokine-inducible SH2-containing protein (CISH), acetyl-CoA acetyltransferase 1 (ACAT1), cytochrome P450 family 11 subfamily A member 1 (Cyp11a1), GATA binding protein 3 (GATA3), nuclear receptor subfamily 4 group A member 1 (NR4A1), nuclear receptor subfamily 4 group A member 2 (NR4A2), nuclear receptor subfamily 4 group A member 3 (NR4A3), methylation-regulated J protein (MCJ), Fas cell surface death receptor (FAS), and selectin P ligand / P-selectin glycoprotein ligand-1 (SELPG / PSGL1).

19. 19. The method of any one of claims 14 to 18, wherein the modified immune cells produced by the method express a chimeric antigen receptor.

20. 1. An in vitro or ex vivo method for producing an engineered immune cell, the method comprising expressing in or introducing into an immune cell a nucleobase editor polypeptide and contacting the cell with two or more guide RNAs capable of targeting a nucleic acid molecule encoding at least one polypeptide selected from the group consisting of T cell receptor alpha constant (TRAC), beta-2 microglobulin (B2M), programmed cell death 1 (PD1), cluster of differentiation 7 (CD7), cluster of differentiation 5 (CD5), cluster of differentiation 33 (CD33), cluster of differentiation 123 (CD123), Cbl proto-oncogene B (CBLB), and class II major histocompatibility complex transactivator (CIITA) polypeptide, thereby reducing expression of the at least one polypeptide compared to a wild-type immune cell, wherein the nucleobase editor polypeptide comprises at least one adenosine deaminase variant domain inserted into a nucleic acid programmable DNA binding protein (napDNAbp), and wherein the adenosine deaminase variant domain has the amino acid sequence MSEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWNRAIGLHDPTAHAEIMALRQGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIGRVVFGVRNAKTGAAGSLMDVLHYPGMNHRVEITEGILADECAALLCYFFRMPRQVFNAQKKAQSSTD (SEQ ID NO: 3) or a fragment thereof lacking only the N-terminal methionine, and having a total of up to 10 modifications, wherein the total of up to 10 modifications are i) a combination of modifications selected from any of the following with reference to SEQ ID NO:3: a) Y147R, Q154R, and Y123H; b) I76Y, Y123H, Y147R, and Q154R; c) V82S and Q154R; d) I76Y, V82S, Y123H, Y147R, and Q154R; e) Y147R, Q154R, Y123H, and V106W; f) I76Y, Y123H, Y147R, Q154R, I76Y, and V106W; g) V82S, Q154R, and V106W; h) I76Y, V82S, Y123H, Y147R, Q154R, and V106W; and ii) one or more amino acid modifications selected from the group consisting of I76Y, V82S, Y123H, Y147R, Y147T, Q154S, Q154R, and T166R with reference to SEQ ID NO:3, and / or S2A, H8Y, T17S, L18E, W23R, W23L, W23G, D24G, E25M, E25D, E25A, E25R, E25V, E25S, E25Y, E25G, R26W, R26G, R26N, R26Q, R26C, R26L, R26K, L34S, H3 6L, N37T, N37S, W45L, P48A, P48S, P48L, P48T, I49F, I49V, R51L, R51H, R52H, A56E, A56S, E59A, E59G, M61I, G67V, L68Q, M70V, M70L, Q71 R, Q71L, N72S, N72D, R74A, R74Q, D77G, L84F, E85K, E85G, A91T, M94L, I95L, H96L, S97C, R98Q, V102A, F104I, F104L, A106V, A106T, R107C , R107H, R107N, R107K, R107P, R107A, R107W, R107S, D108Y, D108N, D108G, D108R, D108Q, D108M, D108L, D108K, D108I, D108F, D108A, D1 08V, A109T, K110I, M118K, H123Y, G125A, N127S, R129Q, E134G, L137M, A138V, A142N, A142G, A142D, A143D, A143G, A143E, A143L, A143W, one or more amino acid modifications selected from the group consisting of A143M, A143S, A143Q, A143R, S146C, S146T, S146R, D147Y, F149Y, M151V, R152P, R152H, R152C, R153C, Q154H, Q154L, Q154R, E155V, E155G, E155D, I156F, I156Y, I156D, K157N, K157R, L157N, Q159L, K160S, K160E, K161Q, K161T, Q163H, and T166P It consists of: I) the napDNAbp is a Cas9 polypeptide and the adenosine deaminase variant domain is inserted between amino acid positions 768 and 769, 791 and 792, 792 and 793, 1015 and 1016, 1022 and 1023, 1026 and 1027, 1029 and 1030, 1040 and 1041, 1052 and 1053, 1054 and 1055, 1067 and 1068, 1068 and 1069, 1247 and 1248, or 1248 and 1249 in the Cas9 reference sequence of SEQ ID NO:1; or II) the napDNAbp is a Cas12 polypeptide and the adenosine deaminase variant domain is: a) between amino acid positions 153 and 154, 255 and 256, 306 and 307, 980 and 981, 1019 and 1020, 534 and 535, 604 and 605, or 344 and 345 of BhCas12b, or the corresponding amino acid residue positions of Cas12a, Cas12c, Cas12d, Casl2e, Casl2g, Casl2h, or Casl2i; b) between amino acid positions 147 and 148, 248 and 249, 299 and 300, 991 and 992, or 1031 and 1032 of BvCasl2b, or the corresponding amino acid residue positions of Cas12a, Cas12c, Cas12d, Casl2e, Casl2g, Casl2h, or Casl2i; or c) between amino acid positions 157 and 158, 258 and 259, 310 and 311, 1008 and 1009, or 1044 and 1045 of AaCasl2b, or the corresponding amino acid residue positions of Cas12a, Cas12c, Cas12d, Casl2e, Casl2g, Casl2h, or Casl2i. Inserted in method.

21. 21. The method of claim 20, wherein the immune cell is a T cell.

22. 22. The method of claim 21, wherein the modified immune cells produced by the method express a chimeric antigen receptor.

23. a polynucleotide programmable DNA binding domain; The amino acid sequence MSEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWNRAIGLHDPTAHAEIMALRQGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIGRVVFGVRNAKTGAAGSLMDVLHYPGMNHRVEITEGILADECAALLCYFFRMPRQVFNAQKKAQSSTD (SEQ ID NO: 3) or a fragment thereof lacking only the N-terminal methionine, and having a total of up to 10 modifications. and two or more guide RNAs that target the nucleobase editor polypeptide to result in an alteration in a nucleic acid molecule encoding at least one polypeptide selected from the group consisting of T cell receptor alpha constant (TRAC), beta-2 microglobulin (B2M), programmed cell death 1 (PD1), cluster of differentiation 7 (CD7), cluster of differentiation 5 (CD5), cluster of differentiation 33 (CD33), cluster of differentiation 123 (CD123), Cbl proto-oncogene B (CBLB), and class II major histocompatibility complex transactivator (CIITA) polypeptides, thereby reducing expression of the at least one polypeptide as compared to a wild-type immune cell. Including, wherein the total of up to 10 modifications is i) a combination of modifications selected from any of the following with reference to SEQ ID NO:3: a) Y147R, Q154R, and Y123H; b) I76Y, Y123H, Y147R, and Q154R; c) V82S and Q154R; d) I76Y, V82S, Y123H, Y147R, and Q154R; e) Y147R, Q154R, Y123H, and V106W; f) I76Y, Y123H, Y147R, Q154R, I76Y, and V106W; g) V82S, Q154R, and V106W; h) I76Y, V82S, Y123H, Y147R, Q154R, and V106W; and ii) one or more amino acid modifications selected from the group consisting of I76Y, V82S, Y123H, Y147R, Y147T, Q154S, Q154R, and T166R with reference to SEQ ID NO:3, and / or S2A, H8Y, T17S, L18E, W23R, W23L, W23G, D24G, E25M, E25D, E25A, E25R, E25V, E25S, E25Y, E25G, R26W, R26G, R26N, R26Q, R26C, R26L, R26K, L34S, H3 6L, N37T, N37S, W45L, P48A, P48S, P48L, P48T, I49F, I49V, R51L, R51H, R52H, A56E, A56S, E59A, E59G, M61I, G67V, L68Q, M70V, M70L, Q71 R, Q71L, N72S, N72D, R74A, R74Q, D77G, L84F, E85K, E85G, A91T, M94L, I95L, H96L, S97C, R98Q, V102A, F104I, F104L, A106V, A106T, R107C , R107H, R107N, R107K, R107P, R107A, R107W, R107S, D108Y, D108N, D108G, D108R, D108Q, D108M, D108L, D108K, D108I, D108F, D108A, D1 08V, A109T, K110I, M118K, H123Y, G125A, N127S, R129Q, E134G, L137M, A138V, A142N, A142G, A142D, A143D, A143G, A143E, A143L, A143W, one or more amino acid modifications selected from the group consisting of A143M, A143S, A143Q, A143R, S146C, S146T, S146R, D147Y, F149Y, M151V, R152P, R152H, R152C, R153C, Q154H, Q154L, Q154R, E155V, E155G, E155D, I156F, I156Y, I156D, K157N, K157R, L157N, Q159L, K160S, K160E, K161Q, K161T, Q163H, and T166P A base editor system consisting of:

24. Two or more guide RNAs; The following array: wherein the bolded sequences represent sequences derived from Cas9, the italicized sequences represent linker sequences, and the underlined sequences represent bipartite nuclear localization sequences; and The amino acid sequence MSEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWNRAIGLHDPTAAHAEIMALRQGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIGRVVFGVRNAKTGAAGSLMDVLHYPGMNHRVEITEGILADECAALLCYFFRMPRQVFNAQKKAQSST or a fragment thereof lacking only the N-terminal methionine, and a fusion protein comprising at least one base editor domain comprising an adenosine deaminase variant comprising the above amino acid sequence or a fragment thereof, having a total of up to 10 modifications.

1. A base editor system comprising: the two or more guide RNAs target the fusion protein to result in an alteration in a nucleic acid molecule encoding at least one polypeptide selected from the group consisting of T cell receptor alpha constant (TRAC), beta-2 microglobulin (B2M), programmed cell death 1 (PD1), cluster of differentiation 7 (CD7), cluster of differentiation 5 (CD5), cluster of differentiation 33 (CD33), cluster of differentiation 123 (CD123), Cbl proto-oncogene B (CBLB), and class II major histocompatibility complex transactivator (CIITA) polypeptides, thereby reducing expression of the at least one polypeptide compared to a wild-type immune cell; The total of up to 10 modifications are: i) a combination of modifications selected from any of the following with reference to SEQ ID NO:3: a) Y147R, Q154R, and Y123H; b) I76Y, Y123H, Y147R, and Q154R; c) V82S and Q154R; d) I76Y, V82S, Y123H, Y147R, and Q154R; e) Y147R, Q154R, Y123H, and V106W; f) I76Y, Y123H, Y147R, Q154R, I76Y, and V106W; g) V82S, Q154R, and V106W; h) I76Y, V82S, Y123H, Y147R, Q154R, and V106W; and ii) one or more amino acid modifications selected from the group consisting of I76Y, V82S, Y123H, Y147R, Y147T, Q154S, Q154R, and T166R with reference to SEQ ID NO:3, and / or S2A, H8Y, T17S, L18E, W23R, W23L, W23G, D24G, E25M, E25D, E25A, E25R, E25V, E25S, E25Y, E25G, R26W, R26G, R26N, R26Q, R26C, R26L, R26K, L34S, H3 6L, N37T, N37S, W45L, P48A, P48S, P48L, P48T, I49F, I49V, R51L, R51H, R52H, A56E, A56S, E59A, E59G, M61I, G67V, L68Q, M70V, M70L, Q71 R, Q71L, N72S, N72D, R74A, R74Q, D77G, L84F, E85K, E85G, A91T, M94L, I95L, H96L, S97C, R98Q, V102A, F104I, F104L, A106V, A106T, R107C , R107H, R107N, R107K, R107P, R107A, R107W, R107S, D108Y, D108N, D108G, D108R, D108Q, D108M, D108L, D108K, D108I, D108F, D108A, D1 08V, A109T, K110I, M118K, H123Y, G125A, N127S, R129Q, E134G, L137M, A138V, A142N, A142G, A142D, A143D, A143G, A143E, A143L, A143W, one or more amino acid modifications selected from the group consisting of A143M, A143S, A143Q, A143R, S146C, S146T, S146R, D147Y, F149Y, M151V, R152P, R152H, R152C, R153C, Q154H, Q154L, Q154R, E155V, E155G, E155D, I156F, I156Y, I156D, K157N, K157R, L157N, Q159L, K160S, K160E, K161Q, K161T, Q163H, and T166P Consists of: Base editor system.

25. 25. An in vitro or ex vivo cell comprising the base editor system of claim 23 or 24, wherein the cell is not a cell of a human embryo.

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