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

Abstract

The invention features genetically modified immune cells comprising a novel adenosine base editor (e.g., ABE8) having enhanced anti-tumor activity, resistance to immunosuppression, and reduced risk of causing graft-versus-host reaction or host-versus-graft reaction, or a combination thereof. The invention also features methods of producing and using these modified immune effector cells.

Description

[0001] Related applications

[0002] This application is an international PCT application claiming priority and interest in U.S. Provisional Application No. 62 / 805,271, filed February 13, 2019; No. 62 / 852,228, filed May 23, 2019; No. 62 / 852,224, filed May 23, 2019; No. 62 / 931,722, filed November 6, 2019; No. 62 / 941,523, filed November 27, 2019; No. 62 / 941,569, filed November 27, 2019; and No. 62 / 966,526, filed January 27, 2020, the entire contents of which are incorporated herein by reference.

[0003] References merged

[0004] All publications, patents, and patent applications mentioned in this specification are incorporated herein by reference as if each individual publication, patent, or patent application were specifically and individually indicated as being incorporated by reference. Unless otherwise stated, all publications, patents, and patent applications mentioned in this specification are incorporated herein by reference in their entirety. Technical Field

[0005] This application relates to genetically modified immune cells containing a novel adenosine base editor, which have enhanced antitumor activity, resistance to immunosuppression, and a reduced risk of inducing graft-versus-host disease or host-versus-graft disease, or a combination thereof. Background Technology

[0006] Autologous and allogeneic immunotherapy are cancer treatments 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 a subject (autologous) or from a donor isolated from a treated subject (allogeneic) and genetically modified to express chimeric antigen receptors. The resulting cells express chimeric antigen receptors on their cell surface (e.g., CAR T cells), and upon administration to the subject, the chimeric antigen receptors bind to markers expressed by tumor cells. This interaction with tumor markers activates CAR-T cells, which then kill the tumor cells. However, for autologous or allogeneic cell therapies to be effective and efficient, significant conditions and cellular responses, such as inhibition of T cell signaling, must be overcome or avoided. For allogeneic cell therapies, graft-versus-host disease (GVHD) and host rejection of CAR-T cells can pose additional challenges. Editing genes involved in these processes can enhance CAR-T cell function and resistance to immunosuppression or suppression, but current methods for performing such editing have the potential to induce significant genomic rearrangements in CAR-T cells, negatively impacting their efficacy. Therefore, there is an urgent need for technologies that can more precisely modify immune cells, especially CAR-T cells. This application addresses this need and other important requirements. Summary of the Invention

[0007] The present invention is characterized by genetically modified immune cells comprising a novel adenosine base editor (e.g., ABE8) that possess enhanced antitumor activity, resistance to immunosuppression, and a reduced risk of inducing graft-versus-host disease or host-versus-graft disease, or a combination thereof. The present invention is further characterized by methods for producing and using these modified immune effector cells.

[0008] On one hand, the present invention provides a method for generating modified immune cells, the method comprising expressing or introducing a nucleobase editor polypeptide into the immune cells and contacting the cells with two or more guide RNAs targeting the nucleobase editor polypeptide to influence alterations in nucleic acid molecules, said nucleic acid molecules encoding at least one polypeptide selected from the group consisting of T cell receptor α constant (TRAC), β-2 microglobulin (B2M), programmed cell death 1 (PD1), differentiation cluster 7 (CD7), differentiation cluster 5 (CD5), differentiation cluster 33 (CD33), differentiation cluster 123 (CD123), Cbl proto-oncogene B (CBLB), and major histocompatibility complex transactivator (CIITA) polypeptides, wherein the nucleobase... The editor polypeptide comprises a nucleic acid programmable DNA-binding protein (napDNAbp) and a base editor domain comprising an adenosine deaminase variant domain, wherein the domain contains a change at amino acid positions 82 and / or 166 of MSEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWNRAIGLHDPTAHAEIMALRQGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIGRVVFGVRNAKTGAAGSLMDVLHYPGMNHRVEITEGILADECAALLCYFFRMPRQVFNAQKKAQSSTD. In one embodiment, the immune cell is a T cell. In one embodiment, the immune cell is derived from a healthy subject.

[0009] In one embodiment, the adenosine deaminase variant domain includes changes at amino acid positions 82 and 166. In one embodiment, the adenosine deaminase variant domain includes a change in V82S. In one embodiment, the adenosine deaminase variant domain includes a change in T166R. In one embodiment, the adenosine deaminase variant domain includes changes in both V82S and T166R. In one embodiment, the adenosine deaminase variant domain further includes one or more of the following changes: Y147T, Y147R, Q154S, Y123H, and / or Q154R. In one embodiment, the adenosine deaminase variant domain comprises altered combinations selected from the group consisting of: Y147T+Q154R; Y147T+Q154S; Y147R+Q154S; V82S+Q154S; V82S+Y147R; V82S+Q154R; V82S+Y123H; I76Y+V82S; V82S+Y123H+Y147T; V82S+Y123 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 one embodiment, the adenosine deaminase variant domain comprises a modified combination: V82S+Q154R. In another embodiment, the adenosine deaminase variant domain comprises a modified combination: Y147R+Q154R+Y123H. In one embodiment, the adenosine deaminase variant domain comprises a modified combination: Y147R+Q154R+Y123H+I76Y. In another embodiment, the adenosine deaminase variant domain comprises a modified combination: I76Y+V82S+Y123H+Y147R+Q154R. In yet another embodiment, the adenosine deaminase variant is TadA*8. In one embodiment, the 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.

[0010] In one embodiment, the adenosine deaminase variant domain comprises a C-terminal deletion starting from 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 an adenosine deaminase variant monomer. 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, and ABE8.24-m.

[0011] 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.

[0012] 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, 16, 17, 18, 19, or 20 N-terminal amino acid residues relative to the full-length adenosine deaminase. In one embodiment, the adenosine deaminase variant domain comprises or is substantially composed of sequences or fragments thereof having adenosine deaminase activity:

[0013] MSEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWNRAIGLHDPTAHAEIMALRQGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIGRVVFGVRNAKTGAAGSLMDVLHYPGMNHRVEITEGILADECAALLCTFFRMPRQVFNAQKKAQSSTD.

[0014] In one implementation, the napDNAbp contains the following sequence:

[0015]

[0016] In this context, bold sequences represent sequences derived from Cas9, italic sequences represent connecting subsequences, and underlined sequences represent dual-core localization sequences.

[0017] In various embodiments of any aspect described herein, the napDNAbp is Staphylococcus aureus Cas9 (SaCas9), Streptococcus thermophilus 1Cas9 (St1Cas9), Streptococcus pyogenes Cas9 (SpCas9), or a variant thereof. In one embodiment, the napDNAbp comprises a variant of SpCas9 having modified protospacer sequence adjacent motif (PAM) specificity or specificity for non-G PAM. In one embodiment, the modified PAM is specific for the nucleic acid sequence 5'-NGC-3'. In one embodiment, the modified SpCas9 comprises amino acid substitutions of D1135M, S1136Q, G1218K, E1219F, A1322R, D1332A, R1335E, and T1337R, or corresponding amino acid substitutions thereof. In various embodiments of any aspect described herein, the napDNAbp comprises nuclease-dead Cas9 (dCas9), Cas9 nickase (nCas9), or nuclease-active Cas9. In one embodiment, the nicking enzyme variant comprises an amino acid substitution D10A or a corresponding amino acid substitution thereof. In various embodiments of any aspect described herein, the base editor polypeptide further comprises a zinc finger domain. In various embodiments of any aspect described herein, the base editor polypeptide further comprises one or more uracil glycosylation 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 modified adenosine deaminase not found in nature. In various embodiments of any aspect described herein, the adenosine deaminase variant is TadA*8. In some embodiments, the 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.

[0018] 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 following amino acid sequence:

[0019] SGGSSGGSSGSETPGTSESATPES.

[0020] In various embodiments of any aspect described herein, the base editor polypeptide further comprises one or more nuclear localization signals (NLS). In one embodiment, the NLS is a bidirectional NLS. In one embodiment, the nuclear base editor polypeptide comprises an N-terminal NLS and a C-terminal NLS. In various embodiments of any aspect described herein, the napDNAbp is modified Staphylococcus aureus Cas9 (SaCas9). In one embodiment, the modified SaCas9 comprises amino acid substitutions of E782K, N968K, and R1015H, or corresponding amino acid substitutions thereof. In one embodiment, the modified SaCas9 comprises the following amino acid sequence:

[0021]

[0022] In various embodiments of any aspect described herein, two or more guide RNAs are expressed or contacted in the cell, each targeting a separate polynucleotide. In various embodiments, multiple base editing involves the 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 genomic sites. In various embodiments of any aspect described herein, two or more guide RNAs are expressed or contacted in the cell, each targeting a B2M or TRAC polynucleotide. In various embodiments of any aspect described herein, three guide RNAs are expressed or contacted in the cell. In various embodiments of any aspect described herein, three guide RNAs are expressed or contacted in the cell, each targeting a B2M, CD7, TRAC, CIITA, PDCD1 and / or CBLC polynucleotide. In various embodiments of any aspect described herein, three guide RNAs are expressed or contacted in the cell, each targeting a B2M, TRAC and PDCD1 polynucleotide. In various embodiments of any aspect described herein, three guide RNAs are expressed or contacted in cells, each targeting B2M, TRAC, and CIITA polynucleotides respectively. In various embodiments of any aspect described herein, four guide RNAs are expressed or contacted in cells, each targeting B2M, CD7, TRAC, CIITA, PDCD1, and / or CBLC polynucleotides respectively. 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 splice acceptor sites or splice donor sites in target polynucleotides. 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 implementations, by introducing a base editor and a guide RNA for one or more genes that target and encode polypeptides, the expression of one or more of the aforementioned 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.

[0023] On the other hand, the present invention provides the expression of chimeric antigen receptors (CARs) in modified immune cells in any aspect described herein. In various embodiments of any aspect described herein, the immune cells are modified in vitro. In various embodiments of any aspect described herein, the immune cells are cytotoxic T cells, regulatory T cells, or T helper cells. In various embodiments of any aspect described herein, the modified immune cells do not contain detectable translocations.

[0024] On the other hand, the present invention provides modified immune cells generated according to methods described in any aspect herein. In various embodiments of any aspect described herein, the cells have reduced immunogenicity and enhanced antitumor activity. In various embodiments of any aspect described herein, the immune cells express chimeric antigen receptors.

[0025] In various embodiments of any aspect described herein, the immune cells are T cells. In various embodiments of any aspect described herein, the cells contain one or more mutations in polynucleotides encoding B2M, CD7, CIITA, PD1, CBLB, and / or TRAC. In one embodiment, the cells contain one or more mutations in polynucleotides encoding B2M, TRAC, and CIITA. In various embodiments of any aspect described herein, the cells contain mutations in one or more polynucleotides encoding TIGIT, TGFBR2, ZAP70, NFATC1, or TET2. In any of the various embodiments described herein, the cells encode V-Set immunomodulatory receptor (VISTA), T cell immunoglobulin mucin 3 (Tim-3), T cell immune receptor with Ig and ITIM domains (TIGIT), transforming growth factor β receptor II (TGFbRII), regulatory factor X-associated ankylosing protein (RFXANK), PVR-associated immunoglobulin domain (PVRIG), lymphocyte activation gene 3 (Lag3), cytotoxic T lymphocyte-associated protein 4 (CTLA-4), chitinase 3-like 1 (Chi3l1), differentiation cluster 96 (CD96), B and T lymphocyte-associated protein (BTLA), Tet methylcytosine dioxygenase 2 (TET2), SproutyRTK signaling antagonist 1 (Spry1). The mutation is present in one or more polynucleotides of RTK signaling antagonist 2 (Spry2), major histocompatibility complex transactivator class II (CIITA), differentiation cluster 7 (CD7), differentiation cluster 33 (CD33), differentiation cluster 52 (CD52), differentiation cluster 123 (CD123), T cell receptor β constant 1 (TRBC1), T cell receptor β 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 4A group member 1 (NR4A1), nuclear receptor subfamily 4A group member 2 (NR4A2), nuclear receptor subfamily 4A group member 3 (NR4A3), methylation-controlled J protein (MCJ), Fas cell surface death receptor (FAS), or selectin P ligand / P-selectin glycoprotein ligand-1 (SELPG / PSGL1).

[0026] In various embodiments of any aspect described herein, the chimeric antigen receptor comprises an extracellular domain having affinity for a tumor-associated biomarker. In one embodiment, the tumor is multiple myeloma. In various embodiments of any aspect described herein, the biomarker is B-cell maturation antigen (BCMA).

[0027] On the other hand, the present invention provides a method for 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.

[0028] On the other hand, the present invention provides a method for treating a tumor in a subject, the method comprising administering an effective amount of modified immune cells according to any aspect described herein.

[0029] On the other hand, the present invention provides a pharmaceutical composition for treating tumors, the pharmaceutical composition comprising an effective amount of modified immune cells according to any aspect described herein.

[0030] On the other hand, the present invention provides a pharmaceutical composition comprising, in a pharmaceutically acceptable excipient, an effective amount of modified immune cells according to any aspect described herein.

[0031] On the other hand, the present invention provides a kit for treating tumors, the kit comprising modified immune cells according to any aspect described herein. In various embodiments of any aspect described herein, the kit includes written instructions for treating tumors using the modified immune effector cells.

[0032] In various embodiments of any aspect described herein, the modified immune cells further comprise a chimeric antigen receptor with affinity for tumor-associated biomarkers. In some embodiments, the chimeric antigen receptor is introduced into the cells via a viral vector, such as a lentiviral vector. In some embodiments, the chimeric antigen receptor is introduced into the cells via a double-stranded DNA template to insert into a nuclease-cleaved locus. In various embodiments of any aspect described herein, the chimeric antigen receptor comprises an extracellular domain with affinity for tumor-associated biomarkers.

[0033] In any of the various embodiments described herein, the tumor is B-cell carcinoma. In any of the various embodiments described herein, the B-cell carcinoma is lymphoma or leukemia. In any of the various embodiments described herein, the B-cell carcinoma is multiple myeloma.

[0034] On the other hand, the present invention provides a method for treating a subject suffering from or with a predisposition to develop graft-versus-host disease (GVHD) using an effective amount of modified immune cells according to any aspect described herein. On the other hand, the present invention provides a pharmaceutical composition for treating GVHD, the pharmaceutical composition comprising an effective amount of modified immune cells according to any aspect described herein. On the other hand, the present invention provides a kit for treating GVHD, the kit comprising modified immune cells according to any aspect described herein. In various embodiments of any aspect described herein, the modified immune cells lack or have reduced levels of functional TRAC.

[0035] On the other hand, the present invention provides a method for treating a subject suffering from or with a predisposition to develop graft-versus-host disease (HVGD) using an effective amount of modified immune cells according to any aspect described herein. On the other hand, the present invention provides a pharmaceutical composition for treating HVGD, the pharmaceutical composition comprising an effective amount of modified immune cells according to any aspect described herein. On the other hand, the present invention provides a kit for treating HVGD, the kit comprising modified immune cells according to any aspect described herein. In various embodiments of any aspect described herein, the modified immune cells lack or have reduced levels of functional B2M.

[0036] On the other hand, the present invention provides a method for generating modified immune cells, the method comprising expressing or introducing a nucleobase editor polypeptide into the immune cells and contacting the cells with two or more guide RNAs capable of targeting nucleic acid molecules, the nucleic acid molecules encoding at least one polypeptide selected from the group consisting of T cell receptor α constant (TRAC), β-2 microglobulin (B2M), programmed cell death 1 (PD1), differentiation cluster 7 (CD7), differentiation cluster 5 (CD5), differentiation cluster 33 (CD33), differentiation cluster 123 (CD123), Cbl proto-oncogene B (CBLB), and 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).

[0037] In one embodiment, the adenosine deaminase variant domain comprises an amino acid sequence:

[0038] MSEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWNRAIGLHDPTAHAEIMALRQGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIGRVVFGVRNAKTGAAGSLMDVLHYPGMNHRVEITEGILADECAALLCYFFRMPRQVFNAQKKAQSSTD, wherein the amino acid sequence contains at least one alteration. In one embodiment, the adenosine deaminase variant domain contains an alteration at amino acid positions 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 one altered combination of the following: Y147T+Q154R; Y147T+Q154S; Y147R+Q154S; V82S+Q154S; V82S+Y147R; V82S+Q154R; V82S+Y123H; I76Y+V82S; V82S+Y123H+Y147T; V82S+Y123H+Y 147R; 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 starting from residues selected from the group consisting of 149, 150, 151, 152, 153, 154, 155, 156, and 157. In one embodiment, the domain of the adenosine deaminase variant is an adenosine deaminase monomer. In one embodiment, the adenosine deaminase variant is an adenosine deaminase 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 containing a TadA domain and an adenosine deaminase variant domain.

[0039] In one embodiment, the napDNAbp is a Cas9 or Cas12 polypeptide. In one embodiment, the adenosine deaminase variant is inserted into a flexible loop, α-helix region, unstructured portion, or solvent-accessible portion of the napDNAbp. In one embodiment, the adenosine deaminase variant is flanked by the N-terminal and C-terminal fragments 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, wherein each instance of "]-[" is an optional linker. In one embodiment, the C-terminus of the N-terminal fragment or the N-terminus of the C-terminal fragment comprises a portion of the flexible loop of the napDNAbp. In one embodiment, the flexible loop comprises an amino acid adjacent to the target nucleobase. In one embodiment, the target nucleobase is 1 to 20 nucleosides from the PAM sequence in the target polynucleotide sequence. In some embodiments, the target nucleobase is 2 to 12 nucleosides upstream of the PAM sequence. In one embodiment, the N-terminal fragment or the C-terminal fragment of the napDNAbp binds to the target polynucleotide sequence.

[0040] In some embodiments, the N-terminal fragment or the C-terminal fragment contains a RuvC domain; the N-terminal fragment or the C-terminal fragment contains an NHN domain; neither the N-terminal fragment nor the C-terminal fragment contains an HNH domain; or neither the N-terminal fragment nor the C-terminal fragment contains a RuvC domain. In some embodiments, the napDNAbp contains a partial or complete deletion in one or more domains, and the deaminase is inserted at the partial or complete deletion site of the napDNAbp. In some embodiments, the deletion is within the RuvC domain; the deletion is within the HNH domain; or the deletion bridges the RuvC domain and the C-terminal domain, the L1 domain and the HNH domain, or the RuvC domain and the L1 domain.

[0041] In another embodiment, the napDNAbp is a Cas9 or Cas12 polypeptide. In one embodiment, the napDNAbp contains a Cas9 polypeptide. In one embodiment, the Cas9 polypeptide is Streptococcus pyogenes Cas9 (SpCas9), Staphylococcus aureus Cas9 (SaCas9), Streptococcus thermophilus 1Cas9 (St1Cas9), or a variant thereof. In one embodiment, the Cas9 polypeptide has the following amino acid sequence (Cas9 reference sequence):

[0042]

[0043] (Single underscore: HNH domain; double underscore: RuvC domain; (Cas9 reference sequence), or the corresponding region thereof.)

[0044] In some embodiments, the Cas9 polypeptide comprises the deletion of amino acids 1017 to 1069 or their corresponding amino acids as numbered in the Cas9 polypeptide reference sequence; the Cas9 polypeptide comprises the deletion of amino acids 792 to 872 or their corresponding amino acids as numbered in the Cas9 polypeptide reference sequence; or the Cas9 polypeptide comprises the deletion of amino acids 792 to 906 or their corresponding amino acids as numbered in the 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 numbered 530 to 537, 569 to 579, 686 to 691, 768 to 793, 943 to 947, 1002 to 1040, 1052 to 1077, 1232 to 1248, and 1298 to 1300 in the Cas9 reference sequence, or their corresponding amino acid positions. In one embodiment, the deaminase is inserted into amino acid positions numbered 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, in the Cas9 reference sequence. In another embodiment, the deaminase is inserted into amino acid positions numbered 768-769, 792-793, 1022-1023, 1026-1027, 1040-1041, 1068-1069, or 1247-1248, or their corresponding amino acid positions, in the Cas9 reference sequence. In one embodiment, the deaminase is inserted into amino acid positions numbered 1016 to 1017, 1023 to 1024, 1029 to 1030, 1040 to 1041, 1069 to 1070, or 1247 to 1248, or corresponding amino acid positions, of the Cas9 reference sequence. In one embodiment, an adenosine deaminase variant is inserted into the Cas9 polypeptide at the locus identified in Table 13A. In one embodiment, the N-terminal fragment comprises amino acid residues 1 to 529, 538 to 568, 580 to 685, 692 to 942, 948 to 1001, 1026 to 1051, 1078 to 1231, and / or 1248 to 1297, or corresponding residues, of the Cas9 reference sequence. In one embodiment, the C-terminal fragment comprises amino acid residues 1301 to 1368, 1248 to 1297, 1078 to 1231, 1026 to 1051, 948 to 1001, 692 to 942, 580 to 685 and / or 538 to 568 of the Cas9 reference sequence, or the corresponding residues thereof.

[0045] In another embodiment, the Cas9 peptide is a modified Cas9 and is specific for the altered PAM. In one embodiment, the Cas9 peptide is a nickase or the Cas9 peptide is nuclease-free. In one embodiment, the Cas9 peptide is a modified SpCas9 peptide. In one embodiment, the modified SpCas9 peptide comprises amino acid substitutions D1135M, S1136Q, G1218K, E1219F, A1322R, D1332A, R1335E, and T1337R (SpCas9-MQKFRAER) and is specific for the altered PAM 5'-NGC-3'.

[0046] In some embodiments, the adenosine deaminase variant is inserted into the 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 inserted between the following amino acid positions: a) 153 to 154, 255 to 256, 306 to 307, 980 to 981, 1019 to 1020, 534 to 535, 604 to 605, or 344 to 345 of BhCas12b, or the corresponding amino acid residues of Cas12a, Cas12c, Cas2d, Cas12e, Cas12g, Cas12h, or Cas12i; b) 147 and 148, 248 and 249, 299 of BvCas12b. The amino acid residues of 300, 991 and 992 or 1031 and 1032 or Cas12a, Cas12c, Cas2d, Cas12e, Cas12g, Cas12h or Cas12i; or c) 157 and 158, 258 and 259, 310 and 311, 1008 and 1009 or 1044 and 1045 or Cas12a, Cas12c, Cas2d, Cas12e, Cas12g, Cas12h or Cas12i of AaCas12b. In one embodiment, the deaminase variant is inserted into the Cas12 polypeptide at the 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.

[0047] On one hand, the present invention provides modified immune cells according to any aspect described herein. In one embodiment, the immune cells are T cells. In one embodiment, the immune cells express chimeric antigen receptors. In one embodiment, the method comprises administering an effective amount of modified immune cells according to any aspect described herein. On another hand, the present invention provides a pharmaceutical composition comprising an effective amount of modified immune cells according to any aspect described herein in a pharmaceutically acceptable excipient. On yet another hand, the present invention provides a kit comprising modified immune cells according to any aspect described herein.

[0048] In one aspect, the present invention provides a base editor system comprising a polynucleotide programmable DNA binding domain and at least one base editor domain, wherein the base editor domain contains...

[0049] MSEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWNRAIGLHDPTAHAEIMALRQGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIGRVVFGVRNAKTGAAGSLMDVLHYPGMNHRVEITEGILADECAALLCYFFRMPRQVFNAQKKAQSSTD, an adenosine deaminase variant altered at amino acid position 82 or 166, and two or more guide RNAs targeting the nucleobase editor polypeptide to influence alterations in nucleic acid molecules, said nucleic acid molecules encoding at least one polypeptide selected from the group consisting of T cell receptor α constant (TRAC), β-2 microglobulin (B2M), programmed cell death 1 (PD1), differentiation cluster 7 (CD7), differentiation cluster 5 (CD5), differentiation cluster 33 (CD33), differentiation cluster 123 (CD123), Cbl proto-oncogene B (CBLB), and major histocompatibility complex transactivator (CIITA) polypeptides. In some embodiments, the adenosine deaminase variant comprises a change in V82S and / or a change in T166R. In some embodiments, the adenosine deaminase variant further comprises one or more of the following changes: Y147T, Y147R, Q154S, Y123H, and Q154R. In some embodiments, the base editor domain is 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 TadA*8 lacking 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 N-terminal amino acid residues relative to the full-length TadA*8. In some embodiments, the adenosine deaminase variant is missing 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 C-terminal amino acid residues relative to the full-length TadA8. In some embodiments, the polynucleotide-programmable DNA-binding domain is Staphylococcus aureus Cas9 (SaCas9), Streptococcus thermophilus 1Cas9 (St1Cas9), Streptococcus pyogenes Cas9 (SpCas9), or a variant thereof. In some embodiments, the polynucleotide-programmable DNA-binding domain is a variant of SpCas9 that has modified protospacer adjacent motif (PAM) specificity or is specific to non-G PAMs. In some embodiments, the polynucleotide-programmable DNA-binding domain is Cas9 without nuclease activity. In some embodiments, the polynucleotide-programmable DNA-binding domain is a Cas9 nickase.

[0050] On one hand, the present invention provides a base editor system comprising two or more guide RNAs and a fusion protein, wherein the fusion protein comprises a multinucleotide programmable DNA-binding domain comprising the following sequence:

[0051]

[0052] The bolded sequences represent sequences derived from Cas9, the italicized sequences represent linker sequences, the underlined sequences represent binuclear localization sequences, and at least one base editor domain is included. This base editor domain is contained within MSEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWNRAIGLHDPTAHAEIMALRQGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIGRVVFGVRNAKTGAAGSLMDVLHYPGMNHRVEITEGILADECAALLCYFFRMPRQV FNAQKKAQSSTD comprises an adenosine deaminase variant altered at amino acid position 82 or 166 and two or more guide RNAs targeting the nucleobase editor polypeptide to influence alterations in nucleic acid molecules that encode at least one polypeptide selected from the group consisting of T cell receptor α constant (TRAC), β-2 microglobulin (B2M), programmed cell death 1 (PD1), differentiation cluster 7 (CD7), differentiation cluster 5 (CD5), differentiation cluster 33 (CD33), differentiation cluster 123 (CD123), Cbl proto-oncogene B (CBLB), and major histocompatibility complex transactivator (CIITA) polypeptides.

[0053] On one hand, a cell comprising any one of the base editor systems described above is provided. Any type of cell is a human cell or a mammalian cell. In some embodiments, the cell is ex vivo, in vivo, or in vitro.

[0054] The descriptions and examples herein illustrate embodiments of this disclosure in detail. It should be understood that this disclosure is not limited to the specific embodiments described herein and therefore can be varied. Those skilled in the art will recognize that numerous changes and modifications are possible with respect to the content of this disclosure, and these changes and modifications are included within its scope.

[0055] Unless otherwise specified, the practice of some of the embodiments disclosed herein employs conventional techniques of immunology, biochemistry, chemistry, molecular biology, microbiology, cell biology, genomics, and recombinant DNA, which are within the scope of the art. See, for example, Sambrook and Green, Molecular Cloning: A Laboratory Manual, 4th Edition (2012); the series Current Protocols in Molecular Biology (FMAusubel et al., eds.); the series Methods In Enzymology (Academic Press, Inc.), PCR 2: A Practical Approach (MJ MacPherson, B.D. Hames, and G.G. 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 (RI Freshney, ed. (2010)).

[0056] The chapter headings used here are for organizational purposes only and should not be construed as limiting the topics described.

[0057] While various features of this disclosure may be described in the context of a single embodiment, these features may also be provided individually or in any suitable combination. Conversely, although this disclosure may be described herein in the context of a single embodiment for clarity, it may also be implemented in a single embodiment. The section headings used herein are for organizational purposes only and should not be construed as limiting the subject matter described.

[0058] The features of this disclosure are specifically set forth in the appended claims. A better understanding of the features and advantages of the invention will be obtained by referring to the following detailed description of illustrative embodiments, in which the principles of this disclosure are utilized, and by taking into account the accompanying drawings.

[0059] definition

[0060] The following definitions supplement those in the art and are specific to the present application, and are not attributable to any related or unrelated cases, such as any co-owned patents or applications. While any methods and materials similar or equivalent to those described herein may be used in the practice of testing this disclosure, 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.

[0061] Unless otherwise defined, all technical and scientific terms used herein have the meanings commonly understood by one of ordinary skill in the art to which this invention pertains. The following references provide general definitions for 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 Harper Collins Dictionary of Biology (1991).

[0062] In this application, unless otherwise specified, the use of the singular includes the plural. It must be noted that the singular forms “a,” “an,” and “the” used in the specification include plural references unless the context clearly specifies otherwise. In this application, unless otherwise specified, the use of “or” means “and / or” and is understood to be inclusive. Furthermore, the use of the term “including” and other forms such as “include,” “includes,” and “included” is not restrictive.

[0063] As used in this specification and claims, the terms “comprising” (and any form of inclusion, such as “comprise” and “comprises”), “having” (and any form of having, such as “have” and “has”), “including” (and any form of inclusion, such as “includes” and “include”), or “containing” (and any form of containing, such as “contains” and “contain”) are inclusive or open-ended and do not exclude additional, unmentioned elements or method steps. It is contemplated that any embodiments discussed in this specification can be implemented with respect to any method or combination of this disclosure, and vice versa. Furthermore, the components of this disclosure can be used to implement the methods of this disclosure.

[0064] The terms "about" or "approximately" refer to a specific value within an acceptable margin of error as determined by someone skilled in the art, which will depend in part on how the value is measured or determined, i.e., the measurement system. For example, according to practice in the art, "about" may mean within or more than one standard deviation. Alternatively, "about" may mean a range of up to 20%, up to 10%, up to 5%, or up to 1% of a given value. Or, particularly for biological systems or processes, the term may mean within an order of magnitude, such as within 5 times or 2 times the value. Where a specific value is described in the application and claims, unless otherwise stated, the meaning of the term "about" should be assumed to be within an acceptable margin of error for the specific value.

[0065] The range provided here should be understood as an abbreviation of all values ​​within that range. For example, the range 1 to 50 is understood to include values ​​from 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.

[0066] References to “some embodiments,” “an embodiment,” “one embodiment,” or “other embodiments” in the specification refer to specific features, structures, or characteristics described in connection with the embodiments that are included in at least some embodiments, but not necessarily all embodiments of this disclosure.

[0067] "Adenosine deaminase" refers to a polypeptide or fragment thereof capable of catalyzing 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 the hydrolytic deamination of 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 derived from any organism, such as bacteria.

[0068] In some embodiments, the adenosine deaminase is a 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 does not exist in nature. 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 naturally occurring deaminases. For example, deaminase domains are described in international PCT applications PCT / 2017 / 045381 (WO 2018 / 027078) and PCT / US2016 / 058344 (WO 2017 / 070632), which are each incorporated herein by reference in their entirety.Also see Komor, AC, et al., “Programmable base editing of a target base ingenomic 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 baseexcision repair inhibition and bacteriophage Mu Gam protein yields C:G-to-T: Abase 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.2018Dec;19(12):770-788.doi:10.1038 / s41576-018-0059-1, the entire contents of which are incorporated herein by reference.

[0069] Wild-type TadA(wt) adenosine deaminase has the following sequence (also known as the TadA reference sequence):

[0070] MSEVEFSHEYWMRHALTLAKRAWDEREVPVGAVLVHNNRVIGEGWNRPIGRHDPTAHAEIMALRQGGLVMQNYRLIDATLYVTLEPCVMCAGAMIHSRIGRVVFGARDAKTGAAGSLMDVLHHPGMNHRVEITEGILADECAALLSDFFRMRRQEIKAQKKAQSSTD

[0071] In some embodiments, the adenosine deaminase comprises the following sequence alterations:

[0072]

[0073] (Also known as TadA*7.10).

[0074] In some embodiments, TadA*7.10 includes at least one modification. In some embodiments, TadA*7.10 includes modifications at amino acid 82 and / or 166. In specific embodiments, variants of the above sequence include one or more of the following modifications: Y147T, Y147R, Q154S, Y123H, V82S, T166R, and / or Q154R. The modification Y123H is also referred to herein as H123H (the modification H123Y in TadA*7.10 reverts to Y123H (wt)). In other embodiments, variants of the TadA*7.10 sequence comprise combinations of changes selected from the following groups: Y147T+Q154R; Y147T+Q154S; Y147R+Q154S; V82S+Q154S; V82S+Y147R; V82S+Q154R; V82S+Y123H; I76Y+V82S; V82S+Y123H+Y147T; V82S+Y12 3H+Y147R; V82S+Y123H+Q154R; Y147R+Q154R+Y123H; Y147R+Q154R+I76Y; Y147R+Q154R+T16 6R; Y123H+Y147R+Q154R+I76Y; V82S+Y123H+Y147R+Q154R and I76Y+V82S+Y123H+Y147R+Q154R.

[0075] In other embodiments, the present invention provides adenosine deaminase variant comprising a deletion, for example, of TadA*8, comprising a C-terminal deletion beginning at residues 149, 150, 151, 152, 153, 154, 155, 156, or 157, corresponding to a corresponding mutation in TadA*7.10, the TadA reference sequence, or another TadA. In other embodiments, the adenosine deaminase variant is a TadA (e.g., TadA*8) monomer comprising one or more of the following alterations: Y147T, Y147R, Q154S, Y123H, V82S, T166R, and / or Q154R, corresponding to a corresponding mutation in TadA*7.10, the TadA reference sequence, or another TadA 7. In other embodiments, the adenosine deaminase variant is a monomer comprising altered combinations selected from the group consisting of: Y147T+Q154R; Y147T+Q154S; Y147R+Q154S; V82S+Q154S; V82S+Y147R; V82S+Q154R; V82S+Y123H; I76Y+V82S; V82S+Y123H+Y147T; V82S+Y123H+Y147R; V82S+Y 123H+Q154R; Y147R+Q154R+Y123H; Y147R+Q154R+I76Y; Y147R+Q154R+T166R; Y123H+Y147R+Q154R+I76Y; V82S+Y123H+Y147R+Q154R and I76Y+V82S+Y123H+Y147R+Q154R, relative to the corresponding mutations in TadA*7.10, the TadA reference sequence, or another TadA 7.

[0076] In other embodiments, the adenosine deaminase variant is a homodimer comprising two adenosine deaminase domains (e.g., TadA*8), each domain having one or more of the following alterations: Y147T, Y147R, Q154S, Y123H, V82S, T166R, and / or Q154R, relative to a corresponding mutation in TadA*7.10, the TadA reference sequence, or another TadA. In other embodiments, the adenosine deaminase variant is a homodimer comprising two adenosine deaminase domains (e.g., TadA*8), each domain having altered combinations selected from the group consisting of: Y147T+Q154R; Y147T+Q154S; Y147R+Q154S; V82S+Q154S; V82S+Y147R; V82S+Q154R; V82S+Y123H; I76Y+V82S; V82S+Y123H+Y147T; V82S+Y12 3H+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, relative to the corresponding mutations in TadA*7.10, the TadA reference sequence, or another TadA 7.

[0077] 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), comprising one or more of the following alterations Y147T, Y147R, Q154S, Y123H, V82S, T166R, and / or Q154R, relative to a corresponding mutation in TadA*7.10, the TadA reference sequence, or another TadA. 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), comprising altered combinations selected from the group consisting of: Y147T+Q154R; Y147T+Q154S; Y147R+Q154S; V82S+Q154S; V82S+Y147R; V82S+Q154R; V82S+Y123H; I76Y+V82S; V82S+Y123H+Y147T; V8 2S+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, corresponding to the corresponding mutations in TadA*7.10, the TadA reference sequence, or another TadA 7.

[0078] 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), comprising one or more of the following alterations Y147T, Y147R, Q154S, Y123H, V82S, T166R, and / or Q154R, relative to a corresponding mutation in TadA*7.10, the TadA reference sequence, or another TadA. 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), comprising altered combinations selected from: 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 or I76Y+V82S+Y123H+Y147R+Q154R, corresponding to the corresponding mutations in TadA*7.10, the TadA reference sequence, or another TadA 7.

[0079] In one embodiment, the adenosine deaminase is TadA*8, which comprises or is substantially composed of the following sequences or fragments thereof having adenosine deaminase activity:

[0080] MSEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWNRAIGLHDPTAHAEIMALRQGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIGRVVFGVRNAKTGAAGSLMDVLHYPGMNHRVEITEGILADECAALLCTFFRMPRQVFNAQKKAQSSTD.

[0081] In some embodiments, the TadA*8 is truncated. In some embodiments, the truncated TadA*8 is missing 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 N-terminal amino acid residues compared to the full-length TadA*8. In some embodiments, the truncated TadA*8 is missing 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 C-terminal amino acid residues compared to the full-length TadA*8. In some embodiments, the adenosine deaminase variant is the full-length TadA*8.

[0082] In a particular embodiment, the adenosine deaminase heterodimer comprises a TadA*8 domain and an adenosine deaminase domain selected from one of the following:

[0083] Staphylococcus aureus (S. aureus) TadA:

[0084] MGSHMTNDIYFMTLAIEEAKKAAQLGEVPIGAIITKDDEVIARAHNLRETLQQPTAHAEHIAIERAAKVLGSWRLEGCTLYVTLEPCVMCAGTIVMSRIPRVVYGADDPKGGCSGSLMNLLQQSNFNHRAIVDKGVLKEACSTLLTTFFKNLRANKKSTN

[0085] Bacillus subtilis (B. subtilis) TadA:

[0086] MTQDELYMKEAIKEAKKAEEKGEVPIGAVLVINGEIIARAHNLRETEEQRSIAHAEMLVIDEACKALGTWRLEGATLYVTLEPCPMCAGAVVLSRVEKVVFGAFDPKGGCSGTLMNLLQEERFNHQAEVVSGVLEEECGGMLSAFFRELRKKKKAARKNLSE

[0087] Salmonella typhimurium (S. typhimurium) TadA:

[0088] MPPAFITGVTSLSDVELDHEYWMRHALTLAKRAWDEREVPVGAVLVHNHRVIGEGWNRPIGRHDPTAHAEIMALRQGGLVLQNYRLLDTTLYVTLEPCVMCAGAMVHSRIGRVVFGARDAKTGAAGSLIDVLHHPGMNHRVEIIEGVLRDECATLLSDFFRMRRQEIKALKKADRAEGAGPAV

[0089] Shewanella putrefaciens TadA:

[0090] MDEYWMQVAMQMAEKAEAAGEVPVGAVLVKDGQQIATGYNLSISQHDPTAHAEILCLRSAGKKLENYRLLDATLYITLEPCAMCAGAMVHSRIARVVYGARDEKTGAAGTVVNLLQHPAFNHQVEVTSGVLAEACSAQLSRFFKRRRDEKKALKLAQRAQQGIE

[0091] Haemophilus influenzae F3031 TadA:

[0092] MDAAKVRSEFDEKMMRYALELADKAEALGEIPVGAVLVDDARNIIGEGWNLSIVQSDPTAHAEIIALRNGAKNIQNYRLLNSTLYVTLEPCTMCAGAILHSRIKRLVFGASDYKTGAIGSRFHFFDDYKMNHTLEITSGVLAEECSQKLSTFFQKRREEKKIEKALLKSLSDK

[0093] Caulobacter crescentus TadA:

[0094] MRTDESEDQDHRMMRLALDAARAAAEAGETPVGAVILDPSTGEVIATAGNGPIAAHDPTAHAEIAAMRAAAAKLGNYRLTDLTLVVTLEPCAMCAGAISHARIGRVVFGADDPKGGAVVHGPKFFAQPTCHWRPEVTGGVLADESADLLRGFFRARRKAKI

[0095] Geobacter sulfurreducens TadA:

[0096] MSSLKKTPIRDDAYWMGKAIREAAKAAARDEVPIGAVIVRDGAVIGRGHNLREGSNDPSAHAEMIAIRQAARRSANWRLTGATLYVTLEPCLMCMGAIILARLERVVFGCYDPKGGAAGSLYDLSADPRLNHQVRLSPGVCQEECGTMLSDFFRDLRRRKKAKATPALFIDERKVPPEP

[0097] TadA*7.10

[0098] MSEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWNRAIGLHDPTAHAEIMALRQGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIGRVVFGVRNAKTGAAGSLMDVLHYPGMNHRVEITEGILADECAALLCYFFRMPRQVFNAQKKAQSSTD

[0099] "Administering" in this document means providing a patient or subject with one or more of the compositions described herein. For example, but not limited to, administering the composition by injection, such as intravenous (iv), subcutaneous (sc), intradermal (id), intraperitoneal (ip), or intramuscular (im) injection. One or more of these routes may be used. Parenteral administration may be, for example, by bolus injection or gradual infusion over time. Alternatively, or concurrently, it may be administered orally.

[0100] "Pharmaceutical" refers to any small molecule compound, antibody, nucleic acid molecule or polypeptide, or fragment thereof.

[0101] As used in this article, "allogeneic" refers to cells of the same species that are genetically different from cells compared to each other.

[0102] "Change" refers to a change (e.g., an increase or decrease) in the structure, expression level, or activity of a gene or polypeptide, as detected by standard methods known in the art, such as those described herein. As used herein, change includes changes in the sequence of a polynucleotide or polypeptide or changes in expression levels, such as 25%, 40%, 50%, or greater.

[0103] "Improvement" refers to reducing, inhibiting, weakening, mitigating, preventing, or stabilizing the development or progression of a disease.

[0104] "Analogous molecules" are molecules that are not identical but have similar functions or structural features. For example, polynucleotide or polypeptide analogs retain the biological activity of the corresponding naturally occurring polynucleotide or polypeptide while having certain modifications that enhance the analog's function relative 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 without altering, for example, ligand binding. Analogs may include non-natural nucleotides or amino acids.

[0105] "Anti-neoplasia activity" refers to the prevention or inhibition of tumor maturation and / or proliferation.

[0106] As used in this article, "autologous" refers to cells derived from the same subject.

[0107] A "base editor (BE)" or "nucleobase editor (NBE)" refers to a reagent that binds to polynucleotides and has nucleobase modification activity. In various embodiments, the base editor comprises a nucleobase-modifying polypeptide (e.g., a deaminase) and a nucleic acid-programmable nucleotide-binding domain that binds to a guide polynucleotide (e.g., guide RNA). In various embodiments, the reagent is a biomolecular complex comprising a protein domain having base-editing activity, i.e., capable of modifying bases (e.g., DNA) within a nucleic acid molecule (e.g., A, T, C, G, or U). In some embodiments, the polynucleotide-programmable DNA-binding domain is fused to or linked to a deaminase domain. In one embodiment, the reagent is a fusion protein comprising a domain having base-editing activity. In another embodiment, the protein domain having base-editing activity is linked to guide RNA (e.g., via an RNA-binding motif on the guide RNA and an RNA-binding domain fused to a deaminase). In some embodiments, the domain having base-editing activity is capable of deaminating bases within a nucleic acid molecule. In some embodiments, the base editor is capable of deaminating one or more bases within a DNA molecule. In some embodiments, the base editor is capable of deaminating one or more bases within a DNA molecule. In some embodiments, the base editor is an adenosine base editor (ABE).

[0108] In some embodiments, the base editor is generated by cloning an adenosine deaminase variant (e.g., TadA*8) into a scaffold comprising a circular substitution Cas9 (e.g., spCAS9 or saCAS9) and a binuclear localization sequence (e.g., ABE8). Circular substitution Cas9s are known in the art and described, for example, in Oakes et al., Cell 176, 254–267, 2019. Exemplary circular arrangements are shown below, where bold sequences denote sequences derived from Cas9, italic sequences denote linker sequences, and underlined sequences denote binuclear localization sequences.

[0109] CP5 (with MSP "NGC = conventional Cas9-like NGG with mutant Pam variant" PID = protein-protein interaction domain and "D10A" nickase):

[0110]

[0111]

[0112] In some embodiments, ABE8 is selected from the base editors listed in Tables 8, 9, 10, or 11 below. In some embodiments, ABE8 contains an adenosine deaminase variant evolved from TadA. In some embodiments, the adenosine deaminase variant of ABE8 is the TadA*8 variant described in Table 9 below. In some embodiments, the adenosine deaminase variant is a TadA*7.10 variant (e.g., TadA*8) containing one or more modifications selected from Y147T, Y147R, Q154S, Y123H, V82S, T166R, and / or Q154R. In various implementations, ABE8 includes a variant of TadA*7.10 (e.g., TadA*8) having a combination of changes selected from the following groups: Y147T+Q154R; Y147T+Q154S; Y147R+Q154S; V82S+Q154S; V82S+Y147R; V82S+Q154R; V82S+Y123H; I76Y+V82S; V82S+Y123H+Y147T; V8 2S+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 some embodiments, ABE8 is a monomeric construct. In some embodiments, ABE8 is a heterodimeric construct. In some embodiments, the ABE8 comprises the sequence:

[0113] MSEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWNRAIGLHDPTAHAEIMALRQGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIGRVVFGVRNAKTGAAGSLMDVLHYPGMNHRVEITEGILADECAALLCTFFRMPRQVFNAQKKAQSSTD。

[0114] In some embodiments, the polynucleotide programmable DNA-binding domain is a CRISPR-related enzyme (e.g., Cas or Cpf1). In some embodiments, the base editor is a non-catalytically inactive Cas9 (dCas9) fused with a deaminase domain. In some embodiments, the base editor is a Cas9 nickase (nCas9) fused with a deaminase domain. Base editors 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 see Komor, AC, et al., “Programmable editing of a target base in genomic DNA without double-strandedDNA 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: Abase 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 ofliving cells." Nat Rev Genet.2018Dec;19(12):770-788.doi:10.1038 / s41576-018-0059-1, the entire contents of which are incorporated herein by reference.

[0115] For example, the adenine base editor (ABE) used in the base editing compositions, systems, and methods described herein has a nucleic acid sequence (8877 base pairs), (Addgene, Watertown, MA.; Komor NM et al., 2017, SciAdv., 30; 3(8): 2017Nov23; 551(7681): 464-471. doi: 10.1038 / nature24644; Koblan LW et al., Nat Biotechnol. 2018Oct; 36(9): 843-846. doi: 10.1038 / nbt.4172.) is provided below. It also includes a polynucleotide sequence having at least 95% or higher identity with the ABE nucleic acid sequence.

[0116]

[0117] "Base editing activity" refers to the use of chemical alterations to bases within 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, for example, converting a target C·G to T·A. In another embodiment, the base editing activity is adenosine or adenine deaminase activity, for example, converting a target A·T to C·G. In yet another embodiment, the base editing activity is cytidine deaminase activity, for example, converting a target C·G to T·A, and adenosine or adenine deaminase activity, for example, converting A·T to G·C. In some embodiments, base editing activity is evaluated by editing efficiency. Base editing efficiency can be measured by any suitable method, for example, by Sanger sequencing or next-generation sequencing. In some embodiments, base editing efficiency is measured as a percentage of total sequencing reads with nucleobase conversions affected by the base editor, for example, a percentage of total sequencing reads with a target AT base pair converted to a GC base pair. In some implementations, when base editing is performed in a cell population, the base editing efficiency is measured by the percentage of total cells with nuclear base transformations affected by the base editor.

[0118] The term "base editor system" refers to a system for editing the nucleobases of a target nucleotide sequence. In various embodiments, the base editor system comprises (1) a polynucleotide-programmable nucleotide-binding domain (e.g., Cas9); (2) a deaminase domain for deamination of the nucleobase (e.g., adenosine deaminase and / or cytidine deaminase); 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.

[0119] In some embodiments, the base editor system may include more than one base editing component. For example, the base editor system may include more than one deaminase. In some embodiments, the base editor system may include one or more adenosine deaminases. In some embodiments, a single guide polynucleotide may be used to target different deaminases to target nucleic acid sequences. In some embodiments, a pair of guide polynucleotides may be used to target different deaminases to target nucleic acid sequences.

[0120] The deaminase domain and polynucleotide-programmable nucleotide-binding component of a base editor system can be covalently or nonvalently associated with each other, or any combination of such associations and interactions. For example, in some embodiments, the deaminase domain can target a target nucleotide sequence via the polynucleotide-programmable nucleotide-binding domain. In some embodiments, the polynucleotide-programmable nucleotide-binding domain can be fused to 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 through nonvalent interaction or association with the deaminase domain. For example, in some embodiments, the deaminase domain may include additional heterologous portions or domains that are capable of interacting, associating, or forming complexes with additional heterologous portions or domains that are part of the polynucleotide-programmable nucleotide-binding domain. In some embodiments, the additional heterologous portion may be capable of binding, interacting, associating, or forming complexes with a polypeptide. In some embodiments, the additional heterologous portion may be capable of binding, interacting, associating, or forming complexes with a polynucleotide. In some embodiments, the additional heterologous portion may be capable of binding a guide polynucleotide. In some embodiments, the additional heterologous portion may be capable of binding a polypeptide linker. In some embodiments, the additional heterologous portion may be capable of binding to a polynucleotide linker. The additional heterologous portion may be a protein domain. In some embodiments, the additional heterologous portion may be a K homology (KH) domain, an MS2 capsid protein domain, a PP7 capsid protein domain, an SfMuCom capsid protein domain, a sterile α motif, a telomerase Ku-binding motif and Ku protein, a telomerase Sm7-binding motif and Sm7 protein, or an RNA recognition motif.

[0121] The base editor system may further include a guide polynucleotide component. It should be understood that components of the base editor system can be associated with each other through covalent bonds, non-covalent interactions, or any combination of associations and interactions. In some embodiments, the deaminase domain can target a target nucleotide sequence via the guide polynucleotide. For example, in some embodiments, the deaminase domain may include an additional heterologous portion or domain (e.g., a polynucleotide-binding domain, such as an RNA or DNA-binding protein) capable of interacting with, associating with, or forming a complex with a portion or segment (e.g., a polynucleotide motif) of the guide polynucleotide. In some embodiments, the additional heterologous portion or domain (e.g., a polynucleotide-binding domain, such as an RNA or DNA-binding protein) may be fused to or linked to the deaminase domain. In some embodiments, the additional heterologous portion may be capable of binding, interacting with, associating with, or forming a complex with a polypeptide. In some embodiments, the additional heterologous portion may be capable of binding, interacting with, associating with, or forming a complex with a polynucleotide. In some embodiments, the additional heterologous portion may be capable of binding the guide polynucleotide. In some embodiments, the additional heterologous portion may be capable of binding a polypeptide linker. In some embodiments, the additional heterologous portion may be capable of binding to a polynucleotide linker. The additional heterologous portion may be a protein domain. In some embodiments, the additional heterologous portion may be a K homology (KH) domain, an MS2 capsid protein domain, a PP7 capsid protein domain, an SfMuCom capsid protein domain, a sterile α motif, a telomerase Ku-binding motif and Ku protein, a telomerase Sm7-binding motif and Sm7 protein, or an RNA recognition motif.

[0122] In some embodiments, the base editor system may further include an inhibitor of the base excision repair (BER) component. It should be understood that components of the base editor system can be associated with each other through covalent bonds, non-covalent interactions, or any combination of associations and interactions. The inhibitor of the BER component may include a BER inhibitor. In some embodiments, the BER inhibitor may be a uracil DNA glycosyltransferase inhibitor (UGI). In some embodiments, the BER inhibitor may be an inosine BER glycosyltransferase inhibitor. In some embodiments, the BER inhibitor can target the target nucleotide sequence via the polynucleotide programmable nucleotide binding domain. In some embodiments, the polynucleotide programmable nucleotide binding domain may be fused to or linked with the BER inhibitor. In some embodiments, the polynucleotide programmable nucleotide binding domain may be fused to or linked with a deaminase domain and the BER inhibitor. In some embodiments, the polynucleotide programmable nucleotide binding domain can target the BER inhibitor to the target nucleotide sequence through non-covalent interaction or association with the BER inhibitor. For example, in some embodiments, the BER inhibitor may include an additional heterologous moiety or domain capable of interacting, associating, or forming a complex with an additional heterologous moiety or domain that is part of the polynucleotide programmable nucleotide binding domain.

[0123] In some embodiments, the inhibitor of BER can target the target nucleotide sequence via the guide polynucleotide. For example, in some embodiments, the BER inhibitor may include an additional heterologous portion or domain (e.g., a polynucleotide-binding domain, such as an RNA or DNA-binding protein) capable of interacting with, associating with, or forming a complex with a portion or segment (e.g., a polynucleotide motif) of the guide polynucleotide. In some embodiments, the additional heterologous portion or domain of the guide polynucleotide (e.g., a polynucleotide-binding domain, such as an RNA or DNA-binding protein) may be fused to or linked to the BER inhibitor. In some embodiments, the additional heterologous portion may be capable of binding, interacting with, associating with, or forming a complex with the polynucleotide. In some embodiments, the additional heterologous portion may be capable of binding the guide polynucleotide. In some embodiments, the additional heterologous portion may be capable of binding a peptide linker. In some embodiments, the additional heterologous portion may be capable of binding a polynucleotide linker. The additional heterologous portion may be a protein domain. In some implementations, the additional heterologous portion may be a K homology (KH) domain, an MS2 capsid protein domain, a PP7 capsid protein domain, an SfMuCom capsid protein domain, a sterile α motif, a telomerase Ku-binding motif and Ku protein, a telomerase Sm7-binding motif and Sm7 protein, or an RNA recognition motif.

[0124] "B cell maturation antigen, or tumor necrosis factor receptor superfamily member 17 polypeptide (BCMA)" refers to a protein identical to NCBI accession number NP_001183 or a fragment thereof expressed on mature B lymphocytes having at least approximately 85% of its amino acid sequence. An exemplary BCMA polypeptide sequence is provided below.

[0125] >NP_001183.2 Tumor necrosis factor receptor superfamily member 17 [Homo sapiens]

[0126] MLQMAGQCSQNEYFDSLLHACIPCQLRCSSNTPPLTCQRYCNASVTNSVKGTNAILWTCLGLSLIISLAVFVLMFLLRKINSEPLKDEFKNTGSGLLGMANIDLEKSRTGDEIILPRGLEYTVEECTCEDCIKSKPKVDSDHCFPLPAMEEGATILVTTKTNDYCKSLPAALSATEIEKSISAR

[0127] This antigen can be used to treat relapsed or refractory multiple myeloma and other hematologic malignancies.

[0128] "B cell maturation antigen, or tumor necrosis factor receptor superfamily member 17, (BCMA) polynucleotide" refers to a nucleic acid molecule encoding the BCMA polypeptide. The BCMA gene encodes a cell surface receptor that recognizes B cell activating factors. An exemplary B2M polypeptide sequence is provided below.

[0129] >NM_001192.2 Homo sapiens TNF receptor superfamily member 17 (TNFRSF17), mRNA

[0130] AAGACTCAAACTTAGAAACTTGAATTAGATGTGGTATTCAAATCCTTAGCTGCCGCGAAGACACAGACAGCCCCCGTAAGAACCCACGAAGCAGGCGAAGTTCATTGTTCTCCAACATTCTAGCTGCTCTTGCTGCATTTGCTCTGGAATTCTTGTAGAGATATTACTTGTCCTTCCAGGCTGTTCTTTCTGTAGCTCCCTTGTTTTCTTTTTGTGATCATGTTGCAGATGGCTGGGCAGTGCTCCCAAAATGAATATTTTGACAGTTTGTTGCATGCTTGCATACCTTGTCAACTTCGATGTTCTTCTAATACTCCTCCTCTAACATGTCAGCGTTATTGTAATGCAAGTGTGACCAATTCAGTGAAAGGAACGAATGCGATTCTCTGGACCTGTTTGGGACTGAGCTTAATAATTTCTTTGGCAGTTTTCGTGCTAATGTTTTTGCTAAGGAAGATAAACTCTGAACCATTAAAGGACGAGTTTAAAAACACAGGA TCAGGTCTCCTGGGCATGGCTAACATTGACCTGGAAAAGAGCAGGACTGGTGATGAAATTATTCTTCCGAGAGGCCTCGAGTACACGGTGGAAGAATGCACCTTGTGAAGACTGCATCAAGAGCA AACCGAAGGTCGACTCTGACCATTGCTTTCCACTCCCAGCTATGGAGGAAGGCGCAACCATTCTTGTCACCACGAAAACGAATGACTATTGCAAGAGCCTGCCAGCTGCTTTGAGTGCTACGGA GATAGAGAAATCAATTTCTGCTAGGTAATTAACCATTTCGACTCGAGCAGTGCCACTTTAAAAATCTTTTGTCAGAATAGATGATGTGTCAGATCTCTTTAGGATGACTGTATTTTTCAGTTGC CGATACAGCTTTTTGTCCTCTAACTGTGGAAACTCTTTATGTTAGATATATTTCTCTAGGTTACTGTTGGGAGCTTAATGGTAGAAACTTCCTTGGTTTCATGATTAAACTCTTTTTTTTCCTGA

[0131] "β-2 microglobulin (B2M) polypeptide" refers to a protein that has at least about 85% amino acid sequence identity with UniProt accession number P61769 or a fragment thereof and has immunomodulatory activity. An exemplary B2M polypeptide sequence is provided below.

[0132] >sp|P61769|B2MG_Human Beta-2 Microglobulin OS=Homo sapiens OX=9606GN=B2M PE=1 SV=1

[0133] MSRSVALAVLALLSLSGLEAIQRTPKIQVYSRHPAENGKSNFLNCYVSGFHPSDIEVDLL

[0134] KNGERIEKVEHSDLSFSKDWSFYLLYYTEFTPTEKDEYACRVNHVTLSQPKIVKWDRDM

[0135] "β-2-microglobulin (B2M) polynucleotide" refers to the nucleic acid molecule encoding the B2M polypeptide. The β-2-microglobulin gene encodes a serum protein associated with the major histocompatibility complex. B2M is involved in the non-self recognition of host CD8+ T cells. An exemplary B2M polynucleotide sequence is provided below.

[0136] >DQ217933.1 Homo sapiens beta-2-microglobulin (B2M) gene, complete codon

[0137]

[0138] The term "Cas9" or "Cas9 domain" refers to an RNA-guided nuclease containing the Cas9 protein or fragments thereof (e.g., proteins containing the active, inactive, or partially active DNA-cutting domains of Cas9, and / or the binding domain of gRNA Cas9). Cas9 nucleases are sometimes also called Casnl nucleases or CRISPR (Regularly Clustered Interspaced Short Palindromic Repeats)-associated nucleases. CRISPR is an adaptive immune system that provides protection against mobile genetic elements (viruses, transposable elements, and conjugating plasmids). A CRISPR cluster contains a spacer sequence, a sequence complementary to the preceding mobile element, and a target invading nucleic acid. The CRISPR cluster is transcribed and processed into CRISPR RNA (crRNA). In type II CRISPR systems, proper processing of pre-crRNA requires a small transcoding RNA (tracrRNA), endogenous ribonuclease 3 (rnc), and the Cas9 protein. tracrRNA acts as a guide for ribonuclease 3 to assist in the processing of pre-crRNA. Subsequently, Cas9 / crRNA / tracrRNA endonucleases linear or circular dsDNA targets complementary to the spacer sequence. Target strands not complementary to crRNA are first cleaved via endonucleation, followed by 3′–5′ exonucleation. In nature, DNA binding and cleavage typically require both proteins and two RNAs. However, single guide RNAs (“sgRNA,” or simply “gRNA”) can be engineered to integrate various aspects of crRNA and tracrRNA 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 within a CRISPR repeat sequence (PAM or protospacer sequence adjacent motif) to help distinguish between self and non-self.The Cas9 nuclease sequence and structure are well known to those skilled in the art (see, for example, “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), all of which are incorporated herein by reference). Cas9 orthologs have been described in a variety of species, including but not limited to Streptococcus pyogenes and Streptococcus thermophilus. Based on this disclosure, other suitable Cas9 nucleases and sequences will be apparent to those skilled in the art, and such Cas9 nucleases and sequences include Cas9 sequences of organisms and loci disclosed in Chylinski, Rhun, and Charpentier, “The tracrRNA and Cas9 families of type IICRISPR-Cas immunity systems” (2013) RNA Biology 10:5, 726-737, the entire contents of which are incorporated herein by reference.

[0139] An exemplary Cas9 is Streptococcus pyogenes Cas9 (spCas9), whose amino acid sequence is provided below:

[0140]

[0141]

[0142] (Single underscore: HNH domain; Double underscore: RuvC domain)

[0143] Nuclease-inactivated Cas9 proteins are interchangeably referred to as “dCas9” proteins (for nuclease-“dead” Cas9) or catalytically inactivated Cas9. Methods for generating Cas9 proteins (or fragments thereof) with inactive DNA-cutting domains are known (see, for example, 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-cutting domain of Cas9 is known to comprise 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 silence the nuclease activity of Cas9. For example, mutations in D10A and H840A completely inactivate the nuclease activity of Streptococcus 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-cutting domain, i.e., Cas9 is a nicking enzyme, referred to as an “nCas9” protein (for “nicking enzyme” 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) a gRNA-binding domain of Cas9; or (2) a DNA-cutting domain of Cas9. In some embodiments, proteins comprising Cas9 or fragments thereof are referred to as “Cas9 variants”. Cas9 variants are homologous to Cas9 or fragments thereof. For example, the Cas9 variant is at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, at least about 99.5%, 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 variations 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-cutting domain) such that the fragment is at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, at least about 99.5%, 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.

[0144] In some embodiments, the fragment is at least 100 amino acids long. In some embodiments, the fragment is at least 100, 150, 200, 250, 300, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1050, 1100, 1150, 1200, 1250, or 1300 amino acids long.

[0145] In some embodiments, wild-type Cas9 corresponds to Cas9 from Streptococcus pyogenes (NCBI reference sequence: NC_017053.1, nucleotide and amino acid sequences are as follows).

[0146]

[0147]

[0148] (Single underscore: HNH domain; Double underscore: RuvC domain)

[0149] In some embodiments, the wild-type Cas9 corresponds to or comprises the following nucleotide and / or amino acid sequence:

[0150]

[0151]

[0152]

[0153] (Single underscore: HNH domain; Double underscore: RuvC domain)

[0154] In some embodiments, wild-type Cas9 corresponds to Cas9 from Streptococcus pyogenes (NCBI reference sequence: NC_002737.2 (nucleotide sequence below); and Uniprot reference sequence: Q99ZW2 (amino acid sequence below).

[0155]

[0156]

[0157] (SEQ ID NO:1. Single underscore: HNH domain; Double underscore: RuvC domain)

[0158] In some implementations, Cas9 refers to Cas9 derived from the following: Corynebacterium ulcerans (NCBI Refs: NC_015683.1, NC_017317.1); Corynebacterium diphtheria (NCBI Refs: NC_016782.1, NC_016786.1); Spiroplasma syrphidicola (NCBI Refs: NC_021284.1); Prevotella intermedia (NCBI Refs: NC_017861.1); Spiroplasma taiwanense, China (NCBI Refs: NC_021846.1); Streptococcus iniae (NCBI Refs: NC_021314.1); Belliella balolaensis. Cas9 from any other organism (NCBI Refs: YP_018010.1); Psychroflexus torquisI (NCBI Refs: YP_018721.1); Streptococcus thermophilus (NCBI Refs: YP_820832.1); Listeria innocua (NCBI Refs: NP_472073.1); Campylobacter jejuni (NCBI Refs: YP_002344900.1); Neisseria meningitidis (NCBI Refs: YP_002342100.1).

[0159] In some embodiments, Cas9 is derived from Neisseria meningitidis (Nme). In some embodiments, the Cas9 is Nme1, Nme2, or Nme3. In some embodiments, the PAM interaction 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 aDinucleotide PAM for In Vivo Genome Editing, Molecular Cell (2018)). The exemplary Neisseria meningitidis Cas9 protein Nme1Cas9 (NCBI Reference: WP_002235162.1; Type II CRISPR RNA-guided endonuclease Cas9) has the following amino acid sequence:

[0160]

[0161] Another exemplary meningococcal Cas9 protein, Nme2Cas9 (NCBI Reference: WP_002230835; type II CRISPR RNA-guided endonuclease Cas9), has the following amino acid sequence:

[0162]

[0163] In some embodiments, dCas9 corresponds to or contains, partially or entirely, a Cas9 amino acid sequence having one or more mutations that inactivate Cas9 nuclease activity. For example, in some embodiments, the dCas9 domain contains the D10A and H840A mutations or a corresponding mutation in another Cas9. In some embodiments, dCas9 contains the amino acid sequence of dCas9 (D10A and H840A):

[0164]

[0165]

[0166] (Single underscore: HNH domain; Double underscore: RuvC domain).

[0167] In some embodiments, the Cas9 domain contains a D10A mutation, while the residue at position 840 remains histidine at the corresponding position in the amino acid sequence provided above or in any amino acid sequence provided herein.

[0168] In other embodiments, dCas9 variants with mutations other than D10A and H840A are provided, such as Cas9 (dCas9) that leads to nuclease inactivation. For example, such mutations include other amino acid substitutions at D10 and H840, or other substitutions within the Cas9 nuclease domain (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%, at least about 80%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, at least about 99.5%, or at least about 99.9% identical. In some embodiments, variants or homologs of about 5, 10, 15, 20, 25, 30, 40, 50, 75, 100, or more amino acids are provided, either shorter or longer.

[0169] In some embodiments, the Cas9 fusion proteins provided herein comprise the full-length amino acid sequence of the 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 only one or more fragments thereof. Exemplary amino acid sequences of suitable Cas9 domains and Cas9 fragments are provided herein, and other suitable sequences of Cas9 domains and fragments will be apparent to those skilled in the art.

[0170] It should be understood that additional Cas9 proteins (e.g., nuclease-dead Cas9 (dCas9), Cas9 nickase (nCas9), or nuclease-active Cas9), including their variants and homologues, 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 nuclease-inactive Cas9 (dCas9). In some embodiments, the Cas9 protein is a Cas9 nickase (nCas9). In some embodiments, the Cas9 protein is a Cas9 with nuclease activity.

[0171] Exemplary catalytically inactive Cas9 (dCas9):

[0172]

[0173] Exemplary catalytically active Cas9 nickase (nCas9):

[0174]

[0175] Exemplary catalytic activity of Cas9:

[0176]

[0177] In some implementations, Cas9 refers to Cas9 derived from archaea (e.g., nanoarchaea), which constitute domains and kingdoms of single-celled prokaryotic microorganisms. In some embodiments, Cas9 refers to CasX or CasY, which has been described, for example, in Burstein et al., "New CRISPR-Cas systems from uncultivated microbes." CellRes. 2017 Feb 21. doi:10.1038 / cr.2017.21, the entire contents of which are incorporated herein by reference. Metagenomics using genome resolution has identified numerous CRISPR-Cas systems, including Cas9, which was first reported in the field of archaea. This divergent Cas9 protein has been found in rarely studied nanoarchaea as part of an active CRISPR-Cas system. 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 refers to CasX, or a variant of CasX. In some embodiments, Cas9 refers to CasY, or a variant of CasY. It should be understood that other RNA-guided DNA-binding proteins can be used as nucleic acid-programmable DNA-binding proteins (napDNAbp) and are within the scope of this disclosure.

[0178] In certain embodiments, the napDNAbps that can be used in the methods of the present invention include circular substitutions known and described in the art, such as those by Oakes et al., Cell 176, 254–267, 2019. Exemplary circular substitutions are shown below, where bold sequences represent sequences derived from Cas9, italic sequences represent linker sequences, and underlined sequences represent binuclear localization.

[0179] CP5 (with MSP "NGC = conventional Cas9-like NGG with mutant Pam variant" PID = protein-protein interaction domain and "D10A" nickase):

[0180]

[0181]

[0182] Non-limiting examples of multinucleotide programmable nucleotide-binding domains that can be incorporated into a base editor include CRISPR protein-derived domains, restriction nucleases, broad-spectrum nucleases, TAL nucleases (TALEN), and zinc finger nucleases (ZFN).

[0183] In some embodiments, the nucleic acid programmable DNA-binding protein (napDNAbp) or any fusion protein provided herein may be a CasX or CasY protein. In some embodiments, napDNAbp is a CasY protein. In some embodiments, napDNAbp is a CasY protein. In some embodiments, napDNAbp contains 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 that of a naturally occurring CasX or CasY protein. In some embodiments, napDNAbp is a naturally occurring CasX or CasY protein. In some embodiments, napDNAbp contains 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 that of any CasX or CasY protein described herein. It should be understood that Cas12b / C2c1, CasX and CasY from other bacterial species may also be used according to this disclosure.

[0184] Cas12b / C2c1(uniprot.org / uniprot / T0D7A2#2)

[0185] sp|T0D7A2|C2C1_ALIAG CRISPR-associated endo-nuclease C2c1 OS=Alicyclobacillus acido-terrestris (strain ATCC 49025 / DSM 3922 / CIP106132 / NCIMB 13137 / GD3B)GN=c2c1 PE=1 SV=1

[0186]

[0187] CasX (uniprot.org / uniprot / F0NN87; uniprot.org / uniprot / F0NH53)

[0188] >tr|F0NN87|F0NN87_SULIH CRISPR - associated Casx protein OS = Sulfolobus islandicus (strain HVE10 / 4) GN = SiH_0402 PE = 4 SV = 1

[0189] MEVPLYNIFGDNYIIQVATEAENSTIYNNKVEIDDEELRNVLNLAYKIAKNNEDAAAERRGKAKKKKGEEGETTTSNIILPLSGNDKNPWTETLKCYNFPTTVALSEVFKNFSQVKECEEVSAPSFVKPEFYEFGRSPGMVERTRRVKLEVEPHYLIIAAAGWVLTRLGKAKVSEGDYVGVNVFTPTRGILYSLIQNVNGIVPGIKPETAFGLWIARKVVSSVTNPNVSVVRIYTISDAVGQNPTTINGGFSIDLTKLLEKRYLLSERLEAIARNALSISSNMRERYIVLANYIYEYLTGSKRLEDLLYFANRDLIMNLNSDDGKVRDLKLISAYVNGELIRGEG

[0190] >tr|F0NH53|F0NH53_SULIR CRISPR - associated protein, Casx OS = Sulfolobus islandicus (strain REY15A) GN = SiRe_0771 PE = 4 SV = 1

[0191] MEVPLYNIFGDNYIIQVATEAENSTIYNNKVEIDDEELRNVLNLAYKIAKNNEDAAAERRGKAKKKKGEEGETTTSNIILPLSGNDKNPWTETLKCYNFPTTVALSEVFKNFSQVKECEEVSAPSFVKPEFYKFGRSPGMVERTRRVKLEVEPHYLIMAAAGWVLTRLGKAK VSEGDYVGVNVFTPTRGILYSLIQNVNGIVPGIKPETAFGLWIARKVVSSVTNPNVSVVSIYTISDAVGQNPTTINGGFSIDLTKLLEKRDLLSERLEAIARNALSISSNMRERYIVLANYIYEYLTGSKRLEDLLYFANRDLIMNLNSDDGKVRDLKLISAYVNGELIRGEG

[0192] Delta bacteriumCasX

[0193] MEKRINKIRKKLSADNATKPVSRSGPMKTLLVRVMTDDLKKRLEKRRKKPEVMPQVISNNAANNLRMLLDDYTKMKEAILQVYWQEFKDDHVGLMCKFAQPASKKIDQNKLKPEMDEKGNLTTAGFACSQCGQPLFVYKLEQVSEKGKAYTNYFGRCNVAEHEKLILLAQLKPVKDSDEAVTYSLGKFGQRALDFYSIHVTKESTHPVKPLAQIAGNRYASGPVGKALSDACMGTIASFLSKYQDIIIEHQKVVKGNQKRLESLRELAGKENLEYPSVTLPPQPHTKEGVDAYNEVIARVRMWVNLLWQKLKLSRDDAKPLLRLKGFPSFPVVERRENEVDWWNTINEVKKLIDAKRDMGRVFWSGVTAEKRNTILEGYNYLPNENDHKKREGSLENPKKPAKRQFGDLLLYLEKKYAGDWGKVFDEAWERIDKKIAGLTSHIEREEARNAEDAQSKAVLTDWLRAKASFVLERLKEMDEKEFYACEIQLQK WYGDLRGNPFAVEAENRVVDISGFSIGSDGHSIQYRNLLAWKYLENGKREFYLLMNYGKKGRIRFTDGTDIKKSGKWQGLLYGGGAKVIDLTFDPDDEQLIILPLAFGTRQGREFIWNDLLSLETGLIKLANGRVIEKTIYNKKIGRDEPALFVALTFERREVVDPSNIKPVNLIGVARGENIPAVIALTDPEGCPLPEFKDSSGGPTDILRIGEGYKEKQRAIQAAKEVEQRRAGGYSRKFASKSRNLADDMVRNSARDLFYHAVTHDAVLVFANLSRGFGRQGKRTFMTERQYTKMEDWLTAKLAYEGLTSKTYLSKTLAQYTSKTCSNCGFTITYADMDVMLVRLKKTSDGWATTLNNKELKAEYQITYYNRYKRQTVEKELSAELDRLSEESGNNDISKWTKGRRDEALFLLKKRFSHRPVQEQFVCLDCGHEVHAAEQAALNIARSWLFLNSNSTEFKSYKSGKQPFVGAWQAFYKRRLKEVWKPNA

[0194] CasY(ncbi.nlm.nih.gov / protein / APG80656.1)

[0195] >APG80656.1 CRISPR-associated protein CasY [uncultured Parcubacteria]

[0196]

[0197] The term "Cas12" or "Cas12 domain" refers to an RNA-guided nuclease containing the Cas12 protein or a fragment thereof (e.g., a protein containing the active, inactive, or partially active DNA-cutting domain of Cas12, and / or the binding domain of gRNA Cas12). Cas12 belongs to the class 2V CRISPR / Cas system. Cas12 nucleases are sometimes also referred to as CRISPR (regularly clustered short palindromic repeats)-associated nucleases. An exemplary sequence of the Cas12 domain of *Bacillus cereus* Cas12b (BhCas12b) is provided below:

[0198]

[0199] Amino acid sequences having at least 85% or higher identity with the BhCas12b amino acid sequence can also be used in the methods of this invention.

[0200] "Cbl proto-oncogene B (CBLB) polypeptide" refers to a protein that has at least approximately 85% amino acid sequence identity with the gene bank accession number ABC86700.1 or its fragment involved in the regulation of immune responses. An exemplary CBLB polypeptide sequence is provided below.

[0201] >ABC86700.1 CBL-B [Homo sapiens]

[0202] MANSMNGRNPGGRGGNPRKGRILGIIDAIQDAVGPPKQAAADRRTVEKTWKLMDKVVRLCQNPKLQLKNSPPYILDILPDTYQHLRLILSKYDDNQKLAQLSENEYFKIYIDSLMKKSKRAIRLFKEGKERMYEEQSQDRRNLTKLSLIFSHMLAEIKAIFPNGQFQGDNFRITKADAAEFWRKFFGDKTIVPWKVFRQCLHEVHQISSGLEAMALKSTIDLTCNDYISVFEFDIFTRLFQPWGSILRNWNFLAVTHPGYMAFLTYDEVKARLQKYSTKPGSYIFRLSCTRLGQWAIGYVTGDGNILQTIPHNKPLFQALIDGSREGFYLYPDGRSYNPDLTGLCEPTPHDHIKVTQEQYELYCEMGSTFQLCKICAENDKDVKIEPCGHLMCTSCLTAWQESDGQGCPFCRCEIKGTEPIIVDPFDPRDEGSRCCSIIDPFGMPMLDLDDDDDREESLMMNRLANVRKCTDRQNSPVTSPGSSPLAQRRKPQPDPLQIPHLSLPPVPPRLDLIQKGIVRSPCGSPTGSPKSSPCMVRKQDKPLPAPPPPLRDPPPPPPERPPPIPPDNRLSRHIHHVESVPSRDPPMPLEAWCPRDVFGTNQLVGCRLLGEGSPKPGITASSNVNGRHSRVGSDPVLMRKHRRHDLPLEGAKVFSNGHLGSEEYDVPPRLSPPPPVTTLLPSIKCTGPLANSLSEKTRDPVEEDDDEYKIPSSHPVSLNSQPSHCHNVKPPVRSCDNGHCMLNGTHGPSSEKKSNIPDLSIYLKGDVFDSASDPVPLPPARPPTRDNPKHGSSLNRTPSDYDLLIPPLGEDAFDALPPSLPPPPPPARHSLIEHSKPPGSSSRPSSGQDLFLLPSDPFVDLASGQVPLPPARRLPGENVKTNRTSQDYDQLPSCSDGSQAPARPPKPRPRRTAPEIHHRKPHGPEAALENVDAKIAKLMGEGYAFEEVKRALEIAQNNVEVARSILREFAFPPPVSPRLNL

[0203] "Cbl proto-oncogene B (CBLB) polynucleotide" refers to the nucleic acid molecule encoding the CBLB polypeptide. The CBLB gene encodes the E3 ubiquitin ligase. An exemplary CBLB nucleic acid sequence is provided below.

[0204] >DQ349203.1 Homo sapiens CBL-B mRNA, complete codon

[0205]

[0206] "Chimeric antigen receptor" or "CAR" refers to a synthetic receptor that includes an extracellular antigen-binding domain, a transmembrane domain, and an intracellular signaling domain that confers antigen specificity to immune cells.

[0207] "Class II major histocompatibility complex trans-activator (CIITA) peptide" refers to a protein that has at least about 85% amino acid sequence identity with the NCBI reference sequence: NP_000237.2 or a fragment thereof, or as a transcriptional coactivator. An exemplary CIITA peptide sequence is provided below.

[0208]

[0209] "Class II major histocompatibility complex transactivator (CIITA) polynucleotide" refers to a nucleic acid molecule encoding a CIITA polypeptide. An exemplary CIITA nucleic acid sequence is provided below.

[0210]

[0211]

[0212]

[0213] "Cluster 7 (CD7) polypeptide" refers to a protein that has at least approximately 85% amino acid sequence identity with the NCBI reference sequence: NP_006128.1 or a fragment thereof, and is involved in T cell and T cell / B cell interactions. An exemplary CD7 polypeptide sequence is provided below.

[0214]

[0215]

[0216] "Cluster 7 (CD7) polynucleotide" refers to a nucleic acid molecule encoding a CD7 polypeptide. The CD7 gene encodes a transmembrane protein. An exemplary CD7 nucleic acid sequence is provided below.

[0217]

[0218] "Cluster 5 (CD5) polypeptide" refers to a protein that has at least about 85% amino acid sequence identity with the NCBI reference sequence: NP_001333385.1 or a fragment thereof, and is expressed on the surface of T cells. An exemplary CD5 polypeptide sequence is provided below.

[0219]

[0220] "Cluster 5 (CD5) polynucleotide" refers to a nucleic acid molecule encoding a CD5 polypeptide. The CD5 gene encodes a transmembrane protein. An exemplary CD5 nucleic acid sequence is provided below.

[0221]

[0222]

[0223] The term "conserved amino acid substitution" or "conserved mutation" refers to the substitution of one amino acid for another amino acid that shares a common characteristic. One functional approach to defining the common characteristic between individual amino acids is to analyze the normalized frequency of amino acid changes between corresponding proteins from homologous organisms (Schulz, GE and Schirmer, RH, Principles of Protein Structure, Springer-Verlag, New York (1979)). Based on such analysis, amino acid groups can be defined where amino acids within a group preferentially exchange with each other and are therefore most similar to each other in their effect on the overall protein structure (Schulz, GE and Schirmer, RH, supra). Non-restrictive examples of conserved mutations include amino acid substitutions, such as arginine for lysine and vice versa, thus maintaining a positive charge; glutamate for aspartic acid and vice versa, to maintain a negative charge; threonine for serine, thus maintaining a free -OH; and glutamine for asparagine, thus maintaining a free -NH2.

[0224] As used interchangeably in this article, the terms "coding sequence" or "protein-coding sequence" refer to a polynucleotide fragment that encodes a protein. This region or sequence has a start codon near the 5' end and a stop codon near the 3' end. A coding sequence may also be referred to as an open reading frame.

[0225] "Cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) polypeptide" refers to a protein that has at least approximately 85% sequence identity with NCBI accession number EAW70354.1 or a fragment thereof. An exemplary amino acid sequence is provided below:

[0226] >EAW70354.1 Cytotoxic T-lymphocyte-associated protein 4 [Homo sapiens] MACLGFQRHKAQLNLATRTWPCTLLFFLLFIPVFCKAMHVAQPAVVLASSRGIASFVCEYASPGKATEVRVTVLRQADSQVTEVCAATYMMGNELTFLDDSICTGTSSGNQVNLTIQGLRAMDTGLYICKVELMYPPPYYLGIGNGTQIYVIDPEPCPDSDFLLWILAAVSSGLFFYSFLLTAVSLSKMLKKRSPLTTGVYVKMPPTEPECEKQFQPYFIPIN

[0227] "Cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) polynucleotide" refers to a nucleic acid molecule encoding the CTLA-4 polypeptide. The CTLA-4 gene encodes an immunoglobulin superfamily and a protein that transmits inhibitory signals to T cells. An exemplary CTLA-4 nucleic acid sequence is provided below.

[0228] >BC074842.2 Homo sapiens cytotoxic T lymphocyte-associated protein 4, mRNA (cDNA clone MGC:104099IMAGE:30915552), complete cds

[0229] GACCTGAACACCGCTCCCATAAAGCCATGGCTTGCCTTGGATTTCAGCGGCACAAGGCTCAGCTGAACCTGGCTACCAGGACCTGGCCCTGCACTCTCCTGTTTTTTCTTCTCTTCATCCCTGTCTTCTGCAAAGCAATGCACGTGGCCCAGCCTGCTGTGGTACTGGCCAGCAGCCGAGGCATCGCCAGCTTTGTGTGTGAGTATGCATCTCCAGGCAAAGCCACTGAGGTCCGGGTGACAGTGCTTCGGCAGGCTGACAGCCAGGTGACTGAAGTCTGTGCGGCAACCTACATGATGGGGAATGAGTTGACCTTCCTAGATGATTCCATCTGCACGGGCACCTCCAGTGGAAATCAAGTGAACCTCACTATCCAAGGACTGAGGGCCATGGACACGGGACTCTACATCTGCAAGGTGGAGCTCATGTACCCACCGCCATACTACCTGGGCATAGGCAACGGAACCCAGATTTATGTAATTGATCCAGAACCGTGCCCAGATTCTGACTTCCTCCTCTGGATCCTTGCAGCAGTTAGTTCGGGGTTGTTTTTTTATAGCTTTCTCCTCACAGCTGTTTCTTTGAGCAAAATGCTAAAGAAAAGAAGCCCTCTTACAACAGGGGTCTATGTGAAAATGCCCCCAACAGAGCCAGAATGTGAAAAGCAATTTCAGCCTTATTTTATTCCCATCAATTGAGAAACCATTATGAAGAAGAGAGTCCATATTTCAATTTCCAAGAGCTGAGG

[0230] 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 that catalyzes the hydrolysis and deamination of adenine to hypoxanthine. In some embodiments, the adenosine deaminase catalyzes the hydrolysis and deamination of adenosine or adenine (A) to inosine (I). In some embodiments, the deaminase or deaminase domain is an adenosine deaminase that catalyzes the hydrolysis and deamination of adenosine or deoxyadenosine to inosine or deoxyinosine, respectively. In some embodiments, the adenosine deaminase catalyzes the hydrolysis and 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 bacteria. In some embodiments, the adenosine deaminase is derived from bacteria, such as *Escherichia coli*, *Staphylococcus aureus*, *Salmonella typhimurium*, *Shewanella putrefactive*, *Haemophilus influenzae*, and *Stenobacterium*.

[0231] In some embodiments, the adenosine deaminase is a 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 does not exist in nature. 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 naturally occurring deaminases. For example, deaminase domains are described in international PCT applications PCT / 2017 / 045381 (WO 2018 / 027078) and PCT / US2016 / 058344 (WO 2017 / 070632), which are each incorporated herein by reference in their entirety.Also see Komor, AC, et al., “Programmable base editing of a target base ingenomic 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 baseexcision repair inhibition and bacteriophage Mu Gam protein yields C:G-to-T: Abase 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.2018Dec;19(12):770-788.doi:10.1038 / s41576-018-0059-1, the entire contents of which are incorporated herein by reference.

[0232] "Detection" refers to identifying the presence, absence, or amount of an analyte to be detected. In one embodiment, it involves detecting sequence alterations in polynucleotides or peptides. In another embodiment, it involves detecting the presence of insertions or deletions.

[0233] "Detectable label" refers to a composition that, when linked to a molecule of interest, makes the molecule detectable by spectroscopic, photochemical, biochemical, immunochemical, or chemical means. Useful labels include radioactive isotopes, magnetic beads, metal beads, colloidal particles, fluorescent dyes, electron-dense reagents, enzymes (e.g., commonly used in ELISA), biotin, digoxigenin, or haptens.

[0234] "Disease" refers to any symptom or condition that impairs or interferes with the normal function of cells, tissues, or organs. In one implementation, a disease is a tumor or cancer.

[0235] As used herein, the term "effective amount" refers to an amount of bioactive agent sufficient to elicit the desired biological response. The effective amount of the bioactive agent used to practice the contents of this invention to treat a disease varies depending on the route of administration, the subject's age, weight, and general health. Ultimately, the attending physician or veterinarian will determine the appropriate amount and dosage regimen. This amount is referred to as the "effective" amount. In one embodiment, the effective amount is an amount sufficient to introduce the altered base editor of the invention (e.g., a fusion protein comprising a programmable DNA-binding protein, a nucleobase editor, and gRNA) into the gene of interest in cells (e.g., in vitro or in vivo cells). In another embodiment, the effective amount is the amount of base editor required to achieve a therapeutic effect (e.g., to reduce or control the disease or its symptoms or condition). This therapeutic effect does not require alteration of the gene of interest in all cells of the subject, tissue, or organ, but only about 1%, 5%, 10%, 25%, 50%, 75%, or more of the gene of interest present in the cells of the subject, tissue, or organ.

[0236] As used in this article, "epipote" refers to an antigenic determinant. An epitope is a part of an antigen molecule that, through its structure, determines the specific antibody molecule that recognizes and binds to it.

[0237] A “fragment” refers to a portion of a polypeptide or nucleic acid molecule. This portion contains at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of the full length of a reference nucleic acid molecule or polypeptide. A fragment may contain 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.

[0238] Graft-versus-host disease (GVHD) is a pathological condition in which transplanted cells from a donor produce an immune response against the host cells.

[0239] "Guide RNA" or "gRNA" refers to a polynucleotide that is specific to 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 guide RNA (gRNA). gRNA can exist as a complex of two or more RNAs or as a single RNA molecule. gRNA existing as a single RNA molecule may be called single guide RNA (sgRNA), but "gRNA" is used interchangeably to refer to guide RNA existing as a single molecule or a complex of two or more molecules. Typically, gRNA existing as a single RNA species contains two domains: (1) a domain homologous to the target nucleic acid (e.g., guiding the 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 called tracrRNA and contains a stem-loop structure. For example, in some embodiments, domain (2) is identical or homologous to the tracrRNA 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 including domain 2) can be found in U.S. Provisional Patent Application, USSN 61 / 874,682, filed September 6, 2013, entitled “Switchable Cas9 Nucleases and Uses Thereof,” and U.S. Provisional Patent Application, USSN 61 / 874,746, filed September 6, 2013, entitled “Delivery System For Functional Nucleases,” the entire contents of which are incorporated herein by reference. In some embodiments, the gRNA comprises two or more of domains (1) and (2) and may be referred to as an “extended gRNA.” As described herein, an extended gRNA will bind two or more Cas9 proteins and bind target nucleic acids in two or more different regions. gRNA contains a nucleotide sequence complementary to the target site, which mediates the binding of the nuclease / RNA complex to the target site, providing sequence specificity for the nuclease:RNA complex. As those skilled in the art will understand, RNA polynucleotide sequences, such as gRNA sequences, include the nucleobase uracil (U), a pyrimidine derivative, rather than the nucleobase thymine (T) contained in the DNA polynucleotide sequence. In RNA, uracil pairs with adenine bases and substitutes for thymine during DNA transcription.

[0240] "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 variants of the TadA domain (e.g., TadA*7.10 and TadA*8 or two TadA*8 domains).

[0241] Host-resistant graft disease (HVGD) refers to a pathological condition in which the host's immune system produces an immune response to transplanted cells from a donor.

[0242] "Hybridization" refers to hydrogen bonds between complementary nucleobases, which can be Watson-Crick, Hoogsteen, or reverse Hoogsteen hydrogen bonds. For example, adenine and thymine are complementary nucleobases that pair up by forming hydrogen bonds.

[0243] "Immune cells" refer to immune system cells that are capable of generating immune responses.

[0244] "Immune effector cells" are lymphocytes that, once activated, can generate an immune response against target cells. T cells are an example of immune effector cells.

[0245] The term "base repair inhibitor" or "IBR" refers to a protein capable of inhibiting the activity of nucleic acid repair enzymes such as base excision repair (BER) enzymes. In some embodiments, the IBR is an inhibitor of inosine base excision repair. Exemplary inhibitors of base repair include inhibitors of APE1, Endo III, Endo IV, Endo V, Endo VIII, Fpg, hOGG1, hNEIL1, T7 Endol, T4PDG, UDG, hSMUG1, and hAAG. In some embodiments, the IBR is an inhibitor of Endo V or hAAG. In some embodiments, the 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 some embodiments, the base repair inhibitor is catalytically inactive EndoV or catalytically inactive hAAG.

[0246] In some embodiments, the base repair inhibitor is a uracil glycosylase inhibitor (UGI). UGI refers to a protein capable of inhibiting uracil-DNA glycosylase base excision repair enzymes. In some embodiments, the UGI domain comprises wild-type UGI or a fragment of wild-type UGI. In some embodiments, the UGI protein provided herein comprises a fragment of UGI and a protein homologous to UGI or a UGI fragment. 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 a “dead inosine-specific nuclease.” Not wishing to be bound by any particular theory, a catalytically inactive inosine glycosylation enzyme (e.g., alkyladenine glycosylation enzyme (AAG)) can bind inosine but cannot create a base-deficient site or remove inosine, thereby spatially blocking the newly formed inosine moiety from DNA damage / repair mechanisms. In some embodiments, a catalytically inactive inosine-specific nuclease can bind inosine in nucleic acids but does not cleave the nucleic acids. Non-limiting exemplary catalytically inactive inosine-specific nucleases include catalytically inactive alkyladenine glycosylation enzymes (AAG nucleases), such as those from humans, and catalytically inactive endonuclease V (EndoV nucleases), such as those from *E. coli*. In some embodiments, the catalytically inactive AAG nuclease contains an E125Q mutation or a corresponding mutation in another AAG nuclease.

[0247] "Increase" refers to a positive change of at least 10%, 25%, 50%, 75%, or 100%.

[0248] An intein is a protein fragment capable of self-excision and joining the remaining fragment (exein) with peptide bonds in a process called protein splicing. Inteins are also called protein introns. The process by which an intein self-excises and joins the remaining portion of the protein is referred to herein as "protein splicing" or "intein-mediated protein splicing." In some embodiments, the intein of a precursor protein (the protein containing the intein prior to intein-mediated protein splicing) originates 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, the DnaE of 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."

[0249] Other intein systems may also be used. For example, synthetic inteins based on dnaE inteins, Cfa-N (e.g., split intein-N), and Cfa-C (e.g., split intein-C) intein pairs have been described (e.g., in Stevens et al., J Am Chem Soc. 2016 Feb. 24; 138(7):2162-5, which is incorporated herein by reference). Non-limiting examples of intein pairs that may be used according to this disclosure include: Cfa DnaE inteins, Ssp GyrB inteins, Ssp DnaX inteins, Ter DnaE3 inteins, Ter ThyX inteins, Rma DnaB inteins, and Cne Prp8 inteins (e.g., as described in U.S. Patent No. 8,394,604, which is incorporated herein by reference).

[0250] Exemplary nucleotide and amino acid sequences of inteins are provided.

[0251] DnaE Intein-N DNA:

[0252] TGCCTGTCATACGAAACCGAGATACTGACAGTAGAATATGGCCTTTGCCAATCGGGAAGATTGTGGAGAAACGGATAGAATGCACAGTTTACTCTGTCGATAACAATGGTAACATTTATACTCAGCCAGTTGCCCAGTGGCACGACCGGGGA GAGCAGGAAGTATTCGAATACTGTCTGGAGGATGGAAGTCTCATTAGGGCCACTAAGGACCACAAATTTATGACAGTCGATGGCCAGATGCTGCCTATAGACGAAATCTTTGAGCGAGAGTTGGACCTCATGCGAGTTGACAACCTTCCTAAT

[0253] DnaE Intein-N protein:

[0254] CLSYETEILTVEYGLLPIGKIVEKRIECTVYSVDNNGNIYTQPVAQWHDRGEQEVFEYCLEDGSLIRATKDHKFMTVDGQMLPIDEIFERELDLMRVDNLPN

[0255] DnaE Intein-C DNA:

[0256] ATGATCAAGATAGCTACAAGGAAGTATCTTGGCAAACAAACGTTTATGATATTGGAGTCGAAAGAGATCACACTTTGCTCTGAAGAACGGATTCATAGCTTCTAAT

[0257] Intein-C:MIKIATRKYLGKQNVYDIGVERDHNFALKNGFIASN

[0258] Cfa-N DNA:

[0259] TGCCTGTCTTATGATACCGAGATACTTACCGTTGAATATGGCTTCTTGCCTATTGGAAAGATTGTCGAAGAGAGAATTGAATGCACAGTATATACTGTAGACAAGAATGGTTTCGTTTACACACAGCCCATTGCTCAATGCACAATCGCGGCACAAGAAGTATTTGAGTACTGTCTCGAGGATGGAAGCATCATACGAGCAACTAAAGATCATAAATTCATGACCACTGACGGGCAGATGTTGCCAAATAGATGAGATATTCGAGCGGGGCTTGGATCTCAAACAAGTGGATGGATTGCCA

[0260] Cfa-N protein:

[0261] CLSYDTEILTVEYGFLPIGKIVEERIECTVYTVDKNGFVYTQPIAQWHNRGEQEVFEYCLEDGSIIRATKDHKFMTTDGQMLPIDEIFERGDLLKQVDGLP

[0262] Cfa-C DNA:

[0263] ATGAAGAGGACTGCCGATGGATCAGAGTTTGAATCTCCCAAGAAGAAGAGGAAAGTAAAGATAATATCTCGAAAAGTCTTGGTACCCAAAATGTCTATGATATTGGAGTGGAGAAAGATCACAACTTCCTTCTCAAGAACGGTCTCGTAGCCAGCAAC

[0264] Cfa-C protein:

[0265] MKRTADGSEFESPKKKRKVKIISRKSLGTQNVYDIGVEKDHNFLLKNGLVASN

[0266] Intein-N and intein-C can be fused to the N-terminal and C-terminal portions of cleaved Cas9, respectively, to connect the N-terminal and C-terminal portions of cleaved Cas9. For example, in some embodiments, intein-N is fused to the C-terminus of the N-terminal portion of cleaved Cas9, forming an N-[N-terminal portion of cleaved Cas9]-[intein-N]--C structure. In some embodiments, intein-C is fused to the N-terminus of the C-terminal portion of cleaved Cas9, forming an N-[intein-C]-[C-terminal portion of cleaved Cas9]-C structure. Intein-mediated protein splicing mechanisms for connecting proteins to which inteins are fused (e.g., cleaved Cas9) are known in the art, for example, as in Shah et al., Chem Sci. 2014; 5(1):446-461, which is incorporated herein by reference. Methods for designing and using inteptides are known in the art and described, for example, WO2014004336, WO2017132580, US20150344549 and US20180127780, each of which is incorporated herein by reference in its entirety.

[0267] The terms "isolated," "purified," or "biologically pure" refer to materials that, to varying degrees, do not contain components typically associated with their native state. "Isolated" indicates the degree of separation from the original source or surrounding environment. "Purified" indicates a degree of separation beyond isolation. "Purified" or "biologically pure" proteins are sufficiently free of other materials such that any impurities do not materially affect the protein's biological properties or cause other adverse consequences. That is, a nucleic acid or peptide of the present invention is purified if it is substantially free of cellular material, viral material, or culture medium when produced using recombinant DNA technology, or substantially free of chemical precursors or other chemicals during chemical synthesis. Purity and homogeneity are typically determined using analytical chemistry techniques, such as polyacrylamide gel electrophoresis or high-performance liquid chromatography. The term "purified" can also mean that the nucleic acid or protein produces essentially a single band in the electrophoretic gel. For proteins that can be modified (e.g., phosphorylation or glycosylation), different modifications may produce different isolated proteins that can be purified individually.

[0268] "Separated polynucleotide" refers to a nucleic acid (e.g., DNA) that does not contain a gene, which is located flanking the gene in the naturally occurring genome of the organism from which the nucleic acid molecules of this invention are derived. Therefore, this term includes, for example, recombinant DNA integrated into a vector; DNA incorporated into an autonomously replicating plasmid or virus; or genomic DNA incorporated into a prokaryotic or eukaryotic organism; or DNA existing as an independent molecule separate from other sequences (e.g., cDNA or a genomic or cDNA fragment produced by PCR or restriction endonuclease digestion). Furthermore, this term includes RNA molecules transcribed from DNA molecules, and recombinant DNA as part of a heterozygous gene encoding an additional polypeptide sequence.

[0269] "Isolated polypeptide" refers to the polypeptide of the present invention that has been separated from its naturally occurring associated components. Typically, a polypeptide is isolated when it is at least 60% (by weight) free of proteins and naturally occurring organic molecules. Preferably, the polypeptide of the present invention is prepared at least 75% by weight, more preferably at least 90% by weight, and most preferably at least 99% by weight. The isolated polypeptide of the present 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 chemical synthesis of the protein. Purity can be measured by any suitable method, such as column chromatography, polyacrylamide gel electrophoresis, or analysis by HPLC.

[0270] As used herein, the term "linker" 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 parts (e.g., two components of a protein complex or ribonucleoprotein complex), or two domains of a fusion protein, such as 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). Linkers can connect different components or different parts of a base editor system. For example, in some embodiments, a linker can connect a guide polynucleotide-binding domain of a polynucleotide-programmable nucleotide-binding domain and a catalytic domain of a deaminase. In some embodiments, a linker can connect a CRISPR peptide 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, the linker may connect a deamination component and a polynucleotide-programmable nucleotide-binding component of the base editor system. In some embodiments, the linker may connect the RNA-binding portion of the deamination component and the polynucleotide-programmable nucleotide-binding component of the base editor system. In some embodiments, the linker may connect the RNA-binding portion of the deamination component and the RNA-binding portion of the polynucleotide-programmable nucleotide-binding component of the base editor system. The linker may be located between or on either side of two groups, molecules, or other portions and is connected to each via a covalent bond or non-covalent interaction, thereby connecting the two. In some embodiments, the linker may be an organic molecule, group, polymer, or chemical portion. In some embodiments, the linker may be a polynucleotide. In some embodiments, the linker may be a DNA linker. In some embodiments, the linker may be an RNA linker. In some embodiments, the linker may include an aptamer capable of binding a ligand. In some embodiments, the ligand may be a carbohydrate, peptide, protein, or nucleic acid. In some embodiments, the linker may include an aptamer that can be derived from a riboswitch. The riboswitch of the derived aptamer can be selected from theophylline riboswitch, thiamine pyrophosphate (TPP) riboswitch, adenosylcobalamin (AdoCbl) riboswitch, S-adenosylmethionine (SAM) riboswitch, SAH riboswitch, flavin mononucleotide (FMN) riboswitch, tetrahydrofolate riboswitch, lysine riboswitch, glycine riboswitch, purine riboswitch, GlmS riboswitch, or pre-queosine1 (PreQ1) riboswitch. In some embodiments, the linker may comprise an aptamer that binds to a polypeptide or protein domain, such as a polypeptide ligand.In some embodiments, the peptide ligand may be a K homology (KH) domain, an MS2 capsid protein domain, a PP7 capsid protein domain, an SfMuCom capsid protein domain, a sterile α 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 peptide ligand may be part of a base editor system component. For example, the nucleobase editing component may include a deaminase domain and an RNA recognition motif.

[0271] In some embodiments, the linker can be one or more amino acids (e.g., a peptide or protein). In some embodiments, the linker can be about 5-100 amino acids long, for example, 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 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 long. Longer or shorter linkers are also considered.

[0272] In some embodiments, the linker connects the gRNA-binding domain of an RNA-programmable nuclease, including a Cas9 nuclease domain and a catalytic domain of a nucleic acid editing protein (e.g., cytidine or adenosine deaminase). In some embodiments, the linker connects dCas9 and the nucleic acid editing protein. For example, the linker is located between or on either side of two groups, molecules, or other parts and is covalently linked to each, thereby connecting the two. In some embodiments, the linker may be one or more amino acids (e.g., a peptide or a protein). In some embodiments, the linker may be approximately 5-200 amino acids long, for example, 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 more amino acid lengths. Longer or shorter linkers may also be considered.

[0273] In some embodiments, the domain of the nucleobase editor is fused via a linker comprising the following amino acid sequence: SGGSSGSETPGTSESATPESSGGS, SGGSSGGSSGSETPGTSESATPESSGGSSGGS, or GGSGGSPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSTE PSEGSAPGTSTEPSEGSAPGTSESATPESGPGSEPATSGGSGGS. In some embodiments, the domain of the nucleobase editor is fused via a linker comprising the amino acid sequence SGSETPGTSESATPES, which may also be referred to as an XTEN linker. In some embodiments, the linker comprises the amino acid sequence SGGS. In some embodiments, the linker comprises (SGGS)n, (GGGS)n, (GGGGS)n, (G)n, (EAAAK)n, (GGS)n, SGSETPGTSESATPES, or (XP)n motifs, or any one of these, wherein n is independently an integer between 1 and 30, and wherein 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.

[0274] In some embodiments, the linker is 24 amino acids long. In some embodiments, the linker comprises the amino acid sequence SGGSSGGSSGSETPGTSESATPES. In some embodiments, the linker is 40 amino acids long. In some embodiments, the linker comprises the amino acid sequence SGGSSGGSSGSETPGTSESATPESSGGSSGGSSGGSSGGS. In some embodiments, the linker is 64 amino acids long. In some embodiments, the linker comprises the amino acid sequence SGGSSGGSSGSETPGTSESATPESSGGSSGGSSGGSSGGSSGSETPGTSESATPESSGGS SGGS. In some embodiments, the linker is 92 amino acids long. In some embodiments, the linker comprises the amino acid sequence PGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGSEPATS.

[0275] A “marker” is any protein or polynucleotide that has altered expression levels or activity in relation to a disease or condition.

[0276] As used herein, the term “mutation” refers to the substitution of a residue within a sequence (e.g., a nucleic acid or amino acid sequence) by another residue, or the deletion or insertion of one or more residues within a sequence. Mutations are generally described herein by identifying the original residue, followed by its position in the sequence, and by the identity of the newly substituted residue. Various methods for performing 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, currently disclosed base editors can efficiently generate “desired mutations,” such as point mutations, in nucleic acids (e.g., nucleic acids within a subject’s genome) without generating a large number of undesired mutations, such as accidental point mutations. In some embodiments, the desired mutation is a mutation generated by a specific base editor (e.g., a cytidine base editor or an adenosine base editor) that binds to a guide polynucleotide (e.g., gRNA) specifically designed to generate the desired mutation.

[0277] Typically, mutations generated or identified in a sequence (e.g., an amino acid sequence as 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.

[0278] "Neoplasia" refers to cells or tissues that exhibit abnormal growth or proliferation. The term neoplasm includes both cancer and solid tumors.

[0279] The term "non-conservative mutation" refers to an amino acid substitution between different groups, such as tryptophan replaced with lysine, or serine replaced with phenylalanine. In this case, the non-conservative amino acid substitution preferably does not interfere with or inhibit the biological activity of the functional variant. Non-conservative amino acid substitution can enhance the biological activity of the functional variant, thereby increasing the biological activity of the functional variant compared to the wild-type protein.

[0280] "Activated T cell nuclear factor 1 (NFATc1) polypeptide" refers to a protein that has at least about 85% amino acid sequence identity with NCBI accession number NM_172390.2 or a fragment thereof, and is a component of the activated T cell DNA-binding transcription complex. An exemplary amino acid sequence is provided below.

[0281] >NP_765978.1 Activated T cell nuclear factor, cytoplasmic isoform A [Homo sapiens]

[0282] MPSTSFPVPSKFPLGPAAAVFGRGETLGPAPRAGGTMKSAEEEHYGYASSNVSPALPLPTAHSTLPAPCHNLQTSTPGIIPPADHPSGYGAALDGGPAGYFLSSGHTRPDGAPALESPRIEITSCLGLYHNNNQFFHDVEVEDVLPSSKRSPSTATLSLPSLEAYRDPSCLSPASSLSS RSCNSEASSYESNYSYPYASPQTSPWQSPCVSPKTTDPEEGFPRGLGACTLLGSPRHSPSTSPRASVTEESWLGARSSRPASPCNKRKYSLNGRQPPYSPHHSPTPSPHGSPRVSVTDDSWLGNTTQYTSSAIVAAINALTTDSSLDLGDGVPVKSRKTTLEQPPSVALKVEPVGEDLG SPPPPADFAPEDYSSFQHIRKGGFCDQYLAVPQHPYQWAKPKPLSPTSYMSPTLPALDWQLPSHSGPYELRIEVQPKSHHRAHYETEGSRGAVKASAGGHPIVQLHGYLENEPLMLQLFIGTADDRLLRPHAFYQVHRITGKTVSTTSHEAILSNTKVLEIPLLPENSMRAVIDCAGIL KLRNSDIELRKGETDIGRKNTRVRLVFRVHVPQPSGRTLSLQVASNPIECSQRSAQELPLVEKQSTDSYPVVGGKKMVLSGHNFLQDSKVIFVEKAPDGHHVWEMEAKTDRDLCKPNSLVVEIPPFRNQRITSPVHVSFYVCNGKRKRSQYQRFTYLPANGNAIFLTVSREHERVGCFF

[0283] "Activated T cell nuclear factor 1 (NFATc1) polynucleotide" refers to a nucleic acid molecule encoding the NFATc1 polypeptide. The NFATc1 gene encodes a protein involved in the inducible expression of cytokine genes, particularly IL-2 and IL-4, in T cells. Exemplary nucleic acids that have been sequenced are provided below.

[0284] >NM_172390.2 Homo sapiens activated T cell nuclear factor 1 (NFATC1), transcript variant 1, mRNA

[0285]

[0286] The terms “nuclear localization sequence,” “nuclear localization signal,” or “NLS” refer to an amino acid sequence that facilitates protein importation into the cell nucleus. Nuclear localization sequences are known in the art and described, for example, in Plank et al.’s international PCT application, PCT / EP2000 / 011690, filed November 23, 2000, and published as WO / 2001 / 038547 on May 31, 2001, 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, such as that 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 MDSLLMNRRKFLYQFKNVRWAKGRRETY.

[0287] As used herein, the terms "nucleic acid" and "nucleic acid molecule" refer to compounds comprising nucleobases and acidic moieties, such as nucleosides, nucleotides, or polymers of nucleotides. Typically, polymerized nucleic acids, such as nucleic acid molecules comprising three or more nucleotides, are linear molecules in which adjacent nucleotides are linked together by phosphodiester bonds. In some embodiments, "nucleic acid" refers to a single nucleic acid residue (e.g., a nucleotide and / or a nucleoside). In some embodiments, "nucleic acid" refers to an oligonucleotide chain comprising three or more individual nucleotide residues. As used herein, the terms "oligonucleotide" and "polynucleotide" are used interchangeably to refer to polymers of nucleotides (e.g., a string of at least three nucleotides). In some embodiments, "nucleic acid" includes RNA as well as single-stranded and / or double-stranded DNA. Nucleic acids can be naturally occurring, for example, in the context of genomes, transcripts, mRNA, tRNA, rRNA, siRNA, snRNA, plasmids, viscera, chromosomes, chromatids, or other naturally occurring nucleic acid molecules. On the other hand, nucleic acid molecules can be non-naturally occurring molecules, such as recombinant DNA or RNA, artificial chromosomes, engineered genomes or fragments thereof, or synthetic DNA, RNA, DNA / RNA hybrids, or including non-naturally occurring nucleotides or nucleosides. Furthermore, the terms "nucleic acid," "DNA," "RNA," and / or similar terms include nucleic acid analogs, such as analogs having a backbone other than a phosphodiester backbone. Nucleic acids can be purified from natural sources, produced and optionally purified using recombinant expression systems, chemically synthesized, etc. Where appropriate, such as in the case of chemically synthesized molecules, nucleic acids may contain nucleoside analogs, such as analogs with chemically modified bases or sugar and backbone modifications. Unless otherwise specified, nucleic acid sequences are presented in a 5' to 3' orientation. In some embodiments, the nucleic acid is or comprises a natural nucleoside (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-propynyluridine, C5-propynylcytidine, C5- Methylcytidine, 2-aminoadenosine, 7-deadenosine, 7-deadenosine, 8-oxoadenosine, 8-oxoguanosine, O(6)-methylguanosine and 2-thiocytidine); chemically modified bases; biologically modified bases (e.g. methylated bases); inserted bases; modified sugars (2'-e.g. fluororibose, ribose, 2'-deoxyribose, arabinose and hexose); and / or modified phosphate groups (e.g. thiophosphates and 5'-N-phosphoramide bonds).

[0288] The terms "nucleic acid programmable DNA-binding protein" or "napDNAbp" are used interchangeably with "polynucleotide programmable nucleotide-binding domain" to refer to a protein associated with a nucleic acid (e.g., DNA or RNA), such as a guide nucleic acid or guide polynucleotide (e.g., gRNA), that guides napDNAbp to a specific nucleic acid sequence. In some embodiments, the polynucleotide programmable acid-binding domain is a polynucleotide programmable DNA-binding domain. In some embodiments, the polynucleotide programmable acid-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 may be associated with a guide RNA that guides the Cas9 protein to a specific DNA sequence complementary to the guide RNA. In some embodiments, napDNAbp is a Cas9 domain, such as an active nuclease Cas9, a Cas9 nickase (nCas9), or a nuclease-inactivated 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, and Cas12i.Non-restricted examples of Cas enzymes include Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas5d, Cas5t, Cas5h, Cas5a, Cas6, Cas7, Cas8, Cas8a, Cas8b, Cas8c, Cas9 (also known as Csn1 or Csx12), Cas10, Cas10d, Cas12a / Cpfl, Cas12b / C2cl, Cas12c / C2c3, Cas12d / CasY, Cas12e / CasX, Cas12g, Cas12h, Cas12i, Csy1, Csy2, Csy3, Csy4, Cse1, Cse2, Cse3, Cse4, Cse5e, Csc1, C sc2, 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 Cas effector proteins, type V Cas effector proteins, type VI proteins, CARF, DinG, their homologs or their modified or engineered versions. Other nucleic acid-programmable DNA-binding proteins are also within the scope of this disclosure, although 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 which are incorporated herein by reference.

[0289] The terms “nucleobase,” “nitrogenous base,” or “base” are used interchangeably herein to refer to nitrogenous biological compounds that form nucleosides, which are components of nucleotides. The ability of nucleobases to form base pairs and stack on top of each other directly results in long-chain helical structures, such as ribonucleic acid (RNA) and deoxyribonucleic acid (DNA). 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 may 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; both are produced by deamination (replacing 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 pentose sugar (ribose or deoxyribose). Examples of nucleosides include adenosine, guanosine, uridine, cytidine, 5-methyluridine (m5U), deoxyadenosine, deoxyguanosine, thymidine, deoxyuridine, and deoxycytidine. Examples of nucleosides with modified nucleobases include inosine (I), xanthine (X), 7-methylguanosine (m7G), dihydrouridine (D), 5-methylcytidine (m5C), and pseudouridine (Ψ). A "nucleotide" consists of a nucleobase, a pentose sugar (ribose or deoxyribose), and at least one phosphate group.

[0290] As used herein, the terms "nucleoside base editing domain" or "nucleoside base editing protein" refer to proteins or enzymes that catalyze nucleoside base modifications in RNA or DNA, such as cytosine (or cytidine) to uracil (or uridine) or thymidine (or thymidine) and adenine (or adenosine) to hypoxanthine (or inosine), as well as non-template-based nucleotide addition and insertion. In some embodiments, the nucleoside editing domain is a deaminase domain (e.g., adenine deaminase or adenosine deaminase; or cytidine deaminase or cytosine deaminase). In some embodiments, the nucleoside editing domain is multiple deaminase domains (e.g., adenine deaminase or adenosine deaminase and cytidine or cytosine deaminase). In some embodiments, the nucleoside editing domain may be a naturally occurring nucleoside editing domain. In some embodiments, the nucleoside editing domain may be a modified or evolved nucleoside editing domain derived from a naturally occurring nucleoside editing domain. The nucleobase editing domain can be derived from any organism, such as bacteria, humans, chimpanzees, gorillas, monkeys, cattle, dogs, rats, or mice.

[0291] As used herein, “obtain” includes, in the case of “obtaining a medicine”, synthesizing, purchasing, or otherwise acquiring the medicine.

[0292] As used herein, "patient" or "subject" means a mammalian subject or individual diagnosed with, at risk of having or developing, or suspected of having or developing a disease or condition. In some embodiments, the term "patient" refers to a mammalian subject with a higher-than-average likelihood of developing a disease or condition. Exemplary patients may be humans, non-human primates, cats, dogs, pigs, cattle, horses, camels, llamas, goats, sheep, rodents (e.g., mice, rabbits, rats, or guinea pigs), and other mammals that may benefit from the therapies disclosed herein. Exemplary human patients may be male and / or female.

[0293] "Patients in need" or "subjects in need" in this article refers to patients who are diagnosed with, at risk of having, are expected to have, or are suspected of having a disease or condition.

[0294] The terms “pathogenic mutation,” “pathogenic variant,” “disease coat mutation,” “pathogenic variation,” “harmful mutation,” or “susceptibility mutation” refer to genetic alterations or mutations that increase an individual’s susceptibility or predisposition to a certain disease or disorder. In some embodiments, a pathogenic mutation comprises the substitution of at least one wild-type amino acid for at least one pathogenic amino acid in a protein encoded by the gene.

[0295] The terms “protein,” “peptide,” “polypeptide,” and their grammatical equivalents are used interchangeably herein to refer to a polymer of amino acid residues linked together by peptide (amide) bonds. These terms refer to proteins, peptides, or polypeptides of any size, structure, or function. Typically, proteins, peptides, or polypeptides are at least three amino acids in length. A protein, peptide, or polypeptide can refer to a single protein or a collection of proteins. One or more amino acids in a protein, peptide, or polypeptide can be modified, for example, by adding chemical entities such as carbohydrate groups, hydroxyl groups, phosphate groups, farnesyl, isofarnesyl, fatty acid groups, linkers for conjugation, functionalization, or other modifications. Proteins, peptides, or polypeptides can also be single molecules or can be multi-molecular complexes. Proteins, peptides, or polypeptides can simply be fragments of naturally occurring proteins or peptides. Proteins, peptides, or polypeptides can be naturally occurring, recombinant, or 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. A protein may be located at the N-terminal (N-terminal) portion of a fusion protein or at the C-terminal (C-terminal) portion of a fusion protein, thereby forming an N-terminal fusion protein or a C-terminal fusion protein, respectively. The protein may contain various domains, such as nucleic acid-binding domains (e.g., the gRNA-binding domain of Cas9, which guides the binding of a protein to a target site) and nucleic acid-cutting domains, or catalytic domains of nucleic acid-editing proteins. In some embodiments, the protein comprises a protein moiety, 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 forms a complex or associates with a nucleic acid, such as RNA or DNA. Any protein provided herein can be produced by any method known in the art. For example, the proteins provided herein can be produced by recombinant protein expression and purification, which is particularly suitable for fusion proteins containing peptide linkers. Methods for recombinant protein expression and purification are well known and include those described in 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.

[0296] The polypeptides and proteins disclosed herein (including their functional portions and functional variants) may 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, leucine, α-aminodecanoic acid, homoserine, S-acetaminomethylcysteine, trans-3- and trans-4-hydroxyproline, 4-aminophenylalanine, 4-nitrophenylalanine, 4-chlorophenylalanine, 4-carboxyphenylalanine, β-phenylserine, β-hydroxyphenylalanine, phenylglycine, α-naphthylalanine, cyclohexylalanine, cyclohexylglycine, dihydroindole-2-tetrahydrotetrahydro, 3- Quinoline, 3-hydroxyphenylalanine, 3-2-hydroxyphenylalanine-carboxylic acid, aminomalonic acid, aminomalonic acid monoamide, N'-benzyl-N'-methyl-lysine, N',N'-dibenzyl-lysine, 6-hydroxylysine, ornithine, α-aminocyclopentanecarboxylic acid, α-aminocyclohexanecarboxylic acid, α-aminocycloheptanecarboxylic acid, α-(2-amino-2-norbornene)carboxylic acid, α,γ-diaminobutyric acid, α,β-diaminopropionic acid, homophenylalanine, and α-tert-butylglycine. Peptides and proteins can be associated with post-translational modifications of one or more amino acids in the peptide construct. Non-limiting examples of post-translational modifications include phosphorylation, acylation (including acetylation and formylation), glycosylation (including N-linking and O-linking), amidation, hydroxylation, alkylation (including methylation and ethylation), ubiquitination, addition of pyrrolidone carboxylic acid, formation of disulfide bonds, sulfation, myristylation, palmitoylation, isoprenelation, farnesylation, geraniylation, glycosylation, esterification, and iodination.

[0297] "Programmed cell death 1 (PDCD1 or PD-1) peptide" refers to a protein that has at least about 85% amino acid sequence identity with NCBI accession number AJS10360.1 or a fragment thereof. The PD-1 protein is believed to be involved in immune responses and the regulation of T cell function under tolerance conditions. An exemplary B2M peptide sequence is provided below.

[0298] AJS10360.1 Programmed cell death 1 protein [Homo sapiens]

[0299] MQIPQAPWPVVWAVLQLGWRPGWFLDSPDRPWNPPTFSPALLVVTEGDNATFTCSFSNTSESFVLNWYRMSPSNQTDKLAAFPEDRSQPGQDCRFRVTQLPNGRDFHMSVVRARRNDSGTYLCGAISLAPKAQIKESLRAELRV TERRAEVPTAHSPSPRPAGQFQTLVVGVVGGLLGSLVLLVWVLAVICSRAARGTIGARRTGQPLKEDPSAVPVFSVDYGELDFQWREKTPEPPVPCVPEQTEYATIVFPSGMGTSSPARRGSADGPRSAQPLRPEDGHCSWPL

[0300] "Programmed cell death 1 (PDCD1 or PD-1) polynucleotide" refers to a nucleic acid molecule encoding a PD-1 polypeptide. The PDCD1 gene encodes an inhibitory cell surface receptor that inhibits T cell effector function in an antigen-specific manner. An exemplary PDCD1 nucleic acid sequence is provided below.

[0301] >AY238517.1 Homo sapiens programmed cell death 1 (PDCD1) mRNA, complete cds

[0302] ATGCAGATCCCACAGGCGCCCTGGCCAGTCGTCTGGGCGGTGCTACAACTGGGCTGGCGGCCAGGATGGTTCTTAGACTCCCCAGACAGGCCCTGGAACCCCCCCACCTTCTCCCCAGCCCTGCTCGTGGTGACCGAAGGGGACAACGCCACCTTCACCTGCAGCTTCTCCAACACATCGGAGAGCTTCGTGCTAAACTGGTACCGCATGAGCCCCAGCAACCAGACGGACAAGCTGGCCGCCTTCCCCGAGGACCGCAGCCAGCCCGGCCAGGACTGCCGCTTCCGTGTCACACAACTGCCCAACGGGCGTGACTTCCACATGAGCGTGGTCAGGGCCCGGCGCAATGACAGCGGCACCTACCTCTGTGGGGCCATCTCCCTGGCCCCCAAGGCGCAGATCAAAGAGAGCCTGCGGGCAGAGCTCAGGGTGACAGAGAGAAGGGCAGAAGTGCCCACAGCCCACCCCAGCCCCTCACCCAGGCCAGCCGGCCAGTTCCAAACCCTGGTGGTTGGTGTCGTGGGCGGCCTGCTGGGCAGCCTGGTGCTGCTAGTCTGGGTCCTGGCCGTCATCTGCTCCCGGGCCGCACGAGGGACAATAGGAGCCAGGCGCACCGGCCAGCCCCTGAAGGAGGACCCCTCAGCCGTGCCTGTGTTCTCTGTGGACTATGGGGAGCTGGATTTCCAGTGGCGAGAGAAGACCCCGGAGCCCCCCGTGCCCTGTGTCCCTGAGCAGACGGAGTATGCCACCATTGTCTTTCCTAGCGGAATGGGCACCTCATCCCCCGCCCGCAGGGGCTCAGCTGACGGCCCTCGGAGTGCCCAGCCACTGAGGCCTGAGGATGGACACTGCTCTTGGCCCCTCTGA

[0303] As used herein in the context of proteins or nucleic acids, the term “recombinant” refers to a protein or nucleic acid that is not naturally occurring but is a product of human engineering. For example, in some embodiments, recombinant protein or nucleic acid molecules comprise 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.

[0304] "Decrease" refers to a negative change of at least 10%, 25%, 50%, 75% or 100%.

[0305] "Reference" refers to standard or control conditions. In one embodiment, the reference is wild-type or healthy cells. In other embodiments, and not limited to, the reference is untreated cells that have not been subjected to the test conditions, or to placebo or saline, culture medium, buffer, and / or control vectors that do not contain the target polynucleotide.

[0306] A "reference sequence" is a defining sequence used as the basis for sequence comparison. The reference sequence can be a subset or the entirety of a specific sequence; for example, a fragment of a full-length cDNA or gene sequence, or the complete cDNA or gene sequence. For peptides, the reference peptide sequence is typically of 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 in length. For nucleic acids, the reference nucleic acid sequence is typically of at least about 50 nucleotides, at least about 60 nucleotides, at least about 75 nucleotides, about 100 nucleotides, or about 300 nucleotides in length, or any integer between or near these values. In some embodiments, the reference sequence is the wild-type sequence of the target protein. In other embodiments, the reference sequence is a polynucleotide sequence encoding a wild-type protein.

[0307] The terms “RNA-programmable nuclease” and “RNA-guided nuclease” are used in conjunction with one or more RNAs that are not cleaving targets (e.g., binding or associating). In some embodiments, when forming a complex with RNA, the RNA-programmable nuclease may be referred to as a nuclease:RNA complex. Typically, the bound RNA is referred to as guide RNA (gRNA). gRNA can exist as a complex of two or more RNAs or as a single RNA molecule. gRNA existing as a single RNA molecule may be referred to as single guide RNA (sgRNA), although “gRNA” is used interchangeably to refer to guide RNA existing as a single molecule or a complex of two or more molecules. Typically, gRNA existing as a single RNA species contains two domains: (1) a domain homologous to the target nucleic acid (e.g., guiding the binding of the Cas9 complex to the target); and (2) a domain that binds the Cas9 protein. In some embodiments, domain (2) corresponds to a sequence called tracrRNA and contains a stem-loop structure. For example, in some embodiments, domain (2) is identical or homologous to the tracrRNA 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 including domain 2) can be found in U.S. Provisional Patent Application, USSN 61 / 874,682, filed September 6, 2013, entitled “Switchable Cas9 Nucleases and Uses Thereof,” and U.S. Provisional Patent Application, USSN 61 / 874,746, filed September 6, 2013, entitled “Delivery System For Functional Nucleases,” the entire contents of which are incorporated herein by reference. In some embodiments, the gRNA comprises two or more of domains (1) and (2) and may be referred to as an “extended gRNA.” For example, as described herein, an extended gRNA will bind two or more Cas9 proteins and bind target nucleic acids in two or more different regions. gRNA contains a nucleotide sequence complementary to the target site, which mediates the binding of the nuclease / RNA complex to the target site, providing sequence specificity for the nuclease:RNA complex.

[0308] In some implementations, the RNA-programmable nuclease is a Cas9 endonuclease (of a CRISPR-related system), such as Cas9 (Casn1) from Streptococcus pyogenes (see, for example, “Complete genome sequence of an Mlstrain of Streptococcus pyogenes.” Ferretti JJ, McShan WM, Ajdic DJ, Savic D.J., 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); "CRISPR RNA maturation by trans-encoded small RNA and host factor RNase"). III."Deltcheva E.,Chylinski K.,Sharma CM.,Gonzales K.,Chao Y.,PirzadaZ.A.,Eckert MR,Vogel J.,Charpentier E.,Nature 471:602-607(2011).

[0309] Because RNA-programmable nucleases (such as Cas9) use RNA:DNA hybridization to target DNA cleavage sites, these proteins can, in principle, target any sequence specified by the guide RNA. Methods for site-specific cleavage (e.g., genome modification) using RNA-programmable nucleases (e.g., Cas9) are known in the art (see, for example, 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 which are incorporated herein by reference.

[0310] The term "single nucleotide polymorphism (SNP)" is a variation of a single nucleotide occurring at a specific location in the genome, where each variation is present to a certain degree (e.g., >1%) in the population. For example, at a specific base position in the human genome, the C nucleotide may be present in most individuals, but in a minority of individuals, that position is occupied by A. This means that an SNP exists at that specific location, and the two possible nucleotide variations, C or A, are called alleles at that location. SNPs are the basis for differences in disease susceptibility. The severity of disease and how our bodies respond to treatment are also manifestations of genetic variation. SNPs can fall into the coding region of a gene, the non-coding region of a gene, or the intergenic region (the region between genes). In some implementations, due to the degeneracy of the genetic code, SNPs within the coding sequence do not necessarily change the amino acid sequence of the resulting protein. There are two types of SNPs in coding regions: synonymous SNPs and non-synonymous SNPs. Synonymous SNPs do not affect the protein sequence, while non-synonymous SNPs change the amino acid sequence of the protein. The non-synonymous SNPs are of two types: missense and nonsense. SNPs not located in protein-coding regions can still affect gene splicing, transcription factor binding, messenger RNA degradation, or the sequence of non-coding RNA. Gene expression affected by such SNPs is called an eSNP (expressed SNP), which can be located upstream or downstream of the gene. Single nucleotide variants (SNVs) are variations of a single nucleotide, with no frequency limit, and can occur in somatic cells. Somatic single nucleotide variants are also called single nucleotide alterations.

[0311] "Specific binding" refers to 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 the polypeptides and / or nucleic acid molecules of the present invention, but substantially does not recognize and bind to other molecules in the sample, such as biological samples.

[0312] Nucleic acid molecules that can be used in the methods of this invention include any nucleic acid molecule encoding a polypeptide or fragment thereof of the invention. Such nucleic acid molecules do not need to be 100% identical to an endogenous nucleic acid sequence, but generally exhibit substantially the same identity. Polynucleotides having “substantially the same” identity with an endogenous sequence are generally capable of hybridizing with at least one strand of a double-stranded nucleic acid molecule. “Hybridization” refers to the pairing of complementary polynucleotide sequences (e.g., genes described herein) or portions thereof under various stringent conditions to form a double-stranded molecule. (See, for example, Wahl, GM and SLBerger (1987) Methods Enzymol. 152:399; Kimmel, AR (1987) Methods Enzymol. 152:507).

[0313] For example, stringent salt concentrations are typically below about 750 mM NaCl and 75 mM trisodium citrate, preferably below about 500 mM NaCl and 50 mM trisodium citrate, and more preferably below about 250 mM NaCl and 25 mM trisodium citrate. Low stringent hybridization can be obtained in the absence of organic solvents such as formamide, while high stringent hybridization can be obtained in the presence of at least about 35% formamide, more preferably at least about 50% formamide. Stringent temperature conditions typically include temperatures of at least about 30°C, more preferably at least about 37°C, and most preferably at least about 42°C. For example, sodium dodecyl sulfate (SDS), and the inclusion or exclusion of vector DNA, are well known to those skilled in the art. Different degrees of stringency are achieved by combining these different conditions as needed. In one embodiment, hybridization will occur at 30°C in 750 mM NaCl, 75 mM trisodium citrate, and 1% SDS. In another embodiment, hybridization will occur 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 one embodiment, hybridization will occur 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 apparent to those skilled in the art.

[0314] For most applications, the stringency of the post-hybridization wash step will vary. The stringency of the wash can be defined by salt concentration and temperature. As mentioned above, the stringency of the wash can be increased by decreasing the salt concentration or increasing the temperature. For example, the stringent salt concentration for the wash step is preferably less than about 30 mM NaCl and 3 mM trisodium citrate, most preferably less than about 15 mM NaCl and 1.5 mM trisodium citrate. The stringent temperature conditions for the wash step 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, the wash step will occur at 25°C in 30 mM NaCl, 3 mM trisodium citrate, and 0.1% SDS. In a more preferred embodiment, the wash step will be carried out at 42°C in 15 mM NaCl, 1.5 mM trisodium citrate, and 0.1% SDS. In an even more preferred embodiment, the wash step will be carried out at 68°C in 15 mM NaCl, 1.5 mM trisodium citrate, and 0.1% SDS. Other variations of these conditions will be apparent to those skilled in the art. Hybridization techniques are well known to those skilled in the art and have been described, for example, by 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.

[0315] "Split" means to divide into two or more segments.

[0316] "Split Cas9 protein" or "split Cas9" refers to a Cas9 protein provided as an N-terminal and C-terminal fragment encoded by two separate nucleotide sequences. The polypeptides corresponding to the N-terminal and C-terminal portions of the Cas9 protein can be spliced ​​to form a "reconstructed" Cas9 protein. In certain embodiments, the Cas9 protein is split into two fragments within a disordered region of the protein, for example, as described in Nishimasu et al., Cell, Volume 156, Issue 5, pp. 935-949, 2014, or as in Jiang et al. (2016) Science 351:867-871. PDB file:5F9R, each of which is incorporated herein by reference. In some embodiments, the protein is split into two fragments at any C, T, A, or S position within the SpCas9 region, approximately between amino acids A292-G364, F445-K483, or E565-T637, or in any other Cas9, Cas9 variant (e.g., nCas9, dCas9), or other nap DNAbp. In some embodiments, the protein is split into two fragments at SpCas9 T310, T313, A456, S469, or C574. In some embodiments, the process of splitting the protein into two fragments is referred to as “split” the protein.

[0317] In other embodiments, the N-terminal portion of the Cas9 protein contains amino acids 1-573 or 1-637 of wild-type Streptococcus pyogenes Cas9 (SpCas9) (NCBI Reference Sequence: NC_002737.2, Uniprot Reference Sequence: Q99ZW2) and the C-terminal portion of the Cas9 protein contains amino acids 574-1368 or 638-1368 of wild-type SpCas9 or their corresponding positions.

[0318] The C-terminal portion of a split Cas9 protein can be linked to the N-terminal portion of another split Cas9 protein to form the 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. Therefore, in some embodiments, the C-terminal portion of the split Cas9 contains a portion of amino acid (551-651)-1368 of spCas9. “(551-651)-1368” refers to the amino acid sequence from amino acid 551-651 (inclusive) to amino acid 1368.For example, the C-terminal portion of a split Cas9 may contain a subset of any amino acid from spCas9: 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, 57 3-1368, 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, 625-13 68, 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 contains a portion of 574-1368 or 638-1368 of the SpCas9 protein.

[0319] "Subject" refers to mammals, including but not limited to human or non-human mammals such as cattle, horses, dogs, sheep, or cats. Subjects include livestock and domesticated animals raised to produce labor and provide goods such as food, including but not limited to cattle, goats, chickens, horses, pigs, rabbits, and sheep.

[0320] "Substantially identical" means that the polypeptide or nucleic acid molecule is identical to a reference amino acid sequence (e.g., any of the amino acid sequences described herein) or nucleic acid sequence (e.g., any of the nucleic acid sequences described herein). In one embodiment, such a sequence is at least 60%, 80%, 85%, 90%, 95%, or even 99% identical to the sequence used for comparison at the amino acid level or nucleic acid level.

[0321] Sequence identity is typically determined using sequence analysis software (e.g., Sequence Analysis Software Package of the Genetics Computer Group, University of Wisconsin Biotechnology Center, 1710 University Avenue, Madison, Wis. 53705, BLAST, BESTFIT, GAP, or PILEUP / PRETTYBOX programs). Such software matches identical or similar sequences by specifying degrees of homology for various substitutions, deletions, and / or other modifications. Conserved substitutions typically include substitutions within the following groups: glycine, alanine; valine, isoleucine, leucine; aspartic acid, glutamic acid, asparagine, glutamine; serine, threonine; lysine, arginine; and phenylalanine, tyrosine. In an exemplary method for determining the degree of identity, the BLAST program can be used, where probability scores between e-3 and e-100 represent closely related sequences.

[0322] For example, COBALT is used with the following parameters:

[0323] a)alignment parameters:Gap penalties-11,-1and End-Gap penalties-5,-1,

[0324] b)CDD Parameters: Use RPS BLAST on; Blast E-value 0.003; Find Conservedcolumns and Recompute on, and

[0325] c) Query Clustering Parameters: Use query clusters on; Word Size 4; Maxcluster distance 0.8; Alphabet Regular.

[0326] For example, EMBOSS is used with the following parameters:

[0327] a)Matrix:BLOSUM62;

[0328] b) GAP OPEN: 10;

[0329] c) GAP EXTEND: 0.5;

[0330] d)OUTPUT FORMAT:pair;

[0331] e)END GAP PENALTY:false;

[0332] f)END GAP OPEN:10; and

[0333] g)END GAP EXTEND:0.5.

[0334] The term "target site" refers to a sequence within a nucleic acid molecule modified by a nucleobase editor. In one embodiment, the target site is deaminated by a deaminase or a fusion protein containing a deaminase (e.g., cytidine or adenine deaminase).

[0335] "tet methylcytosine dioxygenase 2 (TET2) polypeptide" refers to a protein that shares at least about 85% amino acid sequence identity with NCBI accession number FM992369.1 or a fragment thereof and possesses catalytic activity in converting methylcytosine to 5-hydroxymethylcytosine. Defects in this gene are associated with myeloproliferative disorders, and the enzyme's ability to methylate cytosine contributes to transcriptional regulation. An exemplary TET2 amino acid sequence is provided below.

[0336] CAX30492.1 tet oncogene family member 2 [Homo sapiens]

[0337]

[0338] "TET methylcytosine dioxygenase 2 (TET2) polynucleotide" refers to a nucleic acid molecule encoding a TET2 polypeptide. The TET polypeptide encodes a methylcytosine dioxygenase and has transcriptional regulatory activity. Exemplary TET2 nucleic acids are presented below.

[0339]

[0340] "Transforming growth factor receptor 2 (TGFBRII) polypeptide" refers to a protein that shares at least approximately 85% sequence identity with NCBI accession number ABG65632.1 or a fragment thereof and possesses immunosuppressive activity. An exemplary amino acid sequence is provided below:

[0341] ABG65632.1 Transforming growth factor β receptor II [Homo sapiens]

[0342] MGRGLLRGLWPLHIVLWTRIASTIPPHVQKSVNNDMIVTDNNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSD ECNDNIIFSEEYNTSNPDLLLVIFQVTGISLLPPLGVAISVIIIFYCYRVNRQQKLSSTWETGKTRKLMEFSEHCAIILEDDRSDISSTCANNINHNTELLPIELDTLVGKGRFAEVYKAKLKQNTSEQFETVAVKIFPYEE YASWKTEKDIFSDINLKHENILQFLTAEERKTELGKQYWLITAFHAKGNLQEYLTRHVISWEDLRKLGSSLARGIAHLHSDHTPCGRPKMPIVHRDLKSSNILVKNDLTCCLCDFGLSLRLDPTLSVDDLANSGQVGTARYM APEVLESRMNLENVESFKQTDVYSMALVLWEMTSRCNAVGEVKDYEPPFGSKVREHPCVESMKDNVLRDRGRPEIPSFWLNHQGIQMVCETLTECWDHDPEARLTAQCVAERFSELEHLDRLSGRSCSEEKIPEDGSLNTTK

[0343] "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 provided below.

[0344] >M85079.1 Human TGF-β II receptor mRNA, complete cds

[0345]

[0346] "T-cell immune receptor (TIGIT) polypeptide with Ig and ITIM domains" refers to a protein that has at least about 85% sequence identity with NCBI accession number ACD74757.1 or a fragment thereof and has immunomodulatory activity. An exemplary TIGIT amino acid sequence is provided below.

[0347] >ACD74757.1 T-cell immune receptor with Ig and ITIM domains [Homo sapiens] MRWCLLLIWAQGLRQAPLASGMMTGTIETTGNISAEKGGSIILQCHLSSTTAQVTQVNWEQQDQLLAICNADLGWHISPSFKDRVAPGPGLGLTLQSLTVNDTGEYFCIYHTYPDGTYTGRIFLEVLESSVAEHGARFQIPLLGAMAATLVVICTAVIVVVALTRKKKALRIHSVEGDLRRKSAGQEEWSPSAPSPPGSCVQAEAAPAGLCGEQRGEDCAELHDYFNVLSYRSLGNCSFFTETG

[0348] "T-cell immune receptors with Ig and ITIM domains (TIGIT) polynucleotides" refers to nucleic acids encoding the TIGIT polypeptide. The TIGIT gene encodes an inhibitory immune receptor associated with tumorigenesis and T-cell exhaustion. Exemplary nucleic acid sequences are provided below:

[0349] >EU675310.1 Homo sapiens T cell immune receptor (TIGIT) mRNA with Ig and ITIM domains, completecds

[0350] CGTCCTATCTGCAGTCGGCTACTTTCAGTGGCAGAAGAGGCCACATCTGCTTCCTGTAGGCCCTCTGGGCAGAAGCATGCGCTGGTGTCTCCTCCTGATCTGGGCCCAGGGGCTGAGGCAGGCTCCCCTCGCCTCAGGAATGATGACAGGCACAATAGAAACAACGGGGAACATTTCTGCAGAGAAAGGTGGCTCTATCATCTTACA ATGTCACCTCTCCTCCACCACGGCACAAGTGACCCAGGTCAACTGGGAGCAGCAGGACCAGCTTCTGGCCATTTGTAATGCTGACTTGGGGTGGCACATCTCCCCATCCTTCAAGGATCGAGTGGCCCCAGGTCCCGGCCTGGGCCTCACCCTCCAGTCGCTGACCGTGAACGATACAGGGGAGTACTTCTGCATCTATCACACCTAC CCTGATGGGACGTACACTGGGAGAATCTTCCTGGAGGTCCTAGAAAGCTCAGTGGCTGAGCACGGTGCCAGGTTCCAGATTCCATTGCTTGGAGCCATGGCCGCGACGCTGGTGGTCATCTGCACAGCAGTCATCGTGGTGGTCGCGTTGACTAGAAAGAAGAAAGCCCTCAGAATCCATTCTGTGGAAGGTGACCTCAGGAGAAAAT CAGCTGGACAGGAGGAATGGAGCCCCAGTGCTCCCTCACCCCCAGGAAGCTGTGTCCAGGCAGAAGCTGCACCTGCTGGGCTCTGTGGAGAGCAGCGGGGAGAGGACTGTGCCGAGCTGCATGACTACTTCAATGTCCTGAGTTACAGAAGCCTGGGTAACTGCAGCTTCTTCACAGAGACTGGTTAGCAACCAGAGGCATCTTCTGG

[0351] "T-cell receptor α constant (TRAC) polypeptide" refers to a protein that has at least about 85% amino acid sequence identity with NCBI accession number P01848.2 or a fragment thereof and has immunomodulatory activity. An exemplary amino acid sequence is provided below.

[0352] >sp|P01848.2|TRAC_HUMAN RecName:Full=T cell receptor α constant

[0353] IQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKTVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRILLLKVAGFNLLMTLRLWSS

[0354] "T cell receptor α constant (TRAC) polynucleotide" refers to the nucleic acid encoding the TRAC polypeptide. An example TRAC nucleic acid sequence is provided below.

[0355] >X02592.1 Human mRNA of the T cell receptor α chain (TCR-alpha)

[0356]

[0357] As used in this article, "transduction" refers to the transfer of genes or genetic material into cells via viral vectors.

[0358] As used in this article, “transformation” refers to the process of introducing genetic changes into cells produced by introducing exogenous nucleic acids.

[0359] "Transfection" refers to the transfer of genes or genetic material into cells through chemical or physical means.

[0360] "Translocation" refers to the rearrangement of nucleic acid fragments between non-homologous chromosomes.

[0361] As used herein, the terms “treat,” “treating,” “treatment,” etc., refer to reducing or improving a condition and / or related symptoms or achieving a desired pharmacological and / or physiological effect. It should be understood that, although not excluded, treating a condition or symptom does not require the complete elimination of the condition, symptom, or related symptoms. In some embodiments, the effect is therapeutic, i.e., but not limited to, partially or completely reducing, weakening, eliminating, alleviating, reducing, lessening, lowering the intensity of the disease and / or unfavorable symptoms attributable to the disease, or curing the disease and / or unfavorable symptoms. In some embodiments, the effect is preventative, i.e., the effect protects against or prevents the occurrence or recurrence of the disease or symptom. For this purpose, currently disclosed methods include administering a therapeutically effective amount of the composition as described herein.

[0362] "Uracil glycosylase inhibitor" or "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, the UGI is a protein, fragment thereof, or domain capable of inhibiting the uracil-DNA glycosylase base excision repair enzyme. In some embodiments, the UGI domain comprises a wild-type UGI or a modified version thereof. In some embodiments, the UGI domain comprises a fragment of the exemplary amino acid sequence set forth below. In some embodiments, the UGI fragment 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%, or at least 99%, or 100% identical to the exemplary UGI sequences provided below. In some embodiments, the UGI comprises an amino acid sequence homologous to the exemplary UGI amino acid sequence or a fragment thereof, as described below. In some embodiments, the UGI or a portion thereof is 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 at least 99% or 100% identical to a wild-type UGI or a portion thereof, as described below. An exemplary UGI comprises the following amino acid sequence:

[0363] >splP14739IUNGI_BPPB2 Uracil-DNA glycosylation enzyme inhibitor MTNLSDIIEKETGKQLVIQESILMLPEEVEEVIGNKPESDILVHTAYDESTDENVMLLTSD APEYKPWALVIQDSNGENKIKML.

[0364] The term "vector" refers to a means of introducing a nucleic acid sequence into a cell to produce transformed cells. Vectors include plasmids, transposons, bacteriophages, viruses, liposomes, and episomes. An "expression vector" is a nucleic acid sequence containing a nucleotide sequence to be expressed in a recipient cell. Expression vectors may include additional nucleic acid sequences to promote and / or facilitate the expression of the introduced sequence, such as initiation, termination, enhancer, promoter, and secretion sequences.

[0365] "The zeta chain of the T-cell receptor-associated protein kinase 70 (ZAP70) polypeptide" refers to a protein that has at least about 85% amino acid sequence identity with NCBI accession number AAH53878.1 and possesses kinase activity. An exemplary amino acid sequence is provided below.

[0366] >AAH53878.1 Zeta-chain (TCR)-associated protein kinase 70kDa [Homo sapiens]

[0367] MPDPAAHLPFFYGSISRAEAEEHLKLAGMADGLFLLRQCLRSLGGYVLSLVHDVRFHHFPIERQLNGTYAIAGGKAHCGPAELCEFYSRDPDGLPCNLRKPCNRPSGLEPQPGVFDCLRDAMVRDYVRQTWKLEGEALEQAIISQAPQVEKLIA TTAHERMPWYHSSLTREEAERKLYSGAQTDGKFLLRPRKEQGTYALSLIYGKTVYHYLISQDKAGKYCIPEGTKFDTLWQLVEYLKLKADGLIYCLKEACPNSSASNASGAAAPTLPAHPSTLTHPQRRIDTLNSDGYTPEPARITSPDKPRPMP MDTSVYESPYSDPEELKDKKLFLKRDNLLIADIELGCGNFGSVRQGVYRMRKKQIDVAIKVLKQGTEKADTEEMMREAQIMHQLDNPYIVRLIGVCQAEALMLVMEMAGGGPLHKFLVGKREEIPVSNVAELLHQVSMGMKYLEEKNFVHRDLAA RNVLLVNRHYAKISDFGLSKALGADDSYYTARSAGKWPLKWYAPECINFRKFSSRSDVWSYGVTMWEALSYGQKPYKKMKGPEVMAFIEQGKRMECPPECPPELYALMSDCWIYKWEDRPDFLTVEQRMRACYYSLASKVEGPPGSTQKAEAACA

[0368] "The zeta chain of the T cell receptor-associated protein kinase 70 (ZAP70) polynucleotide" refers to the nucleic acid encoding the 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 provided below.

[0369] >BC053878.1 Homo sapiens zeta-chain (TCR)-related protein kinase 70kDa, mRNA (cDNA clone MGC:61743IMAGE:5757161), complete cds

[0370]

[0371] Any composition or method provided herein may be combined with one or more of any other compositions and methods provided herein.

[0372] DNA editing has become a viable means of altering 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 determine product outcomes in a semi-random manner, resulting in complex populations of genetic products. While precise, user-defined repair outcomes can be achieved through the homology-directed repair (HDR) pathway, several challenges have prevented efficient repair using HDR in treatment-related cell types. In practice, this pathway is inefficient compared to the competitive, error-prone non-homologous end joining pathway. Furthermore, HDR is strictly limited to the G1 and S phases of the cell cycle, preventing precise repair of DSBs in post-mitotic cells. Therefore, it has proven difficult or impossible to efficiently alter genome sequences in these populations in a user-defined, programmable manner. Attached Figure Description

[0373] Figure 1A and 1B This is a diagram of three proteins that affect T cell function. Figure 1A This is a diagram of the TRAC protein, a key component of graft-versus-host disease. Figure 1B This is a diagram of the B2M protein, a component of the MHC class 1 antigen-presenting complex found on nucleated cells, which can be recognized by the host's CD8+ T cells. Figure 1C This is a diagram illustrating T cell signaling that leads to PDCD1 gene expression, and the resulting PD-1 protein inhibits T cell signaling.

[0374] Figures 2A to 2D The transformation from A·T to G·C and the phenotypic results were described in primary cells. Figure 2A This is a violin plot depicting the reduction in protein expression, as measured by flow cytometry, after electroporation of primary human T cells with specified mRNAs and 41 single sgRNAs targeting 6 genes. The individual values ​​shown represent the average percentage of cells with reduced protein expression from two duplicate cells edited with the specified mRNA and one of the 41 sgRNAs tested. Figure 2B This is a heatmap depicting an NGS analysis of A·T to G·C conversion of eight ABE8 mRNAs and ABE7.10-m / d at six target sites. The values ​​shown reflect the average of three independent biological replicates. The location of the edited nucleotide at each target site is shown above the heatmap. Figure 2CThis is an NGS analysis plot depicting the A·T to G·C conversion in multiple-edited T cells at sites 21 (B2M), 25 (TRAC), and 24 (CIITA) after primary human T cells were electroporated, showing the mRNA and three sgRNAs in multiple-edit format. Figure 2D (Above) is a protein expression map of B2M, CIITA, and TRAC proteins, as measured by flow cytometry on the cell population in Figure 1. Five days after 2C electroporation. Values ​​shown are from representative donors. Figure 2D (The image below) is a table describing the percentage of cell expression measured by flow cytometry after using the specified ABE edit.

[0375] Figure 3 This is a heatmap depicting the protein knockdown of the ABE editor in primary T cells as measured by flow cytometry. Eight mRNAs encoding the ABE8 editor and two mRNAs encoding ABE7.10-m / d were transfected into T cells containing 41 sgRNAs targeting six genes, and their effects on protein expression were measured using flow cytometry. The values ​​shown are the mean of n=2 independent replicates.

[0376] Figure 4 This is a graph depicting the effective cytotoxic activity of ABE-edited CAR-T cells against antigen-positive tumor cells. Fluorescently labeled RPMI-8226 cells were seeded at time = 0 hours, and their growth was monitored using the IncuCyte live-cell imaging system for 28 hours prior to CAR-T cell introduction. The specified ABE (…) was used… Figure 1C Multiple-edited T cells were transduced with a lentivirus encoding an anti-BCMACAR molecule and introduced into RPMI-8226 cells at time = 28 hours. Growth of RPMI-8226 cells was monitored over an additional 68 hours. Values ​​shown are the mean of n = 3 independent biological replicates.

[0377] Figure 5A and 5B RNA amplicon sequencing was used to detect cellular A-to-I editing in RNA associated with ABE treatment. Individual data points are shown, with error bars representing standard deviations. Biological replicates of n=3 were performed on different days. Figure 5A This is a graph depicting the A-to-I editing frequency in the targeted RNA amplicon of the core ABE 8 construct compared to the ABE7 and Cas9(D10A) nickase controls. Figure 5B This is a graph depicting the A-to-I editing frequency in the target RNA amplicon of ABE8, which has mutations reported to improve RNA off-target editing.

[0378] Figure 6A and6B This is a diagram illustrating examples of gating mechanisms used to assess protein knockdown in T cells. It represents a representative gating strategy used for population analysis of live, single lymphocytes to determine reductions in surface proteins via flow cytometry.

[0379] Figure 7 It is a diagram depicting alleles generated by ABE across eight different genomic loci in HEK293T cells.

[0380] Figure 8A and 8B The study depicted whole transcriptome and whole genome sequencing data from cells processed with base editor mRNA. Figure 8A This is a banding diagram depicting the whole transcriptome sequencing of HEK293T cells treated with specified mRNA. Variant allele frequencies of A->G mutations across the RNA transcriptome were observed in replicate HEK293T cell experiments. Total A->G mutations are shown above each sample. Figure 8B This is a banding plot depicting the whole transcriptome sequencing in T cells treated with specified mRNAs. Variant allele frequencies of A-to-G mutations across the RNA transcriptome were observed in three different T cell donors. Total A-to-G mutations are shown above each sample.

[0381] Figure 9A and 9B Representative examples of gates for flow sorting of B2M-positive and B2M-negative cells prior to whole-genome sequencing are depicted. Figure 9A Representative diagrams and phylogenetic trees of live B2M-positive HEK293T cells sorted into single-cell clones under untreated conditions are depicted. Figure 9B Representative diagrams and phylogenetic images of live, B2M-negative HEK293T cells sorted for all treatment conditions (ABE, CBE, or Cas9-treated cells) are depicted.

[0382] Figure 10 This is a table describing the Cas9 variants used to access all possible PAMs within the NRNN PAM space. Only Cas9 variants that require identification of three or fewer defining nucleotides in their PAM are listed. Non-G PAM variants include SpCas9-NRRH, SpCas9-NRTH, and SpCas9-NRCH. Detailed Implementation

[0383] The present invention is characterized by genetically modified immune cells comprising a novel adenosine base editor (e.g., ABE8) that possess enhanced antitumor activity, resistance to immunosuppression, and a reduced risk of inducing graft-versus-host disease or host-versus-graft disease, or a combination thereof. The present invention is further characterized by methods for producing and using these modified immune effector cells (e.g., immune effector cells such as T cells). The present invention is also characterized by methods for treating subjects who have or are predisposed to developing tumors, graft-versus-host disease (GVHD), or host-versus-graft disease (HVGD) with an effective amount of modified immune effector cells (e.g., CAR-T cells).

[0384] Base editor systems containing adenosine deaminase, as described herein, can be used to modify immune effector cells to express chimeric antigen receptors (CARs) and knock out or weaken specific genes to reduce the negative impact their expression may have on immune cell function.

[0385] Autologous, patient-derived chimeric antigen receptor-T-cell (CAR-T) therapy has shown significant efficacy in treating some blood cancers. While these products offer significant clinical benefits to patients, the need for personalized therapies presents substantial manufacturing challenges and financial burdens. Allogeneic CAR-T therapy has been developed as a potential solution to these challenges, offering similar clinical efficacy characteristics to autologous products while using cells from a single healthy donor to treat many patients, thus significantly reducing commodity costs and batch-to-batch variability.

[0386] Most first-generation allogeneic CAR-T cells use nucleases to introduce two or more targeted genomic DNA double-strand breaks (DSBs) into a target T cell population, relying on error-prone DNA repair to generate a knockout mutation in the target gene in a semi-random manner. This nuclease-based gene knockout strategy aims to reduce the risk of graft-versus-host disease and host rejection of CAR-T. However, the simultaneous induction of multiple DSBs results in the final cell product containing large-scale genomic rearrangements, such as balanced and unbalanced translocations, as well as a relatively high abundance of local rearrangements, including inversions and large deletions. Furthermore, with increasing amounts of synchronous genetic modification being performed by induced DSBs, considerable genotoxicity has been observed in the treated cell population. This has the potential to significantly reduce the cell expansion potential per production run, thereby reducing the number of patients that each healthy donor can treat.

[0387] Base editors (BEs) are an emerging class of gene-editing agents capable of efficiently, user-defined, targeted genomic DNA modification without the creation of DSBs. Here, an alternative approach to producing allogeneic CAR-T cells is proposed, utilizing base editing technology to reduce or eliminate detectable genomic rearrangements while simultaneously increasing cell expansion. As illustrated herein, simultaneous modification of three loci via base editing produces highly efficient gene knockouts without detectable translocation events, compared to nuclease-only editing strategies. In one embodiment, the base editor (e.g., ABE8) is used for multiplex base editing of at least one cell surface target (e.g., including but not limited to TRAC, B2M, CD7, PDCD1, CBLB, and / or CITA) in T cells. In one embodiment, ABE8 is used for multiplex base editing of TRAC, B2M, and CIITA in T cells. Multiplex gene editing may facilitate the creation of CAR-T cell therapies with improved therapeutic properties. This approach addresses known limitations of multiplexed T cell products and represents a promising step towards next-generation cell-based precision therapies.

[0388] Chimeric antigen receptors and CAR-T cells

[0389] This invention provides immune cells modified using a nucleobase editor for expressing chimeric antigen receptors (CARs) as described herein. Modifying immune cells to express chimeric antigen receptors enhances their immune activity, wherein the chimeric antigen receptors have an affinity for epitopes on antigens, and where the antigens are associated with adaptive alterations in the organism. For example, chimeric antigen receptors can have an affinity for epitopes on proteins expressed in tumor cells. Because CAR-T cells can function independently of the major histocompatibility complex (MHC), activated CAR-T cells can kill tumor cells expressing antigens. The direct action of CAR-T cells evades tumor cell defense mechanisms that have evolved in response to MHC-mediated antigen presentation to immune cells.

[0390] 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 an individual expressing antibodies against the individual's own tissues).

[0391] Some implementations include autologous immune cell immunotherapy, wherein the immune cells are obtained from an individual suffering from a disease or adaptive alteration, characterized by cancerous or other altered cells expressing surface markers. The obtained immune cells are genetically modified to express chimeric antigen receptors and are effectively redirected to specific antigens. Therefore, in some implementations, the immune cells are obtained from an individual requiring CAR-T immunotherapy. In some implementations, these autologous immune cells are cultured and modified shortly after being obtained from the individual. In other implementations, the autologous cells are obtained and then stored for future use. This practice may be desirable for individuals who may be receiving parallel treatments that will reduce immune cell counts in the future. In allogeneic immune cell immunotherapy, the immune cells can be obtained from a donor other than the individual to be treated. After modification to express chimeric antigen receptors, the immune cells are administered to the individual to treat the tumor. In some implementations, the immune cells to be modified to express chimeric antigen receptors can be obtained from a pre-existing primary culture of immune cells.

[0392] Immune cells and / or immune effector cells can be isolated or purified from samples collected from individuals 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 mononuclear blood cells by centrifugation. Immune effector cells can be further isolated or purified using selective purification methods based on cell-specific markers such as CD25, CD3, CD4, CD8, CD28, CD45RA, or CD45RO. In one embodiment, CD25+ is used as a marker for selecting regulatory T cells. In another embodiment, the present invention provides T cells with a targeted gene knockout at the TCR constant region (TRAC) responsible for TCRαβ surface expression. TCRαβ-deficient CAR T cells are compatible with allogeneic immunotherapy (Qasim et al., Sci. Transl. Med. 9, eaaj 2013 (2017); Valton et al., Mol Ther. 2015 Sep; 23(9): 1507–1518). If necessary, residual TCRαβT cells can be removed using the CliniMACS magnetic bead removal method to minimize the risk of GVHD. In another embodiment, the present invention provides ex vivo selected donor T cells to recognize minor histocompatibility antigens expressed on recipient hematopoietic cells, thereby minimizing the risk of graft-versus-host disease (GVHD), a leading 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 fluorescently activated cell sorting, fluorescently labeled antibodies with affinity for immune effector cell markers are used to label immune effector cells in a sample. Gating strategies suitable for cells expressing the labels are used to isolate the cells. For example, T lymphocytes can be isolated from other cells in a sample using, for example, fluorescently labeled antibodies specific to immune effector cell markers (e.g., CD4, CD8, CD28, CD45) and corresponding gating strategies. In one embodiment, a CD45 gating strategy is employed. In some implementations, a gating strategy targeting other biomarkers specific to immune effector cells is used instead of or in combination with the CD45 gating strategy.

[0393] The immune effector cells considered in this invention are effector T cells. In some embodiments, the effector T cells are naïve CD8 cells. + T cells, cytotoxic T cells, or regulatory T (Treg) cells. In some embodiments, effector T cells are thymocytes, immature T lymphocytes, mature T lymphocytes, resting T lymphocytes, or activated T lymphocytes. In some embodiments, immune effector cells are CD4+ cells. + CD8+ T cells or CD4 - CD8 - T cells. In some embodiments, immune effector cells are helper T cells. In some embodiments, helper T cells are helper T cell 1 (Th1), helper T cell 2 (Th2), or CD4-expressing helper T cells (CD4+ T cells). In some embodiments, immune effector cells are any other subset of T cells. In addition to chimeric antigen receptors, modified immune effector cells may also express exogenous cytokines, different chimeric receptors, or any other agents that enhance immune effector cell signaling or function. For example, co-expression of chimeric antigen receptors and cytokines can enhance the ability of CAR-T cells to lyse target cells.

[0394] The chimeric antigen receptor considered in this invention comprises an extracellular binding domain, a transmembrane domain, and an intracellular domain. Binding of the antigen to the extracellular binding domain can activate CAR-T cells and produce effector responses, including CAR-T cell proliferation, cytokine production, and other processes leading to the death of antigen-expressing cells. In some embodiments of this invention, the chimeric antigen receptor further comprises a linker.

[0395] The extracellular binding domain of the chimeric antigen receptors considered herein contains 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).

[0396] In various embodiments, the CAR specifically binds to alpha-fetoprotein. Exemplary anti-alpha-fetoprotein 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).

[0397] In various implementation schemes, CAR specifically binds to BCMA. Exemplary anti-BCMACARs 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 (bluebird 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 The following are listed: 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-TherapyBio-medicine Technology Co Ltd), and BCMA02 / bb2121 (bluebird bio Inc.). The polypeptide sequence of the BCMA02 / bb2121 CAR is as follows:

[0398] MALPVTALLLPLALLLHAARPDIVLTQSPPSLAMSLGKRATISCRASESVTILGSHLIHWYQQKPGQPPTLLIQLASNVQTGVPARFSGSGSRTDFTLTIDPVEEDDVAVYYCLQSRTIPRTF GGGTKLEIKGSTSGSGKPGSGEGSTKGQIQLVQSGPELKKPGETVKISCKASGYTFTDYSINWVKRAPGKGLKWMGWINTETREPAYAYDFRGRFAFSLETSASTAYLQINNLKYEDTATYFC ALDYSYAMDYWGQGTSVTVSSAAATTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSC RFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR

[0399] In various implementations, the CAR specifically binds to CCK2R. Exemplary anti-CCK2R CAR

[0400] Including but not limited to anti-CCK2R CAR-T adaptor molecule (CAM) + anti-FITC CAR T cell therapy (cancer), Endocyte / Purdue (Purdue University).

[0401] In various embodiments, the CAR specifically binds to the 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), RNACART123 (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 (General Hospital of the Chinese People's Liberation Army). 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 Institute of Gene Immunology), ALLO-501 (Pfizer Inc.), ATA-190 (QIMR Berghofer Institute for Medical Research), AUTO-1 (University College London), AVA-008 (Avacta) Ltd., axicabtageneciloleucel (Kite Pharma Inc.), BG-T19 (Guangzhou Bio-gene Technology Co Ltd.), and BinD-19 (Shenzhen BinDeBio Ltd.).), BPX-401 (Bellicum Pharmaceuticals Inc), CAR19h28TM41BBz (Westmead Institute for Medical Research), C-CAR-011 (General Hospital of the Chinese People's Liberation Army), 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 Hengrui Biotechnology Co., Ltd.), 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 (MingJuTherapeutics (Shanghai) Co., Ltd), KD-C-19 (Nanjing Kaedi Biotech Inc), LinCART19 (iCellGene Therapeutics), lisocabtagene maraleucel (Juno Therapeutics Inc), MatchCART (Shanghai Hrain 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 Life Science Technology Co., Ltd.), RB-1916 (Refuge Biotechnologies Inc), SKLB-083019 (Chengdu Yinhe Biomedical Co., Ltd.), spCART-19 (Shanghai Unica-Therapeutic Biomedical 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). 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 (Wugen Inc.). In 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 (General Hospital of the Chinese People's Liberation Army), MB-106 (Fred Hutchinson Cancer Research Center), and MB-CART20.1 (Miltenyi Biotec GmbH).

[0402] 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 Unica-Therapeutic Biomedical Technology Co., Ltd.), JCAR-018 (Opus Bio Inc.), MendCART (Shanghai Hengrun Biotechnology Co., Ltd.), 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 (UNCLineberger Comprehensive Cancer Center), CBM-C30.1 (General Hospital of the Chinese People's Liberation Army), 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 γδ T cell therapy (acute myeloid leukemia), TC BioPharm / University College London, CAR33VH (Opus Bio Inc), CART-33 (General Hospital of the Chinese People's Liberation Army), CIK-CAR.CD33 (Formula Pharmaceuticals Inc), UCART-33 (Cellectis SA), and VOR-33 (Columbia University).

[0403] 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 CD38A2. Exemplary anti-CD38A2 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 HSan 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 CART cells (Baylor College of Medicine), UCART7 (Washington University in St Louis).

[0404] 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-β receptor (CTBR). Exemplary anti-chimeric TGF-β receptor (CTBR) CARs include, but are not limited to, CAR-CTBR T cells (bluebirdbio Inc). In various embodiments, the CAR specifically binds to Claudin18.2. Exemplary anti-Claudin18.2 CARs include, but are not limited to, CAR-CLD18 T cells (Carsgen Therapeutics Ltd) and KD-022 (Nanjing KaediBiotech Inc).

[0405] 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. Exemplary anti-dual CLL1 / CD33 CARs include, but are not limited to, ICG-136 (iCell Gene Therapeutics). In various embodiments, the CAR specifically binds to dual EpCAM / CD3. Exemplary anti-dual EpCAM / CD3 CARs include, but are not limited to, IKT-701 (Icell Kealex Therapeutics). In various embodiments, the CAR specifically binds to dual ErbB / 4ab. Exemplary anti-dual ErbB / 4ab CARs include, but are 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).

[0406] In various implementations, 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 a monoclonal antibody expressing a CTLA-4 checkpoint inhibitor + PD-1 checkpoint inhibitor (EGFR-positive advanced solid tumors), Shanghai Cell Therapy Institute (Shanghai Cell Therapy Institute), CSG-EGFR (Carsgen Therapeutics Ltd.), and EGFR-IL12-CART (Pregene (Shenzhen) Biotechnology Co., Ltd.).

[0407] 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 therapy (G250-scFV-transferred renal cell carcinoma) and Erasmus Medical Center (Daniel den Hoed Cancer Center).

[0408] In various implementations, the CAR specifically binds to GD2. Exemplary anti-GD2 CARs include, but are not limited to, 1RG-CART (University College London), 4SCAR-GD2 (Shenzhen Institute of Gene Immunology), 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-15T cells (UNCLineberger Comprehensive Cancer Center), and IKT-703 (Icell Kealex Therapeutics). In various implementations, the CAR specifically binds to both GD2 and MUC1. Exemplary anti-GD2 / MUC1 CARs include, but are not limited to, PSMACAR-T (University of Pennsylvania).

[0409] 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 β-7. Exemplary anti-integrin β-7 CARs include, but are not limited to, MMG49CAR T-cell therapy (Osaka University). In various embodiments, the CAR specifically binds to LC antigens. Exemplary anti-LC antigen CARs include, but are not limited to, VM-803 (ViroMed Co Ltd) and VM-804 (ViroMed Co Ltd).

[0410] In various implementations, 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 Institute of Biomedical and Health Sciences), MesoCART (University of Pennsylvania), meso-CAR-T+PD-78 (MirImmune LLC), RB-M1 (Refuge Biotechnologies Inc.), and TC-210 (TCR2 Therapeutics Inc.).

[0411] In various implementations, 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 implementations, 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).

[0412] 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 (F1Oncology 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).

[0413] In various implementations, CAR is 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).

[0414] 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 a fragment thereof) portion of the extracellular binding domain recognizes and binds to the epitope of the antigen. In some embodiments, the antibody fragment portion of the chimeric antigen receptor is a single-chain variable fragment (scFV). The scFV comprises a light fragment and a variable fragment of a monoclonal antibody. In other embodiments, the antibody fragment portion of the chimeric antigen receptor is a multi-chain variable fragment, which may contain more than one extracellular binding domain and thus bind more than one antigen simultaneously. In the multi-chain variable fragment embodiments, a hinge region may separate the different variable fragments, providing the necessary spatial arrangement and flexibility.

[0415] 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 linked by disulfide bonds, wherein each light chain is connected to one of the heavy chains via a disulfide bridge. In some embodiments, the light chain comprises a constant region and a variable region. The complementarity-determining region located in the variable region of the antibody is responsible for the antibody's affinity for a specific antigen. Therefore, antibodies that recognize different antigens comprise different complementarity-determining regions. The complementarity-determining region is located in a variable domain of the extracellular binding domain, and the variable domain (i.e., the variable heavy chain and the variable light chain) may be linked to a linker, or in some embodiments, linked to a disulfide bond.

[0416] In some embodiments, the antigen recognized and bound by the extracellular domain is a protein or peptide, nucleic acid, lipid, or polysaccharide. The antigen can be heterologous, such as an antigen expressed in pathogenic bacteria or viruses. The antigen can also be synthetic; for example, some people are extremely allergic to synthetic latex, and exposure to such an antigen can lead to an extreme immune response. In some embodiments, the antigen is autologous and expressed on diseased or otherwise altered cells. For example, in some embodiments, the antigen is expressed in tumor cells. In some embodiments, the tumor cells are solid tumor cells. In other embodiments, the tumor cells are blood cancers, such as B-cell carcinoma. In some embodiments, B-cell carcinoma is lymphoma (e.g., Hodgkin's lymphoma or non-Hodgkin's lymphoma) or 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, B-cell carcinoma is multiple myeloma.

[0417] Antibody-antigen interactions are non-covalent interactions caused by hydrogen bonds, electrostatic or hydrophobic interactions, or van der Waals forces. The affinity of the extracellular binding domain of a chimeric antigen receptor for the antigen can be calculated using the following formula:

[0418] KA = [antibody-antigen] / [antibody][antigen], where

[0419] [Ab] = the molar concentration of the antibody that does not occupy binding sites;

[0420] [Ag] = the molar concentration of unoccupied binding sites on the antigen; and

[0421] [Ab-Ag] = the molar concentration of the antibody-antigen complex.

[0422] Antibody-antigen interactions can also be characterized based on the dissociation of antigen and antibody. The dissociation constant (KD) is the ratio of binding rate to dissociation rate, and is inversely proportional to the affinity constant. Therefore, KD = 1 / KA. Those skilled in the art will be familiar with these concepts and will know that conventional methods, such as ELISA assays, can be used to calculate these constants.

[0423] The chimeric antigen receptor described herein has a transmembrane domain that spans the lipid bilayer of CAR-T cells and separates the extracellular binding domain from the intracellular signaling domain. In some embodiments, the domain is derived from other receptors having a transmembrane domain, while in other embodiments, the domain is synthetic. In some embodiments, the transmembrane domain may be derived from a non-human transmembrane domain, and in some embodiments, it is humanized. “Humanization” refers to optimizing the nucleic acid sequence encoding the transmembrane domain so that it is expressed more reliably or efficiently in human individuals. 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, subunits of the T cell receptor (TCR) complex, PD1, or any differentiated protein cluster or other protein that are expressed in immune effector cells and have a transmembrane domain. In some embodiments, the transmembrane domain will be synthetic, and such sequences will contain a number of hydrophobic residues.

[0424] In some embodiments, the chimeric antigen receptor is designed to include a spacer sequence between a transmembrane domain and an extracellular domain, an intracellular domain, or both. The length of such a spacer sequence 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 some embodiments, the linker sequence can be 20, 30, 40, 50, 60, 70, 80, 90, or 100 amino acids long. In other embodiments, the length of the spacer sequence can be between 100 and 500 amino acids. The spacer sequence can be any polypeptide that links one domain to another and is used to position such a linker domain to enhance or optimize the function of the chimeric antigen receptor.

[0425] The intracellular signaling domains of the chimeric antigen receptors considered herein include a primary signaling domain. In some embodiments, the chimeric antigen receptor includes a primary signaling domain and a secondary or co-stimulatory signaling domain. In some embodiments, the domains include one or more tyrosine-based immune receptor activation motifs or ITAMs. In some embodiments, the primary signaling domain includes more than one ITAM. The ITAM integrated into the chimeric antigen receptor may be derived from the ITAMs of other cellular receptors. In some embodiments, the primary signaling domain containing the ITAM may be derived from a subunit of the TCR complex, such as CD3γ, CD3ε, CD3ζ, or CD3δ (see [link to documentation]). Figure 1A In some implementations, the primary signal structure domain containing ITAM may be derived from FcRγ, FcRβ, CD5, CD22, CD79a, CD79b, or CD66d. In some implementations, the secondary signal structure domain is derived from CD28. In other implementations, the secondary signal structure domain is derived from CD2, CD4, CDS, CD8α, CD83, CD134, CD137, ICOS, or CD154.

[0426] This document also provides nucleic acids encoding the chimeric antigen receptor described herein. In some embodiments, the nucleic acid is isolated or purified. In vitro delivery of the nucleic acid can be accomplished using methods known in the art. For example, immune cells obtained from an individual can be transformed with a nucleic acid vector encoding a chimeric antigen receptor. Receptor immune cells can then be transformed using said vector, thereby causing these cells to express the chimeric antigen receptor. Effective methods for transforming immune cells include transfection and transduction. Such methods are well known in the art. For example, suitable methods for delivering nucleic acid molecules encoding chimeric antigen receptors (and nucleic acids encoding base editors) can be found in International Patent 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, those methods and vectors described herein for delivering nucleic acids encoding base editors (e.g., ABE8) are suitable for delivering nucleic acids encoding chimeric antigen receptors.

[0427] Some aspects of the present invention provide immune cells comprising chimeric antigens and altered endogenous genes that enhance immune cell function, resistance to immunosuppression or suppression, 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 α component of the immune cell receptor complex is encoded by the TRAC gene, and in some embodiments, said gene is edited such that the α subunit of the TCR complex is nonfunctional or absent. Since said subunit is essential for endogenous immune cell signaling, editing said gene can reduce the risk of graft-versus-host disease caused by allogeneic immune cells.

[0428] Host immune cells can potentially recognize allogeneic CAR-T cells as non-self cells and trigger an immune response to eliminate them. B2M is expressed in almost all nucleated cells and is associated with MHC class I complexes. Figure 1B ). Circulating host CD8 + T cells can recognize this B2M protein as non-self and kill allogeneic cells. To overcome this transplant rejection, in some implementations, the B2M gene is edited to be knocked out or knocked down.

[0429] In some embodiments of the present invention, the PDCD1 gene is edited in CAR-T cells to knock out or weaken its expression. The PDCD1 gene encodes the cell surface receptor PD-1, an immune system checkpoint expressed in immune cells that participates in reducing autoimmunity by promoting apoptosis of antigen-specific immune cells. By knocking out or weakening 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.

[0430] The CBLB gene encodes an E3 ubiquitin ligase that plays an important role in suppressing the activation of immune effector cells. (Reference) Figure 1C As shown, the CBLB protein facilitates signaling pathways that lead to immune effector cell tolerance and actively inhibits signaling pathways that lead to immune effector cell activation. Because immune effector cell activation is essential for CAR-T cell proliferation in vivo after transplantation, in some embodiments of this invention, CBLB is edited to be knocked out or attenuated.

[0431] In some embodiments, gene editing can be performed on immune cells to enhance immune cell function or reduce immunosuppression or inhibition before cells are converted to express the chimeric antigen receptor. In other embodiments, gene editing can be performed on CAR-T cells to enhance immune cell function or reduce immunosuppression or inhibition, i.e., after the immune cells are converted to express the chimeric antigen receptor. In some embodiments, the immune cells comprise the chimeric antigen receptor and edited TRAC, B2M, PDCD1, CD7, CIITA, CBLB genes, or combinations thereof, wherein the expression of the edited genes is knocked out or knocked down.

[0432] In some embodiments, the immune cells comprise a chimeric antigen receptor and an edited TRAC gene, wherein the 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, as well as one or more of the B2M, PDCD1, CD7, CIITA, and / or CBLB genes, wherein the 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, as well as a B2M gene, wherein the 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, as well as a PDCD1 gene, wherein the 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, as well as a CD7 gene, wherein 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 TRAC and CIITA genes, wherein the 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, wherein the 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, wherein the 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, wherein the 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, wherein the 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, wherein the 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, wherein the 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, wherein the 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, wherein the 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, wherein the expression of the edited genes is knocked out or knocked down.

[0433] In some embodiments, the immune cells comprise a chimeric antigen receptor and edited TRAC, B2M, PDCD1, and CBLB genes, wherein the 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, PDCD1, and CD7 genes, wherein the 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, CD7, and CIITA genes, wherein the 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, PDCD1, and CIITA genes, wherein the 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, CBLB, and CIITA genes, wherein the 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, CD7, and CIITA genes, wherein the 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, CD7, and CBLB genes, wherein the 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, CD7, and CBLB genes, wherein the 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, CD7, and CBLB genes, wherein the expression of the edited genes is knocked out or knocked down.

[0434] In some embodiments, the immune cells comprise a chimeric antigen receptor and edited TRAC, B2M, PDCD1, CD7, and CIITA genes, wherein the 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, PDCD1, CD7, and CBLB genes, wherein the 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, PDCD1, CIITA, and CBLB genes, wherein the 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, CD7, CIITA, and CBLB genes, wherein the 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, CD7, CIITA, and CBLB genes, wherein the expression of the edited genes is knocked out or knocked down. In some implementations, the immune cells contain chimeric antigen receptors and edited TRAC, B2M, PDCD1, CD7, and CBLB genes, wherein the expression of the edited genes is knocked out or knocked down.

[0435] In some embodiments, the immune cells comprise a chimeric antigen receptor and an edited B2M gene, wherein the 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, as well as one or more of the following genes: CBLB, PDCD1, CD7, CIITA, and / or TRAC, wherein the 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, as well as the PDCD1 gene, wherein the 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, as well as the CIITA gene, wherein the 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, as well as the CD7 gene, wherein 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 B2M, CIITA, and CBLB genes, wherein the 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, wherein the 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, wherein the 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, wherein the 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, wherein the 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, wherein the 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, wherein the 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, wherein the 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, wherein the expression of the edited genes is knocked out or knocked down.

[0436] In some embodiments, the immune cells comprise a chimeric antigen receptor and an edited PDCD1 gene, wherein the 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, as well as one or more of the following genes: B2M, CBLB, CD7, CIITA, and / or TRAC, wherein the 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 and CBLB gene, wherein the 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 and CIITA gene, wherein 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 PDCD1, CIITA, and CBLB genes, wherein the expression of the edited gene is knocked out or knocked down.

[0437] In some embodiments, the immune cells comprise a chimeric antigen receptor and an edited CD7 gene, wherein the 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, wherein the 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 CD7 gene, as well as a CIITA gene, wherein 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 CD7, PDCD1, and CIITA genes, wherein 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 CD7, PDCD1, CIITA, and CBLB genes, wherein the expression of the edited gene is knocked out or knocked down.

[0438] In some embodiments, the immune cells comprise a chimeric antigen receptor and an edited CBLB gene, wherein the 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, as well as one or more of the following genes: B2M, PDCD1, CD7, CIITA, and / or TRAC, wherein the 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, as well as the CIITA gene, wherein the expression of the edited gene is knocked out or knocked down.

[0439] In some embodiments, the immune cells comprise a chimeric antigen receptor and an edited CIITA gene, wherein the 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, as well as one or more of the B2M, PDCD1, CD7, CBLB, and / or TRAC genes, wherein the expression of the edited gene is knocked out or knocked down.

[0440] In some embodiments, immune cells, including but not limited to any immune cells containing any of the above-described gene edits, can be edited to produce mutations in other genes, thereby enhancing CAR-T function or reducing immunosuppression or inhibition of said cells. For example, in some embodiments, the immune cells comprise a chimeric antigen receptor and edited TGFBR2, ZAP70, NFATC1, TET2 genes, or combinations thereof, wherein the expression of the edited genes is knocked out or knocked down. In some embodiments, the immune cells comprise a chimeric antigen receptor and an edited TGFBR2 gene, wherein 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 ZAP70 genes, wherein the 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 ZAP70 genes, wherein the 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 NFATC1 genes, wherein the 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, wherein the 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, wherein the 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, wherein the 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, wherein the 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, NFATC1, and TET2 genes, wherein the expression of the edited genes is knocked out or knocked down. In some embodiments, the immune cells comprise a chimeric antigen receptor and edited ZAP70 and NFATC1 genes, wherein the expression of the edited genes is knocked out or knocked down. In some embodiments, the immune cells comprise a chimeric antigen receptor and edited ZAP70 and TET2 genes, wherein the expression of the edited genes is knocked out or knocked down. In some embodiments, the immune cells comprise a chimeric antigen receptor and edited ZAP70, PDCD1, and TET2 genes, wherein the expression of the edited genes is knocked out or knocked down.In some embodiments, the immune cells comprise a chimeric antigen receptor and an edited PDCD1 gene, wherein the 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, as well as a TET2 gene, wherein the expression of the edited gene is knocked out or knocked down. And in some embodiments, the immune cells comprise a chimeric antigen receptor and an edited TET2 gene, wherein the expression of the edited gene is knocked out or knocked down.

[0441] In some embodiments, the chimeric antigen receptor is inserted into the TRAC gene. This has advantages. First, since TRAC is highly expressed in immune cells, when its construct is designed to insert the chimeric antigen receptor into the TRAC gene, the chimeric antigen receptor will be similarly expressed, such that receptor expression is driven by the TRAC promoter. Second, inserting the chimeric antigen receptor into the TRAC gene will knock out TRAC expression. In some embodiments, the gene editing system described herein can be used to insert the chimeric antigen receptor into the TRAC gene locus. A gRNA specific to the TRAC gene locus can guide the gene editing system to the locus and initiate double-stranded DNA cleavage. In a particular embodiment, the gRNA is used in conjunction with Cas12b. In various embodiments, the gene editing system is used in conjunction with a nucleic acid having a sequence encoding a CAR receptor. Exemplary guide RNAs are provided in Table 1A below.

[0442] Table 1A: TRAC Guide RNAs

[0443]

[0444] A DNA construct encoding a chimeric antigen receptor and nucleic acid contains an extended TRAC DNA fragment flanking a gRNA targeting sequence. Unbound by theory, the construct binds a complementary TRAC sequence, and then the chimeric antigen receptor DNA located near the TRAC sequence on the construct is inserted into the lesion site, effectively knocking out the TRAC gene and the chimeric antigen receptor nucleic acid. Table 1B provides guide RNAs for the TRAC gene, which can direct the base editing mechanism to the TRAC locus, enabling insertion into the chimeric antigen receptor nucleic acid. The first 11 gRNAs are used for the BhCas12b nuclease. The second group of 11 is used for the BvCas12b nuclease. These are intended for CAR insertion into the TRAC by creating double-strand breaks, not for base editing.

[0445] Table 1B: TRAC Guide RNAs

[0446]

[0447] In some embodiments, ABE8 can be used to target the TRAC locus with the nucleic acid encoding the chimeric antigen receptor of the present invention. In some embodiments, the chimeric antigen receptor is targeted at the TRAC locus using a CRISPR / Cas9 base editing system. To produce the above-described gene edit, 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, wherein 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. In some embodiments, the gRNA comprises a nucleotide analog. These nucleotide analogs can inhibit the degradation of the gRNA during cellular processes. Table 2 provides the target sequences for the gRNA.

[0448] Table 2: Exemplary Target Sequences

[0449]

[0450]

[0451]

[0452] The adenosine deaminase nucleobase editors (e.g., ABE8) used in this invention can act on DNA, including single-stranded DNA. Methods for generating modifications in target nucleobase sequences in immune cells using them are described. In some embodiments, the fusion proteins provided herein contain one or more features that improve the base-editing activity of the fusion protein. For example, any fusion protein provided herein may contain a Cas9 domain with reduced nuclease activity. In some embodiments, any fusion protein provided herein may have a Cas9 domain without nuclease activity (dCas9), or a Cas9 domain that cleaves one strand of a double-stranded DNA molecule, referred to as a Cas9 cleavage enzyme (nCas9). Without wishing to be bound by any particular theory, the presence of a catalytic residue (e.g., H840) maintains the Cas9 activity of cleaving the non-edited (e.g., unmethylated) strand opposite the target nucleobase. Mutations in catalytic residues (e.g., D10 to A10) can prevent cleavage of the edited strand containing the target A residue. These Cas9 variants can generate single-strand DNA breaks (nicks) at specific locations based on target sequences defined by gRNA, thereby repairing the unedited strand and ultimately leading to nucleobase changes on the unedited strand.

[0453] Nucleotide base editor

[0454] This document discloses a base editor or nucleobase editor for editing, modifying, or altering target nucleotide sequences of polynucleotides. It describes a nucleobase editor or base editor comprising a polynucleotide programmable nucleotide-binding domain and a nucleobase editing domain (e.g., adenosine deaminase). The polynucleotide programmable nucleotide-binding domain, when bound to a guide polynucleotide (e.g., gRNA), can specifically bind to the target polynucleotide sequence (i.e., through complementary base pairing between the bases of the bound guide nucleic acid and the target polynucleotide), thereby positioning the base editor to the target nucleic acid sequence to be edited. In some embodiments, the target polynucleotide sequence comprises single-stranded 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.

[0455] Polynucleotide programmable nucleotide binding domain

[0456] It should be understood that polynucleotide-programmable nucleotide-binding domains can also include nucleic acid-programmable proteins that bind RNA. For example, a polynucleotide-programmable nucleotide-binding domain can be associated with a nucleic acid that directs the polynucleotide-programmable nucleotide-binding domain to RNA. Other nucleic acid-programmable DNA-binding proteins are also within the scope of this disclosure, although they are not specifically listed herein.

[0457] The multinucleotide-programmable nucleotide-binding domain of the base editor may itself contain one or more domains. For example, the multinucleotide-programmable nucleotide-binding domain may contain one or more nuclease domains. In some embodiments, the nuclease domain of the multinucleotide-programmable nucleotide-binding domain may contain an endonuclease or an exonuclease. In this document, the term "exonuclease" refers to a protein or polypeptide capable of digesting nucleic acids (e.g., RNA or DNA) from their free ends, and the term "endonuclease" refers to a protein or polypeptide capable of catalyzing (e.g., cleaving) the interior of nucleic acids (e.g., DNA or RNA). In some embodiments, the endonuclease can cleave a single strand of a double-stranded nucleic acid. In some embodiments, the endonuclease can cleave the double strand of a double-stranded nucleic acid. In some embodiments, the multinucleotide-programmable nucleotide-binding domain may be a deoxyribonuclease. In some embodiments, the multinucleotide-programmable nucleotide-binding domain may be a ribonuclease.

[0458] In some embodiments, the nuclease domain of the polynucleotide programmable nucleotide binding domain can cleave zero, one, or both strands of the target polynucleotide. In some embodiments, the polynucleotide programmable nucleotide binding domain may include a nicking enzyme domain. Hereinafter, the term "nicking enzyme" refers to a polynucleotide programmable nucleotide binding domain containing a nuclease domain capable of cleaving only one of the two strands in a double-stranded nucleic acid molecule (e.g., DNA). In some embodiments, the nicking enzyme can be derived from a fully catalytically active (e.g., native) form of the polynucleotide programmable nucleotide binding domain by introducing one or more mutations into the active polynucleotide programmable nucleotide binding domain. For example, in the case where the polynucleotide programmable nucleotide binding domain includes a Cas9-derived nicking enzyme domain, the Cas9-derived nicking enzyme domain may contain a D10A mutation and histidine at position 840. In such embodiments, residue H840 retains catalytic activity and thereby can cleave a single strand of the nucleic acid duplex. In another example, the Cas9-derived nicking enzyme domain may contain an H840A mutation, while the amino acid residue at position 10 remains D. In some embodiments, the nicking enzyme can be derived from a polynucleotide programmable nucleotide-binding domain in a fully catalytically active (e.g., native) form by removing all or part of the nuclease domains that are not required for nicking enzyme activity. For example, in cases where the polynucleotide programmable nucleotide-binding domain contains a nicking enzyme domain derived from Cas9, the Cas9-derived nicking enzyme domain may contain all or part of the RuvC domain or the HNH domain.

[0459] The amino acid sequence of Cas9 with exemplary catalytic activity is as follows:

[0460]

[0461] A base editor comprising a polynucleotide programmable nucleotide-binding domain containing a nicking enzyme domain is thus capable of generating single-strand DNA breaks (nicks) at specific polynucleotide target sequences (e.g., determined by the complementary sequence of the bound guide nucleic acid). In some embodiments, the strand of the target polynucleotide sequence of the nucleic acid duplex cleaved by a base editor containing a nicking enzyme domain (e.g., a Cas9-derived nicking enzyme domain) is the unedited strand (i.e., the strand cleaved by the base editor is the opposite of the strand containing the base to be edited). In other embodiments, a base editor containing a nicking enzyme domain (e.g., a Cas9-derived nicking enzyme domain) can cleave the strand of the DNA molecule targeted for editing. In such embodiments, the non-target strand is not cleaved.

[0462] This document also provides a base editor comprising a catalytically dead polynucleotide programmable nucleotide-binding domain (i.e., unable to cleave target polynucleotide sequences). In this document, the terms "catalytically dead" and "nuclease dead" are used interchangeably to refer to a polynucleotide programmable nucleotide-binding domain having one or more mutations and / or deletions that prevent it from cleaving a nucleic acid strand. In some embodiments, the catalytically dead polynucleotide programmable nucleotide-binding domain base editor may lack nuclease activity due to specific point mutations in one or more nuclease domains. For example, in the case where the base editor comprises a Cas9 domain, Cas9 may contain both D10A and H840A mutations. Such mutations inactivate both nuclease domains, resulting in loss of nuclease activity. In other embodiments, the catalytically dead polynucleotide programmable nucleotide-binding domain may comprise one or more deletions of all or part of a catalytic domain (e.g., RuvC1 and / or HNH domains). In a further embodiment, the catalytically dead polynucleotide programmable nucleotide-binding domain comprises a point mutation (e.g., D10A or H840A) and the deletion of all or part of the nuclease domain.

[0463] This document also considers mutations capable of generating catalytically dead polynucleotide-programmable nucleotide-binding domains from prior functional versions of polynucleotide-programmable nucleotide-binding domains. For example, in the case of catalytically dead Cas9 (“dCas9”), variants with mutations other than D10A and H840A are provided, resulting in nuclease-inactivating Cas9. Such mutations include, for example, other amino acid substitutions at D10 and H840, or other substitutions within the Cas9 nuclease domain (e.g., substitutions in the HNH nuclease subdomain and / or the RuvC1 subdomain). Based on this disclosure and knowledge in the art, other suitable nuclease-inactive dCas9 domains will be apparent to those skilled in the art and are within the scope of this disclosure. Such additional exemplary suitable nuclease-free Cas9 domains include, but are not limited to, the D10A / H840A, D10A / D839A / H840A, and D10A / D839A / H840A / N863A mutant domains (see, for example, Prashant et al., CAS9 transcriptional activators for target specificity screening and paired nickases for cooperative genome engineering. Nature Biotechnology. 2013; 31(9):833-8338, the entire contents of which are incorporated herein by reference).

[0464] Non-limiting examples of multinucleotide programmable nucleotide-binding domains that can be incorporated into a base editor include CRISPR protein-derived domains, restriction nucleases, broad-spectrum nucleases, TAL nucleases (TALENs), and zinc finger nucleases (ZFNs). In some embodiments, the base editor comprises a multinucleotide programmable nucleotide-binding domain containing a natural or modified protein or a portion thereof, which, through binding to a guide nucleic acid, is capable of binding nucleic acid sequence (i.e., regularly clustered, short palindromic repeats)-mediated nucleic acid modifications during CRISPR. Such a protein is referred to herein as a "CRISPR protein". Therefore, this paper discloses a base editor comprising a polynucleotide programmable nucleotide-binding domain, wherein the polynucleotide programmable nucleotide-binding domain comprises all or a portion of a CRISPR protein (i.e., a base editor comprising all or a portion of a CRISPR protein as a domain, also referred to as a "CRISPR protein" base editor-derived domain). The CRISPR protein-derived domain incorporating the base editor can be modified compared to the wild-type or natural version of the CRISPR protein. For example, as described below, the CRISPR protein-derived domain may comprise one or more mutations, insertions, deletions, rearrangements, and / or recombinations relative to the wild-type or natural form of the CRISPR protein.

[0465] CRISPR is an adaptive immune system that provides protection against mobile genetic elements (viruses, transposable elements, and conjugating plasmids). A CRISPR cluster contains a spacer sequence, a sequence complementary to the preceding mobile element, and a target invading nucleic acid. The CRISPR cluster is transcribed and processed into CRISPR RNA (crRNA). In type II CRISPR systems, proper processing of pre-crRNA requires a small RNA (tracrRNA), endogenous ribonuclease 3 (rnc), and the Cas9 protein. tracrRNA acts as a guide for ribonuclease 3-assisted processing of pre-crRNA. Subsequently, Cas9 / crRNA / tracrRNA endonuclease cleaves the linear or circular dsDNA target complementary to the spacer sequence. The target strand not complementary to crRNA is first cleaved by endonuclease and then trimmed 3′–5′ by exonuclease. In nature, DNA binding and cleavage typically require both proteins and two RNAs. However, a single guide RNA (“sgRNA,” or simply “gRNA”) can be engineered to integrate various aspects of 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 identifies a short motif in a CRISPR repeat sequence (PAM or protospacer sequence adjacent motif) to help distinguish between self and non-self.

[0466] In some implementations, the methods described herein can utilize engineered Cas proteins. Guide RNA (gRNA) is a short synthetic RNA consisting of a scaffold sequence required for Cas binding and a user-defined ~20 nucleotide spacer sequence that defines the genomic target to be modified. Therefore, technicians can alter the genomic target-specific portion of the Cas protein based on the specificity of the gRNA targeting sequence for the genomic target compared to the rest of the genome.

[0467] In some embodiments, the gRNA scaffold sequence is as follows: GUUUUAGAGC UAGAAAUAGC AAGUUAAAAUAAGGCUAGUC CGUUAUCAAC UUGAAAAAGU GGCACCGAGU CGGUGCUUUU.

[0468] In some embodiments, the CRISPR protein-derived domain incorporated with the base editor is a nuclease (e.g., deoxyribonuclease or ribonuclease) capable of binding the target polynucleotide when bound to a guide nucleic acid. In some embodiments, the CRISPR protein-derived domain incorporated with the base editor is a nicking enzyme capable of binding the target polynucleotide when bound to a guide nucleic acid. In some embodiments, the CRISPR protein-derived domain incorporated with the base editor is a catalytic death domain capable of binding the target polynucleotide when bound to a guide nucleic acid. In some embodiments, the target polynucleotide bound by the CRISPR protein-derived domain of the base editor is DNA. In some embodiments, the target polynucleotide bound by the CRISPR protein-derived domain of the base editor is RNA.

[0469] The Cas proteins used in this paper include those of classes 1 and 2. Non-limiting examples of Cas proteins include Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas5d, Cas5t, Cas5h, Cas5a, Cas6, Cas7, Cas8, Cas9 (also known as Csn1 or Csx12), Cas10, Csy1, Csy2, Csy3, Csy4, Cse1, 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, 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 modified versions thereof. Unmodified CRISPR enzymes can possess DNA cleavage activity, such as Cas9, which has two functional endonuclease domains: RuvC and HNH. CRISPR enzymes can direct the cleavage of one or both strands at a target sequence, such as within the target sequence and / or within the complementary sequence of the target sequence. For example, a CRISPR enzyme can direct the cleavage of one or both strands within approximately 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.

[0470] A vector encoding a CRISPR enzyme may be used, wherein the CRISPR enzyme is mutated relative to the corresponding wild-type enzyme, such that the mutated CRISPR enzyme lacks the ability to cleave one or both strands of a target polynucleotide containing a target sequence. Cas9 may refer to a polypeptide having 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 with a wild-type exemplary Cas9 polypeptide (e.g., Cas9 from Streptococcus pyogenes). Cas9 may refer to a Cas9 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 with a wild-type exemplary Cas9 polypeptide (e.g., from Streptococcus pyogenes). Cas9 may refer to a wild-type or modified form of the Cas9 protein, which may include amino acid variations, such as deletions, insertions, substitutions, variants, mutations, fusions, chimeras, or any combination thereof.

[0471] In some implementations, the CRISPR protein-derived domain of the base editor may include those 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 Refs: NC_021284.1); Prevotella intermedia (NCBI Refs: NC_017861.1); Spiroplasma taiwanense, China (NCBI Refs: NC_021846.1); Streptococcus iniae (NCBI Refs: NC_021314.1); Belliella baltica (NCBI Refs: NC_021314.1). Refs: NC_018010.1); Psychroflexus torquis I (NCBI Ref: NC_018721.1); Streptococcus thermophilus (NCBI Refs: YP_820832.1); Listeria innocua (NCBI Refs: NP_472073.1); Campylobacter jejuni (NCBI Refs: YP_002344900.1); Neisseria meningitidis (NCBI Refs: YP_002342100.1), Streptococcus pyogenes, or Staphylococcus aureus.

[0472] Cas9 domains of nucleobase editors

[0473] The Cas9 nuclease sequence and structure are well known to those skilled in the art (see, for example, “Completegenome 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), all of which are incorporated herein by reference). Cas9 orthologs have been described in a variety of species, including but not limited to Streptococcus pyogenes and Streptococcus thermophilus. Based on this disclosure, other suitable Cas9 nucleases and sequences will be apparent to those skilled in the art, and such Cas9 nucleases and sequences include Cas9 sequences of organisms and loci disclosed in Chylinski, Rhun, and Charpentier, “The tracrRNA and Cas9 families of type IICRISPR-Cas immunity systems” (2013) RNA Biology 10:5, 726-737, the entire contents of which are incorporated herein by reference.

[0474] In some embodiments, the nucleic acid programmable DNA-binding protein (napDNAbp) is a Cas9 domain. This document provides non-limiting exemplary Cas9 domains. The Cas9 domain can be a nuclease-active Cas9 domain, a nuclease-inactive Cas9 domain (dCas9), or a Cas9 cleavage enzyme (nCas9). In some embodiments, the Cas9 domain is a domain with nuclease activity. 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 of the amino acid sequences described herein. In some embodiments, the amino acid sequence comprised of the Cas9 domain is identical to 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%, or at least 99% or 99.5% of any of the amino acid sequences described herein. In some embodiments, the amino acid sequence comprising the Cas9 domain has mutations of 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 compared to any of the amino acid sequences described herein. In some embodiments, the Cas9 domain comprises 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 consecutive amino acid residues compared to any of the amino acid sequences described herein.

[0475] In some embodiments, a protein comprising a Cas9 fragment is provided. For example, in some embodiments, the protein comprises one of two Cas9 domains: (1) a gRNA-binding domain of Cas9; or (2) a DNA-cutting domain of Cas9. In some embodiments, a protein comprising Cas9 or a fragment thereof is referred to as a "Cas9 variant". The Cas9 variant is homologous to Cas9 or a fragment thereof. For example, the Cas9 variant is at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, at least about 99.5%, 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 variations 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-cutting domain) such that the fragment is at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, at least about 99.5%, 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. In some embodiments, the fragment is at least 100 amino acids long. In some embodiments, the length of the fragment is at least 100, 150, 200, 250, 300, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1050, 1100, 1150, 1200, 1250, or 1300 amino acids.

[0476] In some embodiments, the Cas9 fusion proteins provided herein comprise the full-length amino acid sequence of the 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 only one or more fragments thereof. Exemplary amino acid sequences of suitable Cas9 domains and Cas9 fragments are provided herein, and other suitable sequences of Cas9 domains and fragments will be apparent to those skilled in the art.

[0477] The Cas9 protein can be associated with a guide RNA that guides the Cas9 protein to a specific DNA sequence complementary to the guide RNA. In some embodiments, the polynucleotide-programmable nucleotide-binding domain is the Cas9 domain, such as the nuclease-active Cas9, the Cas9 nickase (nCas9), or the nuclease-inactivated 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.

[0478] In some embodiments, wild-type Cas9 corresponds to Cas9 from Streptococcus pyogenes (NCBI reference sequence: NC_017053.1, nucleotide and amino acid sequences are as follows).

[0479]

[0480]

[0481] (Single underscore: HNH domain; Double underscore: RuvC domain)

[0482] In some embodiments, the wild-type Cas9 corresponds to or comprises the following nucleotide and / or amino acid sequence:

[0483]

[0484]

[0485]

[0486] (Single underscore: HNH domain; Double underscore: RuvC domain).

[0487] In some embodiments, wild-type Cas9 corresponds to Cas9 from Streptococcus pyogenes.

[0488] (NCBI reference sequence: NC_002737.2 (nucleotide sequence below); and Uniprot reference sequence: Q99ZW2 (amino acid sequence below):)

[0489]

[0490]

[0491] (Single underscore: HNH domain; Double underscore: RuvC domain)

[0492] In some implementations, Cas9 refers to Cas9 derived from the following: Corynebacterium ulcerans (NCBI Refs: NC_015683.1, NC_017317.1); Corynebacterium diphtheria (NCBI Refs: NC_016782.1, NC_016786.1); Spiroplasma syrphidicola (NCBI Refs: NC_021284.1); Prevotella intermedia (NCBI Refs: NC_017861.1); Spiroplasma taiwanense, China (NCBI Refs: NC_021846.1); Streptococcus iniae (NCBI Refs: NC_021314.1); Belliella balolaensis. Cas9 from any other organism (NCBI Refs: YP_018010.1); Psychroflexus torquis I (NCBI Refs: YP_018721.1); Streptococcus thermophilus (NCBI Refs: YP_820832.1); Listeria innocua (NCBI Refs: YP_472073.1); Campylobacter jejuni (NCBI Refs: YP_002344900.1); Neisseria meningitidis (NCBI Refs: YP_002342100.1).

[0493] It should be understood that additional Cas9 proteins (e.g., nuclease-dead Cas9 (dCas9), Cas9 nickase (nCas9), or nuclease-active Cas9), including their variants and homologues, 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 nuclease-inactive Cas9 (dCas9). In some embodiments, the Cas9 protein is a Cas9 nickase (nCas9). In some embodiments, the Cas9 protein is a Cas9 with nuclease activity.

[0494] In some embodiments, the Cas9 domain is a nuclease-inactivating 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-inactivating dCas9 domain contains the D10X and H840X mutations in the amino acid sequence described herein, or the corresponding mutations in any amino acid sequence provided herein, where X is any amino acid change. In some embodiments, the nuclease-inactivating dCas9 domain contains the D10A and H840A mutations in the amino acid sequence described herein, or the corresponding mutations in any amino acid sequence provided herein. As an example, a nuclease-free Cas9 domain contains the amino acid sequence listed in the cloning vector pPlatTET-gRNA2 (accession number BAV54124).

[0495] An exemplary amino acid sequence of catalytically inactive Cas9 (dCas9) is as follows:

[0496]

[0497] Based on this disclosure and knowledge in the art, other suitable nuclease-free dCas9 domains will be apparent to those skilled in the art and are within the scope of this disclosure. Such additional exemplary suitable nuclease-free Cas9 domains include, but are not limited to, the D10A / H840A, D10A / D839A / H840A, and D10A / D839A / H840A / N863A mutant domains (see, for example, Prashant et al., CAS9 transcriptional activators for target specificity screening and paired nickases for cooperative genome engineering. Nature Biotechnology. 2013; 31(9):833-8338, the entire contents of which are incorporated herein by reference).

[0498] In some implementations, the Cas9 nuclease has an inactive (e.g., inactivated) DNA cleavage domain, i.e., Cas9 is a cleavage enzyme, referred to as the “nCas9” protein (for “cleavage enzyme” Cas9). The nuclease-inactivated Cas9 protein is interchangeably referred to as the “dCas9” protein (for nuclease-“dead” Cas9) or catalytically inactivated Cas9. Methods for generating Cas9 proteins (or fragments thereof) with an inactive DNA cleavage domain are known (see, for example, 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 comprise 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 silence the nuclease activity of Cas9. For example, mutations in D10A and H840A completely inactivate the nuclease activity of Streptococcus pyogenes Cas9 (Jinek et al., Science. 337:816-821 (2012); Qi et al., Cell. 28:152(5):1173-83 (2013)).

[0499] In some embodiments, the amino acid sequence contained in the dCas9 domain is identical to that of any Cas9 domain described herein by 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%, or at least 99% or 99.5%. In some embodiments, the amino acid sequence comprising the Cas9 domain has mutations of 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 compared to any of the amino acid sequences described herein. In some embodiments, the Cas9 domain comprises 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 consecutive amino acid residues compared to any of the amino acid sequences described herein.

[0500] In some embodiments, dCas9 corresponds to or contains, partially or entirely, a Cas9 amino acid sequence with one or more mutations that inactivate Cas9 nuclease activity. For example, in some embodiments, the dCas9 domain contains D10A and H840A mutations or corresponding mutations in another Cas9.

[0501] In some embodiments, dCas9 comprises the amino acid sequence of dCas9 (D10A and H840A):

[0502] (Single underscore: HNH domain; Double underscore: RuvC domain).

[0503] In some embodiments, the Cas9 domain contains a D10A mutation, while the residue at position 840 remains histidine at the corresponding position in the amino acid sequence provided above or in any amino acid sequence provided herein.

[0504] In other embodiments, dCas9 variants with mutations other than D10A and H840A are provided, such as Cas9 (dCas9) that leads to nuclease inactivation. For example, such mutations include other amino acid substitutions at D10 and H840, or other substitutions within the Cas9 nuclease domain (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%, at least about 80%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, at least about 99.5%, or at least about 99.9% identical. In some embodiments, variants or homologs of about 5, 10, 15, 20, 25, 30, 40, 50, 75, 100, or more amino acids are provided, either shorter or longer.

[0505] In some embodiments, the Cas9 domain is a Cas9 cleavage enzyme. The Cas9 cleavage enzyme can be a Cas9 protein capable of cleaving only one strand of a double-stranded nucleic acid molecule (e.g., a double-stranded DNA molecule). In some embodiments, the Cas9 cleavage enzyme cleaves the target strand of the double-stranded nucleic acid molecule, meaning that the Cas9 cleavage enzyme cleaves the strand that is base-paired (complementary) to the gRNA (e.g., sgRNA) bound to Cas9. In some embodiments, the Cas9 cleavage enzyme contains a D10A mutation and has a histidine residue at position 840. In some embodiments, the Cas9 cleavage enzyme cleaves the non-target, non-base-editing strand of the double-stranded nucleic acid molecule, meaning that the Cas9 cleavage enzyme cleaves the strand that is not base-paired to the gRNA (e.g., sgRNA) bound to Cas9. In some embodiments, the Cas9 cleavage enzyme contains an H840A mutation and has an aspartic acid residue at position 10 or a corresponding mutation. In some embodiments, the Cas9 nicking enzyme 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%, or at least 99%, or 99.5% identical to any of the Cas9 nicking enzymes described herein. Other suitable Cas9 nicking enzymes will be apparent to those skilled in the art based on this disclosure and knowledge of the art, and are within the scope of this disclosure.

[0506]

[0507] In some implementations, Cas9 refers to Cas9 derived from archaea (e.g., nanoarchaea), which constitute domains and kingdoms of single-celled prokaryotic microorganisms. In some embodiments, the programmable nucleotide-binding protein may be a CasX or CasY protein, which has been 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. Metagenomics using genome resolution has identified numerous CRISPR-Cas systems, including Cas9, which was first reported in the field of archaea. This divergent Cas9 protein has been found in rarely studied nanoarchaea as part of an active CRISPR-Cas system. 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 system described herein, Cas9 is replaced by CasY or a variant of CasY. It should be understood that other RNA-guided DNA-binding proteins can be used as nucleic acid-programmable DNA-binding proteins (napDNAbp) and are within the scope of this disclosure.

[0508] In some embodiments, the programmable DNA-binding protein (napDNAbp) or any fusion protein provided herein may be a CasX or CasY protein. In some embodiments, napDNAbp is a CasY protein. In some embodiments, napDNAbp is a CasY protein. In some embodiments, napDNAbp contains 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 that of 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 contains 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 that of any CasX or CasY protein described herein. It should be understood that CasX and CasY from other bacterial species may also be used according to this disclosure.

[0509] Example CasX((uniprot.org / uniprot / F0NN87; uniprot.org / uniprot / F0NH53)

[0510] tr|F0NN87|F0NN87_SULIHCRISPR-associatedCasx protein OS=Icelandic sulfur leaf (strain HVE10 / 4)GN=SiH_0402PE=4SV=1) The amino acid sequence is as follows:

[0511] MEVPLYNIFGDNYIIQVATEAENSTIYNNKVEIDDEELRNVLNLAYKIAKNNEDAAAERRGKAKKKKGEEGETTTSNIILPLSGNDKNPWTETLKCYNFPTTVALSEVFKNFSQVKECEEVSAPSFVKPEFYEFGRSPGMVERTRRVKLEVEPHYLIIAAAGWVLTRLGKAKV SEGDYVGVNVFTPTRGILYSLIQNVNGIVPGIKPETAFGLWIARKVVSSVTNPNVSVVRIYTISDAVGQNPTTINGGFSIDLTKLLEKRYLLSERLEAIARNALSISSNMRERYIVLANYIYEYLTGSKRLEDLLYFANRDLIMNLNSDDGKVRDLKLISAYVNGELIRGEG.

[0512] An exemplary amino acid sequence of CasX (>tr|F0NH53|F0NH53_SULIR CRISPR associated protein, CasxOS=Icelandic sulfur leaf (strain REY15A)GN=SiRe_0771PE=4SV=1) is as follows:

[0513] MEVPLYNIFGDNYIIQVATEAENSTIYNNKVEIDDEELRNVLNLAYKIAKNNEDAAAERRGKAKKKKGEEGETTTSNIILPLSGNDKNPWTETLKCYNFPTTVALSEVFKNFSQVKECEEVSAPSFVKPEFYKFGRSPGMVERTRRVKLEVEPHYLIMAAAGWVLTRLGKAKV SEGDYVGVNVFTPTRGILYSLIQNVNGIVPGIKPETAFGLWIARKVVSSVTNPNVSVVSIYTISDAVGQNPTTINGGFSIDLTKLLEKRDLLSERLEAIARNALSISSMRERYIVLANYIYEYLTGSKRLEDLLYFANRDLIMNLNSDDGKVRDLKLISAYVNGELIRGEG.

[0514] Delta bacteriumCasX

[0515] MEKRINKIRKKLSADNATKPVSRSGPMKTLLVRVMTDDLKKRLEKRRKKPEVMPQVISNNAANNLRMLLDDYTKMKEAILQVYWQEFKDDHVGLMCKFAQPASKKIDQNKLKPEMDEKGNLTTAGFACSQCGQPLFVYKLEQVSEKGKAYTNYFGRCNVAEHEKLILLAQLKPVKDSDEAVTYSLGKFGQRALDFYSIHVTKESTHPVKPLAQIAGNRYASGPVGKALSDACMGTIASFLSKYQDIIIEHQKVVKGNQKRLESLRELAGKENLEYPSVTLPPQPHTKEGVDfAYNEVIARVRMWVNLLWQKLKLSRDDAKPLLRLKGFPSFPVVERRENEVDWWNTINEVKKLIDAKRDMGRVFWSGVTAEKRNTILEGYNYLPNENDHKKREGSLENPKKPAKRQFGDLLLYLEKKYAGDWGKVFDEAWERIDKKIAGLTSHIEREEARNAEDAQSKAVLTDWLRAKASFVLERLKEMDEKEFYACEIQLQ KWYGDLRGNPFAVEAENRVVDISGFSIGSDGHSIQYRNLLAWKYLENGKREFYLLMNYGKKGRIRFTDGTDIKKSGKWQGLLYGGGGKAKVIDLTFDPDDEQLIILPLAFGTRQGREFIWNDLLSLETGLIKLANGRVIEKTIYNKKIGRDEPALFVALTFERREVVDPSNIKPVNLIGVARGENIPAVIALTDPEGCPLPEFKDSSGGPTDILRIGEGYKEKQRAIQAAKEVEQRRAGGYSRKFASKSRNLADDMVRNSARDLFYHAVTHDAVLVFANLSRGFGRQGKRTFMTERQYTKMEDWLTAKLAYEGLTSKTYLSKTLAQYTSKTCSNCGFTITYADMDVMLVRLKKTSDGWATTLNNKELKAEYQITYYNRYKRQTVEKELSAELDRLSEESGNNDISKWTKGRRDEALFLLKKRFSHRPVQEQFVCLDCGHEVHAAEQAALNIARSWLFLNSNSTEFKSYKSGKQPFVGAWQAFYKRRLKEVWKPNA

[0516] An exemplary amino acid sequence of CasY ((ncbi.nlm.nih.gov / protein / APG80656.1)>APG80656.1 CRISPR-associated protein CasY [uncultured Parcubacteria]) is as follows:

[0517]

[0518] The Cas9 nuclease has two functional endonuclease domains: RuvC and HNH. Upon target binding, Cas9 undergoes a conformational change, positioning its nuclease domains to cleave the opposite strand of the target DNA. The final result of Cas9-mediated DNA cleavage is a double-strand break (DSB) in the target DNA (approximately 3-4 nucleotides upstream of the PAM sequence). The resulting DSB is then repaired via one of two general repair pathways: (1) the efficient but error-prone non-homologous end joining (NHEJ) pathway; or (2) the less efficient but high-fidelity homology-directed repair (HDR) pathway.

[0519] The “efficiency” of non-homologous end joining (NHEJ) and / or homologous directed repair (HDR) can be calculated by any convenient method. For example, in some embodiments, efficiency can be expressed as a percentage of successful HDR. For example, a Surveyor nuclease assay can be used to generate cleavage products, and the product-to-substrate ratio can be used to calculate the percentage. For example, a Surveyor nuclease can be used to directly cleave DNA containing a newly integrated restriction sequence as a result of successful HDR. More cleaved substrate indicates a higher HDR percentage (higher HDR efficiency). As an illustrative example, the HDR fraction (percentage) can be calculated using the following equation [(cleavage products) / (substrate plus cleavage products)] (e.g., (b+c) / (a+b+c), where “a” is the band intensity of the DNA substrate, and “b” and “c” are the cleavage products).

[0520] In some embodiments, efficiency can be expressed as a percentage of successful NHEJs. For example, a T7 endonuclease I assay can be used to generate cleavage products, and the product-to-substrate ratio can be used to calculate the NHEJ percentage. T7 endonuclease I cleaves mismatched heteroduplex DNA resulting from hybridization of wild-type and mutant DNA strands (NHEJ produces small random insertions or deletions at the original break site). More cleavages indicate a higher NHEJ percentage (higher NHEJ efficiency). As an illustrative example, the NHEJ fraction (percentage) can be calculated using the following equation: (1 - (1 - (b+c) / (a+b+c))) 1 / 2 )×100, where “a” is the band intensity of the DNA substrate, and “b” and “c” are the cleavage products (Ran et al., Cell. 2013 Sep. 12; 154(6):1380-9; and Ran et al., Nat Protoc. 2013 Nov.; 8(11):2281–2308).

[0521] The NHEJ repair pathway is the most active repair mechanism, frequently resulting in small nucleotide insertions or deletions (indels) at DSB sites. The stochastic nature of NHEJ-mediated DSB repair has significant practical implications because cell populations expressing Cas9 and gRNA or guide polynucleotides can lead to a variety of mutations. In most implementations, NHEJ generates small insertions or deletions in the target DNA, resulting in amino acid deletions, insertions, or frameshift mutations, leading to premature stop codons within the target gene's open reading frame (ORF). The ideal end result is a loss-of-function mutation within the target gene.

[0522] Although NHEJ-mediated DSB repair often disrupts open reading frames of genes, homology-directed repair (HDR) can be used to produce specific nucleotide changes, ranging from single nucleotide changes to large insertions, such as the addition of fluorophores or tags.

[0523] To utilize HDR for gene editing, a DNA repair template containing the desired sequence can be delivered to the cell type of interest using gRNA and Cas9 or the Cas9 nickase. The repair template can contain the desired edit along with other homologous sequences immediately upstream and downstream of the target (called left and right homologous arms). The length of each homologous arm depends on the size of the introduced change; larger insertions require longer homologous arms. The repair template can be a single-stranded oligonucleotide, a double-stranded oligonucleotide, or a double-stranded DNA plasmid. Even in cells expressing Cas9, gRNA, and a foreign repair template, HDR efficiency is typically low (<10% of modified alleles). HDR efficiency can be improved by synchronizing cells, as HDR occurs during the S and G2 phases of the cell cycle. Chemical or genetic repressor genes involved in NHEJ can also increase HDR frequency.

[0524] In some embodiments, Cas9 is a modified Cas9. A given gRNA target sequence can have additional sites throughout the genome where partial homology exists. These sites are called off-target sites and need to be considered when designing gRNAs. Besides optimizing gRNA design, CRISPR specificity can be improved by modifying Cas9. Cas9 generates double-strand breaks (DSBs) through the combined activity of its two nuclease domains, RuvC and HNH. The Cas9 nickase is a D10A mutant of SpCas9 that retains one nuclease domain and generates a DNA nick instead of a DSB. Nickase systems can also be combined with HDR-mediated gene editing for specific gene editing.

[0525] In some embodiments, Cas9 is a variant Cas9 protein. The variant Cas9 polypeptide has an amino acid sequence that differs from the wild-type Cas9 protein by one amino acid (e.g., deletion, insertion, substitution, fusion). In some cases, the variant Cas9 polypeptide has amino acid changes that reduce the nuclease activity of the Cas9 polypeptide (e.g., deletion, insertion, or substitution). For example, in some cases, the variant Cas9 polypeptide has less than 50%, less than 40%, less than 30%, less than 20%, less than 10%, less than 5%, or less than 1% of the nuclease activity of the corresponding wild-type Cas9 protein. In some embodiments, the variant Cas9 protein has no substantial nuclease activity. When the subject Cas9 protein is a variant Cas9 protein without substantial nuclease activity, it can be referred to as "dCas9".

[0526] In some implementations, the variant Cas9 protein has reduced nuclease activity. For example, the variant Cas9 protein exhibits less than about 20%, less than about 15%, less than about 10%, less than about 5%, less than about 1%, or less than about 0.1% of the endonuclease activity of the wild-type Cas9 protein, such as the wild-type Cas9 protein.

[0527] In some embodiments, the variant Cas9 protein can cleave the complementary strand of the guide target sequence, but its ability to cleave the non-complementary strand of the double-stranded guide target sequence is reduced. For example, the variant Cas9 protein may have a mutation (amino acid substitution) that reduces the function of the RuvC domain. As a non-limiting example, in some embodiments, the variant Cas9 protein has D10A (aspartic acid to alanine at amino acid position 10) and therefore can cleave the complementary strand of the double-stranded guide target sequence but cleave the complementary strand of the non-double-stranded guide target sequence (thus causing a single-strand break (SSB) instead of a double-strand break (DSB) when the variant Cas9 protein cleaves the double-stranded target nucleic acid) (see, for example, Jinek et al., Science. 2012 Aug. 17; 337(6096):816-21).

[0528] In some embodiments, the variant Cas9 protein can cleave the non-complementary strand of the double-stranded guide target sequence, but its ability to cleave the complementary strand of the guide target sequence is reduced. For example, the variant Cas9 protein may have a mutation (amino acid substitution) that reduces the function of the HNH domain (RuvC / HNH / RuvC domain motif). As a non-limiting example, in some embodiments, the variant Cas9 protein has an H840A mutation (histidine to alanine at amino acid position 840), thus enabling it to cleave the non-complementary strand of the guide target sequence, but reducing its ability to cleave the complementary strand of the guide target sequence (thus resulting in the use of SSB instead of DSB when the variant Cas9 protein cleaves the double-stranded guide target sequence). Such Cas9 proteins have reduced ability to cleave guide target sequences (e.g., single-stranded guide target sequences) but retain the ability to bind to guide target sequences (e.g., single-stranded guide target sequences).

[0529] In some embodiments, the variant Cas9 protein has a reduced ability to cleave both the complementary and non-complementary strands of the double-stranded target DNA. As a non-limiting example, in some embodiments, the variant Cas9 protein contains both D10A and H840A mutations, resulting in a reduced ability of the peptide to cleave both the complementary and non-complementary strands of the double-stranded target DNA. Such Cas9 proteins have reduced ability to cleave target DNA (e.g., single-stranded target DNA) but retain the ability to bind to target DNA (e.g., single-stranded target DNA).

[0530] As another non-limiting example, in some embodiments, variant Cas9 proteins contain mutations of W476A and W1126A, which reduce the ability of the peptide to cleave target DNA. Such Cas9 proteins have reduced ability to cleave target DNA (e.g., single-stranded target DNA) but retain the ability to bind to target DNA (e.g., single-stranded target DNA).

[0531] As another non-limiting example, in some embodiments, variant Cas9 proteins contain mutations of P475A, W476A, N477A, ​​D1125A, W1126A, and D1127A, which reduce the ability of the peptide to cleave target DNA. Such Cas9 proteins have reduced ability to cleave target DNA (e.g., single-stranded target DNA) but retain the ability to bind to target DNA (e.g., single-stranded target DNA).

[0532] As another non-limiting example, in some embodiments, the variant Cas9 protein contains mutations of H840A, W476A, and W1126A, resulting in a reduced ability of the peptide to cleave target DNA. Such Cas9 proteins have reduced ability to cleave target DNA (e.g., single-stranded target DNA) but retain the ability to bind to target DNA (e.g., single-stranded target DNA). As another non-limiting example, in some embodiments, the variant Cas9 protein contains mutations of H840A, D10A, W476A, and W1126A, resulting in a reduced ability of the peptide to cleave target DNA. Such Cas9 proteins have reduced ability to cleave target DNA (e.g., single-stranded target DNA) but retain the ability to bind to target DNA (e.g., single-stranded target DNA). In some embodiments, the variant Cas9 restores the catalytic His residue at position 840 in the Cas9HNH domain (A840H).

[0533] As another non-limiting example, in some embodiments, the variant Cas9 protein contains mutations of H840A, P475A, W476A, N477A, ​​D1125A, W1126A, and D1127A, which reduces the ability of the peptide to cleave target DNA. Such Cas9 proteins have reduced ability to cleave target DNA (e.g., single-stranded target DNA) but retain the ability to bind to target DNA (e.g., single-stranded target DNA). As another non-limiting example, in some embodiments, the variant Cas9 protein contains mutations of D10A, H840A, P475A, W476A, N477A, ​​D1125A, W1126A, and D1127A, which reduces the ability of the peptide to cleave target DNA. Such Cas9 proteins have reduced ability to cleave target DNA (e.g., single-stranded target DNA) but retain the ability to bind to target DNA (e.g., single-stranded target DNA). In some embodiments, when the variant Cas9 protein contains mutations of W476A and W1126A or when the variant Cas9 protein contains mutations of P475A, W476A, N477A, ​​D1125A, W1126A, and D1127A, the variant Cas9 protein does not bind effectively to the PAM sequence. Therefore, in some such embodiments, when such variant Cas9 proteins are used in a binding method, the method does not require a PAM sequence. In other words, in some embodiments, when such variant Cas9 proteins are used in a binding method, the method may include a guide RNA, but the method can be performed in the absence of a PAM sequence (and the specificity of binding is therefore provided by the targeting fragment of the guide RNA). Other residues can be mutated to achieve the above effect (i.e., inactivate one or more nuclease moieties). As a non-limiting example, residues D10, G12, G17, E762, H840, N854, N863, H982, H983, A984, D986, and / or A987 can be altered (i.e., substituted). In addition, mutations other than alanine substitution are also suitable.

[0534] In some implementations, variant Cas9 proteins with reduced catalytic activity (e.g., when the Cas9 protein has mutations such as D10, G12, G17, E762, H840, N854, N863, H982, H983, A984, D986, and / or A987, e.g., D10A, G12A, G17A, E762A, H840A, N854A, N863A, H982A, H983A, A984A, and / or D986A) can still bind to target DNA in a site-specific manner (because it is still guided to the target DNA sequence by the guide RNA), as long as it retains the ability to interact with the guide RNA.

[0535] In some embodiments, the variant Cas protein can be spCas9, spCas9-VRQR, spCas9-VRER, xCas9(sp), saCas9, saCas9-KKH, spCas9-MQKSER, spCas9-LRKIQK, or spCas9-LRVSQL.

[0536] In some embodiments, SpCas9 is used, which includes amino acid substitutions for D1135M, S1136Q, G1218K, E1219F, A1322R, D1332A, R1335E, and T1337R (SpCas9-MQKFRAER) and has specific modifications to altered PAM 5'-NGC.

[0537] Alternatives to Cas9 for Streptococcus pyogenes can include RNA-guided endonucleases from the Cpf1 family, which exhibit cleaving activity in mammalian cells. CRISPR from Prevotella and Francisella 1 (CRISPR / Cpf1) is a DNA editing technology similar to the CRISPR / Cas9 system. Cpf1 is an RNA-guided endonuclease in the class II CRISPR / Cas system. This acquired immune mechanism is present in Prevotella and Francisella. The Cpf1 gene is associated with a CRISPR locus and encodes an endonuclease that uses guide RNA to locate and cleave viral DNA. Cpf1 is a smaller and simpler endonuclease than Cas9, overcoming some limitations of the CRISPR / Cas9 system. Unlike Cas9 nucleases, Cpf1-mediated DNA cleavage results in double-strand breaks with short 3' overhangs. The staggered cleavage pattern of Cpf1 opens up possibilities for targeted gene transfer, similar to traditional restriction enzyme cloning, potentially increasing the efficiency of gene editing. Similar to the aforementioned Cas9 variants and orthogonal homologs, Cpf1 expands the number of CRISPR-targetable sites to AT-rich regions or AT-rich genomes lacking the NGG PAM sites favored by SpCas9. The Cpf1 locus contains a hybrid alpha / beta domain, a RuvC-I followed by a helical region, a RuvC-II, and a zinc finger-like domain. The Cpf1 protein possesses a RuvC-like endonuclease domain similar to the RuvC domain of Cas9. Furthermore, Cpf1 lacks an HNH endonuclease domain, and its N-terminus lacks the α-helical recognition leaflet of Cas9. The Cpf1 CRISPR-Cas domain architecture indicates that Cpf1 is functionally unique and is classified as a type V CRISPR system. The Cas1, Cas2, and Cas4 proteins encoded by the Cpf1 locus are more similar to type I and type III systems than to type II systems. Functional Cpf1 does not require trans-activation CRISPR RNA (tracrRNA), therefore, only CRISPR (crRNA) is needed. This is advantageous for genome editing because Cpf1 is not only smaller than Cas9, but its sgRNA molecule is also smaller (approximately half the nucleotide size of Cas9). Compared to the G-rich PAM targeted by Cas9, the Cpf1-crRNA complex cleaves target DNA or RNA by recognizing the 5'-YTN-3' motif adjacent to the protospacer sequence. Upon PAM identification, Cpf1 introduces a sticky-end-like DNA double-strand break with 4 or 5 nucleotide overhangs.

[0538] In some embodiments, Cas9 is a Cas9 variant specific to the altered PAM sequence. In some embodiments, additional Cas9 variants and PAM sequences are described in Miller, SM et al., Continuous evolution of SpCas9 variants compatible with non-G PAMs, Nat. Biotechnol. (2020), the entire contents of which are incorporated herein by reference. In some embodiments, the Cas9 variant does not have specific PAM requirements. In some embodiments, the Cas9 variant, such as the SpCas9 variant, is specific to NRNH PAM, where R is A or G and H is A, C, or T. In some embodiments, the SpCas9 variant is specific to the PAM sequence AAA, TAA, CAA, GAA, TAT, GAT, or CAC. In some embodiments, the SpCas9 variant contains amino acid substitutions at positions 1114, 1134, 1135, 1137, 1139, 1151, 1180, 1188, 1211, 1218, 1219, 1221, 1249, 134, 137, 127, 137, 1320, 1321, 1323, 1332, 1333, 1335, 1337, or 1339 of SEQ ID NO:1, or at corresponding positions. In some embodiments, the SpCas9 variant contains amino acid substitutions at positions 1114, 1134, 1135, 1137, 1139, 1151, 1180, 1188, 1211, 1219, 1221, 1256, 1264, 1290, 1318, 1317, 1320, 1323, 1333 or corresponding positions of SEQ ID NO:1. In some embodiments, the SpCas9 variant contains amino acid substitutions at positions 1114, 1131, 1135, 1150, 1156, 1180, 1191, 1218, 1219, 1221, 1227, 1249, 1253, 1286, 1293, 1320, 1321, 1332, 1335, 1339 or corresponding positions of SEQ ID NO:1.In some embodiments, the SpCas9 variants contain amino acid substitutions at positions 1114, 1127, 1135, 1180, 1207, 1219, 1234, 1286, 1301, 1332, 1335, 1337, 1338, 1349 or corresponding positions of SEQ ID NO:1. Exemplary amino acid substitutions and PAM specificity of the SpCas9 variants are shown in Tables 3A-3D.

[0539] Table 3A

[0540]

[0541]

[0542] Table 3B

[0543]

[0544] Table 3C

[0545]

[0546] Table 3D

[0547]

[0548] In some embodiments, Cas9 is meningococcal Cas9 (NmeCas9) or a variant thereof. In some embodiments, NmeCas9 is specific for NNNNGAYW PAM, where Y is C or T and W is A or T. In some embodiments, NmeCas9 is specific for NNNNGYTTPAM, where Y is C or T. In some embodiments, NmeCas9 is specific for NNNNGTCTPAM. In some embodiments, the NmeCas9 is Nme1Cas9. In some embodiments, NmeCas9 is specific for NNNGATT PAM, NNNCCTA PAM, NNNCCTC PAM, NNNCCTT PAM, NNNCCTG PAM, NNNCCGTPAM, NNNCCGGPAM, NNNCCCA PAM, NNNCCCT PAM, NNNCCCC PAM, NNNNCCAT PAM, NNNNCCAGPAM, NNNNCCAT PAM, or NNNNGATT PAM. In some embodiments, Nme1Cas9 is specific for NNNNGATTPAM, NNNCCTA PAM, NNNCCTC PAM, NNNCCTT PAM, or NNNCCTG PAM. In some embodiments, NmeCas9 is specific for CAA PAM, CAAA PAM, or CCA PAM. In some embodiments, the NmeCas9 is Nme2Cas9. In some embodiments, NmeCas9 is specific for NNNCC(N4CC) PAM, where N is any one of A, G, C, or T. In some embodiments, NmeCas9 is specific for NNNCCGT PAM, NNNCCGGPAM, NNNCCCA PAM, NNNCCCT PAM, NNNCCCC PAM, NNNNCCAT PAM, NNNCCAG PAM, NNNNCCAT PAM, or NNNNGATT PAM. In some embodiments, the NmeCas9 is Nme3Cas9. In some embodiments, NmeCas9 is specific for NNNNCAAA PAM, NNNCC PAM, or NNNCNNN PAM. Additional NmeCas9 features and PAM sequences, as described by Edraki et al. Mol. Cell. (2019) 73(4): 714-726, are incorporated herein by reference in their entirety.

[0549] The following provides an exemplary amino acid sequence of Nme1Cas9:

[0550] Type II CRISPR RNA-guided endonuclease Cas9 [Neisseria meningitidis] WP_002235162.1

[0551]

[0552] The following provides an exemplary amino acid sequence of Nme2Cas9:

[0553] Type II CRISPR RNA-guided endonuclease Cas9 [Neisseria meningitidis] WP_002230835.1

[0554]

[0555] Cas12 domain of nucleobase editors

[0556] Microbial CRISPR-Cas systems are typically classified into Class 1 and Class 2 systems. Class 1 systems have multi-subunit effector complexes, while Class 2 systems have single protein effectors. For example, Cas9 and Cpf1 are Class 2 effectors, although they are of different types (Type II and Type V, respectively). In addition to Cpf1, Class 2 Type V CRISPR-Cas systems also include Cas12a / Cpfl, Cas12b / C2cl, Cas12c / C2c3, Cas12d / CasY, Cas12e / CasX, Cas12g, Cas12h, and Cas12i. See, for example, Shmakov et al., “Discovery and Functional Characterization of Diverse Class 2 CRISPR Cas Systems,” Mol. Cell, 2015 Nov. 5; 60(3):385-397; Makarova et al., Classification and Nomenclature of CRISPR-Cas Systems: Where from Here? The full contents of these articles are described in “CRISPR Journal, 2018, 1(5):325-336; and Yan et al., “Functionally diverse type V CRISPR-Cas systems,” Science, 2019 Jan. 4; 363:88-91, and are incorporated herein by reference. Type V Cas proteins contain a RuvC (or RuvC-like) endonuclease domain. While the production of mature CRISPR RNA (crRNA) is generally independent of tracrRNA, Cas12b / C2c1, for example, requires tracrRNA to produce crRNA. Cas12b / C2c1 relies on both crRNA and tracrRNA for DNA cleavage.

[0557] The nucleic acid programmable DNA-binding proteins considered in this invention include Cas proteins classified as type 2V (Cas12 proteins). Non-limiting examples of Cas2V proteins include Cas12a / Cpfl, Cas12b / C2cl, Cas12c / C2c3, Cas12d / CasY, Cas12e / CasX, Cas12g, Cas12h, and Cas12i, their homologs, or modified forms thereof. As used herein, Cas12 proteins may also be referred to as Cas12 nucleases, Cas12 domains, or Cas12 protein domains. In some embodiments, the Cas12 proteins of this invention comprise an amino acid sequence interrupted by an internal fusion protein domain, such as a deaminase domain.

[0558] In some embodiments, the Cas12 domain is a non-nuclease-active Cas12 domain or a Cas12 nickase. In some embodiments, the Cas12 domain is a domain with nuclease activity. For example, the Cas12 domain may be a Cas12 domain that forms a nick on one strand of a double-stranded nucleic acid (e.g., a double-stranded DNA molecule). In some embodiments, the Cas12 domain comprises any of the amino acid sequences described herein. In some embodiments, the amino acid sequence comprised of the Cas12 domain is identical to 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%, or at least 99%, or 99.5% of any of the amino acid sequences described herein. In some embodiments, the amino acid sequence comprising the Cas12 domain has mutations of 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 compared to any of the amino acid sequences described herein. In some embodiments, the Cas12 domain comprises 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 consecutive amino acid residues compared to any of the amino acid sequences described herein.

[0559] In some embodiments, a protein comprising a Cas12 fragment is provided. For example, in some embodiments, the protein comprises one of two Cas12 domains: (1) a gRNA-binding domain of Cas12; or (2) a DNA-cutting domain of Cas12. In some embodiments, a protein comprising Cas12 or a fragment thereof is referred to as a "Cas12 variant". The Cas12 variant is homologous to Cas12 or a fragment thereof. For example, the Cas12 variant is at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, at least about 99.5%, or at least about 99.9% identical to wild-type Cas12. In some embodiments, the Cas12 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 variations compared to wild-type Cas12. In some embodiments, the Cas12 variant comprises a fragment of Cas12 (e.g., a gRNA-binding domain or a DNA-cutting domain) such that the fragment is at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, at least about 99.5%, or at least about 99.9% identical to a corresponding fragment of wild-type Cas12. 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 Cas12. In some embodiments, the fragment is at least 100 amino acids long. In some embodiments, the length of the fragment is at least 100, 150, 200, 250, 300, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1050, 1100, 1150, 1200, 1250, or 1300 amino acids.

[0560] In some embodiments, Cas12 corresponds to or comprises, in part or in whole, a Cas12 amino acid sequence having one or more mutations that alter the activity of the Cas12 nuclease. For example, such mutations include amino acid substitutions within the RuvC nuclease domain of Cas12. In some embodiments, variants or homologs of Cas12 are provided that are at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, at least about 99.5%, or at least about 99.9% identical to wild-type Cas12. In some embodiments, variants of Cas12 are provided having shorter or longer amino acids of about 5, about 10, about 15, about 20, about 25, about 30, about 40, about 50, about 75, about 100, or more amino acids.

[0561] In some embodiments, the Cas12 fusion proteins provided herein comprise the full-length amino acid sequence of the Cas12 protein, such as one of the Cas12 sequences provided herein. However, in other embodiments, the fusion proteins provided herein do not comprise the full-length Cas12 sequence, but only one or more fragments thereof. Exemplary amino acid sequences of suitable Cas12 domains are provided herein, and other suitable sequences of Cas12 domains and fragments will be apparent to those skilled in the art.

[0562] Typically, type 2 V Cas proteins possess a single functional RuvC endonuclease domain (see, for example, Chen et al., “CRISPR-Cas12a target binding unleashes indiscriminate-stranded DNase activity,” Science 360:436-439 (2018)). In some embodiments, the Cas12 is a variant Cas12b protein (see Strecker et al., Nature Communications, 2019, 10(1): Art. No.: 212). In one embodiment, the variant Cas12 polypeptide has an amino acid sequence that differs from the wild-type Cas12 protein by 1, 2, 3, 4, 5 or more amino acids (e.g., deletions, insertions, substitutions, fusions). In some cases, the variant Cas12 polypeptide has amino acid changes (e.g., deletions, insertions, or substitutions) that reduce the activity of the Cas12 polypeptide....

Claims

1. A method for producing modified immune cells ex vivo, the method comprising expressing or introducing a nucleobase editor polypeptide in an immune cell and contacting the cell with a guide RNA mediating the binding of the nucleobase editor polypeptide to a target site to affect the change of the splice acceptor site or splice donor site in a nucleic acid molecule encoding selected from the T cell receptor alpha constant, β-2 microglobulin, programmed cell death 1, differentiation cluster 7 , polypeptides of a group of major histocompatibility complex transactivator polypeptides of Cbl proto-oncogene B and class II major histocompatibility complex transactivator polypeptides to reduce expression of the polypeptide relative to wild-type immune cells, wherein the nucleobase editor polypeptide comprises the Cas9 nickase nucleic acid programmable DNA binding protein napDNAbp and adenosine deaminase variant domain, which consists of the following amino acid sequences or fragments thereof lacking only N-terminal methionine: MSEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWNRAIGLHDPTA HAEIMALRQGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIGRVVFGVRNAKTGAAGS LMDVLHYPGMNHRVEITEGILADECAALLCYFFRMPRQVFNAQKKAQSSTD (SEQ ID NO:3), which has a change, wherein the change is any of the following combinations of changes referring to SEQ ID NO:3: Y147R, Q154R and Y123H; I76Y, Y123H, Y147R and Q154R; V82S and Q154R; or I76Y, V82S, Y123H, Y147R and Q154R.

2. The method of claim 1, wherein the nucleobase editor polypeptide comprises a single adenosine deaminase domain.

3. The method of claim 1, wherein the nucleobase editor polypeptide further comprises a wild-type adenosine deaminase domain.

4. The method of claim 1, wherein the nucleobase editor polypeptide further comprises an adenosine deaminase domain, which is composed of MSEVEFSHEY WMRHALTLAK RARDEREVPVGAVLVLNNRVIGEGWNRAIGLHDPTAHAEI MALRQGGLVM QNYRLIDATL YVTFEPCVMCAGAMIHSRIG RVVFGVRNAKTGAAGSLMDV LHYPGMNHRVEITEGILADE CAALLCYFFR MPRQVFNAQK KAQSSTD (SEQ ID The amino acid sequence of NO:3) or its variant lacking only the N-terminal methionine.

5. The method of claim 1, wherein the napDNAbp is composed of the following sequence composition: EIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFMQPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKDLIIKLPKYSL FELENGRKRMLASAKFLQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPRAFKYFDTTIARKEYRSTKEVLDATLIHQSITGLYETRIDLSQLGGDGGSGGSGG SGGSGGSGGMDKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGETAEATRLKRTARRRYTRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRGH FLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLP EKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTV YNELTKVKYVTEGMRKPAFLSGEQKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLFEDREMIEERLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLI HDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMARENQTTQKQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPSE EVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEGADKRTADGSEFESPKKKRKV.

6. The method of claim 1, wherein the napDNAbp is Staphylococcus aureus Cas9, Streptococcus thermophilus 1Cas9 or Streptococcus pyogenes Cas9.

7. The method of claim 1, wherein the Cas9 nickase napDNAbp is specific for the pro-spacer adjacent motif with the nucleic acid sequence 5'-NGC-3'.

8. The method of claim 1, wherein the nucleobase editor polypeptide further comprises a zinc finger domain.

9. The method of claim 1, wherein the adenosine deaminase variant domain is capable of deaminating adenine in the deoxyribonucleic acid.

10. The method of claim 1, wherein the adenosine deaminase variant domain is not present in nature.

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

12. The method of claim 11, wherein the linker comprises an amino acid sequence: SGGSSGGSSGSETPGTSESATPES.

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

14. The method of claim 13, wherein the NLS is a bidirectional NLS.

15. The method of claim 13, wherein the nucleobase editor polypeptide comprises an N-terminal NLS and a C-terminal NLS.

16. The method of claim 6, wherein the napDNAbp is Staphylococcus aureus Cas9.

17. The method of claim 16, wherein the Staphylococcus aureus Cas9 consists of the following amino acid sequence: KRNYILGLAIGITSVGYGIIDYETRDVIDAGVRLFKEANVENNEGRRSKRGARRLKRRRHRIQRVKKLLFDYNLLTDHSELSGINPYEARVKGLSQKLSEEEFSAALLHLAKRRGVHNVNEVEEDTGNEL STKEQISRNSKALEEKYVAELQLERLKKDGEVRGSINRFKTSDYVKEAKQLLKVQKAYHQLDQSFIDTYIDLLETRRTYYEGPGEGSPFGWKDIKEWYEMLMGHCTYFPEELRSVKYAYNADLYNALNDLNN LVITRDENEKLEYYEKFQIIENVFKQKKPTLKQIAKEILVNEEDIKGYRVTSTGKPEFTNLKVYHDIKDITARKEIIENAELLDQIAKILTIYQSSEDIQEELTNLNSELTQEEIEQISNLKGYTGTHNL SLKAINLILDELWHTNDNQIAIFNRLKLVPKKVDLSQQKEIPTTLVDDFILSPVVKRSFIQSIKVINAIIKKYGLPNDIIIELAREKNSKDAQKMINEMQKRNRQTNERIEEIIRTTGKENAKYLIEKIKLH DMQEGKCLYSLEAIPLEDLLNNPFNYEVDHIIPRSVSFDNSFNNKVLVKQEENSKKGNRTPFQYLSSSDSKISYETFKKHILNLAKGGRISKTKKEYLLEERDINRFSVQKDFINRNLVDTRYATRGLMN LLRSYFRVNNLDVKVKSINGGFTSFLRRKWKFKKERNKGYKHHAEDALIIANADFIFKEWKKLDKAKKVMENQMFEEKQAESMPEIETEQEYKEIFITPHQIKHIKDFKDYKYSHRVDKKPNRKLINDTLYS TRKDDKGNTLIVNNLNGLYDKDNDKLKKLINKSPEKLLMYHHDPQTYQKLKLIMEQYGDEKNPLYKYYEETGNYLTKYSKKDNGPVIKKIKYYGNKLNAHLDITDDYPNSRNKVVKLSLKPYRFDVYLDNGV YKFVTVKNLDVIKKENYYEVNSKCYEEAKKLKKISNQAEFIASFYKNDLIKINGELYRVIGVNNDLLNRIEVNMIDITYREYLENMNDKRPPHIIKTIASKTQSIKKYSTDILGNLYEVKSKKHPQIIKKG.

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

19. The method of claim 1, wherein the immune cells are taken from a healthy subject.

20. The method of claim 1, wherein the guide RNA is expressed in a cell or contacts a cell.

21. The method of claim 1, wherein three guide RNAs are expressed or contacted in a cell, wherein the three guide RNAs each mediate binding of the nucleobase editor polypeptide to a target site in each β-2 microglobulin, T cell receptor alpha constant and Class II major histocompatibility complex transactivator polynucleotide to affect changes in each of the β-2 microglobulin, T cell receptor alpha constant and Class II major histocompatibility complex transactivator polynucleotides, and wherein the altered combination consists of I76Y, V82S, Y123H, Y147R and Q154R of reference SEQ ID NO:

3.

22. The method of claim 21, wherein the three guide RNA mediate binding of the nucleobase editor polypeptide to the target site to affect changes in the T cell receptor alpha constant exon 4 splice acceptor site, β-2 microglobulin exon 1 splice donor site, and / or programmed cell death 1 exon 1 splice donor site.

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

24. The method of claim 1, further comprising expressing a chimeric antigen receptor in the modified immune cell.

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

26. The method of claim 1, wherein the modified immune cell does not contain detectable translocations.

27. A base editor system that includes: a) A nucleobase editor polypeptide comprising a Cas9 nickase polynucleotide programmable DNA binding domain and an adenosine deaminase variant domain consisting of the following amino acid sequence or fragments thereof lacking only N-terminal methionine composition: MSEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWNRAIGLHDPTAHAEI MALRQGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIGRVVFGVRNAKTGAAGSLMDV LHYPGMNHRVEITEGILADECAALLCYFFRMPRQVFNAQKKAQSSTD (SEQ ID NO:3), which has a change, wherein the change is any of the following combinations of changes referring to SEQ ID NO:3: Y147R, Q154R and Y123H; I76Y, Y123H, Y147R and Q154R; V82S and Q154R; or I76Y, V82S, Y123H, Y147R and Q154R; and b) Guide RNA mediating the binding of the nucleobase editor polypeptide to the target site to affect the change of the splice acceptor site or splice donor site in a nucleic acid molecule encoding a polypeptide selected from the group consisting of T cell receptor alpha constant, β-2 microglobulin, programmed cell death 1, cluster of differentiation 7, Cbl proto-oncogene B and class II major histocompatibility complex transactivator polypeptides, thereby reducing the expression of the polypeptide relative to wild-type immune cells.

28. The base editor system of claim 27, wherein the nucleobase editor polypeptide further comprises a wild-type adenosine deaminase domain.

29. The base editor system of claim 27, wherein the polynucleotide programmable DNA binding domain is Staphylococcus aureus Cas9, Streptococcus thermophilus 1Cas9 or Streptococcus pyogenes Cas9.

30. A base editor system comprising a guide RNA and a fusion protein comprising a Cas9 nickase polynucleotide programmable DNA binding domain consisting of the following sequences: EIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFMQPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKDLIIKLPKYSLFELENG RKRMLASAKFLQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPRAFKYFDTTIARKEYRSTKEVLDATLIHQSITGLYETRIDLSQLGGDGGSGGSGGSGGSGGSGGMDK KYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLAHMIKFRGHFLIEGDLNPDNSDVDKLF IQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGA SQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIV DLLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLFEDREMIEERLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSP AIKKGILQTVKVVDELVKVMGRHKPENIVIEMARENQTTQKQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDK AGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEGADKRTADGSEFESPKKKRKV, and adenosine deaminase variant domains, which are composed of the following amino acid sequences or fragments thereof that lack only N-terminal methionine. composition: MSEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWNRAIGLHDPTAHAEI MALRQGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIGRVVFGVRNAKTGAAGSLMDV LHYPGMNHRVEITEGILADECAALLCYFFRMPRQVFNAQKKAQSSTD (SEQ ID NO:3), which has a change, wherein the change is any of the following combinations of changes referring to SEQ ID NO:3: Y147R, Q154R and Y123H; I76Y, Y123H, Y147R and Q154R; V82S and Q154R; or I76Y, V82S, Y123H, Y147R and Q154R; and wherein the guide RNA mediates the binding of the fusion protein to a target site to affect the change of the splice acceptor site or splice donor site in a nucleic acid molecule encoding a polypeptide selected from the group consisting of T cell receptor alpha constant, β-2 microglobulin, programmed cell death 1, cluster of differentiation 7, Cbl proto-oncogene B and class II major histocompatibility complex transactivator polypeptides, thereby reducing the expression of the polypeptide relative to wild-type immune cells.

31. A cell comprising the base editor system of claim 27 or 30.

32. The cell of claim 31, wherein the cell is a mammalian cell.

Images