Compositions and methods for treatment of liquid cancers

Abstract

To provide genetically modified immune cells having enhanced anti-neoplasia activity, resistance to immune suppression, and decreased risk of eliciting a graft versus host reaction, or a combination thereof.SOLUTION: Provided is a composition comprising two or more immune cells, each immune cell comprising a) a different chimeric antigen receptor that targets an antigen selected from the group consisting of CD5, CD7, CD3, CD33, and CD123, wherein the immune cells comprise mutations that reduce or eliminate expression of the targeted antigens; and b) one or more mutations that reduce or eliminate the expression of an immunogenic polypeptide selected from the group consisting of TRAC, LAG-3, FAS, CIITA, TRBC1, TRBC2, CD52, B2M, and PD1.SELECTED DRAWING: None

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application is a continuation of U.S. Provisional Application No. 62 / 907,254, filed September 27, 2019. International PCT applications claiming priority and benefit, the entire contents of which are incorporated herein by reference in their entirety. The body is incorporated herein by reference. [Background technology]

[0002] Autoimmunotherapy and alloimmunotherapy are tumor treatment approaches that involve administering chimeric antigen receptors to the target tumor. Immune cells expressing a chimeric antigen receptor (CAR) are administered. To generate the cells, immune cells are first isolated from the subject (self) or from the subject receiving treatment. They are collected from individual donors (of the same species) and genetically modified (modified) to express chimeric antigen receptors. The resulting cells carry a chimeric antigen receptor (CAR) on their cell surface (e.g., CAR T cells). When administered to a subject, the chimeric antigen receptor binds to the marker expressed by the tumor cells. This interaction with the tumor marker activates the CAR-T cells, which then However, autologous or allogeneic cell therapy is effective and efficient. To be effective, significant conditions and cellular responses, such as inhibition of T cell signaling, must be overcome or Graft-versus-host disease and host rejection of CAR-T cells must be avoided. Editing genes involved in these processes may present additional challenges for therapeutics. This enhances CAR-T cell function and increases tolerance to immunosuppression or immunosuppression. While it is possible to perform such editing, current methods for achieving this involve large amounts of DNA fragments within CAR-T cells. It may induce genome rearrangements, thereby adversely affecting its efficacy. Therefore, technologies for more precisely modifying immune cells (especially CAR-T cells) are needed. This application targets this and other important needs. Let's say. Summary of the Invention

[0003] As described below, the present invention features genetically modified immune cells and The cells exhibited enhanced antitumor activity, resistance to immunosuppression, and enhanced host CD8+ T cell engraftment. When the transplant recipient recognizes the organ as non-self (e.g., when the transplant recipient develops an immune response to the transplanted organ) (producing graft-versus-host or host-versus-graft reactions), reduced risk of inducing graft-versus-host reactions or a combination thereof. In one embodiment, graft-versus-host disease (GVHD) is treated with A subject who has or is prone to develop a disorder characterized by a lack of functional TRAC or The CAR-T cells are administered in a reduced level. The present invention relates to administering functional β2 myelin to subjects with or prone to develop human vein vein vein disease (HVGD). CAR-T cells lacking or with reduced levels of B2M The present invention also provides methods for producing and using these modified immune cells. The method is also featured.

[0004] In one aspect, the invention provides a composition comprising two or more immune cells, each immune cell comprising a ) an antigen selected from the group consisting of CD5, CD7, CD3, CD33, and CD123 different chimeric antigen receptors targeting different antigens, wherein immune cells express the targeted antigens. a) different chimeric antigen receptors containing mutations that reduce or eliminate the expression of TRAC, LAG-3, and , FAS, CIITA, TRBC1, TRBC2, CD52, B2M, and PD1 one or more mutations that reduce or eliminate expression of an immunogenic polypeptide selected from the group consisting of: In some embodiments, one of the immune cells is a chimeric antibody that targets CD5. Contains antigen receptors, and other immune cells are composed of CD7, CD3, CD33, and CD123. In some embodiments, the chimeric antigen receptor targets an antigen selected from the group consisting of: The composition comprises at least three immune cells, each containing a chimeric antibody targeting a different antigen. The antigens targeted, including the primary receptors, are CD3, CD5, and CD7. In an embodiment, one of the immune cells expresses a chimeric antigen receptor that targets CD7; Another immune cell expresses an antigen selected from the group consisting of CD3, CD33, and CD123. In some embodiments, one of the immune cells expresses a chimeric antigen receptor that targets the immune cell. Another immune cell expresses a chimeric antigen receptor that targets CD3, CD33, and CD 123. In embodiments, one of the immune cells expresses a chimeric antigen receptor that targets CD33. However, other immune cells express chimeric antigen receptors that target CD123. In this form, one immune cell may be capable of receiving two, three, four, or more different chimeric antigens. In some embodiments, one of the chimeric antigen receptors targets CD5. and another chimeric antigen receptor consisting of CD7, CD3, CD33, and CD123. In some embodiments, one of the chimeric antigen receptors targets an antigen selected from the group One targets CD7, and another targets CD3, CD33, and CD12. 3. In some embodiments, the chimeric antigen receptor targets an antigen selected from the group consisting of: One of the chimeric antigen receptors targets CD3, and another targets CD33 and CD12. 3. In some embodiments, the chimeric antigen receptor targets an antigen selected from the group consisting of: One of the chimeric antigen receptors targets CD33, and another targets CD123. do.

[0005] In another aspect, the present invention provides a composition comprising at least three immune cells, each of which comprises: It contains different chimeric antigen receptors, one of which targets CD3 and one of which targets One targets CD5, and the third targets CD7, allowing the cells to express the targeted antigen. Further, mutations that reduce or eliminate expression of TRAC, LAG-3, FAS, CIIT A, TRBC1, TRBC2, CD52, B2M, and PD1. The polypeptide further comprises one or more mutations that reduce or eliminate expression of the polypeptide.

[0006] In yet another aspect, the present invention provides a composition comprising at least two immune cells, each Each contains a different chimeric antigen receptor, one of which targets CD3 and Another receptor targets CD7, and cells reduce or eliminate expression of the targeted antigen. Further mutations include TRAC, LAG-3, FAS, CIITA, TRBC1, and TRB Expression of a polypeptide selected from the group consisting of C2, CD52, B2M, and PD1 It further includes one or more mutations that reduce or eliminate the

[0007] In one aspect, the present invention provides a composition comprising at least two immune cells, each of which is a different The chimeric antigen receptors include two chimeric antigen receptors, one of which targets CD5 and another of which targets The body targets CD7, causing cells to harbor mutations that reduce or eliminate expression of the targeted antigen. Also includes TRAC, LAG-3, FAS, CIITA, TRBC1, TRBC2, C reducing or inhibiting the expression of a polypeptide selected from the group consisting of D52, B2M, and PD1; further comprises one or more mutations that eliminate

[0008] In another aspect, the present invention provides a composition comprising at least two immune cells, each of which comprises: It contains different chimeric antigen receptors, one of which targets CD3 and another The inhibitor targets CD5 and induces cell proliferation by mutating the target antigen, reducing or eliminating its expression. Further includes TRAC, LAG-3, FAS, CIITA, TRBC1, TRBC2, reducing or decreasing the expression of a polypeptide selected from the group consisting of CD52, B2M, and PD1 The nucleic acid sequence may further comprise one or more mutations that alter or eliminate the sequence.

[0009] In yet another aspect, the present invention provides a composition comprising at least two immune cells, each Each contains a different chimeric antigen receptor, and one chimeric antigen receptor targets CD33. Another receptor, CD123, is targeted, and the cells reduce expression of the targeted antigen or Further mutations that exclude TRAC, LAG-3, FAS, CIITA, TRBC1, A polypeptide selected from the group consisting of TRBC2, CD52, B2M, and PD1 It further comprises one or more mutations that reduce or eliminate expression.

[0010] In some embodiments, the mutation silences the gene or adds a termination codon to the gene. In some embodiments, the mutation is a C to T or A to G mutation that introduces a nucleotide. To introduce a stop codon or to introduce a splice donor or splice acceptor site In some embodiments, the mutation alters the base site comprising the deaminase domain. In some embodiments, the deaminase is produced by a cytidine or alpha deaminase. In some embodiments, the base editor is BE4. In some embodiments, the mutation increases the expression of the encoded polypeptide compared to a corresponding control cell lacking the mutation. In some embodiments, the composition reduces expression of the polypeptide by about 50% or more in immune cells. In some embodiments, at least 50% of the population comprises a targeted antigen and and / or contain one or more mutations that reduce or eliminate expression of an immunogenic polypeptide. In this embodiment, the immune cells are fratricide resistant. In some embodiments, the immune cells have increased anti-tumor activity. In some embodiments, the immune cells contain less than 1% of the immune cells. In some embodiments, the immune cell is a mammalian cell. In some embodiments, the mammalian cell is a human cell or a rodent cell. In this study, immune cells include cytotoxic T cells, regulatory T cells, T helper cells, dendritic cells, and B cells. In some embodiments, the progenitor cells are NK cells, NK cells, or their precursor cells. are hematopoietic stem cells.

[0011] In one aspect, the present invention provides a pharmaceutical composition, comprising an effective amount of the composition provided herein. and a pharmaceutically acceptable excipient.

[0012] In another aspect, the present invention provides a base editor system, comprising: a nucleic acid programmable (programmable) DNA binding protein (napDNAbp: nucleic acid programmable) fusion proteins containing a DNA binding protein and a deaminase domain, and and at least two guide polynucleotides targeting antigens, the antigens being CD4+ and CD5+. 5, CD7, CD3, CD33, and CD123. In an embodiment, one guide polynucleotide targets CD5 and another guide polynucleotide targets CD6. The protease inhibits an antibody selected from the group consisting of CD7, CD3, CD33, and CD123. In some embodiments, the system includes three guide polynucleotides. Each of these targets one of the antigens CD3, CD5, and CD7. In embodiments, one guide polynucleotide targets CD7 and another guide polynucleotide targets CD8. The nucleotide targets an antigen selected from the group consisting of CD3, CD33, and CD123. In some embodiments, one guide polynucleotide targets CD3, Another guide polynucleotide is an antibody selected from the group consisting of CD33 and CD123. In some embodiments, one guide polynucleotide targets CD33. The other guide polynucleotide targets CD123.

[0013] In some embodiments, the guide polynucleotides each comprise a nucleic acid sequence selected from Table 26. In some embodiments, the guide polynucleotide comprises a sequence of a nucleic acid selected from AAGAAGAG or CAUACCAGCUGAGCCGUCCG In some embodiments, the fusion protein comprises one or more uracil glycosylators. The protein further comprises an enzyme inhibitor (UGI) and / or one or more nuclear localization sequences (NLS). In some embodiments, the napDNAbp is selected from the group consisting of Cas9, Cas12a / Cpfl, Cas 12b / C2cl, Cas12c / C2c3, Cas12d / CasY, Cas12e / CasX, Cas12g, Cas12h, Cas12i, or Cas12j / Cas In some embodiments, the napDNAbp comprises a Ca polypeptide or a portion thereof. In some embodiments, the napDNAbp comprises an s12 polypeptide or a fragment thereof. In some embodiments, the Cas9 comprises an inactive Cas9 polypeptide or a fragment thereof. dead Cas9 (dCas9) or Cas9 nickase (nCas9) In some embodiments, Cas9 is a modified Staphylococcus aureus Cas9(SaCas9), Streptococcus thermophilus 1 Cas9 (St1Cas9), or modified Streptococcus pyoge In some embodiments, the Cas9 is a modified promoter. In some embodiments, the modified PAM comprises a spacer adjacent motif (PAM) specificity. , which has specificity for the nucleic acid sequence 5'-NGC-3'.

[0014] In some embodiments, the deaminase domain deaminates cytidine or adenosine. In some embodiments, the deaminase domain can be substituted with cytidine or amide. In some embodiments, the cytidine deaminase is a cytidine deaminase domain. In some embodiments, the adenosine deaminase is a T In some embodiments, the TadA variant is a TadA*8 variant. In some embodiments, the TadA*8 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*8.1 0, TadA*8.11, TadA*8.12, TadA*8.13, TadA*8.1 4, TadA*8.15, TadA*8.16, TadA*8.17, TadA*8.1 8, TadA*8.19, TadA*8.20, TadA*8.21, TadA*8.2 2, TadA*8.23, or TadA*8.24. The dA variant is the TadA*9 variant.

[0015] In one aspect, the invention includes any of the base editor systems provided herein. The present invention provides a pharmaceutical composition comprising:

[0016] In another aspect, the invention relates to the base editor systems and guides provided herein. A polynucleotide encoding any of the polynucleotides is provided.

[0017] In yet another aspect, the present invention provides a method for the preparation of any of the polynucleotides provided herein. In some embodiments, the vector is a viral vector. In some embodiments, the viral vector is a retroviral vector, an adenoviral vector, or a vector, lentiviral vector, herpesvirus vector, or adeno-associated vector It is a viral vector (AAV).

[0018] In one aspect, the present invention provides a method for the preparation of a polynucleotide or vector as provided herein. The present invention provides a pharmaceutical composition comprising any one of the following:

[0019] In one aspect, the present invention provides a method for producing CAR-expressing immune cells with reduced immunogenicity. In some embodiments, the method comprises: Contains a grammable DNA-binding protein (napDNAbp) and a deaminase domain A fusion protein containing a polynucleotide encoding a different antigen was used to target two different antigens. and a guide polynucleotide, the antigen is selected from the group consisting of CD5, CD7, CD3, CD33, and CD123; This produces CAR-expressing immune cells with reduced immunogenicity. In this form, immune cells express or are in contact with the guide polynucleotide and are then transduced into the guide. The target polynucleotides are TRAC, LAG-3, FAS, CIITA, TRBC1, and T A polypeptide selected from the group consisting of RBC2, CD52, B2M, and PD1 is included. The target polynucleotide is the one that is read.

[0020] In some embodiments, the method comprises (a) administering to a CAR-expressing immune cell a nucleic acid programmable DNA fragment. Fusion protein containing NA-binding protein (napDNAbp) and deaminase domain and (b) expressing a base editor system comprising a CAR-expressing immune cell. At least two guide polypeptides each targeting a polynucleotide encoding a different antigen are included. and contacting the antigen with a nucleotide, wherein the antigen is selected from the group consisting of CD5, CD7, CD3, CD3 3, and CD123, thereby having reduced immunogenicity. The resulting CAR-expressing immune cells are then used to treat the disease.

[0021] In some embodiments, the method further comprises the step of determining whether the CAR-expressing immune cells are TRAC, LAG-3, FA, or other CAR-expressing immune cells. A group consisting of S, CIITA, TRBC1, TRBC2, CD52, B2M, and PD1 A guide polynucleotide targeting a polynucleotide encoding a polypeptide selected from In some embodiments, the immune cells further comprise contacting the immune cells with a CD5, C Targeting an antigen selected from the group consisting of D7, CD3, CD33, and CD123 In some embodiments, the method comprises administering to the immune cells: TRAC, LAG-3, FAS, CIITA, TRBC1, TRBC2, CD52, B2 M, and PD1. Mutations that reduce or eliminate the immunogenicity of CAR-expressing antibodies are introduced, thereby producing CAR-expressing antibodies with reduced immunogenicity. The method further comprises producing a population of immune cells.

[0022] In one aspect, the present invention provides a method for producing a population of CAR-expressing immune cells with reduced immunogenicity. In some embodiments, the method includes: a) injecting a single immune cell with C Expression of an antigen selected from the group consisting of D5, CD7, CD3, CD33, and CD123. A mutation is introduced that reduces or eliminates expression of one of the antigens, and a second immune cell is then injected with a different one of the antigens. and b) introducing mutations that induce TRAC, LAG-3, FAS, and CIITA in immune cells. , TRBC1, TRBC2, CD52, B2M, and PD1. introducing a mutation that reduces or eliminates expression of at least one polypeptide, thereby and producing a population of CAR-expressing immune cells with reduced immunogenicity. In some embodiments, the chimeric antigen receptor expressed by the immune cell is selected from the group consisting of CD3, CD5, Targets an antigen selected from the group consisting of CD7, CD33, and CD123. In this embodiment, the immune cells produced by the method are CD3, CD5, and / or targets the CD7 antigen and fails to express CD3, CD5, and / or CD7 antigens or expressing reduced levels of CD3, CD5, and / or CD7 antigens. In some embodiments, the immune cells produced by the method express the mel antigen receptor. , targeting CD33 and CD123 antigens and capable of expressing CD33 and CD123 antigens. Chimeric antigen recipients expressing absent or reduced levels of CD33 and CD123 antigens. In some embodiments, the CAR is a CD5 chimeric antigen receptor (CAR). In some embodiments, the CD5 CAR is a CD5 CAR construct presented in Table 28. It is coded by things.

[0023] In another aspect, the present invention provides a method for producing immune cells with reduced immunogenicity. In some embodiments, the methods include: a) reducing or eliminating expression of CD5; b) introducing mutations into the gene sequence or regulatory elements of the endogenous CD5 gene; and and expressing a CD5 CAR construct as presented in Table 28. In one embodiment, the CD5 CAR construct comprises or comprises an amino acid sequence selected from the following: Encoding a CD5 CAR polypeptide consisting of: a) 1 MEFGLSWLFLVAILKGVQCIDAMGNIQLVQSGPELKKPGETVKISCKASGYTFTNYGMNW 60 61 VKQAPGKGLRWMGWINTHTGEPTYADDFKGRFAFSLETSASTAYLQINNLKNEDTATYFC 120 121 TRRGYDWYFDVWGAGTTVTVSSGGGGSGGGGSGGGGSDIKMTQSPSSMYASLGERVTITC 180 181 KASQDINSYLSWFHHKPGKSPKTLIYRANRLVDGVPSRFSGSGSGQDYSLTISSLDYEDM 240 241 GIYYCQQYDESPWTFGGGTKLEMKGSGDPAEPKSPDKTHTCPGQPREPQVYTLPPSRDEL 300 301 TKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQ 360 361 QGNVFSCSVMHEALHNHYTQKSLSLSPGKKDPKFWVLVVVGGVLACYSLLVTVAFIIFWV 420 421 RSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSRVKFSRSADAPAYQQGQNQ 480 481 LYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGE 540 541 RRRGKGHDGLYQGLSTATKDTYDALHMQALPPR 573; b) 1 MEFGLSWLFLVAILKGVQCIDAMGNIQLVQSGPELKKPGETVKISCKASGYTFTNYGMNW 60 61 VKQAPGKGLRWMGWINTHTGEPTYADDFKGRFAFSLETSASTAYLQINNLKNEDTATYFC 120 121 TRRGYDWYFDVWGAGTTVTVSSGGGGSGGGGSGGGGSDIKMTQSPSSMYASLGERVTITC 180 181 KASQDINSYLSWFHHKPGKSPKTLIYRANRLVDGVPSRFSGSGSGQDYSLTISSLDYEDM 240 241 GIYYCQQYDESPWTFGGGTKLEMKGSGDPATTTPAPRPPTPAPTIASQPLSLRPEACRPA 300 301 AGGAVHTRGLDFACDFWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPR 360 361 RPGPTRKHYQPYAPPRDFAAYRSRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDK 420 421 RRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTAT 480 481 KDTYDALHMQALPPR 495; c) 1 MEFGLSWLFLVAILKGVQCIDAMGNIQLVQSGPELKKPGETVKISCKASGYTFTNYGMNW 60 61 VKQAPGKGLRWMGWINTHTGEPTYADDFKGRFAFSLETSASTAYLQINNLKNEDTATYFC 120 121 TRRGYDWYFDVWGAGTTVTVSSGGGGSGGGGSGGGGSDIKMTQSPSSMYASLGERVTITC 180 181 KASQDINSYLSWFHHKPGKSPKTLIYRANRLVDGVPSRFSGSGSGQDYSLTISSLDYEDM 240 241 GIYYCQQYDESPWTFGGGTKLEMKGSGDPAIEVMYPPPYLDNEKSNGTIIHVKGKHLCPS 300 301 PLFPGPSKPFWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTR 360 361 KHYQPYAPPRDFAAYRSRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDP 420 421 EMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDA 480 481 LHMQALPPR 489; d) 1 MEFGLSWLFLVAILKGVQCIDAMGNIQLVQSGPELKKPGETVKISCKASGYTFTNYGMNW 60 61 VKQAPGKGLRWMGWINTHTGEPTYADDFKGRFAFSLETSASTAYLQINNLKNEDTATYFC 120 121 TRRGYDWYFDVWGAGTTVTVSSGGGGSGGGGSGGGGSDIKMTQSPSSMYASLGERVTITC 180 181 KASQDINSYLSWFHHKPGKSPKTLIYRANRLVDGVPSRFSGSGSGQDYSLTISSLDYEDM 240 241 GIYYCQQYDESPWTFGGGTKLEMKGSGDPAEPKSPDKTHTCPGQPREPQVYTLPPSRDEL 300 301 TKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQ 360 361 QGNVFSCSVMHEALHNHYTQKSLSLSPGKKDPKPTTTPAPRPPTPAPTIASQPLSLRPEA 420 421 CRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMR 480 481 PVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVL 540 541 DKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLST 600 601 ATKDTYDALHMQALPPR 617; or e) 1 MEFGLSWLFLVAILKGVQCIDAMGNIQLVQSGPELKKPGETVKISCKASGYTFTNYGMNW 60 61 VKQAPGKGLRWMGWINTHTGEPTYADDFKGRFAFSLETSASTAYLQINNLKNEDTATYFC 120 121 TRRGYDWYFDVWGAGTTVTVSSGGGGSGGGGSGGGSDIKMTQSPSSMYASLGERVTITC 180 181 KASQDINSYLSWFHHKPGKSPKTLIYRANRLVDGVPSRFSGSGSGQDYSLTISSLDYEDM 240 241 GIYYCQQYDESPWTFGGGTKLEMKGSGDPATTTPAPRPPTPAPTIASQPLSLRPEACRPA 300 301 AGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQT 360 361 TQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRR 420 421 DPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKD 480 481 DALHMQALPPRX 494.

[0024] In some embodiments, the immune cells produced by the method are compared to corresponding control cells. and exhibiting fratricide resistance and / or increased anti-tumor activity. In some embodiments, the method is performed in vivo or ex vivo. The immune cells thus produced do not contain detectable translocations. In some embodiments, the immune cells produced by this method contain less than 1% indels. Immune cells produced by the method contain less than 5% non-targeted editing. In some embodiments, Immune cells produced by this method contain less than 5% off-target editing. In embodiments, the mutation is generated by a nucleobase modification. In some embodiments, the mutation is in an exon that reduces or eliminates protein expression. In some embodiments, the mutation results in a splice donor site or splice codon. In some embodiments, the one or more mutations are in the price acceptor site of the target polynucleotide. The nucleotides are then transferred to a nucleic acid programmable DNA binding protein (napDNAbp), deamidation a base editor containing a fusion protein comprising an enzyme and one or more guide polynucleotides; It is produced by contacting the compound with a filter system.

[0025] In some embodiments, the deaminase is an adenosine deaminase or a cytidine deaminase. In some embodiments, the cytidine deaminase is BE4. In embodiments, the mutation increases the expression of the encoded polypeptide compared to a corresponding control cell lacking the mutation. In some embodiments, the expression of the guide polynucleotide is reduced by at least about 50%. The guide comprises a sequence selected from those provided in Table 26. The polynucleotide is AGCGACUGCAGAAAGAAGAG or CAUACC In some embodiments, the nucleic acid sequence is selected from the group consisting of: AGCUGAGCCGUCCG; Each of the one or more guide nucleic acid sequences is selected from the group consisting of CD5, FAS, LAG-3, CD52, TRAC, B2M, CIITA, TRBC1, TRBC2, and / or PDC1 / PD-1 genetic In some embodiments, the base editor and the target gene are a nucleic acid sequence that targets a target gene or a regulatory element. One or more guide nucleic acid sequences can be introduced by electroporation, nucleofection, cationic Transduced into immune cells via lipid-mediated methods, viral transduction, or a combination thereof. In some embodiments, the method includes expanding immune cells in culture to induce proliferation of immune cells. In some embodiments, the expression of the antigen or polypeptide further comprises generating a population. is reduced in at least about 50% of the population of immune cells. The method further includes depleting TCRα / β+ cells from the population of modified immune cells.

[0026] In one aspect, the present invention provides a method for producing a compound according to any of the methods provided herein. In addition, CAR-expressing immune cells with reduced immunogenicity are provided.

[0027] In another aspect, the present invention provides a pharmaceutical composition comprising any of the immune cells provided herein. In some embodiments, the pharmaceutical composition comprises a pharmaceutically acceptable excipient. Includes.

[0028] In yet another aspect, the present invention provides a method for killing tumor cells. In embodiments, the method includes screening for CD5, CD7, CD3, CD33, and CD123. The method comprises contacting tumor cells expressing an antigen selected from the group consisting of: Each immune cell comprises a different targeting two of the antigens expressed by the cell. The chimeric antigen receptor (CAR) is expressed by an immune cell, which is capable of expressing one or more CARs that reduce or eliminate expression of the target antigen. Mutations in TRAC, LAG-3, FAS, CIITA, TRBC1, and TRBC2 , reducing the expression of a polypeptide selected from the group consisting of CD52, B2M, and PD1 In some embodiments, the method comprises one or more mutations that result in or eliminate a mutation in an in vitro or is performed in vivo. In some embodiments, the tumor cells are derived from a tumor.

[0029] In one aspect, the present invention provides a method for treating a tumor in a subject. In embodiments, the method includes administering to a subject two or more immune cells, each of the immune cells is a target protein selected from the group consisting of CD5, CD7, CD3, CD33, and CD123 The mice express different chimeric antigen receptors that target antigens expressed by tumor cells in the mice, and the immune system The immune cells contain one or more mutations that reduce or eliminate expression of the antigen, and each of the immune cells TRAC, LAG-3, FAS, CIITA, TRBC1, TRBC2, CD52, B2 1. Reduce or eliminate the expression of a polypeptide selected from the group consisting of PD1, PD2, and PD3. and further comprising one or more mutations.

[0030] In some embodiments, the tumor is a blood cancer. In some embodiments, the tumor is a blood cancer. In some embodiments, the hematological cancer is leukemia, myeloma, and / or lymphoma. In some embodiments, the hematological cancer is a B-cell cancer. The liquid cancer is selected from at least one of the following: T-cell acute lymphoblastic leukemia (T-ALL), mycosis fungoides (MF), Sézary syndrome (SS), peripheral T / NK cell remodeling. Lymphoma, anaplastic large cell lymphoma ALK +, primary cutaneous T-cell lymphoma, T-cell large granular lymphoma pacytic leukemia, angioimmunoblastic T / NK cell lymphoma, hepatosplenic T cell lymphoma, primary cutaneous CD30 + Lymphoproliferative disorders, extranodal NK / T-cell lymphoma, adult T-cell leukemia / lymphoma tumor, T-cell prolymphocytic leukemia, subcutaneous panniculitis-like T-cell lymphoma, primary cutaneous γδ T-cell lymphoma, aggressive NK-cell leukemia, and enteropathy-associated T-cell lymphoma. In some cases, the blood cancer is T-cell acute lymphoblastic leukemia (T-ALL). In embodiments, the hematological cancer is acute myeloid leukemia (AML).

[0031] In another aspect, the present invention provides a method for treating a tumor in a selected subject. In some embodiments, the method comprises administering to a selected subject two or more immune cells. each immune cell targets a different antigen expressed by the tumor cells of the subject. It expresses chimeric antigen receptors (CARs) and recognizes CD5, CD7, CD3, CD33, and CD1. 23, and the immune cells are selected from the group consisting of one or more Each of the immune cells contains a mutation in TRAC, LAG-3, FAS, CIITA, and TRBC. 1, TRBC2, CD52, B2M, and PD1. The subject further comprises one or more mutations that reduce or eliminate expression of CD5, CD7, A subject with a tumor expressing an antigen selected from the group consisting of CD3, CD33, and CD123. In some embodiments, the hematological cancer is selected from T-cell acute lymphoblastic In some embodiments, the blood cancer is acute myeloid leukemia (T-ALL). In some embodiments, two or more chimeric antigen receptors expressing different chimeric antigen receptors (AML) are The immune cells are administered sequentially. In some embodiments, the immune cells express different chimeric antigen receptors. The two or more immune cells are administered simultaneously.

[0032] In yet another aspect, the present invention provides a method for antigen-dependent killing of tumor cells in a subject. In some embodiments, the method provides a method for detecting CD5, CD7, CD3, CD33, and and CD123. and administering immune cells, each immune cell targeting a different antigen. The immune cells express chimeric antigen receptors that reduce or eliminate expression of the target antigen. Mutations in the above, as well as TRAC, LAG-3, FAS, CIITA, TRBC1, and TRBC 2. Reducing the expression of a polypeptide selected from the group consisting of CD52, B2M, and PD1. Contains one or more mutations that reduce or eliminate

[0033] In one aspect, the present invention provides a method for the antigen-dependent death of acute myeloid leukemia (AML) cells in a subject. In some embodiments, the method comprises the step of: administering two or more immune cells to a subject with AML that expresses an antigen, The immune cells express different chimeric antigen receptors that target one of the antigens, and The cells contain one or more mutations that reduce or eliminate expression of the target antigen, as well as TRAC, LAG, and -3, FAS, CIITA, TRBC1, TRBC2, CD52, B2M, and PD1 and one or more mutations that reduce or eliminate expression of a polypeptide selected from the group consisting of: nothing.

[0034] In one aspect, the present invention provides a method for treating T-cell acute lymphoblastic leukemia (T-ALL) cells in a subject. In some embodiments, the method comprises administering to a subject a CD3, Subjects with T-ALL expressing CD5 and CD7 antigens received at least three immunizations. The method comprises administering immune cells to a subject, each immune cell expressing a different antigen that targets one of the antigens. Immune cells express one or more antigen receptors that reduce or eliminate expression of the target antigen. Mutations, as well as TRAC, LAG-3, FAS, CIITA, TRBC1, TRBC2, reducing or decreasing the expression of a polypeptide selected from the group consisting of CD52, B2M, and PD1 It contains one or more mutations that enhance or eliminate the

[0035] In another aspect, the present invention provides a method for treating cancer in a selected subject. In some embodiments, the method includes administering to the subject at least two immune cells. Each immune cell contains a chimeric antigen receptor that targets either the CD33 or CD123 antigen. and the immune cells express one or more mutations that reduce or eliminate expression of the target antigen, as well as T RAC, LAG-3, FAS, CIITA, TRBC1, TRBC2, CD52, B2M and one that reduces or eliminates expression of a polypeptide selected from the group consisting of PD1. These mutations are included, and the cancer is considered to express CD33 and CD123 antigens. In some embodiments, the target antigen is selected by characterizing it as CD33 and CD1. Cancers expressing 23 antigens are AML.

[0036] In yet another aspect, the present invention provides a method for treating cancer in a selected subject. In some embodiments, the method includes administering to the subject three or more immune cells. Each immune cell contains a different chimeric antibody targeting the CD3, CD5, and CD7 antigens. The immune cells express the target antigen receptor and harbor one or more mutations that reduce or eliminate expression of the target antigen. as well as TRAC, LAG-3, FAS, CIITA, TRBC1, TRBC2, and CD5 and reducing or eliminating the expression of a polypeptide selected from the group consisting of PD1, B2M, B2C1, and PD2. and the subject has a cancer that contains one or more mutations that inhibit the expression of CD3, CD5, and CD7 antigens. In some embodiments, the CD3 Cancers that express CD1, CD5, and CD7 antigens are T-ALL.

[0037] In some embodiments, the immune cells are cytotoxic T cells, regulatory T cells, T helper cells, In some embodiments, the subject has previously received lymphocytes, dendritic cells, B cells, or NK cells. In some embodiments, lymphodepletion can be treated with cyclophosphamide. amide, fludarabine, and / or alemtuzumab (Cy / Flu / Campath In some embodiments, the subject is refractory to chemotherapy or In some embodiments, the subject subsequently undergoes allogeneic hematopoietic stem cell transplantation ( In some embodiments, the immune cells are treated with a single human HSCT. In some embodiments, the immune cells are autologous to the subject. In some embodiments, the immune cells are allogeneic to the subject. In some embodiments, the subject is a human subject or a rodent subject. In some embodiments, the subject is a pediatric human subject.

[0038] In one aspect, the present invention provides a method for treating cancer comprising administering to a subject a composition comprising the composition provided herein. In another aspect, the present invention provides a kit comprising any of the above. A base provided herein for use in generating a CAR-expressing immune cell having In some embodiments, the present invention provides a kit comprising any of the editor systems described herein. Any of the kits provided to include written instructions for use of the kit. .

[0039] The description and examples herein particularly exemplify embodiments of the present disclosure. It is to be understood that the present invention is not limited to the particular embodiments described herein, as such may vary. Those skilled in the art will recognize that there are many variations and modifications of this disclosure that are encompassed within the scope of this disclosure. You will recognize it.

[0040] The practice of some embodiments disclosed herein requires, unless otherwise indicated, a skill in the art. Within the scope of technology, immunology, biochemistry, chemistry, molecular biology, microbiology, cell biology, genomics Conventional techniques of genetic engineering, genetic engineering, and recombinant DNA are used. nd Green,Molecular Cloning:A Laboratory Manual,4th Edition(2012), the series Curr ent Protocols in Molecular Biology(FMA usubel,et al.eds.);the series Methods In Enzymology (Academic Press, Inc.), PCR 2:A Practical Approach(MJMacPherson,BDH ames and GRTaylor eds.(1995)), Harlow a nd Lane, eds. (1988) Antibodies, A Laborator y Manual, and Culture of Animal Cells:A Ma nual of Basic Technique and Specialized Applications,6th Edition(RIFreshney,ed (2010)).

[0041] Various features of the disclosure may be described in the context of a single embodiment, but the features may also be used in conjunction with other embodiments. The present disclosure may be modified in various ways, for the sake of clarity, by providing the following: Although described herein in the context of separate embodiments, the present disclosure also applies to a single embodiment. The section headings used herein are for organizational purposes only. These terms are used for illustrative purposes only and should not be construed as limiting the subject matter described.

[0042] The features of the present disclosure are set forth with particularity in the appended claims. By reference to the following detailed description which describes exemplary embodiments, and by reference to the following detailed description, A better understanding of the features and advantages of the present invention may be obtained by considering the accompanying drawings, which are set forth in: It should be possible.

[0043] definition The following definitions supplement those in the art and are intended for the purposes of this application: Attributable to any related or unrelated cases (e.g., any co-owned patents or applications) Any methods and methods similar or equivalent to those described herein are not intended to be limiting. Although various materials and methods can be used to carry out the tests of the present disclosure, preferred materials and methods are Accordingly, the terminology used herein refers to specific embodiments. For illustrative purposes only and not intended to be limiting.

[0044] Unless otherwise defined, all technical and scientific terms used herein are defined by the It has the meaning commonly understood by a person skilled in the art to which the invention pertains. , provides those skilled in the art with general definitions of many of the terms used in this invention: Singlet on et al.,Dictionary of Microbiology and Molecular Biology (2nd ed.1994), The Camb ridge Dictionary of Science and Technology gy (Walker ed., 1988), The Glossary of Gene tics,5th Ed.,R.Rieger et al.(eds.),Sprin ger Verlag (1991), and Hale & Marham, The Harp er Collins Dictionary of Biology (1991).

[0045] In this application, the use of the singular includes the plural unless specifically stated otherwise. where the terms "a," "an," and "the" are used in the singular, unless the context clearly indicates otherwise. Please note that plural referents are included unless otherwise indicated. The use of "or" means "and / or" and is inclusive unless otherwise stated. It is understood that the terms "including" and " "include," "includes," and "inc The use of other forms such as "unintelligible" is not limiting.

[0046] As used in this specification and claims, "comprising" (As well as "comprise" and "comprises" etc.) any form of it), "having" (as well as "have" and any form thereof, such as "has"), "including" (As well as words such as "includes" and "include" any form thereof), or "containing" (as well as "including" the word "contains" and any of its forms such as "contain" is inclusive or open-ended and may contain additional unenumerated elements or method steps. Any embodiment discussed herein does not exclude any method or composition of the present disclosure. It is contemplated that the same may be practiced with respect to compositions of the present disclosure, and vice versa. The articles can be used to achieve the methods of the present disclosure.

[0047] The terms "about" or "approximately" refer to an acceptable range for a particular value as determined by one of ordinary skill in the art. This means that the value is within a certain tolerance, which depends in part on how it is measured or measured. is determined (i.e., depends on the limitations of the measurement system). For example, "about" As is customary in the art, this means within 1 standard deviation or within more than 1 standard deviation. Alternatively, "about" can mean up to 20%, up to 10%, up to 5%, or up to or, particularly with respect to biological systems or processes, The term can mean within an order of magnitude, such as within 5-fold or within 2-fold of a value. Where specific values ​​are recited in the application and claims, unless otherwise stated, the specific values This means that the value is within the tolerance range for a given value.

[0048] It is understood that ranges provided herein are shorthand for all values ​​within the range. For example, the range 1 to 50 is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 1 3, 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, The group consisting of 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 It is understood that the range includes any number, combination of numbers, or subrange from

[0049] In this specification, the terms "some embodiments," "embodiments," "one embodiment," or "other embodiments" may be used. References to an "embodiment" mean that a particular feature, structure, or characteristic described in connection with an embodiment Included in at least some, but not necessarily all, embodiments of the present disclosure This means that it is not.

[0050] "Adenosine deaminase" refers to the hydrolytic deamination of adenine or adenosine. In some embodiments, the term "protein" refers to a polypeptide or fragment thereof that is capable of catalyzing the synthesis of a protein. The deaminase or deaminase domain is an adenosine deaminase, hydrolytic deamination of adenosine to inosine, or deoxyadenosine to deoxyinosine In some embodiments, adenosine deamination catalyzes the hydrolytic deamination of adenosine deamination to adenosine deamination. The enzyme hydrolytically deacetylates adenine or adenosine in deoxyribonucleic acid (DNA). The adenosine deaminases provided herein (e.g., engineered Adenosine deaminase (adenosine deaminase, evolved adenosine deaminase) is a nucleotide of any organism, such as bacteria. It may be of origin.

[0051] In some embodiments, the deaminase or deaminase domain is selected from the group consisting of human, chimpanzee, and the like. Naturally occurring proteins from organisms such as gorilla, monkey, cow, dog, rat, or mouse In some embodiments, the deaminase or deaminase The deaminase domain is not naturally occurring. For example, in some embodiments, The aminase domain has at least 50% similarity to a naturally occurring deaminase, 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 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 less In some embodiments, the adenosine deaminase is at least 99.9% identical to the adenosine deaminase of a bacterial (e.g., E. coli, S. aureus, B. subtilis, S. typhi, S.putrefaciens, H.influenzae, C.crescentus In some embodiments, the adenovirus is derived from G. In some embodiments, the syndeaminase is TadA deaminase. The deaminase is E. coli TadA (ecTadA) deaminase or a fragment thereof. do.

[0052] In some embodiments, the ecTadA deaminase is a truncated ecTadA. For example, truncated ecTadA lacks one or more N-terminal amino acids compared to full-length ecTadA. In some embodiments, the truncated ecTadA may have a length of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 3 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 6, 17, 1 The 8, 19, or 20 N-terminal amino acid residues may be deleted. Type ecTadA has 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10 sequences relative to the full-length ecTadA. 10, 11, 12, 13, 14, 15, 6, 17, 18, 19, or 20 C-terminal amino acids In some embodiments, the ecTadA deaminase may lack an N-terminal amino acid residue. In some embodiments, the TadA deaminase is an N-terminal truncated TadA deaminase. In certain embodiments, TadA is a TadA polypeptide as described in PCT / US2017 / 045381. and any one of the TadAs described in (the entirety of which is incorporated herein by reference). (Incorporated herein).

[0053] In some embodiments, the TadA deaminase is a TadA variant. In some embodiments, the TadA variant is TadA*7.10. The TadA variant is TadA*8. In some embodiments, TadA*8 is adA*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. In the embodiment, TadA*8 is TadA*8a, TadA*8b, TadA*8c, Ta In some embodiments, TadA*8 is TadA*8. In some embodiments, the TadA variant is TadA*9.

[0054] "Adenosine deaminase base editor 8 (ABE8) polypeptide" or "AB "E8" refers to an adenovirus containing modifications at amino acid positions 82 and / or 166 of the following reference sequence: "base editor" refers to a base editor as defined herein, including variants of ribozyme deaminase. MSEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNN RVIGEGWNRAIGLHDPTAHAEIMALRQGGLVMQNYRLIDA TLYVTFEPCVMCAGAMIHSRIGRVVFGVRNAKTGAAGSLM DVLHYPGMNHRVEITEGILADECAALLCYFFRMPRQVFNA QKKAQSSTD

[0055] In some embodiments, ABE8 is a nucleotide sequence selected from the group consisting of nucleotides ... , including further modifications.

[0056] "Adenosine deaminase base editor 8 (ABE8) polynucleotide" means a It refers to a polynucleotide encoding BE8.

[0057] "Adenosine deaminase base editor 9 (ABE9) polypeptide" or "AB "E9" means a base editor, as defined herein, that has one or more of the following modifications: Variants of adenosine deaminase including: R21N in the following reference sequences: , R23H, E25F, N38G, L51W, P54C, M70V, Q71M, N72K , Y73S, V82T, M94V, P124W, T133K, D139L, D139M, C146R, and A158K. JPEG2025066723000001.jpg57161

[0058] The relevant bases that are modified in the reference sequence are underlined and in bold. ABE9 contains additional changes compared to the reference sequence, as described herein. For more information about the ABE9 base editor, see International PCT Application No. PCT / 2020 / 049975 (which is incorporated herein by reference in its entirety).

[0059] "Adenosine deaminase base editor 9 (ABE9) polynucleotide" means a It refers to a polynucleotide encoding BE9.

[0060] "Administering" as used herein refers to administering one or more of the compositions described herein to a patient or or providing to a subject. Examples include, but are not limited to, administering a composition (e.g., injection) injections) are intravenous (iv) injections, subcutaneous (sc) injections, intradermal (id) injections, and intraperitoneal injections. It can be administered by intravenous (ip) or intramuscular (im) injection. Any of these routes can be used. Parenteral administration can be by, for example, bolus injection or In some embodiments, this can be achieved by parenteral perfusion. Oral administration can be intravascular, intravenous, intramuscular, intraarterial, intrathecal, intratumoral, intradermal, intraperitoneal, transtracheal, Subcutaneous, subcuticular, intra-articular, subcapsular, subarachnoid, and intrasternal Alternatively, or concurrently, administration may be by oral route. can.

[0061] "Drug" means any small molecule chemical compound, antibody, nucleic acid molecule, or polypeptide, or or fragments thereof.

[0062] "Alteration" refers to any method known in the art, such as those described herein, that can be used to modify the Changes in the structure, expression level, or activity of a gene or polypeptide detected by As used herein, a change (e.g., an increase or decrease) refers to a change (e.g., an increase or decrease). The addition or decrease of a polynucleotide or polypeptide sequence is a change in the expression Level change (e.g., 10% change, 25% change, 40% change, and 50% change) This includes changes in the structure (or even further changes).

[0063] "Allogeneic," as used herein, refers to cells of the same species that are genetically different compared to cells Refers to...

[0064] "Ameliorate" means to decrease, suppress, attenuate, reduce, arrest, or stabilize the onset or progression of a disease. This means to determine

[0065] An "analog" is a molecule that is not identical but has similar functional or structural characteristics. For example, an analog of a polynucleotide or polypeptide is an analog of the corresponding naturally occurring The polynucleotide or polypeptide of interest may be a polynucleotide or polypeptide that is not naturally occurring, while retaining the biological activity of the polynucleotide or polypeptide. A specific polynucleotide or polypeptide that enhances the function of the analog compared to the existing polynucleotide or polypeptide. Such modifications can, for example, alter the D of the analog without altering ligand binding. Affinity for NA, efficiency, specificity, protease or nuclease resistance, membrane permeability The analogs can be non-natural nucleotides, which can increase transient and / or half-life. or may contain unnatural amino acids.

[0066] "Anti-tumor activity" means preventing or inhibiting tumor maturation and / or growth. do.

[0067] As used herein, "autologous" refers to cells derived from the same subject.

[0068] As used herein, the term "antibody" refers to an antibody that specifically binds to a particular antigen. or the immunoglobulin molecules that react immunologically with it, and the polyclonal form of antibodies including recombinant, monoclonal, genetically engineered, and otherwise modified forms. Examples of antibodies include, but are not limited to, chimeric antibodies, humanized antibodies, heteroconjugate antibodies (e.g., bispecific antibodies), and the like. , tria- and tetraspecific antibodies, diabodies, triabodies, and tetrabodies) , and antigen-binding fragments of antibodies (e.g., Fab', F(ab')2, Fab, Fv, rl Unless otherwise indicated, "monoclonal" refers to antibodies that are specifically targeted to a particular target cell type (including human IgG, human IgG, and scFv fragments). The term "mAb" (monomeric antibody) refers to an antibody capable of specifically binding to a target protein. Both intact molecules and antibody fragments (including, for example, Fab and F(ab')2 fragments) As used herein, Fab and F(ab')2 fragments are meant to include: "antibody fragments" refers to antibody fragments that lack the Fc fragment of an intact antibody. Examples of these antibody fragments are described herein. is described in.

[0069] "B cell maturation antigen or tumor necrosis factor receptor superfamily member 17 polypeptide" "BCMA" is a gene expressed in mature B lymphocytes. NCBI accession number NP_0011 83 or a fragment thereof. Exemplary BCMA polypeptide sequences are provided below.

[0070] >NP_001183.2 Tumor necrosis factor receptor superfamily member 17 [H omo sapiens]

[0071] MLQMAGQCSQNEYFDSLLHACIPCQLRCSSNTPPLTCQR YCNASVTNSVKGTNAILWTCLGLSLIISLAVFVLMFLLRK INSEPLKDEFKNTGSGLLGMANIDLEKSRTGDEIILPRGL EYTVEECTCEDCIKSKPKVDSDHCFPLPAMEEGATILVTT KTNDYCKSLPAALSATEIEKSISAR

[0072] This antigen is targeted in the treatment of relapsed or refractory multiple myeloma and other hematological malignancies. It can be said that.

[0073] "B-cell maturation antigen or tumor necrosis factor receptor superfamily member 17 (BCM) A) "Polynucleotide" means a nucleic acid molecule that encodes a BCMA polypeptide. The BCMA gene encodes a cell surface receptor that recognizes B cell activating factors. The B2M polynucleotide sequence is provided below. >NM_001192.2 Homo sapiens TNF receptor superfamily - Member 17 (TNFRSF17), mRNA AAGACTCAAACTTAGAAACTTGAATTAGATGTGGTATTCA AATCCTTAGCTGCCGCGAAGACACAGACAGCCCCGTAAG AACCCACGAAGCAGGCGAAGTTCATTGTTCTCAACATTCT AGCTGCTCTTGCTGCATTTGCTCTGGAATTCTTGTAGAGA TATTACTTGTCCTTCCAGGCTGTTCTTTCTGTAGCTCCCT TGTTTTCTTTTTGTGATCATGTTGCAGATGGCTGGGCAGT GCTCCCAAAATGAATATTTTGACAGTTTGTTGCATGCTTG CATACCTTGTCAACTTCGATGTTCTTCTAATACTCCTCCT CTAACATGTCAGCGTTATTGTAATGCAAGTGTGACCAATT CAGTGAAAGGAACGAATGCGATTCTCTGGACCTGTTTGGG ACTGAGCTTAATAATTTCTTTGGCAGTTTTCGTGCTAATG TTTTTGCTAAGGAAGATAAACTCTGAACCATTAAAGGACG AGTTTAAAAACACAGGATCAGGTCTCCTGGGCATGGCTAA CATTGACCTGGAAAAGAGCAGGACTGGTGATGAAATTATT CTTCCGAGAGGCCTCGAGTACACGGTGGAAGAATGCACCT GTGAAGACTGCATCAAGAGCAAACCGAAGGTCGACTCTGA CCATTGCTTTCCACTCCCAGCTATGGAGGAAGGCGCAACC ATTCTTGTCACCACGAAAACGAATGACTATTGCAAGAGCC TGCCAGCTGCTTTGAGTGCTACGGAGATAGAGAAATCAAT TTCTGCTAGGTAATTAACCATTTCGACTCGAGCAGTGCCA CTTTAAAAAATCTTTTGTCAGAATAGATGATGTGTCAGATC TCTTTAGGATGACTGTATTTTTCAGTTGCCGATACAGCTT TTTGTCCTCTAACTGTGGAAACTCTTTATGTTAGATATAT TTCTCTAGGTTACTGTTGGGAGCTTAATGGTAGAAACTTC CTTGGTTTCATGATTAAACTCTTTTTTTTCCTGA

[0074] A "base editor (BE)" or "nucleobase editor (NBE)" is a In one embodiment, the agent is a compound that binds to a nucleotide and has nucleobase modifying activity. Nucleic acid programmable DNA binding proteins are used to bind to polynucleotides with specific sequences. In another embodiment, the base editor binds to a sequence within a nucleic acid molecule (e.g., DNA). In some embodiments, the base editor is an enzyme capable of modifying a base. In some embodiments, the base editor can deaminate bases within a nucleic acid molecule. can deaminate bases within a DNA molecule. In some embodiments, the enzyme is capable of deaminating cytidines in DNA. The editors are fusion proteins containing cytidine deaminase or adenosine deaminase. In some embodiments, the base editor is a cytidine deaminase or an adenosine deaminase. In some embodiments, the Cas9 protein is fused to a base endaminase. Determination of the Cas9 fusion protein with cytidine deaminase or adenosine deaminase In some embodiments, the base editor is a base excision repair enzyme. In some embodiments, the fusion protein is fused to a second inhibitor (e.g., a UGI domain). The protein contains a Cas9 nickase fused to a deaminase and an inhibitor of base excision repair ( For example, the UGI domain).

[0075] In some embodiments, a cytidine deaminase or adenosine deaminase nucleobase The editor polypeptide contains the following domains: AB:NH2-[AB]-COOH In the formula, A represents a cytidine deaminase domain, an adenosine deaminase domain, or or an active fragment thereof, and B comprises one or more domains having nucleic acid sequence-specific binding activity. In one embodiment, the cytidine deaminase or adenosine deaminase of the previous aspect The nucleobase editor polypeptides of n -B o ]-COOH, wherein A is a cytidine deaminase domain, an adenosine deaminase domain, or n is an integer: 1, 2, 3, 4, or 5; and B is a nucleic acid sequence-specific The domain has a binding activity, and o is an integer of 1, 2, 3, 4, or 5. In one embodiment, the polypeptide comprises one or more nuclear localization sequences. The peptide comprises at least one of the nuclear localization sequences at the N-terminus or C-terminus. In one embodiment, the polypeptide comprises a nuclear localization signal and is bipartite. ) a nuclear localization signal. In one embodiment, the polypeptides are linked by a linker. Contains one or more domains that have been

[0076] In some embodiments, the base editor is a cytidine base editor (CBE). In some embodiments, the base editor is an adenosine base editor (ABE). In some embodiments, the base editors are adenosine base editors (ABEs) and sigma base editors (SBEs). In some embodiments, the base editor is an adduct base editor (CBE). It is a nuclease-inactive Cas9 (dCas9) fused to dCas9 deaminase. In some embodiments, the Cas9 is a circularly permuted Cas9 (e.g., spCas9 or Circularly permuted Cas9s are known in the art and are described, for example, in Oakes et al., Cell 176, 254-267, 2019 In some embodiments, the base editor is an inhibitor of base excision repair (e.g., UG In some embodiments, the fusion protein is fused to a nucleotide sequence (e.g., a nucleotide sequence of the ... The protein contains a Cas9 nickase fused to a deaminase and an inhibitor of base excision repair (e.g., In other embodiments, the base domain is a UGI domain or a dISN domain. Editor is an abasic base editor.

[0077] In some embodiments, the adenosine deaminase is evolved from TadA. In embodiments, the polynucleotide programmable DNA binding domain is a CRISPR-associated In some embodiments, the base editor is a nucleotide sequence encoding a nucleotide sequence that ... is a catalytically inactive Cas9 (dCas9) fused to a deaminase domain In some embodiments, the base editor is a Cas9 fused to a deaminase domain. In some embodiments, the base editor is a base excision repair enzyme. In some embodiments, the inhibitor of base excision repair (BER) is fused to an inhibitor of base excision repair. , uracil DNA glycosylase inhibitor (UGI). In some embodiments, the base The inhibitor of excision repair is an inosine base excision repair inhibitor.

[0078] In some embodiments, the base editor is a variant of adenosine deaminase (e.g., For example, TadA*7.10) was used in combination with a circularly permuted Cas9 (e.g., spCAS9) and In some embodiments, the fragment is generated by cloning into a scaffold containing a nuclear localization sequence. In this paper, base editors are used to identify variants of adenosine deaminase (e.g., TadA*8). ) with a circularly permuted Cas9 (e.g., spCAS9 or saCAS9) and a bipartite nucleus Generated by cloning into a scaffold containing a localization sequence (e.g., ABE8) Circularly permuted Cas9s are known in the art and are described, for example, in Oakes et al. l., Cell 176, 254-267, 2019.

[0079] In some embodiments, the polynucleotide programmable DNA binding domain comprises a CRI In some embodiments, the base residue is an SPR-related (e.g., Cas or Cpf1) enzyme. Deter is a catalytically inactive Cas9 (dCas9) fused to a deaminase domain. In some embodiments, the base editor is fused to a deaminase domain. In some embodiments, the base editor is a salt In some embodiments, the nucleotide sequence is fused to an inhibitor of base excision repair (BER). In some embodiments, the inhibitor is a uracil DNA glycosylase inhibitor (UGI). The inhibitor of base excision repair is an inosine base excision repair inhibitor.

[0080] For more information on base editors, see International PCT Application No. PCT / 2017 / 045381 (WO 2018 / 027078) and PCT / US2016 / 058344 (WO201 7 / 070632), each of which is incorporated herein by reference in its entirety. (This is incorporated by reference in Komor, AC, et al., "Programmable le editing of a target base in genomic D NA without double-stranded DNA cleavage” Nature 533,420-424(2016), Gaudelli,NM,e t al.,“Programmable base editing of A·T to G·C in genomic DNA without DNA cleava ge”Nature 551,464-471(2017), Komor,AC,e t al.,“Improved base excision repair inh ibition and bacteriophage Mu Gam protein yields C:G-to-T:A base editors with hig her efficiency and product purity”Science e Advances 3:eaao4774(2017), and Rees, H.A. ,et al.,“Base editing:precision chemistry y on the genome and transcriptome of liv ing cells.”Nat Rev Genet.2018 Dec;19(12) See also: 770-788.doi:10.1038 / s41576-018-0059-1 See, e.g., U.S. Pat. No. 6,229,493, the entire contents of which are incorporated herein by reference.

[0081] By way of example, base editor compositions, systems, and methods described herein may be used. The adenine base editor (ABE) is a nucleotide sequence (8877 base pairs) provided below. (Addgene, Watertown, MA; Gaudelli NM, e t al.,Nature.2017 Nov 23;551(7681):464-4 71.doi:10.1038 / nature24644;Koblan LW,et al., Nat Biotechnol.2018 Oct;36(9):843-84 6.doi:10.1038 / nbt.4172.) The nucleic acid sequence of ABE and at least 9 Polynucleotide sequences with 5% or more identity are also encompassed. ATATGCCAAGTACGCCCCCTATTGACGTCATGACGGTAA ATGGCCCGCCTGGCATTATGCCCAGTACATGACCTTATGG GACTTTCCTACTTGGCAGTACATCTACGTATTAGTCCATCG CTATTACCATGGTGATGCGGTTTTGGCAGTACATCAATGG GCGTGGATAGCGGTTTGACTCACGGGGATTTCCAAGTCTC CACCCCATTGACGTCAATGGGAGTTTGTTTTGGCACCAAA ATCAACGGGACTTTCCAAAATGTCGTAACAACTCCGCCCC ATTGACGCAAATGGGCGGTAGGCGTGTACGGTGGGAGGTC TATATAAGCAGAGCTGGTTTAGTGAACCGTCAGATCCGCT AGAGATCCGCGGCCGCTAATACGACTCACTATAGGGGAGAG CCGCCACCATGAAACGGACAGCCGACGGAAGCGAGTTCGA GTCACCAAAGAAGAAGCGGAAAAGTCTCTGAAGTCGAGTTT AGCCACGAGTATTGGATGAGGCACGCACTGACCCTGGCAA AGCGAGCATGGGATGAAAAGAGAAGTCCCCGTGGGCGCCGT GCTGGTGCACAACAATAGAGTGATCGGAGAGGGATGGAAC AGGCCAATCGGCCGCCACGACCTACCGCACACGCAGAGA TCATGGCACTGAGGCAGGGAGGCCTGGTCATGCAGAATTA CCGCCTGATCGATGCCACCCTGTATGTGACACTGGAGCCA TGCGTGATGTGCGCAGGAGCAATGATCCACAGCAGGATCG GAAGAGTGGTGTTCGGAGCACGGGACGCCAAGACCGGCGC AGCAGGCTCCCTGATGGATGTGCTGCACCACCCCGGCATG AACCACCGGGTGGAGATCACAGAGGGAATCCTGGCAGACG AGTGCGCCGCCCTGCTGAGCGATTTCTTTAGAATGCGGAG ACAGGAGATCAAGGCCCAGAAGAAGGCACAGAGCTCCACC GACTCTGGAGGATCTAGCGGAGGATCCTCTGGAAGCGAGA CACCAGGCACAAGCGAGTCCGCCACACCAGAGAGCTCCGG CGGCTCCTCCGGAGGATCCTCTGAGGTGGAGTTTTCCCAC GAGTACTGGATGAGACATGCCCTGACCCTGGCCAAGAGGG CACGCGATGAGAGGGAGGTGCCTGTGGGAGCCGTGCTGGT GCTGAACAATAGAGTGATCGGCGAGGGCTGGAACAGAGCC ATCGGCCTGCACGACCCAACAGCCCATGCCGAAATTATGG CCCTGAGACAGGGCGGCCTGGTCATGCAGAACTACAGACT GATTGACGCCACCCTGTACGTGACATTCGAGCCTTGCGTG ATGTGCGCCGGCGCCATGATCCACTCTAGGATCGGCCGCG TGGTGTTTGGCGTGAGGAACGCAAAAACCGGCGCCGCAGG CTCCCTGATGGACGTGCTGCACTACCCCGGCATGAATCAC CGCGTCGAAATTACCGAGGGAATCCTGGCAGATGAATGTG CCGCCCTGCTGTGCTATTTCTTTCGGATGCCTAGACAGGT GTTCAATGCTCAGAAGAAGGCCCAGAGCTCCACCGACTCC GGAGGATCTAGCGGAGGCTCCTCTGGCTCTGAGACACCTG GCACAAGCGAGAGCGCAACACCTGAAAGCAGCGGGGGCAG CAGCGGGGGGTCAGACAAGAAGTACAGCATCGGCCTGGCC ATCGGCACCAACTCTGTGGGCTGGGCCGTGATCACCGACG AGTACAAGGTGCCCAGCAAGAAATTCAAGGTGCTGGGCAA CACCGACCGGCACAGCATCAAGAAGAACCTGATCGGAGCC CTGCTGTTCGACAGCGGCGAAACAGCCGAGGCCACCCGGC TGAAGAGAACCGCCAGAAGAAGATACACCAGACGGAAGAA CCGGATCTGCTATCTGCAAGAGATCTTCAGCAACGAGATG GCCAAGGTGGACGACAGCTTCTTCCACAGACTGGAAGAGT CCTTCCTGGTGGAAGAGGATAAGAAGCACGAGCGGCACCC CATCTTCGGCAACATCGTGGACGAGGTGGCCTACCACGAG AAGTACCCCACCATCTACCACCTGAGAAAGAAACTGGTGG ACAGCACCGACAAGGCCGACCTGCGGCTGATCTATCTGGC CCTGGCCCACATGATCAAGTTCCGGGGCCACTTCCTGATC GAGGGCGACCTGAACCCCGACAACAGCGACGTGGACAAGC TGTTCATCCAGCTGGTGCAGACCTACAACCAGCTGTTCGA GGAAAACCCCATCAACGCCAGCGGCGTGGACGCCAAGGCC ATCCTGTCTGCCAGACTGAGCAAGAGCAGACGGCTGGAAA ATCTGATCGCCCAGCTGCCCGGCGAGAAGAAGAATGGCCT GTTCGGAAACCTGATTGCCCTGAGCCTGGGCCTGACCCCC AACTTCAAGAGCAACTTCGACCTGGCCGAGGATGCCAAAC TGCAGCTGAGCAAGGACACCTACGACGACGACCTGGACAA CCTGCTGGCCCAGATCGGCGACCAGTACGCCGACCTGTTT CTGGCCGCCAAGAACCTGTCCGACGCCATCCTGCTGAGCG ACATCCTGAGAGTGAACACCGAGATCACCAAGGCCCCCCT GAGCGCCTCTATGATCAAGAGATACGACGAGCACCACCAG GACCTGACCCTGCTGAAAGCTCTCGTGCGGCAGCAGCTGC CTGAGAAGTACAAAGAGATTTTCTTCGACCAGAGCAAGAA CGGCTACGCCGGCTACATTGACGGCGGAGCCAGCCAGGAA GAGTTCTACAAGTTCATCAAGCCCATCCTGGAAAAGATGG ACGGCACCGAGGAACTGCTCGTGAAGCTGAACAGAGAGGA CCTGCTGCGGAAGCAGCGGACCTTCGACAACGGCAGCATC CCCCACCAGATCCACCTGGGAGAGCTGCACGCCATTCTGC GGCGGCAGGAAGATTTTTACCCATTCCTGAAGGACAACCG GGAAAAGATCGAGAAGATCCTGACCTTCCGCATCCCCTAC TACGTGGGCCCTCTGGCCAGGGGAAACAGCAGATTCGCCT GGATGACCAGAAAGAGCGAGGAAACCATCACCCCCTGGAA CTTCGAGGAAGTGGTGGACAAGGGCGCTTCCGCCCAGAGC TTCATCGAGCGGATGACCAACTTCGATAAGAACCTGCCCA ACGAGAAGGTGCTGCCCAAGCACAGCCTGCTGTACGAGTA CTTCACCGTGTATAACGAGCTGACCAAAGTGAAATACGTG ACCGAGGGAATGAGAAAGCCCGCCTTCCTGAGCGGCGAGC AGAAAAAGGCCATCGTGGACCTGCTGTTCAAGACCAACCG GAAAGTGACCGTGAAGCAGCTGAAAGAGGACTACTTCAAG AAAATCGAGTGCTTCGACTCCGTGGAAATCTCCGGCGTGG AAGATCGGTTCAACGCCTCCCTGGGCACATACCACGATCT GCTGAAAATTATCAAGGACAAGGACTTCCTGGACAATGAG GAAAACGAGGACATTCTGGAAGATATCGTGCTGACCCTGA CACTGTTTGAGGACAGAGAGATGATCGAGGAACGGCTGAA AACCTATGCCCACCTGTTCGACGACAAAGTGATGAAGCAG CTGAAGCGGCGGAGATACACCGGCTGGGGCAGGCTGAGCC GGAAGCTGATCAACGGCATCCGGGACAAGCAGTCCGGCAA GACAATCCTGGATTTCCTGAAGTCCGACGGCTTCGCCAAC AGAAACTTCATGCAGCTGATCCACGACGACAGCCTGACCT TTAAAGAGGACATCCAGAAGCCCAGGTGCCGGCCAGG CGATAGCCTGCACGAGCACATTGCCAATCTGGCGGCAGC CCCGCCATTAAGAAGGGCATCCTGCAGACAGTGAAGGTGG TGGACGAGCTCGTGAAAGTGATGGGCCGGCACAAGCCCGA GAACATCGTGATCGAAATGGCCAGAGAGAACCAGACCACC CAGAAGGGACAGAGAACACAGCCGCGAGAGAATGAAAGCGGA TCGAAGAGGGCATCAAAGAGCTGGGCAGCCAGATCCTGAA AGAACACCCCGTGGAAACACCCAGCTGCAGACGAGAAG CTGTACCTGTACTACCTGCAGAATGGGCGGGATATGTACG TGGACCAGGAACTGGACATCAACCGGCTGTCCGACTACGA TGTGGACCATATCGTGCCTCAGAGCTTTCTGAAGGACGAC TCCATCGACAACAAGGTGCTGACCAGAAGCGACAAGAACC GGGGCAAGAGCGACAACGTGCCCTCCGAAGAGGTCGTGAA GAAGATGAAGAACTACTGGCGGCAGCTGCTGAACGCCAAG CTGATTACCCAGAGAAAGTTCGACAATCTGACCAAGGCCG AGAGAGGCGGCCTGAGCGAACTGGATAAGGCCGGCTTCAT CAAGAGACAGCTGGTGGAAACCCGGCAGATCACAAAGCAC GTGGCACAGATCCTGGACTCCCGGATGAACACTAAGTACG ACGAGAATGACAAGCTGATCCGGGAAGTGAAAGTGATCAC CCTGAAGTCCAAGCTGGTGTCCGATTTCCGGAAGGATTTC CAGTTTTACAAAGTGCGCGAGATCAACAACTACCACCACG CCCACGACGCCTACCTGAACGCCGTCGTGGGAACCGCCCT GATCAAAAAGTACCCTAAGCTGGAAAGCGAGTTCGTGTAC GGCGACTACAAGGTGTACGACGTGCGGAAGATGATCGCCA AGAGCGAGCAGGAAATCGGCAAGGCTACCGCCAAGTACTT CTTCTACAGCAACATCATGAACTTTTTCAAGACCGAGATT ACCCTGGCCAACGGCGAGATCCGGAAGCGGCCTCTGATCG AGACAAACGGCGAAACCGGGGAGATCGTGTGGGATAAGGG CCGGGATTTTGCCACCGTGCGGAAAGTGCTGAGCATGCCC CAAGTGAATATCGTGAAAAAGACCGAGGTGCAGACAGGCG GCTTCAGCAAAGAGTCTATCCTGCCCAAGAGGAACAGCGA TAAGCTGATCGCCAGAAAGAAGGACTGGGACCCTAAGAAG TACGGCGGCTTCGACAGCCCCACCGTGGCCTATTCTGTGC TGGTGGTGGCCAAAGTGGAAAAGGGCAAGTCCAAGAAACT G AAGAGTGTGAAAGAGCTGCTGGGGATCACCATCATGGAAA GAAGCAGCTTCGAGAAGAATCCCATCGACTTTCTGGAAGC CAAGGGCTACAAAGAAGTGAAAAAGGACCTGATCATCAAG CTGCCTAAGTACTCCCTGTTCGAGCTGGAAAACGGCCGGA AGAGAATGCTGGCCTGCCGGCGAACTGCAGAAGGGAAA CGAACTGGCCCTGCCCTCCAAATATGTGAACTTCCTGTAC CTGGCCAGCCACTATGAGAAGCTGAAGGGCTCCCCCGAGGG ATAATGAGCAGAAACAGCTGTTTGTGGAACAGCACAAGCA CTACCTGGACGAGATCATCGAGCAGATCAGCGAGTTCTCCC AAGAGAGTGATCCTGGCCGACGCTAATCTGGACAAAGTGC TGTCCGCCTACCAACAAGCACCGGGATAAGCCCATCAGAGA GCAGGCCGAGAATATCATCCACCTGTTTACCCTGACCAAT CTGGGAGCCCCTGCCGCCTTCAAGTACTTTGACACCACCA TCGACCGGAAGAGGTACACCAGCACCAAAGAGGTGCTGGA CGCCACCCTGATCCACCAGAGCATCACCGGCCTGTACGAG ACACGGATCGACCTGTCTCAGCTGGGAGGTGACTCTGGCG GCTCAAAAGAACCGCCGACGGCAGCGAATTCGAGCCCAA GAAGAAGAGGAAAGTCTAACCGGTCATCATCACCATCACC ATTGAGTTTAAACCCGCTGATCAGCCTCGACTGTGCCTTC TAGTTGCCAGCCATCTGTTGTTTGCCCCTCCCCCGTGCCT TCCTTGACCCTGGAAGGTGCCACTCCCACTGTCCTTTCCT AATAAAATGAGGAAATTGCATCGCATTGTCTGAGTAGGGTG TCATTCTATTCTGGGGGGTGGGGTGGGGCAGGACAGCAAG GGGGAGGATTGGGAAGACAATAGCAGGCATGCTGGGGATG CGGTGGGCTCTATGGCTTCTGAGGCGGAAAGAACCAGCTG GGGCTCGATACCGTCGACCTCTAGCTAGAGCTTGGCGTAA TCATGGTCATAGCTGTTTCCTGTGTGAAATTGTTATCCGC TCACAATTCCACACAACATACGAGCCGGAAGCATAAAGTG TAAAGCCTAGGGTGCCTAATGAGTGAGCTAACTCACATTA ATTGCGTTGCGCTCACTGCCCGCTTTCCAGTCGGGAAACC TGTCGTGCCAGCTGCATTAATGAATCGGCCAACGCGCGGG GAGAGGCGGTTTGCGTATTGGGCGCTCTTCCGCTTCCTCG CTCACTGACTCGCTGCGCTCGGTCGTTCGGCTGCGGCGAG CGGTATCAGCTCACTCAAAGGCGGTAATACGGTTATCCAC AGAATCAGGGGATAACGCAGGAAAGAACATGTGAGCAAAA GGCCAGCAAAAGGCCAGGAACCGTAAAAAGGCCGCGTTGC TGGCGTTTTTCCATAGGCTCCGCCCCCCTGACGAGCATCA CAAAAATCGACGCTCAAGTCAGAGGTGGCGAAACCCGACA GGACTATAAAGATACCAGGCGTTTCCCCCTGGAAGCTCCC TCGTGCGCTCTCCTGTTCCGACCCTGCCGCTTACCGGATA CCTGTCCGCCTTTCTCCCTTCGGGAAGCGTGGCGCTTTCT CATAGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCG TTCGCTCCAAGCTGGGCTGTGTGCACGAACCCCCCGTTCA GCCCGACCGCTGCGCCTTATCCGGTAACTATCGTCTTGAG TCCAACCCGGTAAGACACGACTTATCGCCACTGGCAGCAG CCACTGGTAACAGGATTAGCAGAGCGAGGTATGTAGGCGG TGCTACAGAGTTCTTGAAGTGGTGGCCTAACTACGGCTAC ACTAGAAGAACAGTATTTGGTATCTGCGCTCTGCTGAAGC CAGTTACCTTCGGAAAAAGAGTTGGTAGCTCTTGATCCGG CAAACAAACCACCGCTGGTAGCGGTGGTTTTTTTGTTTGC AAGCAGCAGATTACGCGCAGAAAAAAAGGATCTCAAGAAG ATCCTTTGATCTTTTCTACGGGGTCTGACACTCAGTGGAA CGAAAACTCACGTTAAGGGATTTTGGTCATGAGATTATCA AAAAGGATCTTCACCTAGATCCTTTTAAATTAAAAATGAA GTTTTAAATCAATCTAAAGTATATATGAGTAAACTTGGTC TGACAGTTACCAATGCTTAATCAGTGAGGCACCTATCTCA GCGATCTGTCTATTTCGTTCATCCATAGTTGCCTGACTCC CCGTCGTGTAGATAACTACGATACGGGAGGGCTTACCATC TGGCCCCAGTGCTGCAATGATACCGCGAGACCCACGCTCA CCGGCTCCAGATTTATCAGCAATAAACCAGCCAGCCGGAA GGGCCGAGCGCAGAAGTGGTCCTGCAACTTTATCCGCCTC CATCCAGTCTATTAATTGTTGCCGGGAAGCTAGAGTAAGT AGTTCGCCAGTTAATAGTTTGCGCAACGTTGTTGCCATTG CTACAGGCATCGTGGTGTCACGCTCGTCGTTTGGTATGGC TTCATTCAGCTCCGGTTCCCAACGATCAAGGCGAGTTACA TGATCCCCCATGTTGTGCAAAAAAGCGGTTAGCTCCTTCG GTCCTCCGATCGTTGTCAGAAGTAAGTTGGCCGCAGTGTT ATCACTCATGGTTATGGCAGCACTGCATAATTCTCTTACT GTCATGCCATCCGTAAGATGCTTTTCTGTGACTGGTGAGT ACTCAACCAAGTCATTCTGAGAATAGTGTATGCGGCGACC GAGTTGCTCTTGCCCGGCGTCAATACGGGATAATACCGCG CCACATAGCAGAACTTTAAAAGTGCTCATCATTGGAAAAC GTTCTTCGGGGCGAAAACTCTCAAGGATCTTACCGCTGTT GAGATCCAGTTCGATGTAACCCACTCGTGCACCCAACTGA TCTTCAGCATCTTTTACTTTCACCAGCGTTTCTGGGTGAG CAAAAACAGGAAGGCAAAATGCCGCAAAAAAGGGAATAAG GGCGACACGGAAATGTTGAATACTCATACTCTTCCTTTTT CAATATTATTGAAGCATTTATCAGGGTTATTGTCTCATGA GCGGATACATATTTGAATGTATTTAGAAAAATAAACAAAT AGGGGTTCCGCGCACATTTCCCCGAAAAGTGCCACCTGAC GTCGACGGATCGGGAGATCGATCTCCCGATCCCCTAGGGT CGACTCTCAGTACAATCTGCTCTGATGCCGCATAGTTAAG CCAGTATCTGCTCCCTGCTTGTGTGTTGGAGGTCGCTGAG TAGTGCGCGAGCAAAATTTAAGCTACAACAAGGCAAGGCT TGACCGACAATTGCATGAAGAATCTGCTTAGGGTTAGGCG TTTTGCGCTGCTTCGCGATGTACGGGCCAGATATACGCGT TGACATTGATTATTGACTAGTTATTAATAGTAATCAATTA CGGGGTCATTAGTTCATAGCCCATATATGGAGTTCCGCGT TACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCC AACGACCCCCGCCCATTGACGTCAATAATGACGTATGTT CCATAGTAACGCCAATGGGACTTTCCATTGACGTCATG GGTGGAGTATTTACGGTAAACTGCCCACTTGGCAGTACAT CAAGTGTATC

[0082] In some embodiments, the base editor is adenosine deaminase base editor 8 ( In some embodiments, ABE8 is selected from the group consisting of: In some embodiments, ABE8 is selected from the group consisting of TadA, TadB, TadC, TadD, TadE, TadE1, TadE2, TadE3, TadE4, TadE5, TadE6, TadE7, TadE8, TadE9, TadE10, TadE11, TadE12, TadE13, TadE14, TadE15, TadE16, TadE17, In some embodiments, the ABE8 gene is an evolved variant of adenosine deaminase. Variants of adenosine deaminase are listed in Tables 11, 13, or 14 below. In some embodiments, the variant of adenosine deaminase is TadA*8. Ant is Y147T, Y147R, Q154S, Y123H, V82S, T166R, and / or TadA*7 containing one or more modifications selected from the group of Q154R, ... 0.10 variant (e.g., TadA*8). In various embodiments, ABE8 is TadA*7.10 variants (e.g., TadA*7.10) having a combination of modifications selected from the group consisting of: For example, TadA*8) contains: Y147T+Q154R, Y147T+Q154S, Y14 7R+Q154S, V82S+Q154S, V82S+Y147R, V82S+Q154 R, V82S+Y123H, I76Y+V82S, V82S+Y123H+Y147T, V82S+Y123H+Y147R, V82S+Y123H+Q154R, Y147R+ Q154R+Y123H, Y147R+Q154R+I76Y, Y147R+Q154R +T166R, Y123H+Y147R+Q154R+I76Y, V82S+Y123H +Y147R+Q154R, and I76Y+V82S+Y123H+Y147R+Q1 54R. In some embodiments, ABE8 is a monomeric construct. ABE8 is a heterodimeric construct. In some embodiments, the ABE8 base editor contains the following sequence: MSEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNNRV IGEGWNRAIGLHDPTAHAEIMALRQGGLVMQNYRLIDATL YVTFEPCVMCAGAMIHSRIGRVVFGVRNAKTGAAGSLMDV LHYPGMNHRVEITEGILADECAALLCTFFRMPRQVFNAQK KAQSSTD.

[0083] As an example, the sequences used in the base editing compositions, systems, and methods described herein are The CBE has the nucleic acid sequence (8877 base pairs) provided below. (Addgene, Watertown, MA; Komor AC, et al. ,2017,Sci Adv.,30;3(8):eaao4774.doi:10.1 126 / sciadv.aao4774). At least 95% identity with the BE4 nucleic acid sequence. Polynucleotide sequences having identical sequences are also included. 1 ATATGCCAAG TACGCCCCCT ATTGACGTCA ATGACGGTAA ATGGCCCGCC TGGCATTATG 61 CCCAGTACAT GACCTTATGG GACTTTCCTA CTTGGCAGTA CATCTACGTA TTAGTCATCG 121 CTATTACCAT GGTGATGCGG TTTTGGCAGT ACATCAATGG GCGTGGATAG CGGTTTGACT 181 CACGGGGATT TCCAAGTCTC CACCCCATTG ACGTCAATGG GAGTTTGTTT TGGCACCAAA 241 ATCAACGGGA CTTTCCAAAA TGTCGTAACA ACTCCGCCCC ATTGACGCAA ATGGGCGGTA 301 GGCGTGTACG GTGGGAGGTC TATATAAGCA GAGCTGGTTT AGTGAACCGT CAGATCCGCT 361 AGAGATCCGC GGCCGCTAAT ACGACTCACT ATAGGGAGAG CCGCCACCAT GAGCTCAGAG 421 ACTGGCCCAG TGGCTGTGGA CCCCACATTG AGACGGCGGA TCGAGCCCCA TGAGTTTGAG 481 GTATTCTTCG ATCCGAGAGA GCTCCGCAAG GAGACCTGCC TGCTTTACGA AATTAATTGG 541 GGGGGCCGGC ACTCCATTTG GCGACATACA TCACAGAACA CTAACAAGCA CGTCGAAGTC 601 AACTTCATCG AGAAGTTCAC GACAGAAAGA TATTTCTGTC CGAACACAAG GTGCAGCATT 661 ACCTGGTTTC TCAGCTGGAG CCCATGCGGC GAATGTAGTA GGGCCATCAC TGAATTCCTG 721 TCAAGGTATC CCCACGTCAC TCTGTTTATT TACATCGCAA GGCTGTACCA CCACGCTGAC 781 CCCCGCAATC GACAAGGCCT GCGGGATTTG ATCTCTTCAG GTGTGACTAT CCAAATTATG 841 ACTGAGCAGG AGTCAGGATA CTGCTGGAGA AACTTTGTGA ATTATAGCCC GAGTAATGAA 901 GCCCACTGGC CTAGGTATCC CCATCTGTGG GTACGACTGT ACGTTCTTGA ACTGTACTGC 961 ATCATACTGG GCCTGCCTCC TTGTCTCAAC ATTCTGAGAA GGAAGCAGCC ACAGCTGACA 1021 TTCTTTACCA TCGCTCTTCA GTCTTGTCAT TACCAGCGAC TGCCCCCACA CATTCTCTGG 1081 GCCACCGGGT TGAAATCTGG TGGTTCTTCT GGTGGTTCTA GCGGCAGCGA GACTCCCGGG 1141 ACCTCAGGT CCGCCACACC CGAAAGTTCT GGTGGTTCTT CTGGTGGTTC TGATAAAAAAG 1201 TATTCTATTG GTTTAGCCAT CGGCACTAAT TCCGTTGGAT GGGCTGTCAT AACCGATGAA 1261 TACAAAGTAC CTTCAAAGAA ATTTAAGGTG TTGGGGAACA CAGACCGTCA TTCGATTAAA 1321 AAGAATCTTA TCGGTGCCCT CCTATTCGAT AGTGGCGAAA CGGCAGAGGC GACTCGCCTG 1381 AAACGAACCG CTCGGAGAAG GTATACACGT CGCAAGAACC GAATATGTTA CTTACAAGAA 1441 ATTTTTAGCA ATGAGATGGC CAAAGTTGAC GATTCTTTCT TTCACCGTTT GGAAGAGTCC 1501 TTCCTTGTCG AAGAGGACAA GAAACATGAA CGGCACCCCA TCTTTGGAAA CATAGTAGAT 1561 GAGGTGGCAT ATCATGAAAA GTACCCAACG ATTTATCACC TCAGAAAAAA GCTAGTTGAC 1621 TCAACTGATA AAGCGGACCT GAGGTTAATC TACTTGGCTC TTGCCCATAT GATAAAGTTC 1681 CGTGGGCACT TTCTCATTGA GGGTGATCTA AATCCGGACA ACTCGGATGT CGACAAACTG 1741 TTCATCCAGT TAGTACAAAC CTATAATCAG TTGTTTGAAG AGAACCCTAT AAATGCAAGT 1801 GGCGTGGATG CGAAGGCTAT TCTTAGCGCC CGCCTCTCTA AATCCCGACG GCTAGAAAAC 1861 CTGATCGCAC AATTACCCGG AGAGAAGAAA AATGGGTTGT TCGGTAACCT TATAGCGCTC 1921 TCACTAGGCC TGACACCAAA TTTTAAGTCG AACTTCGACT TAGCTGAAGA TGCCAAATTG 1981 CAGCTTAGTA AGGACACGTA CGATGACGAT CTCGACAATC TACTGGCACA AATTGGAGAT 2041 CAGTATGCGG ACTTATTTTT GGCTGCCAAA AACCTTAGCG ATGCAATCCT CCTATCTGAC 2101 ATACTGAGAG TTAATACTGA GATTACCAAG GCGCCGTTAT CCGCTTCAAT GATCAAAAGG 2161 TACGATGAAC ATCACCAAGA CTTGACACTT CTCAAGGCCC TAGTCCGTCA GCAACTGCCT 2221 GAGAAATATA AGGAAATATT CTTTGATCAG TCGAAAAACG GGTACGCAGG TTATATTGAC 2281 GGCGGAGCGA GTCAAGAGGA ATTCTACAAG TTTATCAAAC CCATATTAGA GAAGATGGAT 2341 GGGACGGAAG AGTTGCTTGT AAAACTCAAT CGGAAGATC TACTGCGAAA GCAGCGGACT 2401 TTCGACAACG GTAGCATTCC ACATCAAATC CACTTAGGCG AATTGCATGC TATACTTAGA 2461 AGGCAGGAGG ATTTTTATCC GTTCCTCAAA GACAATCGTG AAAAGATTGA GAAAATCCTA 2521 ACCTTTCGCA TACCTTACTA TGTGGGACCC CTGGCCCGAG GGAACTCTCG GTTCGCATGG 2581 REPEAT AGTCCREACT AACGATTACT CCATGGAATTT TTREPEAT TGTCREADAA 2641 GGTGCGTCAG CTCAATCGTT CATCGAGAGG ATGACCAACT TTGACAAA TTTACCGAAC 2701 GAAAAGTAT TGCCTAXASSOCIATTACTT CHAPTER TGACTACTACATCHACTC 2761 ACGAAAGTTA AGTATGTCAC TGAGGGCATG CGTAAACCCG CCTTTCTAAG CGGAGAACAG 2821 AAGAAAGCAA TAGTAGATCT GTTATTCAAG ACCAACCGCA AAGTGACAGT TAAGCAATTG 2881 AAAGAGGACT ACTTTAAGAA AATTGAATGC TTCGATTCTG TCGAGATCTC CGGGGTAGAA 2941 GATCGATTTA ATGCGTCACT TGGTACGTAT CATGACCCTCC WINDOW INTERFACE 3001 GACTTCCTGG ATTACK REPEAT ATCTCTGG ATTACKGTT GACTCTTACC 3061 CTCTTTGAAG ATCGGGAAAT GATTGAGGAA AGACTAAAAAA CATACGCTCA CCTGTTCGAC 3121 GATAAGGTTA TGAAACAGTT AAAGAGGCGT CGCTATACGG GCTGGGGACG ATTGTCGCGG 3181 AAACTTATCA ACGGGATAAG AGACAAGCAA AGTGGTAAAA CTATTCTCGA TTTTCTAAAG 3241 AGCGACGGCT TCGCCAATAG GAACTTTATG CAGCTGATCC ATGATGACTC TTTAACCTTC 3301 AAAGAGGATA TACAAAAGGC ACAGGTTTCC GGACAAGGGG ACTCATTGCA CGAACATATT 3361 GCGAATCTTG CTGGTTCGCC AGCCATCAAA AAGGGCATAC TCCAGACAGT CAAAGTAGTG 3421 GATGAGCTAG TTAAGGTCAT GGGACGTCAC AAACCGGAAA ACATTGTAAT CGAGATGGCA 3481 CGCGAAAATC AAACGACTCA GAAGGGGCAA AAAAAACAGTC GAGAGCGGAT GAAGAGAATA 3541 GAAGAGGGTA TTAAAGAACT GGGCAGCCAG ATCTTAAAGG AGCATCCTGT GGAAAATACC 3601 CAATTGCAGA ACGAGAAACT TTACCTCTAT TACCTACAAA ATGGAAGGGA CATGTATGTT 3661 GATCAGGAAC TGGACATAAA CCGTTTATCT GATTACGACG TCGATCACAT TGTACCCCAA 3721 TCCTTTTTGA AGGACGATTC AATCGACAAT AAAGTGCTTA CACGCTCGGA TAAGAACCGA 3781 GGGAAAAGTG ACAATGTTCC AAGCGAGGAA GTCGTAAAGA AAATGAGAA CTATTGGCGG 3841 CAGCTCCTAA ATGCGAAACT GATAACGCAA AGAAAGTTCG ATAACTTAAC TAAAGCTGAG 3901 AGGGGTGGCT TGTCTGAACT TGACAAGGCC GGATTTATTA AACGTCAGCT CGTGGAAACC 3961 CGCCAAATCA CAAAGCATGT TGCACAGATA CTAGATTCCC GAATGAATAC GAAATACGAC 4021 GAGAACGATA AGCTGATTCG GGAAGTCAAA GTAATCACTT TAAAGTCAAA ATTGGTGTCG 4081 GACTTCAGAA AGGATTTTCA ATTCTATAAA GTTAGGGAGA TAAATAACTA CCACCATGCG 4141 CACGACGCTT ATCTTAATGC CGTCGTAGGG ACCGCACTCA TTAAGAAATA CCCGAAGCTA 4201 GAAAGTGAGT TTGTGTATGG TGATTACAAA GTTTATGACG TCCGTAAGAT GATCGCGAAA 4261 AGCGAACAGG AGATAGGCAA GGCTACAGCC AAATACTTCT TTTATTCTAA CATTATGAAT 4321 TTCTTTAAGA CGGAAATCAC TCTGGCAAAC GGAGAGATAC GCAAACGACC TTTAATTGAA 4381 ACCAATGGGG AGACAGGTGA AATCGTATGG GATAAGGGCC GGGACTTCGC GACGGTGAGA 4441 AAAGTTTTGT CCATGCCCCA AGTCAACATA GTAAAGAAAA CTGAGGTGCA GACCGGAGGG 4501 TTTTCAAAGG AATCGATTCT TCCAAAAAGG AATAGTGATA AGCTCATCGC TCGTAAAAAG 4561 GACTGGGACC CGAAAAAGTA CGGTGGCTTC GATAGCCCTA CAGTTGCCTA TTCTGTCCTA 4621 GTAGTGGCAA AAGTTGAGAA GGGAAAATCC AAGAAACTGA AGTCAGTCAA AGAATTATTG 4681 GGGATAACGA TTATGGAGCG CTCGTCTTTT GAAAAGAACC CCATCGACTT CCTTGAGGCG 4741 AAAGGTTACA AGGAAGTAAA AAAGGATCTC ATAATTAAAC TACCAAAGTA TAGTCTGTTT 4801 GAGTTAGAAA ATGGCCGAAA ACGGATGTTG GCTAGCGCCG GAGAGCTTCA AAAGGGGAAC 4861 GAACTCGCAC TACCGTCTAA TACCGTGAAT TTCCTGTATT TAGCGTCCCA TTACGAGAAG 4921 TTGAAAGGTT CACCTGAAGA TAACGAACAG AAGCAACTTT TTGTTGAGCA GCACAAACAT 4981 TATCTCGACG AAATCATAGA GCAAATTTCG GAATTCAGTA AGAGAGTCAT CCTAGCTGAT 5041 GCCAATCTGG ACAAAGTATT AAGCGCATAC AACAAGCACA GGGATAAACC CATACGTGAG 5101 CAGGCGGAAA ATTACK TTTGTTTACT CTTACC TCGGCGCTCC AGCCGCATTC 5161 AAGTATTTTG ACACAACGAT AGATCGCAAA CGATACACTT CTACCAAGGA GGTGCTAGAC 5221 GCGACACTGA TTCACCAATC CATCACGGGA TTATGAAA CTCGGATAGA TTTGTCACAG 5281 CTTGGGGGTG ACTCTGGTGG TTCTGGAGGA TCTGGTGGTT CTACTAATCT GTCAGATATT 5341 ATTGAAAAGG AGACCGGTAA GCAACTGGTT ATCCAGGAAT CCATCCTCAT GCTCCCAGAG 5401 GAGGTGGAAG AAGTCATTGG GAACAAGCCG GAAAGCGATA TACTCGTGCA CACCGCCTAC 5461 GACCGAGAGCA CCGACGAGAA TGTCATGCTT CTGACTAGCG ACGCCCCTGA ATACAAGCCT 5521 TGGGCTCTGG TCATACAGGA TAGCAACGGT GAGAACAAGA TTAAGATGCT CTCTGGTGGT 5581 TCTGGAGGAT CTGGTGGTTC TACTAATCTG TCAGATATTA TTGAAAAGGA GACCGGTAAG 5641 CAACTGGTTA TCCAGGAATC CATCCTCATG CTCCCAGAGG AGGTGGAAGA AGTCATTGGG 5701 AACAAGCCGG AAAGCGATAT ACTCGTGCAC ACCGCCTACG ACGAGAGCAC CGACGAGAAT 5761 GTCATGCTTC TGACTAGGCGA CGCCCCTGAA TACAAGCCTT GGGCTCTGGT CATACAGGAT 5821 AGCAACGGTG AGAACAAGAT TAAGATGCTC TCTGGTGGTT CTCCCAAGAA GAAGAGGAAA 5881 GTCTAACCGG TCATCATCAC CATCACCATT GAGTTTAAAC CCGCTGATCA GCCTCGACTG 5941 TGCCTTCTAG TTGCCAGCCA TCTGTTGTTT GCCCCTCCCC CGTGCCTTCC TTGACCCTGG 6001 AAGGTGCCAC TCCCACTGTC CTTTCCTAAT AAAATGAGGA AATTGCATCG CATTGTCTGA 6061 GTAGGTGTCA TTCTATTCTG GGGGGTGGGG TGGGGCAGGA CAGCAAGGGG GAGGATTGGG 6121 AAGACAATAG CAGGCATGCT GGGGATGCGG TGGGCTCTAT GGCTTCTGAG GCGGAAAGAA 6181 CCAGCTGGGG CTCGATACCG TCGACCTCTA GCTAGAGCTT GGCGTAATCA TGGTCATAGC 6241 TGTTTCCTGT GTGAAATTGT TATCCGCTCA CAATTCCACA CAACATACGA GCCGGAAGCA 6301 TAAAGTGTAA AGCCTAGGGT GCCTAATGAG TGAGCTAACT CACATTAATT GCGTTGCGCT 6361 CACTGCCCGC TTTCCAGTCG GGAAACCTGT CGTGCCAGCT GCATTAATGA ATCGGCCAAC 6421 GCGCGGGGAG AGGCGGTTTG CGTATTGGGC GCTCTTCCGC TTCCTCGCTC ACTGACTCGC 6481 TGCGCTCGGT CGTTCGGCTG CGGCGAGCGG TATCAGCTCA CTCAAAGGCG GTAATACGGT 6541 TATCCACAGA ATCAGGGGAT AACGCAGGAA AGAACATGTG AGCAAAAGGC CAGCAAAAGG 6601 CCAGGAACCG TAAAAAGGCC GCGTTGCTGG CGTTTTTCCA TAGGCTCCGC CCCCCTGACG 6661 AGCATCACAA AAATCGACGC TCAAGTCAGA GGTGGCGAAA CCCGACAGGA CTATAAAGAT 6721 ACCAGGCGTT TCCCCCTGGA AGCTCCCTCG TGCGCTCTCC TGTTCCGACC CTGCCGCTTA 6781 CCGGATACCT GTCCGCCTTT CTCCCTTCGG GAAGCGTGGC GCTTTCTCAT AGCTCACGCT 6841 GTAGGTATCT CAGTTCGGTG TAGGTCGTTC GCTCCAAGCT GGGCTGTGTG CACGAACCCC 6901 CCGTTCAGCC CGACCGCTGC GCCTTATCCG GTAACTATCG TCTTGAGTCC AACCCGGTAA 6961 GACACGACTT ATCGCCACTG GCAGCAGCCA CTGGTAACAG GATTAGCAGA GCGAGGTATG 7021 TAGGCGGTGC TACAGAGTTC TTGAAGTGGT GGCCTAACTA CGGCTACACT AGAAGAACAG 7081 TATTTGGTAT CTGCGCTCTG CTGAAGCCAG TTACCTTCGG AAAAAGAGTT GGTAGCTCTT 7141 GATCCGGCAA ACAAACCACC GCTGGTAGCG GTGGTTTTTT TGTTTGCAAG CAGCAGATTA 7201 CGCGCAGAAA AAAAGGATCT CAAGAAGATC CTTTGATCTT TTCTACGGGG TCTGACGCTC 7261 AGTGGAACGA AAACTCACGT TAAGGGATTT TGGTCATGAG ATTATCAAAA AGGATCTTCA 7321 CCTAGATCCT TTTAAATTAA AAATGAAGTT TTAAATCAAT CTAAAGTATA TATGAGTAAA 7381 CTTGGTCTGA CAGTTACCAA TGCTTAATCA GTGAGGCACC TATCTCAGCG ATCTGTCTAT 7441 TTCGTTCATC CATAGTTGCC TGACTCCCCG TCGTGTAGAT AACTACGATA CGGGAGGGCT 7501 TACCATCTGG CCCCAGTGCT GCAATGATAC CGCGAGACCC ACGCTCACCG GCTCCAGATT 7561 TATCAGCAAT AAACCAGCCA GCCGGAAGGG CCGAGCGCAG AAGTGGTCCT GCAACTTTAT 7621 CCGCCTCCAT CCAGTCTATT AATTGTTGCC GGGAAGCTAG AGTAAGTAGT TCGCCAGTTA 7681 ATAGTTTGCG CAACGTTGTT GCCATTGCTA CAGGCATCGT GGTGTCACGC TCGTCGTTTG 7741 GTATGGCTTC ATTCAGCTCC GGTTCCCAAC GATCAAGGCG AGTTACATGA TCCCCCATGT 7801 TGTGCAAAAA AGCGGTTAGC TCCTTCGGTC CTCCGATCGT TGTCAGAAGT AAGTTGGCCG 7861 CAGTGTTATC ACTCATGGTT ATGGCAGCAC TGCATAATTC TCTTACTGTC ATGCCATCCG 7921 TAAGATGCTT TTCTGTGACT GGTGAGTACT CAACCAAGTC ATTCTGAGAA TAGTGTATGC 7981 GGCGACCGAG TTGCTCTTGC CCGGCGTCAA TACGGGATAA TACCGCGCCA CATAGCAGAA 8041 CTTTAAAAGT GCTCATCATT GGAAAACGTT CTTCGGGGCG AAAACTCTCA AGGATCTTAC 8101 CGCTGTTGAG ATCCAGTTCG ATGTAACCCA CTCGTGCACC CAACTGATCT TCAGCATCTT 8161 TTACTTTCAC CAGCGTTTCT GGGTGAGCAA AAACAGGAAG GCAAAATGCC GCAAAAAAGG 8221 GAATAAGGGC GACACGGAAA TGTTGAATAC TCATACTCTT CCTTTTTCAA TATTATTGAA 8281 GCATTTATCA GGGTTATTGT CTCATGAGCG GATACATATT TGAATGTATT TAGAAAAATA 8341 AACAAATAGG GGTTCCGCGC ACATTTCCCC GAAAAGTGCC ACCTGACGTC GACGGATCGG 8401 GAGATCGATC TCCCGATCCC CTAGGGTCGA CTCTCAGTAC AATCTGCTCT GATGCCGCAT 8461 AGTTAAGCCA GTATCTGCTC CCTGCTTGTG TGTTGGAGGT CGCTGAGTAG TGCGCGAGCA 8521 AAATTTAAGC TACAACAAGG CAAGGCTTGA CCGACAATTG CATGAAGAAT CTGCTTAGGG 8581 TTAGGCGTTT TGCGCTGCTT CGCGATGTAC GGGCCAGATA TACGCGTTGA CATTGATTAT 8641 TGACTAGTTA TTAATAGTAA TCAATTACGG GGTCATTAGT TCATAGCCCA TATATGGAGT 8701 TCCGCGTTAC ATAACTTACG GTAAATGGCC CGCCTGGCTG ACCGCCCAAC GACCCCCGCC 8761 CATTGACGTC AATAATGACG TATGTTCCCA TAGTAACGCC AATAGGGACT TTCCATTGAC 8821 GTCAATGGGT GGAGTATTTA CGGTAAACTG CCCACTTGGC AGTACATCAA GTGTATC

[0084] In some embodiments, the cytidine base editor is BE4 and is selected from the group consisting of one of the following: having a nucleic acid sequence selected from:

[0085] Original BE4 nucleic acid sequence: ATGagctcagagactggcccagtggctgtggaccccacat tgagacggcggatcgagccccatgagtttgaggtattctt cgatccgagagagctccgcaaggagacctgcctgctttac gaaattaattgggggggccggcactccatttggcgacata catcacagaacactaacaagcacgtcgaagtcaacttcat cgagaagttcacgacagaaagatatttctgtccgaacaca aggtgcagcattacctggtttctcagctggagccgcgaat gtagtagggccatcactgaattcctgtcaaggtatcccca cgtcactctgtttatttacatcgcaaggctgtaccaccac gctgacccccgcaatcgacaaggcctgcgggatttgatct cttcaggtgtgactatccaaattatgactgagcaggagtc aggatactgctggagaaactttgtgaattatagcccgagt aatgaagcccactggcctaggtatccccatctgtgggtac gactgtacgttcttgaactgtactgcatcatactgggcct gcctccttgtctcaacattctgagaaggaagcagccacag ctgacattctttaccatcgctcttcagtcttgtcattacc agcgactgcccccacacattctctgggccaccgggttgaa atctggtggttcttctggtggttctagcggcagcgagact cccgggacctcagagtccgccacacccgaaagttctggtg gttcttctggtggttctgataaaaagtattctattggttt agccatcggcactaattccgttggatgggctgtcataacc gatgaatacaaagtaccttcaaagaaatttaaggtgttgg ggaacacagaccgtcattcgattaaaaagaatcttatcgg tgccctcctattcgatagtggcgaaacggcagaggcgact cgcctgaaacgaaccgctcggagaaggtatacacgtcgca agaaccgaatatgttacttacaagaaatttttagcaatga gatggccaaagttgacgattctttctttcaccgtttggaa gagtccttccttgtcgaagaggacaagaaacatgaacggc accccatctttggaaacatagtagatgaggtggcatatca tgaaaagtacccaacgatttatcacctcagaaaaaagcta gttgactcaactgataaagcggacctgaggttaatctact tggctcttgcccatatgataaagttccgtgggcactttct cattgagggtgatctaaatccggacaactcggatgtcgac aaactgttcatccagttagtacaaacctataatcagttgt ttgaagagaaccctataaatgcaagtggcgtggatgcgaa ggctattcttagcgcccgcctctctaaatcccgacggcta gaaaacctgatcgcacaattacccggagagaagaaaaatg ggttgttcggtaaccttatagcgctctcactaggcctgac accaaattttaagtcgaacttcgacttagctgaagatgcc aaattgcagcttagtaaggacacgtacgatgacgatctcg acaatctactggcacaaattggagatcagtatgcggactt atttttggctgccaaaaaccttagcgatgcaatcctccta tctgacatactgagagttaatactgagattaccaaggcgc cgttatccgcttcaatgatcaaaaggtacgatgaacatca ccaagacttgacacttctcaaggccctagtccgtcagcaa ctgcctgagaaatataaggaaatattctttgatcagtcga aaaacgggtacgcaggttatattgacggcggagcgagtca agaggaattctacaagtttatcaaacccatattagagaag atggatgggacggaagagttgcttgtaaaactcaatcgcg aagatctactgcgaaagcagcggactttcgacaacggtag cattccacatcaaatccacttaggcgaattgcatgctata cttagaaggcaggaggatttttatccgttcctcaaagaca atcgtgaaaagattgagaaaatcctaacctttcgcatacc ttactatgtgggacccctggcccgagggaactctcggttc gcatggatgacaagaaagtccgaagaaacgattactccat ggaattttgaggaagttgtcgataaaggtgcgtcagctca atcgttcatcgagaggatgaccaactttgacaagaattta ccgaacgaaaaagtattgcctaagcacagtttactttacg agtatttcacagtgtacaatgaactcacgaaagttaagta tgtcactgagggcatgcgtaaacccgcctttctaagcgga gaacagaagaaagcaatagtagatctgttattcaagacca accgcaaagtgacagttaagcaattgaaagaggactactt taagaaaattgaatgcttcgattctgtcgagatctccggg gtagaagatcgatttaatgcgtcacttggtacgtatcatg acctcctaaaagataattaaagataaggacttcctggataa cgaagagaatgaagatatcttagagaatagatgtttgact cttaccctctttgaagaatcgggaaatgattgaggaaagac taaaaacatacgctcacctgttcgacgataaggttatgaa acagttaaagaggcgtcgctatacgggctgggggacgattg tcgcggaaacttatcaacgggataagagacaagcaaagtg gtaaaactattctcgatttctctaaagagcgacggcttgc caataggaactttatgcagctgatccatgatgactcttta accttcaaagaggatatacaaaagggcacaggtttccggac aaggggactcattgcacgaacatattgcgaatcttgctgg ttcgccagccatcaaaaagggcatactccagacagtcaaa gtagtggatgagctagttaaggtcatgggacgtcacaaac cggaaaacattgtaatcgagatggcacgcgaaaatcaaac gactcagaaggggcaaaaaaacagtcgagagcggatgaag agaatagaagagggtattaaagaactgggcagccagatct taaaggagcatcctgtggaaaatacccaattgcagaacga gaaactttacctctattacctacaaaatggaagggacatg tatgttgatcaggaactggacataaaccgtttatctgatt acgacgtcgatcacattgtaccccaatcctttttgaagga cgattcaatcgacaataaagtgcttacacgctcggataag aaccgagggaaaagtgacaatgttccaagcgaggaagtcg taaagaaaatgaagaactattggcggcagctcctaaatgc gaaactgataacgcaaagaaagttcgataacttaactaaa gctgagaggggtggcttgtctgaacttgacaaggccggat ttattaaacgtcagctcgtggaaacccgccaaatcacaaa gcatgttgcacagatactagattcccgaatgaatacgaaa tacgacgagaacgataagctgattcgggaagtcaaagtaa tcactttaaagtcaaaattggtgtcggacttcagaaagga ttttcaattctataaagttagggagataaataactaccac catgcgcacgacgcttatcttaatgccgtcgtagggaccg cactcattaagaaatacccgaagctagaaagtgagtttgt gtatggtgattacaaagtttatgacgtccgtaagatgatc gcgaaaagcgaacaggagataggcaaggctacagccaaat acttcttttattctaacattatgaatttctttaagacgga aatcactctggcaaacggagagatacgcaaacgaccttta attgaaaccaatggggagacaggtgaaatcgtatgggata agggccgggacttcgcgacggtgagaaaagttttgtccat gccccaagtcaacatagtaaagaaaactgaggtgcagacc ggagggttttcaaaggaatcgattcttccaaaaaggaata gtgataagctcatcgctcgtaaaaaggactgggacccgaa aaagtacggtggcttcgatagccctacagttgcctattct gtcctagtagtggcaaaagttgagaagggaaaatccaaga aactgaagtcagtcaaagaattattggggataacgattat ggagcgctcgtcttttgaaaagaaccccatcgacttcctt gaggcgaaaggttacaaggaagtaaaaaaggatctcataa ttaaactaccaaagtatagtctgtttgagttagaaaatgg ccgaaaacggatgttggctagcgccggagagcttcaaaag gggaacgaactcgcactaccgtctaaatacgtgaatttcc tgtatttagcgtcccattacgagaagttgaaaggttcacc tgaagataacgaacagaagcaactttttgttgagcagcac aaacattatctcgacgaaatcatagagcaaatttcggaat tcagtaagagagtcatcctagctgatgccaatctggacaa agtattaagcgcatacaacaagcacagggataaacccata cgtgagcaggcggaaaatattatccatttgtttactctta ccaacctcggcgctccagccgcattcaagtattttgacac aacgatagatcgcaaacgatacacttctaccaaggaggtg ctagacgcgacactgattcaccaatccatcacgggattat atgaaactcggatagatttgtcacagcttgggggtgactc tggtggttctggaggatctggtggttctactaatctgtca gatattattgaaaaggagaccggtaagcaactggttatcc aggaatccatcctcatgctcccagaggaggtggaagaagt cattgggaacaagccggaaagcgatatactcgtgcacacc gcctacgacgagagcaccgacgagaatgtcatgcttctga ctagcgacgcccctgaatacaagccttgggctctggtcat a caggatagcaacggtgagaacaagattaagatgctctctg gtggttctggaggatctggtggttctactaatctgtcaga tattattgaaaaggagaccggtaagcaactggttatccag gaatccatcctcatgctcccagaggaggtggaagaagtca ttgggaacaagccggaaagcgatatactcgtgcacaccgc ctacgacgagagcaccgacgagaatgtcatgcttctgact agcgacgcccctgaatacaagccttgggctctggtcatac aggatagcaacggtgagaacaagattaagatgctctctgg tggttctAAAAGGACGGCGGACGGATCAGAGTTCGAGAGT CCGAAAAAAAAACGAAAGGTCGAAtaa

[0086] BE4 codon-optimized 1 nucleic acid sequence: ATGTCATCCGAAACCGGGCCAGTGGCCGTAGACCCAACAC TCAGGAGGCGGATAGAACCCCATGAGTTTGAAGTGTTCTT CGACCCCAGAGAGCTGCGCAAAGAGACTTGCCTCCTGTAT GAAATAAATTGGGGGGGTCGCCATTCAATTTGGAGGCACA CTAGCCAGAATACTAACAAACACGTGGAGGTAAATTTTAT CGAGAAGTTTACCACCGAAAGATACTTTTGCCCCAATACA CGGTGTTCAATTACCTGGTTTCTGTCATGGAGTCCATGTG GAGAATGTAGTAGAGCGATAACTGAGTTCCTGTCTCGATA TCCTCACGTCACGTTGTTTATATACATCGCTCGGCTTTAT CACCATGCGGACCCGCGGAACAGGCAAGGTCTTCGGGACC TCATATCCTCTGGGGTGACCATCCAGATAATGACGGAGCA AGAGAGCGGATACTGCTGGCGAAACTTTGTTAACTACAGC CCAAGCAATGAGGCACACTGGCCTAGATATCCGCATCTCT GGGTTCGACTGTATGTCCTTGAACTGTACTGCATAATTCT GGGACTTCCGCCATGCTTGAACATTCTGCGGCGGAAACAA CCACAGCTGACCTTTTTCACGATTGCTCTCCAAAGTTGTC ACTACCAGCGATTGCCACCCCACATCTTGTGGGCTACTGG ACTCAAGTCTGGAGGAAGTTCAGGCGGAAGCAGCGGGTCT GAAACGCCCGGAACCTCAGAGAGCGCAACGCCCGAAAGCT CTGGAGGGTCAAGTGGTGGTAGTGATAAGAAATACTCCAT CGGCCTCGCCATCGGTACGAATTCTGTCGGTTGGGCCGTT ATCACCGATGAGTACAAGGTCCCTTCTAAGAAATTCAAGG TTTTGGGCAATACAGACCGCCATTCTATAAAAAAAAACCT GATCGGCGCCCTTTTGTTTGACAGTGGTGAGACTGCTGAA GCGACTCGCCTGAAGCGAACTGCCAGGAGGCGGTATACGA GGCGAAAAAACCGAATTTGTTACCTCCAGGAGATTTTCTC AAATGAAATGGCCAAGGTAGATGATAGTTTTTTTCACCGC TTGGAAGAAAGTTTTCTCGTTGAGGAGGACAAAAAGCACG AGAGGCACCCAATCTTTGGCAACATAGTCGATGAGGTCGC ATACCATGAGAAATATCCTACGATCTATCATCTCCGCAAG AAGCTGGTCGATAGCACGGATAAAGCTGACCTCCGGCTGA TCTACCTTGCTCTTGCTCACATGATTAAATTCAGGGGCCA TTTCCTGATAGAAGGAGACCTCAATCCCGACAATTCTGAT GTCGACAAACTGTTTATTCAGCTCGTTCAGACCTATAATC AACTCTTTGAGGAGAACCCCATCAATGCTTCAGGGGTGGA CGCAAAGGCCATTTTGTCCGCGCGCTTGAGTAAATCACGA CGCCTCGAGAATTTGATAGCTCAACTGCCGGGTGAGAAGA AAAACGGGTTGTTTGGGAATCTCATAGCGTTGAGTTTGGG ACTTACGCCAAACTTTAAGTCTAACTTTGATTTGGCCGAA GATGCCAAATTGCAGCTGTCCAAAGATACCTATGATGACG ACTTGGATAACCTTCTTGCGCAGATTGGTGACCAATACGC GGATCTGTTTCTTGCCGCAAAAAATCTGTCCGACGCCATA CTCTTGTCCGATATACTGCGCGTCAATACTGAGATAACTA AGGCTCCCCTCAGCGCGTCCATGATTAAAAGATACGATGA GCACCACCAAGATCTCACTCTGTTGAAAGCCCTGGTTCGC CAGCAGCTTCCAGAGAAGTATAAGGAGATATTTTTCGACC AATCTAAAACGGCTATGCGGGTTACATTGACGGTGGCGC CTTCCAAGAAGAATTCTACAAGTTTATAAAGCCGATACTT GAGAAAATGGACGGTACAGAGGAATTGTTGGTTAAGCTCA ATCGCGAGGACTTGTTGAGAAAGCAGCGCACATTTGACAA TGGTAGTATTCCACACCAGATTCATCTGGGCGAGTTGCAT GCCATTCTTAGAAGACAAGAAGATTTTTATCCGTTTCTGA AAGATAACAGAGAAAAAGATTGAAAAGATACTTACCTTTCG CATACCGTATTATGTAGGTCCCCTGGCTAGAGGGAACAGT CGCTTCGCTTGGATGACTCGAAAATCAGAAGAAAACAATAA CCCCCTGGAATTTTGAAGAAGTGGTAGATAAAGGTGCGAG TGCCCAATCTTTTATTGAGCGGATGACAAATTTTGACAAG AATCTGCCTAACGAAAAGGTGCTTCCCAAGCATTCCCTTT TGTATGAATACTTTACATGTATAATGAACTGACTAAAGT GAAGTACGTTACCGAGGGGATGCGAAAGCCAGCTTTTCTC AGTGGCGAGCAGAAAAAAAGCAATAGTTGACCTGCTGTTCA AGACGAATAGGAAGGTTACCGTCAAACAGCTCAAAGAAGA TTACTTTAAAAAGATCGAATGTTTTGATTCAGTTGAGATA AGCGGAGTAGAGGATAGATTTAACGCAAGTCTTGGGAACTT ATCATGACCTTTTGAAGATCATCAAGGATAAAGATTTTTT GGACAACGAGGAGAATGAAGATATCCTGGAAGATATAGTA CTTACCTTGACGCTTTTTGAAGATCGAGAGATGATCGAGG AGCGACTTAAGACGTACGCACATCTCTTTGACGATAAGGT TATGAAACAATTGAAACGCCGGCGGTATACTGGCTGGGGC AGGCTTTCTCGAAAGCTGATTAATGGTATCCGCGATAAGC AGTCTGGAAAGACAATCCTTGACTTTCTGAAAAGTGATGG ATTTGCAAATAGAAACTTTATGCAGCTTATACATGATGAC TCTTTGACGTTCAAGGAAGACATCCAGAAGGCACAGGTAT CCGGCCAAGGGGATAGCCTCCATGAACACATAGCCAACCT GGCCGGCTCACCAGCTATTAAAAAGGGAATATTGCAAACC GTTAAGGTTGTTGACGAACTCGTTAAGGTTATGGGCCGAC ACAAACCAGAGAATATCGTGATTGAGATGGCTAGGGAGAA TCAGACCACTCAAAAAGGTCAGAAAAATTCTCGCGAAAGG ATGAAGCGAATTGAAGAGGGAATCAAAGAACTTGGCTCTC AAATTTTGAAAGAGCACCCGGTAGAAAACACTCAGCTGCA GAATGAAAAGCTGTATCTGTATTATCTGCAGAATGGTCGA GATATGTACGTTGATCAGGAGCTGGATATCAATAGGCTCA GTGACTACGATGTCGACCACATCGTTCCTCAATCTTTCCT GAAAGATGACTCTATCGACAACAAAGTGTTGACGCGATCA GATAAGAACCGGGGAAAATCCGACAATGTACCCTCAGAAG AAGTTGTCAAGAAGATGAAAAACTATTGGAGACAATTGCT GAACGCCAAGCTCATAACACAACGCAAGTTCGATAACTTG ACGAAAGCCGAAAGAGGTGGGTTGTCAGAATTGGACAAAG CTGGCTTTATTAAGCGCCAATTGGTGGAGACCCGGCAGAT TACGAAACACGTAGCACAAATTTTGGATTCACGAATGAAT ACCAAATACGACGAAAACGACAAATTGATACGCGAGGTGA AAGTGATTACGCTTAAGAGTAAGTTGGTTTCCGATTTCAG GAAGGATTTTCAGTTTTACAAAGTAAGAGAAATAAACAAC TACCACCACGCCCATGATGCTTACCTCAACGCGGTAGTTG GCACAGCTCTTATCAAAAAATATCCAAAGCTGGAAAGCGA GTTCGTTTACGGTGACTATAAAGTATACGACGTTCGGAAG ATGATAGCCAAATCAGAGCAGGAAATTGGGAAGGCAACCG CAAAATACTTCTTCTATTCAAACATCATGAACTTCTTTAA GACGGAGATTACGCTCGCGAACGGCGAAATACGCAAGAGG CCCCTCATAGAGACTAACGGCGAAACCGGGGAGATCGTAT GGGACAAAGGACGGGACTTTGCGACCGTTAGAAAAGTACT TTCAATGCCACAAGTGAATATTGTTAAAAAGACAGAAGTA CAAACAGGGGGGTTCAGTAAGGAATCCATTTTGCCCAAGC GGAACAGTGATAAATTGATAGCAAGGAAAAAAGATTGGGA CCCTAAGAAGTACGGTGGTTTCGACTCTCCTACCGTTGCA TATTCAGTCCTTGTAGTTGCGAAAGTGGAAAAGGGGAAAA GTAAGAAGCTTAAGAGTGTTAAAGAGCTTCTGGGCATAAC CATAATGGAACGGTCTAGCTTCGAGAAAAATCCAATTGAC TTTCTCGAGGCTAAAGGTTACAAGGAGGTAAAAAAGGACC TGATAATTAAACTCCCAAAGTACAGTCTCTTCGAGTTGGA GAATGGGAGGAAGAGAATGTTGGCATCTGCAGGGGAGCTC CAAAAGGGGAACGAGCTGGCTCTGCCTTCAAAATACGTGA ACTTTCTGTACCTGGCCAGCCACTACGAGAAACTCAAGGG TTCTCCTGAGGATAACGAGCAGAAACAGCTGTTTGTAGAG CAGCACAAGCATTACCTGGACGAGATAATTGAGCAAATTA GTGAGTTCTCAAAAAGAGTAATCCTTGCAGACGCGAATCT GGATAAAGTTCTTTCCGCCTATAATAAGCACCGGGACAAG CCTATACGAGAACAAGCCGAGAACATCATTCACCTCTTTA CCCTTACTAATCTGGGCGCGCCGGCCGCCTTCAAATACTT CGACACCACGATAGACAGGAAAAGGTATACGAGTACCAAA GAAGTACTTGACGCCACTCTCATCCACCAGTCTATAACAG GGTTGTACGAAACGAGGATAGATTTGTCCCAGCTCGGCGG CGACTCAGGAGGGTCAGGCGGCTCCGGTGGATCAACGAAT CTTTCCGACATAATCGAGAAAGAAACCGGCAAACAGTTGG TGATCCAAGAATCAATCCTGATGCTGCCTGAAGAAGTAGA AGAGGTGATTGGCAACAAACCTGAGTCTGACATTCTTGTC CACACCGCGTATGACGAGAGCACGGACGAGAACGTTATGC TTCTCACTAGCGACGCCCCTGAGTATAAACCATGGGCGCT G GTCATCCAAGATTCCAATGGGGAAAACAAGATTAAGATGC TTAGTGGTGGGTCTGGAGGGAGCGGTGGGTCCACGAACCT CAGCGACATTATTGAAAAAGAGACTGGTAAACAACTTGTA ATACAAGAGTCTATTCTGATGTTGCCTGAAGAGGTGGAGG AGGTGATTGGGAACAAACCGGAGTCTGATATACTTGTTCA TACCGCCTATGACGAATCTACTGATGAGAATGTGATGCTT TTaACGTCAGACGCTCCCGAGTACAAACCCTGGGCTCTGG TGATTCAGGACAGCAATGGTGAGAATAAGATTAAAATGTT GAGTGGGGGCTCAAAGCGCACGGCTGACGGTAGCGAATTT GAGAGCCCCAAAAAAAAACGAAAGGTCGAAtaa

[0087] BE4コドン optimization2 nucleic acid sequence: ATGAGCAGCGAGACAGGCCCTGTGGCTGTGGATCCTACAC TGCGGAGAGAATCGAGCCCCACGAGTTCGAGGTGTTCTT CGACCCCAGAGAGCTGCGGAAAGAGACATGCCTGCTGTAC GAGATCAACTGGGGCGGCAGACACTCTATCTGGCGGCACA CAAGCCAGAACACCAACAAGCACGTGGAAGTGAACTTTAT CGAGAAGTTTACGACCGAGCGGTACTTCTGCCCCAACACC AGATGCAGCATCACCTGGTTCTGAGCTGGTCCCCTTGCG GCGAGTGCAGCAGAGCCATCACCGAGTTTCTGTCCAGATA TCCCCACGTGACCCTGTTCATCTATATCGCCCGGCTGTAC CACCACGCCGATCCTAGAAATAGACAGGGACTGCGCGACC TGATCAGCAGCGGAGTGACCATCCAGATCATGACCGAGCA AGAGAGCGGCTACTGCTGGCGGAACTTCGTGAACTACAGC CCCAGCAACGAAGCCCACTGGCCTAGATATCCTCACCTGT GGGTCCGACTGTACGTGCTGGAACTGTACTGCATCATCCT GGGCCTGCCTCCATGCCTGAAACATCCTGAGAAGAAAGCAG CCTCAGCTGACCTTCTTCCACAATCGCCCTGCAGAGCTGCC ACTACCAGAGACTGCCTCCCACACATCCTGTGGGCCACCGG ACTTAAGAGCGGAGGATCTAGCGGCGCTCTAGCGGATCT GAGACACCTGGCACAAGCGAGTCTGCCACACCTGAGAGTA GCGGCGGATCTTCTGGCGGCTCCGACAAGAAGTACTCTAT CGGACTGGCCATCGGCACCAACTCTGTTGGATGGGCCGTG ATCACCGACGAGTACAAGGTGCCCAGCAAGAAATTCAAGG TGCTGGGCAACACCGACCGGCACAGCATCAAGAAGAATCT GATCGGCGCCCTGCTGTTCGACTCTGGCGAAACAGCCGAA GCCACCAGACTGAAGAGAACCGCCAGGCGGAGATACACCC GGCGGAAGAACCGGATCTGCTACCTGCAAGAGATCTTCAG CAACGAGATGGCCAAGGTGGACGACAGCTTCTTCCACAGA CTGGAAGAGTCCTTCCTGGTGGAAGAGGACAAGAAGCACG AGCGGCACCCCATCTTCGGCAACATCGTGGATGAGGTGGC CTACCACGAGAAGTACCCCACCATCTACCACCTGAGAAAG AAACTGGTGGACAGCACCGACAAGGCCGACCTGAGACTGA TCTACCTGGCTCTGGCCCACATGATCAAGTTCCGGGGCCA CTTTCTGATCGAGGGCGATCTGAACCCCGACAACAGCGAC GTGGACAAGCTGTTCATCCAGCTGGTGCAGACCTACAACC AGCTGTTCGAGGAAAACCCCATCAACGCCTCTGGCGTGGA CGCCAAGGCTATCCTGTCTGCCAGACTGAGCAAGAGCAGA AGGCTGGAAAACCTGATCGCCCAGCTGCCTGGCGAGAAGA AGAATGGCCTGTTCGGCAACCTGATTGCCCTGAGCCTGGG ACTGACCCCTAACTTCAAGAGCAACTTCGACCTGGCCGAG GATGCCAAACTGCAGCTGAGCAAGGACACCTACGACGACG ACCTGGACAATCTGCTGGCCCAGATCGGCGATCAGTACGC CGACTTGTTTCTGGCCGCCAAGAACCTGTCCGACGCCATC CTGCTGAGCGATATCCTGAGAGTGAACACCGAGATCACAA AGGCCCCTCTGAGCGCCTCTATGATCAAGAGATACGACGA GCACCACCAGGATCTGACCCTGCTGAAGGCCCTCGTTAGA CAGCAGCTGCCAGAGAAGTACAAAGAGATTTTCTTCGATC AGTCCAAGAACGGCTACGCCGGCTACATTGATGGCGGAGC CAGCCAAGAGGAATTCTACAAGTTCATCAAGCCCATCCTG GAAAAGATGGACGGCACCGAGGAACTGCTGGTCAAGCTGA ACAGAGAGGACCTGCTGCGGAAGCAGCGGACCTTCGACAA TGGCTCTATCCCTCACCAGATCCACCTGGGAGAGCTGCAC GCCATTCTGCGGAGACAAGAGGACTTTTACCCATTCCTGA AGGACAACCGGGAAAAGATCGAGAAGATCCTGACCTTCAG GATCCCCTACTACGTGGGACCACTGGCCAGAGGCAATAGC AGATTCGCCTGGATGACCAGAAAGAGCGAGGAAACCATCA CACCCTGGAACTTCGAGGAAGTGGTGGACAAGGGCGCCAG CGCTCAGTCCTTCATCGAGCGGATGACCAACTTCGATAAG AACCTGCCTAACGAGAAGGTGCTGCCCAAGCACTCCCTGC TGTATGAGTACTTCACCGTGTACAACGAGCTGACCAAAGT GAAATACGTGACCGAGGGAATGAGAAAGCCCGCCTTTCTG AGCGGCGAGCAGAAAAAGGCCATTGTGGATCTGCTGTTCA AGACCAACCGGAAAGTGACCGTGAAGCAGCTGAAAGAGGA CTACTTCAAGAAAATCGAGTGCTTCGACAGCGTGGAAATC AGCGGCGTGGAAGATCGGTTCAATGCCAGCCTGGGCACAT ACCACGACCTGCTGAAAATTATCAAGGACAAGGACTTCCT GGACAACGAAGAGAACGAGGACATTCTCGAGGACATCGTG CTGACCCTGACACTGTTTGAGGACAGAGAGATGATCGAGG AACGGCTGAAAACATACGCCCACCTGTTCGACGACAAAGT GATGAAGCAACTGAAGCGGAGGCGGTACACAGGCTGGGGC AGACTGTCTCGGAAGCTGATCAACGGCATCCGGGATAAGC AGTCCGGCAAGACAATCCTGGATTTCCTGAAGTCCGACGG CTTCGCCAACAGAAACTTCATGCAGCTGATCCACGACGAC AGCCTGACCTTTAAAGAGGACATCCAGAAAGCCCAGGTGT CCGGCCAAGGCGATTCTCTGCACGAGCACATTGCCAACCT GGCCGGATCTCCCGCCATTAAGAAGGGCATCCTGCAGACA GTGAAGGTGGTGGACGAGCTTGTGAAAGTGATGGGCAGAC ACAAGCCCGAGAACATCGTGATCGAAATGGCCAGAGAGAA CCAGACCACACAGAAGGGCCAGAAGAACAGCCGCGAGAGA ATGAAGCGGATCGAAGAGGGCATCAAAGAGCTGGGCAGCC AGATCCTGAAAGAACACCCCGTGGAAAACACCCAGCTGCA GAACGAGAAGCTGTACCTGTACTACCTGCAGAATGGACGG GATATGTACGTGGACCAAGAGCTGGACATCAACCGGCTGA GCGACTACGATGTGGACCATATCGTGCCCCAGAGCTTTCT GAAGGACGACTCCATCGATAACAAGGTCCTGACCAGAAGC GACAAGAACCGGGGCAAGAGCGATAACGTGCCCTCCGAAG AGGTGGTCAAGAAGATGAAGAACTACTGGCGACAGCTGCT GAACGCCAAGCTGATTACCCAGCGGAAGTTCGATAACCTG ACCAAGGCCGAGAGAGGCGGCCTGAGCGAACTTGATAAGG CCGGCTTCATTAAGCGGCAGCTGGTGGAAACCCGGCAGAT CACCAAACACGTGGCACAGATTCTGGACTCCCGGATGAAC ACTAAGTACGACGAGAATGACAAGCTGATCCGGGAAGTGA AAGTCATCACCCTGAAGTCTAAGCTGGTGTCCGATTTCCG GAAGGATTTCCAGTTCTACAAAGTGCGGGAAATCAACAAC TACCATCACGCCCACGACGCCTACCTGAATGCCGTTGTTG GAACAGCCCTGATCAAGAAGTATCCCAAGCTGGAAAGCGA GTTCGTGTACGGCGACTACAAGGTGTACGACGTGCGGAAG ATGATCGCCAAGAGCGAACAAGAGATCGGCAAGGCTACCG CCAAGTACTTTTTCTACAGCAACATCATGAACTTTTTCAA GACAGAGATCACCCTGGCCAACGGCGAGATCCGGAAAAGA CCCCTGATCGAGACAAACGGCGAAACCGGGGAGATCGTGT GGGATAAGGGCAGAGATTTTGCCACAGTGCGGAAAGTGCT GAGCATGCCCCAAGTGAATATCGTGAAGAAAACCGAGGTG CAGACAGGCGGCTTCAGCAAAGAGTCTATCCTGCCTAAGC GGAACAGCGATAAGCTGATCGCCAGAAAGAAGGACTGGGA CCCTAAGAAGTACGGCGGCTTCGATAGCCCTACCGTGGCC TATTCTGTGCTGGTGGTGGCCAAAGTGGAAAAGGGCAAGT CCAAAAAGCTCAAGAGCGTGAAAGAGCTGCTGGGGATCAC CATCATGGAAAGAAGCAGCTTTGAGAAGAACCCGATCGAC TTTCTGGAAGCCAAGGGCTACAAAGAAGTCAAGAAGGACC TCATCATCAAGCTCCCCAAGTACAGCCTGTTCGAGCTGGA AAATGGCCGGAAGCGGATGCTGGCCTCAGCAGGCGAACTG CAGAAAGGCAATGAACTGGCCCTGCCTAGCAAATACGTCA ACTTCCTGTACCTGGCCAGCCACTATGAGAAGCTGAAGGG CAGCCCCGAGGACAATGAGCAAAAGCAGCTGTTTGTGGAA CAGCACAAGCACTACCTGGACGAGATCATCGAGCAGATCA GCGAGTTCTCCAAGAGAGTGATCCTGGCCGACGCTAACCT GGATAAGGTGCTGTCTGCCTATAACAAGCACCGGGACAAG CCTATCAGAGAGCAGGCCGAGAATATCATCCACCTGTTTA CCCTGACCAACCTGGGAGCCCCTGCCGCCTTCAAGTACTT CGACACCACCATCGACCGGAAGAGGTACACCAGCACCAAA GAGGTGCTGGACGCCACACTGATCCACCAGTCTATCACCG GCCTGTACGAAACCCGGATCGACCTGTCTCAGCTCGGCGG CGATTCTGGTGGTTCTGGCGGAAGTGGCGGATCCACCAAT CTGAGCGACATCATCGAAAAAGAGACAGGCAAGCAGCTCG TGATCCAAGAATCCATCCTGATGCTGCCTGAAGAGGTTGA GGAAGTGATCGGCAACAAGCCTGAGTCCGACATCCTGGTG CACACCGCCTACGATGAGAGCACCGATGAGAACGTCATGC TGCTGACAAGCGACGCCCCTGAGTACAAGCCTTGGGCTCT C GTGATTCAGGACAGCAATGGGGAGAACAAGATCAAGATGC TGAGCGGAGGTAGCGGAGGCAGTGGCGGAAGCACAAACCT GTCTGATATCATTGAAAAAGAAACCGGGAAGCAACTGGTC ATTCAAGAGTCCATTCTCATGCTCCCGGAAGAAGTCGAGG AAGTCATTGGAAACAAACCCGAGAGCGATATTCTGGTCCA CACAGCCTATGACGAGTCTACAGACGAAAACGTGATGCTC CTGACCTCTGACGCTCCCGAGTATAAGCCCTGGGCACTTG TTATCCAGGACTCTAACGGGGGAAAACAAAATCAAAATGTT GTCCGGCGGCAGCAAGCGGACAGCCGATGGATCTGAGTTC GAGAGCCCCAAGAAGAAACGGAAGGTgGAGtaa

[0088] "Base editing activity" refers to the ability to chemically change bases within a polynucleotide. In one embodiment, the first base is converted to a second base. In this state, the base editing activity is a cytidine deaminase activity, e.g., converting the target C·G to T· In another embodiment, the base editing activity is adenosine deaminase activity or adenine deaminase activity, e.g., converting A·T to G·C. base editing activity is mediated by cytidine deaminase activity (e.g., converting the target C·G to T·A). ), and adenosine deaminase activity or adenine deaminase activity (e.g., A (converting .T to GC).

[0089] In some embodiments, base editing activity is assessed by the efficiency of editing. is measured by any suitable means (e.g., Sanger sequencing or next-generation sequencing) In some embodiments, the base editing efficiency can be measured by the nucleic acid sequence achieved by the base editor. The percentage of all sequencing reads with acid-base conversion (e.g., target A·T base pair becomes G· Whole sequencing with C base pairs converted or target C·G base pairs converted to T·A base pairs In some embodiments, the base editing efficiency is measured by: When base editing is performed in a cell population, the nucleic acid bases produced by the base editor It is measured by the percentage of total cells that have converted.

[0090] The term "base editor system" refers to a system that edits nucleic acid bases in a target nucleotide sequence. In various embodiments, the base editor system refers to a system for: (1) generating a polynucleotide sequence; (2) a programmable nucleotide binding domain (e.g., Cas9); a deaminase domain for deaminating the nucleic acid base (e.g., adenosine deaminase) (3) one or more guide polynucleotides ( In some embodiments, the polynucleotide programmable The nucleotide binding domain is a polynucleotide programmable DNA binding domain. In some embodiments, the base editor is a cytidine base editor (CBE). In some embodiments, the base editor is an adenine or adenosine base editor ( In some embodiments, the base editor system is an adenosine deaminase (ABE). The first is ABE8, an enzyme base editor.

[0091] In some embodiments, ABE8 is a monomeric construct. 8 is ABE8.1-m, ABE8.2-m, ABE8.3-m, ABE8.4-m, A BE8.5-m, ABE8.6-m, ABE8.7-m, ABE8.8-m, ABE8. 9-m, ABE8.10-m, ABE8.11-m, ABE8.12-m, ABE8.1 3-m, ABE8.14-m, ABE8.15-m, ABE8.16-m, ABE8.1 7-m, ABE8.18-m, ABE8.19-m, ABE8.20-m, ABE8.2 1-m, ABE8.22-m, ABE8.23-m, ABE8.24-m. In embodiments, ABE8 is a heteromeric construct. In some embodiments, ABE8 is , ABE8.1-d, ABE8.2-d, ABE8.3-d, ABE8.4-d, ABE 8.5-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.

[0092] In some embodiments, a base editor system may comprise two or more base editing components. For example, a base editor system can include two or more deaminases. In embodiments, the base editor system may comprise one or more cytidine deaminases In some embodiments, the base editor system comprises one or more adenosine deaminases In some embodiments, a single guide polynucleotide may be utilized to generate a different The deaminase may be targeted to a target nucleic acid sequence. In some embodiments, a pair of guide Polynucleotides can be used to target different deaminases to target nucleic acid sequences. good.

[0093] Nucleic acid base components of base editor systems and polynucleotide programmable nucleases The oxidase-binding moieties may be covalently or non-covalently bound to each other or their association and The interaction can be any combination of the above. For example, in some embodiments, , the deaminase domain is a polynucleotide programmable nucleotide binding domain In some embodiments, the polynucleotide may be targeted to a target nucleotide sequence. The nucleotide-programmable binding domain is fused to a deaminase domain or In some embodiments, the polynucleotide may be linked to a programmable nucleotide linker. The binding domain interacts non-covalently with the deaminase domain or By associating with the deaminase domain, the deaminase domain is targeted to the target nucleotide sequence. For example, in some embodiments, a nucleobase editing component (e.g., , deaminase domain) can contain additional heterologous moieties or domains, additional heterologous moieties that are part of the oligonucleotide programmable nucleotide binding domain or or heterologous domains may interact with, associate with, or form complexes with, a heterologous domain. In some embodiments, the additional heterologous moiety may be attached to the polypeptide or may be coupled to the polypeptide. capable of interacting with, associating with, or forming a complex with a polypeptide In some embodiments, the additional heterologous moiety can be attached to the polynucleotide. or capable of interacting with, associating with, or binding to a polynucleotide. In some embodiments, the additional heterologous moiety may be capable of forming a complex with the oligonucleotide. In some embodiments, the additional heterologous In some embodiments, the moiety may be attached to a polypeptide linker. In some cases, the heterologous moiety may be attached to a polynucleotide linker. In some embodiments, the additional heterologous moiety may be a protein domain. domain, MS2 coat protein domain, PP7 coat protein domain, SfM u Com coat protein domain, steril α motif, telomerase telomerase Ku binding motif and Ku protein, telomerase Sm7 binding motif and S It may be the m7 protein, or an RNA recognition motif.

[0094] The base editor system may further comprise a guide polynucleotide component. The components of the editor system may be linked by covalent bonds, non-covalent interactions, or associations thereof. It is understood that the molecules may associate with each other through any combination of these interactions. In embodiments, the deaminase domain is cleaved by the guide polynucleotide to the target nucleoside. For example, in some embodiments, a base editor system can be used to target a base sequence. The nucleobase editing component (e.g., deaminase domain) of the Polynucleoside receptors such as RNA-binding proteins or DNA-binding proteins A portion or segment of the guide polynucleotide may comprise a nucleic acid sequence (e.g., a nucleic acid sequence), ... can interact with, associate with, or form complexes with a target (e.g., a polynucleotide motif) In some embodiments, additional heterologous moieties or domains (e.g., Polynucleotide-binding domains, such as RNA- or DNA-binding proteins ) may be fused or linked to the deaminase domain. The species moiety may be capable of binding to, interacting with, or interacting with a polypeptide. In some embodiments, the polypeptide may be associated with or complexed with a polypeptide. In this embodiment, the additional heterologous moiety may be attached to or interact with the polynucleotide. Able to act on, associate with, or form a complex with a polynucleotide In some embodiments, the additional heterologous moiety may be a guide polynucleotide. In some embodiments, the additional heterologous moiety may be linked to a polypeptide linker. In some embodiments, the additional heterologous moiety may be capable of binding to a polynucleotide. In some cases, it may be attached to a linker. The additional heterologous moiety may be a protein domain. In some embodiments, the additional heterologous moiety is a K homology (KH) domain, an MS2 coat protein, or a nucleotide sequence. Protein domain, PP7 coat protein domain, SfMu Com coat protein domain, steryl α motif, telomerase Ku binding motif and Ku protein, Telomerase Sm7 binding motif and Sm7 protein or RNA recognition motif could be.

[0095] In some embodiments, the base editor system comprises an inhibitor of base excision repair (BER) components. The components of the base editor system may further comprise a covalently or non-covalently linked agent. can associate with one another through interaction or any combination of these associations and interactions It should be understood that the inhibitor of the BER component may include a BER inhibitor. In its most basic form, the inhibitor of BER is the uracil DNA glycosylase inhibitor (UGI). In some embodiments, the inhibitor of BER may be an inosine BER inhibitor. In some embodiments, the inhibitor of BER is a polynucleotide programmable nucleoside. The nucleotide binding domain can be targeted to a target nucleotide sequence. In this state, the polynucleotide programmable nucleotide binding domain acts as an inhibitor of BER. In some embodiments, the polynucleotide programmable nucleic acid may be fused or linked to a nucleic acid. The nucleotide-binding domain is fused or linked to the deaminase domain and the BER inhibitor. In some embodiments, the polynucleotide programmable nucleotide binding domain The main one is either non-covalently interacting with or binding to the BER inhibitor. By associating with the target nucleotide sequence, the BER inhibitor is targeted to the target nucleotide sequence. For example, in some embodiments, the inhibitor of a BER component can be an additional heterologous moiety or or heterologous domains, and the polynucleotide may comprise a programmable nucleotide binding domain. It may interact with or associate with additional heterologous moieties or heterologous domains that are part of the domain. or may form a complex.

[0096] In some embodiments, the inhibitor of BER is a target nucleic acid sequence that is linked to the target nucleic acid sequence by a guide polynucleotide. For example, in some embodiments, inhibitors of BER can be targeted to nucleotide sequences. may contain additional heterologous moieties or domains (e.g., RNA-binding proteins or DNA-binding The guide polynucleotide can include a polynucleotide binding domain (e.g., a polynucleotide binding domain of a ligation protein), can interact with a portion or segment of a nucleic acid sequence (e.g., a polynucleotide motif) In some embodiments, the guide polynucleotide may be Additional heterologous moieties or heterologous domains of the nucleotides (e.g., RNA-binding proteins or D Polynucleotide-binding domains (e.g., NA-binding proteins) are fused to or attached to inhibitors of BER. In some embodiments, the additional heterologous moiety may be attached to the polynucleotide. can obtain, interact with, associate with, or bind to a polynucleotide; In some embodiments, the additional heterologous The moiety may be capable of binding to a guide polynucleotide. The heterologous moiety may be attached to a polypeptide linker. The heterologous moiety may be attached to the polynucleotide linker. In some embodiments, the additional heterologous moiety may be a K homology (K H) domain, MS2 coat protein domain, PP7 coat protein domain, S fMu Com coat protein domain, steryl α motif, telomerase Ku binding motif and Ku protein, telomerase Sm7 binding motif and Sm7 protein The amino acid sequence may be a protein or an RNA recognition motif.

[0097] "β2 microglobulin (B2M) polypeptide" refers to the polypeptide identified in UniProt accession number P 61769 or a fragment thereof, and Exemplary B2M polypeptide sequences are provided below. . >sp|P61769|B2MG_HUMAN β2 microglobulin OS=Hom o sapiens OX=9606 GN=B2M PE=1 SV=1 MSRSVALAVLALLSLSGLEAIQRTPKIQVYSRHPAENGKS NFLNCYVSGFHPSDIEVDLLKNGERIEKVEHSDLSFSKDW SFYLLYYTEFTPTEKDEYACRVNHVTLSQPKIVKWDRDM

[0098] "β2 microglobulin (B2M) polynucleotide" refers to a polynucleotide that encodes a B2M polypeptide. The β2-microglobulin gene is a nucleic acid molecule encoding a major histocompatibility complex (MCC) B2M encodes a serum protein associated with B2M, which is involved in the recognition of non-self by host CD8+ T cells. Exemplary B2M polynucleotide sequences are provided below. >DQ217933.1 Homo sapiens β2 microglobin (B2M) gene Denji, full-length CDS CATGTCATAAATGGTAAGTCCAAGAAAAATACAGGTATTC CCCCCCAAAGAAAACTGTAAATCGACTTTTTTCTATCTG TACTGTTTTTTATTGGTTTTTAAATTGGTTTTCCAAGTGA GTAAATCAGAATCTATCTGTAATGGATTTTAAATTTAGTG TTTCTCTGTGATGTAGTAAACAAGAAACTAGAGGCAAAAA TAGCCCTGTCCCTTGCTAAACTTCTAAGGCACTTTTCTAG TACAACTCAACACTAACATTTCAGGCCTTTAGTGCCTTAT ATGAGTTTTTAAAAGGGGGAAAAGGGAGGGAGCAAGAGTG TCTTAACTCATACATTTAGGCATAACAATTATTCTCATAT TTTAGTTATTGAGAGGGCTGGTAGAAAAACTAGGTAAATA ATATTAATAATTATAGCGCTTATTAAACACTACAGAACAC TTACTATGTACCAGGCATTGTGGGAGGCTCTCTCTTGTGC ATTATCTCATTTCATTAGGTCCATGGAGAGTATTGCATTT TCTTAGTTTAGGCATGGCCTCCACAATAAAGATTATCAAA AGCCTAAAAATATGTAAAAGAAACCTAGAAGTTATTTGTT GTGCTCCTTGGGGAAGCTAGGCAAATCCTTTCAACTGAAA ACCATGGTGACTTCCAAGATCTCTGCCCCTCCCCATCGCC ATGGTCCACTTCCTCTTCTCACTGTTCCTCTTAGAAAAGA TCTGTGGACTCCACCACCACGAAATGGCGGCACCTTATTT ATGGTCACTTTAGAGGGTAGGTTTTCTTAATGGGTCTGCC TGTCATGTTTAACGTCCTTGGCTGGGTCCAAGGCAGATGC AGTCCAAACTCTCACTAAAATTGCCGAGCCCTTTGTCTTC CAGTGTCTAAAATATTAATGTCAATGGAATCAGGCCAGAG TTTGAATTCTAGTCTCTTAGCCTTTGTTTCCCCTGTCCAT AAAATGAATGGGGGTAATTCTTTCCTCCTACAGTTTATTT ATATATTCACTAATTCATTCATTCATCCATCCATTCGTTC ATTCGGTTTACTGAGTACCTACTATGTGCCAGCCCCTGTT CTAGGGTGGAAACTAAGAGAATGATGTACCTAGAGGGCGC TGGAAGCTCTAAAGCCCTAGCAGTTACTGCTTTTACTATT AGTGGTCGTTTTTTTCTCCCCCCCGCCCCCCGACAAATCA ACAGAACAAAGAAAATTACCTAAACAGCAAGGACATAGGG AGGAACTTCTTGGCACAGAACTTTCCAAACACTTTTTCCT GAAGGGATACAAGAAGCAAGAAAGGTACTCTTTCACTAGG ACCTTCTCTGAGCTGTCCTCAGGATGCTTTTGGGACTATT TTTCTTACCCAGAGAATGGAGAAACCCTGCAGGGAATTCC CAAGCTGTAGTTATAAACAGAAGTTCTCCTTCTGCTAGGT AGCATTCAAAGATCTTAATCTTCTGGGTTTCCGTTTTCTC GAATGAAAAATGCAGGTCCGAGCAGTTAACTGGCTGGGGC ACCATTAGCAAGTCACTTAGCATCTCTGGGGCCAGTCTGC AAAGCGAGGGGGCAGCCTTAATGTGCCTCCAGCCTGAAGT CCTAGAATGAGCGCCCGGTGTCCCAAGCTGGGGCGCGCAC CCCAGATCGGAGGGCGCCGATGTACAGACAGCAAACTCAC CCAGTCTAGTGCATGCCTTCTTAAACATCACGAGACTCTA AGAAAAGGAAACTGAAAACGGGAAAGTCCCTCTCTCTAAC CTGGCACTGCGTCGCTGGCTTGGAGACAGGTGACGGTCCC TGCGGGCCTTGTCCTGATTGGCTGGGCACGCGTTTAATAT AAGTGGAGGCGTCGCGCTGGCGGGCATTCCTGAAGCTGAC AGCATTCGGGCCGAGATGTCTCGCTCCGTGGCCTTAGCTG TGCTCGCGCTACTCTCTCTTTCTGGCCTGGAGGCTATCCA GCGTGAGTCTCTCCTACCCTCCCGCTCTGGTCCTTCCTCT CCCGCTCTGCACCCTCTGTGGCCCTCGCTGTGCTCTCTCG CTCCGTGACTTCCCTTCTCCAAGTTCTCCTTGGTGGCCCG CCGTGGGGCTAGTCCAGGGCTGGATCTCGGGGAAGCGGCG GGGTGGCCTGGGAGTGGGGAAGGGGGTGCGCACCCGGGAC GCGCGCTACTTGCCCCTTTCGGCGGGGAGCAGGGGAGACC TTTGGCCTACGGCGACGGGAGGGTCGGGACAAAGTTTAGG GCGTCGATAAGCGTCAGAGCGCCGAGGTTGGGGGAGGGTT TCTCTTCCGCTCTTTCGCGGGGCCTCTGGCTCCCCCAGCG CAGCTGGAGTGGGGGACGGGTAGGCTCGTCCCAAAGGCGC GGCGCTGAGGTTTGTGAACGCGTGGAGGGGCGCTTGGGGT CTGGGGGAGGCGTCGCCCGGGTAAGCCTGTCTGCTGCGGC TCTGCTTCCCTTAGACTGGAGAGCTGTGGACTTCGTCTAG GCGCCCGCTAAGTTCGCATGTCCTAGCACCTCTGGGTCTA TGTGGGGCCACACCGTGGGGAGGAAACAGCACGCGACGTT TGTAGAATGCTTGGCTGTGATACAAAGCGGTTTCGAATAA TTAACTTATTTGTTCCCATCACATGTCACTTTTAAAAAAT TATAAGAACTACCCGTTATTGACATCTTTCTGTGTGCCAA GGACTTTATGTGCTTTGCGTCATTTAATTTTGAAAACAGT TATCTTCCGCCATAGATAACTACTATGGTTATCTTCTGCC TCTCACAGATGAAGAAACTAAGGCACCGAGATTTTAAGAA ACTTAATTACACAGGGGATAAATGGCAGCAATCGAGATTG AAGTCAAGCCTAACCAGGGCTTTTGCGGGAGCGCATGCCT TTTGGCTGTAATTCGTGCATTTTTTTTTAAGAAAAACGCC TGCCTTCTGCGTGAGATTCTCCAGAGCAAACTGGGCGGCA TGGGCCCTGTGGTCTTTTCGTACAGAGGGCTTCCTCTTTG GCTCTTTGCCTGGTTGTTTCCAAGATGTACTGTGCCTCTT ACTTTCGGTTTTGAAAACATGAGGGGGTTGGGCGTGGTAG CTTACGCCTGTAATCCCAGCACTTAGGGAGGCCGAGGCGG GAGGATGGCTTGAGGTCCGTAGTTGAGACCAGCCTGGCCA ACATGGTGAAGCCTGGTCTCTACAAAAAATAATAACAAAA ATTAGCCGGGTGTGGTGGCTCGTGCCTGTGGTCCCAGCTG CTCCGGTGGCTGAGGCGGGAGGATCTCTTGAGCTTAGGCT TTTGAGCTATCATGGCGCCAGTGCACTCCAGCGTGGGCAA CAGAGCGAGACCCTTGTCTCTCAAAAGAAAAAAAAAA AAAGAAAGAGAAGAAGAAGAAGAAAGAAGAAGTGAAGGTTT GTCAGTCAGGGGAGCTGTAAAACCATTAATAAAGATAATC CAAGATGGTTACCAAGACTGTTGAGGACGCCAGAGATCTT GAGCACTTTCTAAGTACCTGGCAATACACTAAGCGCGCTC ACCTTTTCCTCTGGCAAAACATGATCGAAAGCAGAATGTT TTGATCATGAGAAAATTGCATTTAATTTGAATACAATTTA TTTACAACATAAAGGATAATGTATATATCACCACCATTAC TGGTATTTGCTGGTTATGTTAGATGTCATTTTTAAAAAATA ACAATCTGATATTTAAAAAAATCTTATTTTGAAAATTT CCAAAGTAATACATGCCATGCATAGACCATTTCTGGAAGA TACCACAAGAAACATGTAATGATGATTGCCTTCTGAAGGTC TATTTTCCTCCTCTGACCTGTGTGTGGGTTTTGTTTTTGT TTTACTGTGGGCATAAATTAATTTTTCAGTTAAGTTTTGG AAGCTTAAATAACTCTCCAAAAGTCATAAAGCCAGTAACT GGTTGAGCCCAAATTCAAACCCAGCCTGTCTGATACTTGT CCTCTTCTTAGAAAAGATTACAGTGATGCTCTCACAAAAAT CTTGCCGCCTTCCCTCAAACAGAGAGTTCCAGGCAGGATG AATCTGTGCTCTGATCCTGAGGCATTTAATATGTTCTTA TTATTAGAGCTCAGATGCAAAGAGCTCTCTTAGCTTTTA ATGTTATGAAAAAATCAGGTCTTCATTAGATTCCCCAAT CCACCTCTTGATGGGGCTAGTAGCCTTTCCTTAATGATAG GGTGTTTCTAGAGAGATATATCTGGTCAAGGTGGCCTGGT ACTCCTCCTTCTCCCACAGCCTCCCCAGACAAGGAGGAGT AGCTGCCTTTTAGTGATCATGTACCCTGAATATAAGTGTA TTTAAAGAATTTTATACACATATATTTAGTGTCAATCTG TATATTTAGTAGCACTAACACTTCTCTTCATTTTCAATGA AAAATATAGGTTTATAATATTTTCTTCCCACTTCCCCAT GGATGGTCTAGTCATGCCTCTCATTTTGGAAAGTACTGTT TCTGAAACATTAGGCAATATATTCCCAACCTGGCTAGTTT ACAGCAATCACCTGTGGATGCTAATTAAACGCAAATCCC ACTGTCACATGCATTACTCCATTTGATCATAATGGAAAGT ATGTTCTGTCCCATTTGCCATAGTCCTCACCTATCCCTGT TGTATTTTATCGGGTCCAACTCAACCATTTAAGGTATTTG CCAGCTCTTGTATGCATTTAGGTTTTGTTTCTTTGTTTTT TAGCTCATGAAATTAGGTACAAAGTCAGAGAGGGGTCTGG CATATAAAACCTCAGCAGAAATAAAGAGGTTTTGTTGTTT GGTAAGAACATACCTTGGGTTGGTTGGGCACGGTGGCTCG TGCCTGTAATCCCAACACTTTGGGAGGCCAAGGCAGGCTG ATCACTTGAAGTTGGGAGTTCAAGACCAGCCTGGCCAACA TGGTGAAATCCCGTCTCTACTGAAAATACAAAAATTAACC AGGCATGGTGGTGTGTGCCTGTAGTCCCAGGAATCACTTG AACCCAGGAGGCGGAGGTTGCAGTGAGCTGAGATCTCACC ACTGCACACTGCACTCCAGCCTGGGCAATGGAATGAGATT CCATCCCAAAAAATAAAAAAATAAAAAAATAAAGAACATA C CTTGGGTTGATCCACTTAGGAACCTCAGATAATAACATCT GCCACGTATAGAGCAATTGCTATGTCCCAGGCACTCTACT AGACACTTCATACAGTTTAGAAAATCAGATGGGTGTAGAT CAAGGCAGGAGCAGGAACCAAAAAGAAAGGCATAAACATA AGAAAAAAAATGGAAGGGGTGGAAACAGAGTACAATAACA TGAGTAATTTGATGGGGGCTATTATGAACTGAGAAATGAA CTTTGAAAAGTATCTTGGGGCCAAATCATGTAGACTCTTG AGTGATGTGTTAAGGAATGCTATGAGTGCTGAGAGGGCAT CAGAAGTCCTTGAGAGCCTCCAGAGAAAGGCTCTTAAAAA TGCAGCGCAATCTCCAGTGACAGAAGATACTGCTAGAAAT CTGCTAGAAAAAACAAAAAAGGCATGTATAGAGGAATT ATGAGGGAAAGATACCAAGTCACGGTTTATTCTTCAAAAT GGAGGTGGCTTGTTGGGAAGGTGGAAGCTCATTTGGCCAG AGTGGAAATGGAATTGGGAGAAATCGATGACCAAATGTAA ACACTTGGTGCCTGATATAGCTTGACACCAAGTTAGCCCC AAGTGAAATACCCTGGCAATATTAATGTGTCTTTTCCCGA TATTCCTCAGGTACTCCAAAGATTCAGGTTTACTCACGTC ATCCAGCAGAGAATGGAAAGTCAAATTTCCTGAATTGCTA TGTGTCTGGGTTTCATCCATCCGACATTGAAGTTGACTTA CTGAAGAATGGAGAGAGAATTGAAAAAGTGGAGCATTCAG ACTTGTCTTTCAGCAAGGACTGGTCTTTCTATCTCTTGTA CTACACTGAATTCACCCCCACTGAAAGATGAGTATTGCC TGCCGTGTGAACCATGTGACTTTGTCACAGCCCAAGATAG TTAAGTGGGGTAAGTCTTACATTCTTTTGTAAGCTGCTGA AAGTTGTGTATGAGTAGTCATATCATAAAGCTGCTTTTGAT ATAAAAAAGGTTCTATGGCCATACTACCCTGAATGAGTCCC ATCCCATCTGATATAAACAATCTGCATATTGGGATTGTCA GGGAATGTTCTTAAAGATCAGATTAGTGGCACCTGCTGAG ATACTGATGCACAGCATGGTTTCTGAACCAGTAGTTTCCC TGCAGTTGAGCAGGGAGCAGCAGCAGCACTTGCACAAATA NOTECACTCTTAACACTTCTTACCTACTGGCTTCCTCT AGCTTTTGTGGCAGCTTCAGGTATATTTAGCACTGAACGA ACATCTCAAGAAGGTATAGGCCTTTGTTTGTAAGTCCTGC TGTCCTAGCATCCTATAATCCTGGACTTCTCCAGTACTTT CTGGCTGGATTGGTATCTGAGGCTAGTAGGAAGGGCTTGT TCCTGCTGGGTAGCTCTAAACAATGTATTCATGGGTAGGA ACAGCAGCCTATTCTGCCAGCCTTATTTCTAACCATTTTA GACATTTGTTAGTACATGGTATTTTAAAAAGTAAAACTTAA TGTCTTCCTTTTTTTTCTCCACTGTCTTTTTCATAGATCG AGACATGTAAGCAGCATCATGGAGGTAAGTTTTTGACCTT GAGAAAATGTTTTTGTTTCACTGTCCTGAGGACTATTTAT AGACAGCTCTAACATGATAACCCTCACTATGTGGAGAACA TTGACAGAGTAACATTTTAGCAGGGAAAGAAGAATCCTAC AGGGTCATGTTCCCTTTCCCTGTGGAGTGGCATGAAGAAG GTGTATGGCCCCAGGTATGGCCATAATTACTGACCCTCTAC AGAGAGGGCAAAGGAACTGCCAGTATGGTATTGCAGGATA AAGGCAGGTGGTTACCCACATTACCTGCAAGGCTTTGATC TTTCTTCTGCCATTTCCACATTGGACATCTCTGCTGAGGA GAGAAAATGAACCACTCTTTTCCTTTGTATAATGTTGTTTT TATTCTTCAGACAGAGAGGGATTATACAGCTCTGCAG ACATCCCATTCCTGTATGGGGACTGTGTTTGCCTCTTAGA GGTTCCCAGGCCAACTAGAGGAGATAAAGGGAACAGATTG TTATAACTTGATATAATGATAACTATAATAGATGTAACTAC AAGGAGCTCCAGAAGCAAGAGAGAGGGAGGAGGAACTTGGACT TCTCTGCATCTTTAGTTGGAGTCCAAAGGCTTTTCAATGA AATTCTACTGCCCAGGGTACATTGATGCTGAAACCCCATT CAAATCTCCTGTTATATTCTTAGAACAGGGAATTGATTTGG GAGAGCATCAGGAAGGTGGATGATCTGCCCAGTCACACTG TTAGTAAATTGTAGAGCCAGGACCTGAACTCTAATATAGT CATGTGTTACTTAATGACGGGGACATGTTCTGAGAAATGC TTACACAAACCTAGGTGTTGTAGCCTACTACACGCATAGG CTACATGGTATAGCCTATTGCTCCTAGACTACAAACCTGT ACAGCCTGTTACTGTACTGAATACTGTGGGCAGTTGTAAC ACAATGGTAAGTATTTGTGTATCTAAACATAGAAGTTGCA GTAAAAATATGCTATTTTAATCTTATGAGACCACTGTCAT ATATACAGTCCATCATTGACCAAAACATCATATCAGCATT TTTTCTTCTAAGATTTTGGGAGCACCAAAGGGATACACTA ACAGGATATACTCTTTATAATGGGTTTGGAGAACTGTCTG CAGCTACTTCTTTTAAAAAGGTGATCTACACAGTAGAAAT TAGACAAGTTTGGTAATGAGATCTGCAATCCAAATAAAAT AAATTCATTGCTAACCTTTTTTCTTTTCTTTTCAGGTTTGA AGATGCCGCATTTGGATTGGATGAATTCCAAATTCTGCTT GCTTGCTTTTTAATATTGATATGCTTATACACTTACACTT TATGCACAATTGTAGGGTTATAATAATGTTAACATGGAC ATGATCTTCTTTATAATTCTACTTTGAGTGCTGTCTCCAT GTTTGATGTATCTGAGCAGGTTGCTCCACAGGTAGCTCTA GGAGGGCTGGCAACTTAGAGGTGGGGAGCAGAGAATTCTC TTATCCAACATCAACATCTTGGTCAGATTTGAACTCTTCA ATCTCTTGCACTCAAAGCTTGTTAAGATAGTTAAGCGTGC ATAAGTTAACTTCCAATTTACATACTCTGCTTAGAATTTG GGGGAAAATTTAGAAATATAATTGACAGGATTATTGGAAA TTTGTTATAATGAATGAACATTTTGTCATATAAGATTCA TATTTACTTCTTATACATTTGATAAAGTAAGGCATGGTTG TGGTTAATCTGGTTTATTTTTGTTCCACAAGTTAAATAAA TCATAAAACTTGATGTGTTATCTCTTATATCTCACTCCCA CTATTACCCCTTTATTTTCAAACAGGGAAACAGTCTTCAA GTTCCACTTGGTAAAAATGTGAACCCCTTGTATATAGAG TTTGGCTCACAGTGTAAAGGGCCTCAGTGATTCACATTTT CCAGATTAGGAATCTGATGCTCAAAAGAAGTTAAATGGCAT AGTTGGGGTGACACAGCTGTCTAGTGGGAGGCCAGCCTTC TATATTTTAGCCAGCGTTCTTTCCTGCGGGCCAGGTCATG AGGAGTATGCAGACTCTAAGAGGGAGCAAAAGTATCTGAA GGATTTAATATTTTAGCAAGGAATAGATATACAATCATCC CTTGGTCTCCCTGGGGGATTGGTTTCAGGACCCCTTCTTG GACACCAAATCTATGGATATTTAAGTCCCTTCTATAAAAT GGTATAGTATTTGCATATAACCTATCCACATCCTCCTGTA TACTTTAAATCATTTCTAGATTACTTGTAATACCTAATAC AATGTAAATGCTATGCAAATAGTTGTTATTGTTTAAGGAA TAATGACAAGAAAAAAAAGTCTGTACATGCTCAGTAAAGA CACAACCATCCCTTTTTTTCCCCAGTGTTTTTGATCCATG GTTTGCTGAATCCACAGATGTGGAGCCCCTGGATACGGAA GGCCCGCTGTACTTTGAATGACAAATAACAGATTTAAA

[0099] The term "Cas9" or "Cas9 domain" refers to the Cas9 protein or its fragments of (e.g., active, inactive, or partially active DNA cleavage domains of Cas9) and / or a protein containing the gRNA-binding domain of Cas9) Cas9 nuclease is a nuclease that is similar to Casn1 nuclease or CRI. Also called SPR ("clustered regularly interspaced short palindromic repeats")-related nucleases CRISPR is a technology that can be used to create mobile genetic elements (viruses, transposable elements, and conjugative CRISPR clusters are an adaptive immune system that provides protection against viruses (plasmids). It contains a pacer, a sequence complementary to the leading mobile element, and a target invading nucleic acid. The star is transcribed and processed into CRISPR RNA (crRNA). Type II CRISPR In the PR system, the correct processing of the crRNA precursor (pre-crRNA) requires , trans-coding small RNA (tracrRNA), endogenous ribonuclease 3 (r nc) and Cas9 protein. tracrRNA is a precursor crRNA. It acts as a guide for RNase 3-assisted processing by the host. crRNA / tracrRNA is a linear or circular dsDNA target complementary to the spacer. The target strand that is not complementary to the crRNA is first endonucleolytically cleaved. endonucleolytic cleavage followed by 3'-5' exonucleolytic cleavage Essentially, DNA binding and DNA cleavage are typically achieved by proteins However, single guide RNA ("sgRNA") is required. ", or simply "gRNA") refers to both aspects of the crRNA and tracrRNA. It can be engineered to be incorporated into a single RNA species. For example, see Jinek M., et al. al.,Charpentier E.Science 337:816-821(2 012), the entire contents of which are incorporated herein by reference. 9 is a short motif (PAM or protospacer adjacent motif) in the CRISPR repeat sequence. The sequence and structure of Cas9 nuclease The methods are well known to those skilled in the art (see, for example, "Complete Genome Sequencing"). e of an M1 strain of Streptococcus pyoge nes.”Ferretti et al.,Proc.Natl.Acad.Sci. USA98:4658-4663(2001), “CRISPR RNA mat uration by trans-encoded small RNA and h ost factor RNase III.”Deltcheva E., et al .,Nature 471:602-607(2011), and “A program mable dual-RNA-guided DNA endonuclease i n adaptive bacterial immunity.”Jinek M., See, e.g., J. et al. Science 337:816-821 (2012). The entire contents of each of these are incorporated herein by reference. including, but not limited to, S. pyogenes and S. thermophilus Additional suitable Cas9 nucleases and sequences are described in the present disclosure. Based on the above, it will be apparent to one skilled in the art that such Cas9 nucleases and sequences include those listed in Table 1. hylinski, Rhun, and Charpentier, “The tracr. RNA and Cas9 families of type II CRISPR- Cas immunity systems”(2013)RNA Biology 1 Cas9 sequences derived from the organisms and loci disclosed in (the entire contents of which are incorporated herein by reference).

[0100] The nuclease-inactivated Cas9 protein is interchangeably referred to as the "dCas9" protein ( Nuclease-inactivated (dead) Cas9 or catalytically inactive Cas9 A Cas9 protein (or a fragment thereof) with an inactive DNA cleavage domain may be used. ) are known. See, for example, Jinek et al., Science ce.337:816-821(2012), Qi et al., “Repurpos ing CRISPR as an RNA-Guided Platform for Sequence-Specific Control of Gene Expression ssion” (2013) Cell.28;152(5):1173-83 (the entire contents of each of which are incorporated herein by reference). For example, Cas9 The DNA cleavage domain of is composed of two subdomains (HNH nuclease subdomain and The HNH subdomain is known to contain the RuvC1 subdomain. The RuvC1 subdomain cleaves the complementary strand, while the RuvC2 subdomain cleaves the non-complementary strand. Mutations within these subdomains silence the nuclease activity of Cas9. For example, mutations D10A and H840A can inhibit S. pyogenes Cas9 completely inactivates the nuclease activity of (Jinek et al., Science .337:816-821(2012);Qi et al.,Cell.28;152 (5):1173-83(2013)). In some embodiments, dCas9 is The Cas9 amino acid sequence has one or more mutations that inactivate the nuclease activity. In some embodiments, the dCas9 domain corresponds to or comprises a dCas9 domain. The main one contains the D10A and H840A mutations of another Cas9 or corresponding mutations. In some embodiments, the Cas9 nuclease is an inactive (e.g., inactivated) DNA cleavage domain. Cas9 has a nickase domain, i.e., (in the case of "nickase" Cas9) and is referred to as the "nCas9" protein. Additional Cas9 proteins (e.g., nuclease deacetylase-inactive Cas9 (dCas9), Cas9 nickase (nCas9), or Nuclease-active Cas9, including its variants and homologs, is within the scope of this disclosure. It is understood that the scope of the invention is within the scope of the invention. Exemplary Cas9 proteins include those provided herein. In some embodiments, the Cas9 protein may be a polypeptide, such as a polypeptide of interest, or a polypeptide of interest. The protein is a nuclease-inactive Cas9 (dCas9). The Cas9 protein is Cas9 nickase (nCas9). The Cas9 protein is a nuclease-active form of Cas9.

[0101] In some embodiments, proteins comprising fragments of Cas9 are provided. For example, some In embodiments, the protein comprises one of the following two Cas9 domains: ) the gRNA-binding domain of Cas9, or (2) the DNA-cleavage domain of Cas9. In embodiments, proteins comprising Cas9 or fragments thereof are referred to as "Cas9 variants." Cas9 variants share homology with Cas9 or fragments thereof. For example, a Cas9 variant can be at least about 70% identical to a wild-type Cas9, at least about 80% identical, at least about 90% identical, at least about 95% identical, at least about 96% identical 1. At least about 97% identical, at least about 98% identical, at least about 99% identical, In some embodiments, the sequence is at least about 99.5% identical, or at least about 99.9% identical. Cas9 variants have several advantages over wild-type Cas9: 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, 3 5, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48 In some embodiments, the Ca The s9 variants contain fragments of Cas9 (e.g., the gRNA binding domain or the DNA cleavage domain). The fragment contains a fragment of the corresponding wild-type Cas9 gene (a fragment of the corresponding gene), and therefore the fragment is at least About 70% identical, at least about 80% identical, at least about 90% identical, at least about 95% Identical, at least about 96% identical, at least about 97% identical, at least about 98% identical, at least about 99% identical, at least about 99.5% identical, or at least about 99.9% identical In some embodiments, the fragment is at least the amino acid length of the corresponding wild-type Cas9. 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 at least 75%, at least 80%, at least 85%, at least 90%, at least 9 5% identical, at least 96%, at least 97%, at least 98%, at least 99% , or at least 99.5%.

[0102] In some embodiments, the fragment is at least 100 amino acids in length. The fragment must be at least 100, 150, 200, 250, 300, 350, 40 0, 450, 500, 550, 600, 650, 700, 750, 800, 850, 90 0, 950, 1000, 1050, 1100, 1150, 1200, 1250, or less It is at least 1300 amino acids long.

[0103] In some embodiments, Cas9 is selected from Corynebacterium ulcerans s (NCBI reference: NC_015683.1, NC_017317.1), Coryne bacterium diphtheria (NCBI reference: NC_016782.1, NC_016786.1), Spiroplasma syrphidicola(NC BI reference: NC_021284.1), Prevotella intermedia( NCBI reference: NC_017861.1), Spiroplasma taiwanen se (NCBI reference: NC_021846.1), Streptococcus ini ae (NCBI reference: NC_021314.1), Belliella baltica (NCBI reference: NC_018010.1), Psychroflexus torqu is I (NCBI reference: NC_018721.1), Streptococcus t hermophilus (NCBI reference: YP_820832.1), Listeria innocua (NCBI reference: NP_472073.1), Campylobact er jejuni (NCBI reference: YP_002344900.1) or Neiss eria meningitidis(NCBI reference:YP_002342100.1) This refers to Cas9 derived from, or Cas9 derived from any other organism.

[0104] In some embodiments, Cas9 is expressed in Neisseria meningitidis ( In some embodiments, Cas9 is derived from Nme1, Nme2, or Nm e3. In some embodiments, the PAM interaction of Nme1, Nme2, or Nme3. The domains used are N4GAT, N4CC, and N4CAAA, respectively (e.g., Edraki,A.,et al.,A Compact,High-Accuracy Cas9 with a Dinucleotide PAM for In Viv o See Genome Editing, Molecular Cell (2018) (I want to be).

[0105] In some embodiments, the Cas9 fusion proteins provided herein comprise a Cas9 fusion protein. the full-length amino acid sequence of the protein (e.g., one of the Cas9 sequences provided herein) However, in other embodiments, the fusion proteins provided herein comprise It does not contain the full Cas9 sequence, but only one or more fragments thereof. For example, in some embodiments, The Cas9 fusion proteins provided herein comprise a Cas9 fragment, wherein the fragment is a fragment of a Cas9 gene. Binds to RNA and tracrRNA or sgRNA but does not contain a functional nuclease domain does not contain the nuclease domain (e.g., contains only a truncated version of the nuclease domain, or does not contain any nuclease domain).

[0106] Exemplary amino acid sequences of suitable Cas9 domains and Cas9 fragments are provided herein. Further suitable sequences for Cas9 domains and fragments will be apparent to those skilled in the art. .

[0107] In some embodiments, Cas9 constitutes the domain and kingdom of unicellular prokaryotic microorganisms. refers to Cas9 from archaea (e.g., nanoarchaea). In some embodiments, Cas9 is , CasX, or CasY, which are described, for example, by Burstein et al. ,“New CRISPR-Cas systems from uncultivat ed microbes.”Cell Res.2017 Feb 21.doi:10 .1038 / cr.2017.21 (the entire contents of which are incorporated herein by reference). Using genome-resolution metagenomics, several CRISPR- Cas systems have been identified, with Cas9 being the first reported in the archaeal domain of life. This diverse Cas9 protein is essential for the production of active CRISPR-Cas systems. It was discovered in the little-studied nanoarchaea as part of the microbial community. have identified two previously unknown systems, CRISPR-CasX and CRISP R-CasY was discovered, which is the most compact system discovered to date. In some embodiments, Cas9 is one of the stems. In some embodiments, Cas9 is a variant of CasY, or a variant of CasY. This refers to a variant of other RNA-guided DNA-binding proteins. A-binding proteins (napDNAbp) may also be used, and they are within the scope of this disclosure. Please understand that this is within.

[0108] In certain embodiments, napDNAbps useful in the methods of the present invention include those known in the art. It is known, for example, Oakes et al., Cell 176, 254-267, 2019, including circular permutants.

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

[0110] In some embodiments, a nucleic acid programmable DNA binding protein (napDNAbp) Alternatively, any of the fusion proteins provided herein may contain CasX or Cas In some embodiments, the napDNAbp can be a CasX protein. In some embodiments, the napDNAbp is a CasY protein. In this embodiment, the napDNAbp is a naturally occurring CasX or CasY protein. Quality and 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 In some embodiments, the napDNAbp comprises a naturally occurring Cas In some embodiments, the napDNAbp is a protein selected from the group consisting of the X, XB, XC, XD, XE, XF, XF, XG, XH ...H, XF, XH, XH, XH, XH, XH, XH, XH, XH, XH, XH, XH and 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 In accordance with the present disclosure, the present invention also includes amino acid sequences that are 99%, or at least 99.5%, identical to the amino acid sequences of the present invention. It should be understood that CasX and CasY from other bacterial species may also be used.

[0111] The term "Cas12b" or "Cas12b domain" refers to Cas12b / C2 c1 protein or a fragment thereof (e.g., active, inactive, or partial Cas12b containing a highly active DNA cleavage domain and / or the gRNA binding domain of Cas12b. (The content of each of these is described in reference Cas12b orthologues include Alicyclobacillus llus acidoterrestris, Alicyclobacillus ac idophilus(Teng et al.,Cell Discov.2018 N ov 27;4:63), Bacillus hisashi, and Bacillus Additional suitable Cas12b nucleotides include, but are not limited to, species V3-13. The enzymes and sequences will be apparent to those skilled in the art based on the present disclosure.

[0112] In some embodiments, the protein comprising Cas12b or a fragment thereof is referred to as "Cas12 Cas12b variants are referred to as "Cas12b variants." Cas12b variants are Cas12b or fragments thereof. For example, Cas12b variants share at least one homology with wild-type Cas12b. At least about 70% identical, at least about 80% identical, at least about 90% identical, at least about 9 5% identical, at least about 96% identical, at least about 97% identical, at least about 98% identical , at least about 99% identical, at least about 99.5% identical, or at least about 99.9 In some embodiments, the Cas12b variant is % identical to wild-type Cas12b. Compare: 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, 4 2, 43, 44, 45, 46, 47, 48, 49, 50 or more amino acid mutations In some embodiments, the Cas12b variant may comprise a fragment of Cas12b ( For example, a gRNA binding domain or a DNA cleavage domain) and therefore a fragment thereof is at least about 70% identical to the corresponding fragment of wild-type Cas12b, at least about 80% identical to the corresponding fragment of wild-type Cas12b. % identical, at least about 90% identical, at least about 95% identical, at least about 96% identical, At least about 97% identical, at least about 98% identical, at least about 99% identical, at least In some embodiments, the sequence is at least about 99.5% identical, or at least about 99.9% identical. The fragments should be at least 30% of the amino acid length of the corresponding wild-type Cas12b, at least 35 %, at least 40%, at least 45%, at least 50%, at least 55%, at least at least 60%, at least 65%, at least 70%, at least 75%, at least 8 0%, at least 85%, at least 90%, at least 95% identical, at least 96% , at least 97%, at least 98%, at least 99%, or at least 99.5% %. Exemplary Cas12b polypeptides are listed herein.

[0113] "Cbl proto-oncogene B (CBLB) polypeptide" means a polypeptide involved in the regulation of immune responses. GenBank accession number ABC86700.1 or a fragment thereof and at least about 85% Proteins with amino acid sequence identity. Exemplary CBLB Polypeptide Sequences is provided below. >ABC86700.1 CBL-B[Homo sapiens] MANSMNGRNPGGRGGNPRKGRILGIIDAIQDAVGPPKQAA ADRRTVEKTWKLMDKVVRLCQNPKLQLKNSPPYILDILPD TYQHLRLILSKYDDNQKLAQLSENEYFKIYIDSLMKKSKR AIRLFKEGKERMYEEQSQDRRNLTKLSLIFSHMLAEIKAI FPNGQFQGDNFRITKADAAEFWRKFFGDKTIVPWKVFRQC LHEVHQISSGLEAMALKSTIDLTCNDYISVFEFDIFTRLF QPWGSILRNWNFLAVTHPGYMAFLTYDEVKARLQKYSTKP GSYIFRLSCTRLGQWAIGYVTGDGNILQTIPHNKPLFQAL IDGSREGFYLYPDGRSYNPDLTGLCEPTPHDHIKVTQEQY ELYCEMGSTFQLCKICAENDKDVKIEPCGHLMCTSCLTAW QESDGQGCPFCRCEIKGTEPIIVDPFDPRDEGSRCCSIID PFGMPMLDLDDDDDREESLMMNRLANVRKCTDRQNSPVTS PGSSPLAQRRKPQPDPLQIPHLSLPPVPPRLDLIQKGIVR SPCGSPTGSPKSSPCMVRKQDKPLPAPPPPLRDPPPPPPE RPPPIPPDNRLSRHIHHVESVPSRDPPMPLEAWCPRDVFG TNQLVGCRLLGEGSPKPGITASSNVNGRHSRVGSDPVLMR KHRRHDLPLEGAKVFSNGHLGSEEYDVPPRLSPPPPVTTL LPSIKCTGPLANSLSEKTRDPVEEDDDEYKIPSSHPVSLN SQPSHCHNVKPPVRSCDNGHCMLNGTHGPSSEKKSNIPDL SIYLKGDVFDSASDPVPLPPARPPTRDNPKHGSSLNRTPS DYDLLIPPLGEDAFDALPPSLPPPPPPARHSLIEHSKPPG SSSRPSSGQDLFLLPSDPFVDLASGQVPLPPARRLPGENV KTNRTSQDYDQLPSCSDGSQAPARPPKPRPRRTAPEIHHR KPHGPEAALENVDAKIAKLMGEGYAFEEVKRALEIAQNNV EVARSILREFAFPPPVSPRLNL

[0114] "Cbl proto-oncogene B (CBLB) polynucleotide" means a CBLB polypeptide The CBLB gene encodes an E3 ubiquitin ligase. Exemplary CBLB nucleic acid sequences are provided below. >DQ349203.1 Homo sapiens CBL-B mRNA, full-length C DS ATGGCAAACTCAATGAATGGCAGAAACCCTGGTGGTCGAG GAGGAAATCCCGAAAGGTCGAATTTTGGGTATTATTGA TGCTATTCAGGATGCAGTTGGACCCCCTAAGCAAGCTGCC GCAGATCGCAGGACCGTGGAGAAGACTTGGAAGCTCATGG ACAAAGTGGTAAGACTGTGCCAAAATCCCAAACTTCAGTT GAAAAATAGCCCACCATATATACTTGATATTTTGCCTGAT ACATATCAGCATTTACGACTTATATTGAGTAAATATGATGATG ACAACCAGAAACTTGCCCAACTCAGTGAGAATGAGTACTT TAAAATCTACATTGATAGCCTTATGAAAAAGTCAAAACGG GCAAATAAGACTCTTTAAAGAAGGCAAGGAGAGAATGTATG AAGAACAGTCACAGGACAGACGAAATCTCACAAAACTGTC CCTTATCTTCAGTCACATGCTGGCAGAAATCAAAGCAATC TTTCCCAATGGTCAATTCCAGGGAGATAACTTTCGTATCA CAAAAGCAGATGCTGCTGAATTCTGGAGAAAGTTTTTTGG AGACAAAACTATCGTACCATGGAAAGTATTCAGACAGTGC CTTCATGAGGTCCACCAGATTAGCTCTGGCCTGGAAGCAA TGGCTCTAAAATCAACAATTGATTTAACTTGCAATGATTA CATTTCAGTTTTTGAATTTGATATTTTTACCAGGCTGTTT CAGCCTTGGGGCTCTATTTTGCGGAATTGGAATTTCTTAG CTGTGACACATCCAGGTTACATGGCATTTCTCACATATGA TGAAGTTAAAGCACGACTACAGAAATATAGCACCAAACCC GGAAGCTATATTTTCCGGTTAAGTTGCACTCGATTGGGAC AGTGGGCCATTGGCTATGTGACTGGGGATGGGAATATCTT ACAGACCATACCTCATAACAAGCCCTTATTTCAAGCCCTG ATTGATGGCAGCAGGGAAGGATTTTATCTTTATCCTGATG GGAGGAGTTATAATCCTGATTTAACTGGATTATGTGAACC TACACCTCATGACCATATAAAAGTTACACAGGAACAATAT GAATTATATTGTGAAATGGGCTCCACTTTTCAGCTCTGTA AGATTTGTGCAGAGAATGACAAAGATGTCAAGATTGAGCC TTGTGGGCATTTGATGTGCACCTCTTGCCTTACGGCATGG CAGGAGTCGGATGGTCAGGGCTGCCCTTTCTGTCGTTGTG AAATAAAAGGAACTGAGCCCATAATCGTGGACCCCTTTGA TCCAAGAGATGAAGGCTCCAGGTGTTGCAGCATCATTGAC CCCTTTGGCATGCCGATGCTAGACTTGGACGACGATGATG ATCGTGAGGAGTCCTTGATGATGAATCGGTTGGCAAACGT CCGAAAGTGCACTGACAGGCAGAACTCACCAGTCACATCA CCAGGATCCTCTCCCCTTGCCCAGAGAAGAAAGCCACAGC CTGACCCACTCCAGATCCCACATCTAAGCCTGCCACCCGT GCCTCCTCGCCTGGATCTAATTCAGAAAGGCATAGTTAGA TCTCCCTGTGGCAGCCCAACGGGTTCACCAAAGTCTTCTC CTTGCATGGTGAGAAAACAAGATAAACCACTCCCAGCACC ACCTCCTCCCTTAAGAGATCCTCCTCCACCGCCACCTGAA AGACCTCCACCAATCCCACCAGACAATAGACTGAGTAGAC ACATCCATCATGTGGAAAGCGTGCCTTCCAGAGACCCGCC AATGCCTCTTGAAGCATGGTGCCCTCGGGATGTGTTTGGG ACTAATCAGCTTGTGGGATGTCGACTCCTAGGGGAGGGCT CTCCAAAACCTGGAATCACAGCGAGTTCAAATGTCAATGG AAGGCACAGTAGAGTGGGCTCTGACCCAGTGCTTATGCGG AAACACAGACGCCATGATTTGCCTTTAGAAGGAGCTAAGG TCTTTTCCAATGGTCACCTTGGAAGTGAAGAATATGATGT TCCTCCCCGGCTTTCTCCTCCTCCTCCAGTTACCACCCTC CTCCCTAGCATAAAGTGTACTGGTCCGTTAGCAAATTCTC TTTCAGAGAAAACAAGAGACCCAGTAGAGGAAGATGATGA TGAATACAAGATTCCTTCATCCCACCCTGTTTCCCTGAAT TCACAACCATCTCATTGTCATAATGTAAAACCTCCTGTTC GGTCTTGTGATAATGGTCACTGTATGCTGAATGGAACACA TGGTCCATCTTCAGAGAAGAAATCAAACATCCCTGACTTA AGCATATATTTAAAGGGAGATGTTTTTGATTCAGCCTCTG ATCCCGTGCCATTACCACCTGCCAGGCCTCCAACTCGGGA CAATCCAAAGCATGGTTCTTCACTCAACAGGACGCCCTCT GATTATGATCTTCTCATCCCTCCATTAGGTGAAGATGCTT TTGATGCCCTCCCTCCATCTCTCCCACCTCCCCCACCTCC TGCAAGGCATAGTCTCATTGAACATTCAAAACCTCCTGGC TCCAGTAGCCGGCCATCCTCAGGACAGGATCTTTTTCTTC TTCCTTCAGATCCCTTTGTTGATCTAGCAAGTGGCCAAGT TCCTTTGCCTCCTGCTAGAAGGTTACCAGGTGAAAATGTC AAAACTAACAGAACATCACAGGACTATGATCAGCTTCCTT CATGTTCAGATGGTTCACAGGCACCAGCCAGACCCCCTAA ACCACGACCGCGCAGGACTGCACCAGAAATTCACCACAGA AAACCCCATGGGCCTGAGGCGGCATTGGAAAATGTCGATG CAAAAATTGCAAAACTCATGGGAGAGGGTTATGCCTTTGA AGAGGTGAAGAGAGCCTTAGAGATAGCCCAGAATAATGTC GAAGTTGCCCGGAGCATCCTCCGAGAATTTGCCTTCCCTC CTCCAGTATCCCCACGTCTAAATCTATAG

[0115] "Chimeric antigen receptors" or "CARs" are molecules that express specific antigens in immune effector cells. one or more intracellular signaling domains (e.g., T cell signaling domains) that confer heterogeneity refers to a synthetic or engineered receptor comprising an extracellular antigen-binding domain linked to a nucleotide sequence (e.g., a nucleotide sequence) In some embodiments, the CAR comprises a transmembrane domain.

[0116] "Chimeric antigen receptor T cells" or "CAR-T cells" are T cells that express a CAR. and has antigen specificity determined by the antibody-derived targeting domain of the CAR. As used herein, "CAR-T cells" includes T cells or NK cells. As used herein, "CAR-T cells" refers to cells that express a CAR or T cell receptor (TCR). In some embodiments, the CAR-T cells include cells engineered to express any Optionally, a predetermined percentage of T helper CD4+ cells and / or T effector CD8+ CARs can be cells. Methods for producing CARs (e.g., for the treatment of cancer) are publicly available. (e.g., Park et al., Trends Biotechnol., 2012) 9:550-557,2011, Grupp et al., N Engl J Med .,368:1509-1518,2013, Han et al., J. Hemato. l Oncol.6:47,2013, Haso et al.,(2013)Bloo d,121,1165-1174;PCT Pubs, WO2012 / 079000, W See U.S. Publication No. 2013 / 059593 and U.S. Publication No. 2012 / 0213783. (each of which is incorporated herein by reference in its entirety).

[0117] "Class II major histocompatibility complex transactivator (CIITA)" is a NC BI accession number NP_001273331.1 or a fragment thereof and at least about 85% The amino acid sequence identity of the amino acid sequence of the present invention is a protein having immunomodulatory activity. The sequences are provided below. >NP_001273331.1 MHC class II transactivator isoform Room 1 [Homo sapiens] MRCLAPRPAGSYLSEPQGSSQCATMELGPLEGGYLELLNS DADPLCLYHFYDQMDLAGEEEIELYSEPDTDTINCDQFSR LLCDMEGDEETREAYANIAELDQYVFQDSQLEGLSKDIFI EHIGPDEVIGESMEMPAEVGQKSQKRPFPEELPADLKHWK PAEPTVVTGSLLVGPVSDCSTLPCLPLPALFNQEPASGQ MRLEKTDQIPMPFSSSLSCLNLPEGPIQFVPTISTLPHG LWQISEAGTGVSSIFIYHGEVPQASQVPPPSGFTVHGLPT SPDRPGSTSPFAPSATDLPSMPEPALTSRANMTEHKTSPT QCPAAGEVSNKLPKWPEPVEQFYRSLQDTYGAEPAGPDGI LVEVDLVQARLERSSSKSLERELATPDWAERQLAQGGLAE VLLAAKEHRRPRETRVIAVLGKAGQGKSYWAGAVSRAWAC GRLPQYDFVFSVPCHCLNRPGDAYGLQDLLFSLGPQPLVA ADEVFSHILKRPDRVLLILDGFEELEAQDGFLHSTCGPAP AEPCSLRGLLAGLFQKKLLRGCTLLLTARPRGRLVQSLSK ADALFELSGFSMEQAQAYVMRYFESSGMTEHQDRALTLLR DRPLLLSHSHSPTLCRAVCQLSEALLELGEDAKLPSTLTG LYVGLLGRAALDSPPGALAELAKLAWELGRRHQSTLQEDQ FPSADVRTWAMAKGLVQHPPRAAESELAFPSFLLQCFLGA LWLALSGEIKDKELPQYLALTPRKKRPYDNWLEGVPRFLA GLIFQPPARCLGALLGPSAAASVDRKQKVLARYLKRLQPG TLRARQLLELLHCAHEAEEAGIWQHVVQELPGRLSFLGTR LTPPDAHVLGKALEAAGQDFSLDLRSTGICPSGLGSLVGL SCVTRFRAALSDTVALWESLQQHGETKLLQAAEEKFTIEP FKAKSLKDVEDLGKLVQTQRTRSSSEDTAGELPAVRDLKK LEFALGPVSGPQAFPKLVRILTAFSSLQHLDLDALSENKI GDEGVSQLSATFPQLKSLETLNLSQNNITDLGAYKLAEAL PSLAASLLRLSLYNNCICDVGAESLARVLPDMVSLRVMDV QYNKFTAAGAQQLAASLRRCPHVETLAMWTPTIPFSVQEH LQQQDSRISLR

[0118] "Class II major histocompatibility complex transactivator (CIITA)" refers to CI An exemplary CIITA nucleic acid sequence is shown below: provide. >NM_001286402.1 Homo sapiens Class II Major Histocompatibility Complex transactivator (CIITA), transcript variant 1, mRNA GGTTAGTGATGAGGCTAGTGATGAGGCTGTGTGCTTCTGA GCTGGGCATCCGAAGGCATCCTTGGGGAAGCTGAGGGCAC GAGGAGGGGCTGCCAGACTCCGGGAGCTGCTGCCTGGCTG GGATTCCTACACAATGCGTTGCCTGGCTCCACGCCCTGCT GGGTCCTACCTGTCAGAGCCCCAAGGCAGCTCACAGTGTG CCACCATGGAGTTGGGGCCCCTAGAAGGTGGCTACCTGGA GCTTCTTAACAGCGATGCTGACCCCCTGTGCCTCTACCAC TTCTATGACCAGATGGACCTGGCTGGAGAAGAAGAGATTG AGCTCTACTCAGAACCCGACACAGACACCATCAACTGCGA CCAGTTCAGCAGGCTGTTGTGTGACATGGAAGGTGATGAA GAGACCAGGGAGGCTTATGCCAATATCGCGGAACTGGACC AGTATGTCTTCCAGGACTCCCAGCTGGAGGGCCTGAGCAA GGACATTTTCATAGAGCACATAGGACCAGATGAAGTGATC GGTGAGAGTATGGAGATGCCAGCAGAAGTTGGGCAGAAAA GTCAGAAAAGACCCTTCCCAGAGGAGCTTCCGGCAGACCT GAAGCACTGGAAGCCAGCTGAGCCCCCCACTGTGGTGACT GGCAGTCTCCTAGTGGGACCAGTGAGCGACTGCTCCACCC TGCCCTGCCTGCCACTGCCTGCGCTGTTCAACCAGGAGCC AGCCTCCGGCCAGATGCGCCTGGAGAAAACCGACCAGATT CCCATGCCTTTCTCCAGTTCCTCGTTGAGCTGCCTGAATC TCCCTGAGGGACCCATCCAGTTTGTCCCCACCATCTCCAC TCTGCCCCATGGGCTCTGGCAAATCTCTGAGGCTGGAACA GGGGTCTCCAGTATATTCATCTACCATGGTGAGGTGCCCC AGGCCAGCCAAGTACCCCCTCCCAGTGGATTCACTGTCCA CGGCCTCCCAACATCTCCAGACCGGCCAGGCTCCACCAGC CCCTTCGCTCCATCAGCCACTGACCTGCCCAGCATGCCTG AACCTGCCCTGACCTCCCGAGCAAACATGACAGAGCACAA GACGTCCCCCACCCAATGCCCGGCAGCTGGAGAGGTCTCC AACAAGCTTCCAAAATGGCCTGAGCCGGTGGAGCAGTTCT ACCGCTCACTGCAGGACACGTATGGTGCCGAGCCCGCAGG CCCGGATGGCATCCTAGTGGAGGTGGATCTGGTGCAGGCC AGGCTGGAGAGGAGCAGCAGCAAGAGCCTGGAGCGGGAAC TGGCCACCCCGGACTGGGCAGAACGGCAGCTGGCCCAAGG AGGCCTGGCTGAGGTGCTGTTGGCTGCCAAGGAGCACCGG CGGCCGCGTGAGACACGAGTGATTGCTGTGCTGGGCAAAG CTGGTCAGGGCAAGAGCTATTGGGCTGGGGCAGTGAGCCG GGCCTGGGCTTGTGGCCGGCTTCCCCAGTACGACTTTGTC TTCTCTGTCCCCTGCCATTGCTTGAACCGTCCGGGGGATG CCTATGGCCTGCAGGATCTGCTCTTCTCCCTGGGCCCACA GCCACTCGTGGCGGCCGATGAGGTTTTCAGCCACATCTTG AAGAGACCTGACCGCGTTCTGCTCATCCTAGACGGCTTCG AGGAGCTGGAAGCGCAAGATGGCTTCCTGCACAGCACGTG CGGACCGGCACCGGCGGAGCCCTGCTCCCTCCGGGGGCTG CTGGCCGGCCTTTTCCAGAAGAAGCTGCTCCGAGGTTGCA CCCTCCTCCTCACAGCCCGGCCCCGGGGCCGCCTGGTCCA GAGCCTGAGCAAGGCCGACGCCCTATTTGAGCTGTCCGGC TTCTCCATGGAGCAGGCCCAGGCATACGTGATGCGCTACT TTGAGAGCTCAGGGATGACAGAGCACCAAGACAGAGCCCT GACGCTCCTCCGGGACCGGCCACTTCTTCTCAGTCACAGC CACAGCCCTACTTTGTGCCGGGCAGTGTGCCAGCTCTCAG AGGCCCTGCTGGAGCTTGGGGAGGACGCCAAGCTGCCCTC CACGCTCACGGGACTCTATGTCGGCCTGCTGGGCCGTGCA GCCCTCGACAGCCCCCCCGGGGCCCTGGCAGAGCTGGCCA AGCTGGCCTGGGAGCTGGGCCGCAGACATCAAAGTACCCT ACAGGAGGACCAGTTCCCATCCGCAGACGTGAGGACCTGG GCGATGGCCAAAGGCTTAGTCCAACACCCACCGCGGGCCG CAGAGTCCGAGCTGGCCTTCCCCAGCTTCCTCCTGCAATG CTTCCTGGGGGCCCTGTGGCTGGCTCTGAGTGGCGAAATC AAGGACAAGGAGCTCCCGCAGTACCTAGCATTGACCCCAA GGAAGAAGAGGCCCTATGACAACTGGCTGGAGGGCGTGCC ACGCTTTCTGGCTGGGCTGATCTTCCAGCCTCCCGCCCGC TGCCTGGGAGCCCTACTCGGGCCATCGGCGGCTGCCTCGG TGGACAGGAAGCAGAAGGTGCTTGCGAGGTACCTGAAGCG GCTGCAGCCGGGGACACTGCGGGCGCGGCAGCTGCTGGAG CTGCTGCACTGCGCCCACGAGGCCGAGGAGGCTGGAATTT GGCAGCACGTGGTACAGGAGCTCCCCGGCCGCCTCTCTTT TCTGGGCACCCGCCTCACGCCTCCTGATGCACATGTACTG GGCAAGGCCTTGGAGGCGGCGGGCCAAGACTTCTCCCTGG ACCTCCGCAGCACTGGCATTTGCCCCTCTGGATTGGGGAG CCTCGTGGGACTCAGCTGTGTCACCCGTTTCAGGGCTGCC TTGAGCGACACGGTGGCGCTGTGGGAGTCCCTGCAGCAGC ATGGGGAGACCAAGCTACTTCAGGCAGCAGAGGAGAAGTT CACCATCGAGCCTTTCAAAGCCAAGTCCCTGAAGGATGTG GAAGACCTGGGAAAGCTTGTGCAGACTCAGAGGACGAGAA GTTCCTCGGAAGACACAGCTGGGGAGCTCCCTGCTGTTCG GGACCTAAAGAAACTGGAGTTTGCGCTGGGCCCTGTCTCA GGCCCCCAGGCTTTCCCCAAACTGGTGCGGATCCTCACGG CCTTTTCCTCCCTGCAGCATCTGGACCTGGATGCGCTGAG TGAGAACAAGATCGGGGACGAGGGTGTCTCGCAGCTCTCA GCCACCTTCCCCCAGCTGAAGTCCTTGGAAACCCTCAATC TGTCCCAGAACAACATCACTGACCTGGGTGCCTACAAACT CGCCGAGGCCCTGCCTTCGCTCGCTGCATCCCTGCTCAGG CTAAGCTTGTACAATAACTGCATCTGCGACGTGGGAGCCG AGAGCTTGGCTCGTGTGCTTCCGGACATGGTGTCCCTCCG GGTGATGGACGTCCAGTACAACAAGTTCACGGCTGCCGGG GCCCAGCAGCTCGCTGCCAGCCTTCGGAGGTGTCCTCATG TGGAGACGCTGGCGATGTGGACGCCCACCATCCCATTCAG TGTCCAGGAACACCTGCAACAACAGGATTCACGGATCAGC CTGAGATGATCCCAGCTGTGCTCTGGACAGGCATGTTCTC TGAGGACACTAACCACGCTGGACCTTGAACTGGGTACTTG TGGACACAGCTCTTCTCCAGGCTGTATCCCATGAGCCTCA GCATCCTGGCACCCGGCCCCTGCTGGTTCAGGGTTGGCCC CTGCCCGGCTGCGGAATGAACCACATCTTGCTCTGCTGAC AGACACAGGCCCGGCTCCAGGCTCCTTTAGCGCCCAGTTG GGTGGATGCCTGGTGGCAGCTGCGGTCCACCCAGGAGCCC CGAGGCCTTCTCTGAAGGACATTGCGGACAGCCACGGCCA GGCCAGAGGGAGTGACAGAGGCAGCCCCATTCTGCCTGCC CAGGCCCCTGCCACCCTGGGGAGAAAGTACTTCTTTTTTT TTATTTTTAGACAGAGTCTCACTGTTGCCCAGGCTGGCGT GCAGTGGTGCGATCTGGGTTCACTGCAACCTCCGCCTCTT GGGTTCAAGCGATTCTTCTGCTTCAGCCTCCCGAGTAGCT GGGACTACAGGCACCCACCATCATGTCTGGCTAATTTTTC ATTTTTAGTAGAGACAGGGTTTTGCCATGTTGGCCAGGCT GGTCTCAAACTCTTGACCTCAGGTGATCCACCCACCTCAG CCTCCCAAAGTGCTGGGATTACAAGCGTGAGCCACTGCAC CGGGCCACAGAGAAAGTACTTCTCCACCCTGCTCTCCGAC CAGACACCTTGACAGGGCACACCGGGCACTCAGAAGACAC TGATGGGCAACCCCCAGCCTGCTAATTCCCCAGATTGCAA CAGGCTGGGCTTCAGTGGCAGCTGCTTTTGTCTATGGGAC TCAATGCACTGACATTGTTGGCCAAAGCCAAAGCTAGGCC TGGCCAGATGCACCAGCCCTTAGCAGGGAAACAGCTAATG GGACACTAATGGGGCGGTGAGAGGGGAACAGACTGGAAGC ACAGCTTCATTTCCTGTGTCTTTTTTCACTACATTATAAA TGTCTCTTTAATGTCACAGGCAGGTCCAGGGTTTGAGTTC ATACCCTGTTACCATTTTGGGGTACCCACTGCTCTGGTTA TCTAATATGTAACAAGCCACCCCAAATCATAGTGGCTTAA AACAACACTCACATTTA

[0119] In this disclosure, "comprises," "comprising," ", "containing," and "having" are used in "includes," "compound," "compounds," "compounds" or "compounds" may have the meanings given to them in the United States Patent and Trademark Law. Similarly, "consisting essentially of" can mean "including" or "consisting essentially of." "consisting essentially of" or "consisting essentially of" "sts essentially" has the meaning given to it in United States patent law, The term is open-ended and does not include the essential or novel features of what is being listed. is enumerated unless it is changed by more beings than are enumerated. There may be more than one, but prior art embodiments are excluded.

[0120] "Cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) polypeptide" means NC BI accession number EAW70354.1 or a fragment thereof. Exemplary amino acid sequences are provided below: >EAW70354.1 Cytotoxic T-lymphocyte-associated protein 4 [Homo sapi ens] MACLGFQRHKAQLNLATRTWPCTLLFFLLFIPVFCKAMHV AQPAVVLASSRGIASFVCEYASPGKATEVRVTVLRQADSQ VTEVCAATYMMGNELTFLDDSICTGTSSGNQVNLTIQGLR AMDTGLYICKVELMYPPPYYLGIGNGTQIYVIDPEPCPDS DFLLWILAAVSSGLFFYSFLLTAVSLSKMLKKRSPLTTGV YVKMPPTEPECEKQFQPYFIPIN

[0121] "Cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) polynucleotide" means The CTLA-4 gene refers to a nucleic acid molecule that encodes a CTLA-4 polypeptide. Encodes a protein of the immunoglobulin superfamily that transmits inhibitory signals to T cells Exemplary CTLA-4 nucleic acid sequences are provided below. >BC074842.2 Homo sapiens cytotoxic T lymphocyte-associated protein Substance 4, mRNA (cDNA clone MGC:104099 IMAGE:309155 52), full-length CDS GACCTGAACACCGCTCCCATAAAGCCATGGCTTGCCTTGG ATTTCAGCGGCACAAGGCTCAGCTGAACCTGGCTACCAGG ACCTGGCCCTGCACTCTCCTGTTTTTTCTTCTCTTCATCC CTGTCTTCTGCAAAGCAATGCACGTGGCCCAGCCTGCTGT GGTACTGGCCAGCAGCCGAGGCATCGCCAGCTTTGTGTGT GAGTATGCATCTCCAGGCAAAGCCACTGAGGTCCGGGTGA CAGTGCTTCGGCAGGCTGACAGCCAGGTGACTGAAGTCTG TGCGGCAACCTACATGATGGGGAATGAGTTGACCTTCCTA GATGATTCCATCTGCACGGGCACCTCCAGTGGAAATCAAG TGAACCTCACTATCCAAGGACTGAGGGCCATGGACACGGG ACTCTACATCTGCAAGGTGGAGCTCATGTACCCACCGCCA TACTACCTGGGCATAGGCAACGGAACCCAGATTTATGTAA TTGATCCAGAACCGTGCCCAGATTCTGACTTCCTCCTCTG GATCCTTGCAGCAGTTAGTTCGGGGTTGTTTTTTTATAGC TTTCTCCTCACAGCTGTTTCTTTGAGCAAAATGCTAAAGA AAAGAAGCCCTCTTACAACAGGGGTCTATGTGAAAATGCC CCCAACAGAGCCAGAATGTGAAAAGCAATTTCAGCCTTAT TTTATTCCCATCAATTGAGAAACCATTATGAAGAAGAGAG TCCATATTTCAATTTCCAAGAGCTGAGG

[0122] "Cluster of differentiation 2 (CD2) polypeptide" refers to the polypeptide identified under NCBI accession number NP_00175 8.2 or a fragment thereof, and has at least about 85% amino acid sequence identity with the Exemplary amino acid sequences are provided below. >NP_001758.2 T cell surface antigen CD2 isoform 2 precursor [Homo sapiens] The CD2 cytoplasmic domain (amino acid residues 235 to 351) is shown in bold. 1 shows the architecture of an exemplary CD2 polypeptide from Sapiens.

[0123] A "cluster of differentiation 2 (CD2) polynucleotide" refers to a polynucleotide that encodes a CD2 polypeptide. An exemplary CD2 nucleic acid sequence is provided below: >NM_001767. 5 Homo sapiens CD2 molecule (CD2), transcript variant 2, mRNA 1 agtctcactt cagttccttt tgcatgaaga gctcagaatc aaaagaggaa accaacccct 61 aagatgagct ttccatgtaa atttgtagcc agcttccttc tgattttcaa tgtttcttcc 121 aaaggtgcag tctccaaaga gattacgaat gccttggaaa cctggggtgc cttgggtcag 181 gacatcaact tggacattcc tagttttcaa atgagtgatg atattgacga tataaaatgg 241 gaaaaactt cagacaga aaagattgca cattcagaa aagagaga gactttcaag 301 gaaaagata catataagct atttaaaat ggaactctga aattaagca tctgaagacc 361 gatgatcagg attackaca ggtatcaata tatgataca aaggaaaaaa tgtgttggaa 421 aaatattg atttgagat tcagagagg gtctcaac accaatctc ctggacttgt 481 atcacacaaccctgacctg tgaggtaatg atggactg accccgaatt aaacctgtat 541 siagatggga aacatctaaa actttctcag agggtcatca cacacaagtg gaccaccagc 601 ctgagtgcaa aattcaagtg cacagcaggg aaaaagtca gcaaggaatc cagtgtcgag 661 cctgtcagct gtccagagaa aggtctggac atctatctca tcattggcat atgtggagga 721 ggcagcctct tgatgtctt tgtggcactg ctcgttttct atatcaccaa aaggaaaaaa 781 cagaggagtc gagaaatga tgaggagctg gagacagag cccacagagt agctactgaa 841 gaaaggggcc ggaagcccca ccaattcca gctcaaccc ctcagaatcc agcaacttcc 901 siacatcctc ctccaccacc tggtcatcgt 961 cctggacacc gtgttcagca ccagcctcag aagaggcctc ctgctccgtc gggcacacaa 1021 gttcaccagc agaaaggccc gcccctcccc agacctcgag ttcagccaaa acctccccat 1081 ggggcagcag aaaactcatt gtccccttcc tctaattaaa aaagatagaa actgtctttt 1141 tcaataaaaa gcactgtgga tttctgccct cctgatgtgc atatccgtac ttccatgagg 1201 tgttttctgt gtgcagaaca ttgtcacctc ctgaggctgt gggccacagc cacctctgca 1261 tcttcgaact cagccatgtg gtcaacatct ggagtttttg gtctcctcag agagctccat 1321 cacaccagta aggagaagca atataagtgt gattgcaaga atggtagagg accgagcaca 1381 gaaatcttag agatttcttg tcccctctca ggtcatgtgt agatgcgata aatcaagtga 1441 ttggtgtgcc tgggtctcac tacaagcagc ctatctgctt aagagactct ggagtttctt 1501 atgtgccctg gtggacactt gcccaccatc ctgtgagtaa aagtgaaata aaagctttga 1561 ctaga

[0124] "Cluster of differentiation 5 (CD5) polypeptide" refers to the NCBI accession number NP_00133 3385.1 or a fragment thereof, and By "antibody" is meant a protein having anti-inflammatory activity. Exemplary amino acid sequences are provided below. >NP_001333385.1 T cell surface glycoprotein CD5 isoform 2 [H omo sapiens] MVCSQSWGRSSKQWEDPSQASKVCQRLNCGVPLSLGPFLV TYTPQSSIICYGQLGSFSNCSHSRNDMCHSLGLTCLEPQK TTPPTTRPPPTTTPEPTAPPRLQLVAQSGGQHCAGVVEFY SGSLGGTISYEAQDKTQDLENFLCNNLQCGSFLKHLPETE AGRAQDPGEPREHQPLPIQWKIQNSSCTSLEHCFRKIKPQ KSGRVLALLCSGFQPKVQSRLVGGSSICEGTVEVRQGAQW AALCDSSSARSSLRWEEVCREQQCGSVNSYRVLDAGDPTS RGLFCPHQKLSQCHELWERNSYCKKVFVTCQDPNPAGLAA GTVASIILALVLLVVLLVVCGPLAYKKLVKKFRQKKQRQW IGPTGMNQNMSFHRNHTATVRSHAENPTASHVDNEYSQPP RNSHLSAYPALEGALHRSSMQPDNSSDSDYDLHGAQRL

[0125] A "cluster of differentiation 5 (CD5) polynucleotide" refers to a polynucleotide that encodes a CD5 polypeptide. Exemplary CD5 nucleic acid sequences are provided below. >NM_001346456.1 Homo sapiens CD5 molecule (CD5), Transcript variant 2, mRNA 1 gagtcttgct gatgctcccg gctgaataaa ccccttcctt ctttaacttg gtgtctgagg 61 ggttttgtct gtggcttgtc ctgctacatt tcttggttcc ctgaccagga agcaaagtga 121 ttaacggaca gttgaggcag ccccttaggc agcttaggcc tgccttgtgg agcatccccg 181 cggggaactc tggccagctt gagcgacacg gatcctcaga gcgctcccag gtaggcaatt 241 gccccagtgg aatgcctcgt cagagcagtg catggcaggc ccctgtggag gatcaacgca 301 gtggctgaac acagggaagg aactggcact tggagtccgg acaactgaaa cttgtcgctt 361 cctgcctcgg acggctcagc tggtatgacc cagatttcca ggcaaggctc acccgttcca 421 actcgaagtg ccagggccag ctggaggtct acctcaagga cggatggcac atggtttgca 481 gccagagctg gggccggagc tccaagcagt gggaggaccc cagtcaagcg tcaaaagtct 541 gccagcggct gaactgtggg gtgcccttaa gccttggccc cttccttgtc acctacacac 601 ctcagagctc aatcatctgc tacggacaac tgggctcctt ctccaactgc agccacagca 661 gaaatgacat gtgtcactct ctgggcctga cctgcttaga accccagaag acaacacctc 721 caacgacaag gcccccgccc accacaactc cagagcccac agctcctccc aggctgcagc 781 tggtggcaca gtctggcggc cagcactgtg ccggcgtggt ggagttctac agcggcagcc 841 tggggggtac catcagctat gaggcccagg acaagaccca ggacctggag aacttcctct 901 gcaacaacct ccagtgtggc tccttcttga agcatctgcc agagactgag gcaggcagag 961 cccaagaccc aggggagcca cgggaacacc agcccttgcc aatccaatgg aagatccaga 1021 actcaagctg tacctccctg gagcattgct tcaggaaaat caagccccag aaaagtggcc 1081 gagttcttgc cctcctttgc tcaggtttcc agcccaaggt gcagagccgt ctggtggggg 1141 gcagcagcat ctgtgaaggc accgtggagg tgcgccaggg ggctcagtgg gcagccctgt 1201 gtgacagctc ttcagccagg agctcgctgc ggtgggagga ggtgtgccgg gagcagcagt 1261 gtggcagcgt caactcctat cgagtgctgg acgctggtga cccaacatcc cgggggctct 1321 tctgtcccca tcagaagctg tcccagtgcc acgaactttg ggagagaaat tcctactgca 1381 agaaggtgtt tgtcacatgc caggatccaa accccgcagg cctggccgca ggcacggtgg 1441 caagcatcat cctggccctg gtgctcctgg tggtgctgct ggtcgtgtgc ggcccccttg 1501 1561 cgggaatgaa ccaaaacatg tctttccatc gcaaccacac ggcaaccgtc cgatcccatg 1621 1681 acctgtcagc ttatccagct ctggaagggg ctctgcatcg ctcctccatg cagcctgaca 1741 actcctccga cagtgactat gatctgcatg gggctcagag gctcttaaga actgggatcc 1801 atgagcaaaa agccgagagc cagacctgtt tgtcctgaga aaactgtccg ctcttcactt 1861 1921 acaggcgctg ctgccccgag tggcaggcca gctcacactc tgctgcacaa cagctcggcc 1981 gcccctccac ttgtggaagc tgtggtgggc agagccccaa aacaagcagc cttccaacta 2041 gagactcggg ggtgtctgaa ggggggcccc tttccctgcc cgctgggggag cggcgtctca 2101 gtgaaatcgg ctttctcctc agactctgtc cctggtaagg agtgacaagg aagctcacag 2161 ctgggcgagt gcattttgaa tagttttttg tagtagtgc ttttcctcct tcctgacaaa 2221 tcgagcgctt tggcctcttc tgtgcagcat ccacccctgc ggatccctct ggggaggaca 2281 ggaaggggac tcccggagac ctctgcagcc gtggtggtca gaggctgctc acctgagcac 2341 aaagacagct ctgcacattc accgcagctg ccagccaggg gtctgggtgg gcaccaccct 2401 gacccacagc gtcaccccac tccctctgtc ttatgactcc cctccccaac cccctcatct 2461 aaagacacct tcctttccac tggctgtcaa gcccacaggg caccagtgcc acccagggcc 2521 cggcacaaag gggcgcctag taaaccttaa ccaacttggt tttttgcttc acccagcaat 2581 taaaagtccc aagctgaggt agtttcagtc catcacagtt catcttctaa cccaagagtc 2641 agagatgggg ctggtcatgt tcctttggtt tgaataactc ccttgacgaa aacagactcc 2701 tctagtactt ggagatcttg gacgtacacc taatcccatg gggcctcggc ttccttaact 2761 gcaagtgaga agaggaggtc tacccaggag cctcgggtct gatcaaggga gaggccaggc 2821 gcagctcact gcggcggctc cctaagaagg tgaagcaaca tgggaacaca tcctaagaca 2881 ggtcctttct ccacgccatt tgatgctgta tctcctggga gcacaggcat caatggtcca 2941 agccgcataa taagtctgga agagcaaaag ggagttacta ggatatgggg tgggctgctc 3001 ccagaatctg ctcagctttc tgcccccacc aacaccctcc aaccaggcct tgccttctga 3061 gagcccccgt ggccaagccc aggtcacaga tcttccccg accatgctgg gaatccagaa 3121 acagggaccc catttgtctt cccatatctg gtggaggtga gggggctcct caaaagggaa 3181 ctgagaggct gctcttaggg agggcaaagg ttcggggggca gccagtgtct cccatcagtg 3241 ccttttttaa taaaagctct ttcatctata gtttggccac catacagtgg cctcaaagca 3301 accatggcct acttaaaaac caaaccaaaa ataaagagtt tagttgagga gaaaaaaaaa 3361 aaaaaaaaaa aaaaaa

[0126] "Cluster of differentiation 7 (CD7) polypeptide" refers to the NCBI reference sequence: NP_0061 28.1 or a fragment thereof, and Exemplary amino acid sequences are provided below. >NP_006128.1 T cell antigen CD7 precursor [Homo sapiens] 1 MAGPPRLLLL PLLLALARGL PGALAAQEVQ QSPHCTTVPV GASVNITCST SGGLRGIYLR 61 QLGPQPQDII YYEDGVVPTT DRRFRGRIDF SGSQDNLTIT MHRLQLSDTG TYTCQAITEV 121 NVYGSGTLVL VTEEQSQGWH RCSDAPPRAS ALPAPPTGSA LPDPQTASAL PDPPAASALP 181 AALAVISFLL GLGLGVACVL ARTQIKKLCS WRDKNSAACV VYEDMSHSRC NTLSSPNQYQ

[0127] A "cluster of differentiation 7 (CD7) polynucleotide" refers to a polynucleotide that encodes a CD7 polypeptide. Exemplary CD7 nucleic acid sequences are provided below. >NM_006137.7 Homo sapiens CD7 molecule (CD7), mRN A 1 ctctctgagc tctgagcgcc tgcggtctcc tgtgtgctgc tctctgtggg gtcctgtaga 61 cccagagagg ctcagctgca ctcgcccggc tgggagagct gggtgtgggg aacatggccg 121 ggcctccgag gctcctgctg ctgcccctgc ttctggcgct ggctcgcggc ctgcctgggg 181 ccctggctgc ccaagaggtg cagcagtctc cccactgcac gactgtcccc gtgggagcct 241 ccgtcaacat cacctgctcc accagcgggg gcctgcgtgg gatctacctg aggcagctcg 301 ggccacagcc ccaagacatc atttactacg aggacggggt ggtgcccact acggacagac 361 ggttccgggg ccgcatcgac ttctcagggt cccaggacaa cctgactatc accatgcacc 421 gcctgcagct gtcggacact ggcacctaca cctgccaggc catcacggag gtcaatgtct 481 acggctccgg caccctggtc ctggtgacag aggaacagtc ccaaggatgg cacagatgct 541 cggacgcccc accaagggcc tctgccctcc ctgccccacc gacaggctcc gccctccctg 601 acccgcagac agcctctgcc ctccctgacc cgccagcagc ctctgccctc cctgcggccc 661 tggcggtgat ctccttcctc ctcgggctgg gcctgggggt ggcgtgtgtg ctggcgagga 721 cacagataaa gaaactgtgc tcgtggcggg ataagaattc ggcggcatgt gtggtgtacg 781 aggacatgtc gcacagccgc tgcaacacgc tgtcctcccc caaccagtac cagtgaccca 841 gtgggcccct gcacgtcccg cctgtggtcc ccccagcacc ttccctgccc caccatgccc 901 cccaccctgc cacacccctc accctgctgt cctcccacgg ctgcagcaga gtttgaaggg 961 cccagccgtg cccagctcca agcagacaca caggcagtgg ccaggcccca cggtgcttct 1021 cagtggacaa tgatgcctcc tccgggaagc cttccctgcc cagcccacgc cgccaccggg 1081 aggaagcctg actgtccttt ggctgcatct cccgaccatg gccaaggagg gcttttctgt 1141 gggatgggcc tgggcacgcg gccctctcct gtcagtgccg gcccacccac cagcaggccc 1201 ccaacccca ggcagcccgg cagaggacgg gaggagacca gtcccccacc cagccgtacc 1261 agaaataaag gcttctgtgc ttcc

[0128] "Cluster of differentiation 33 (CD33) polypeptide" refers to the NCBI reference sequence: NP_00 A protein having at least about 85% amino acid sequence identity with 1763.3 or a fragment thereof. CD33 is also known as Siglec-3. The acid sequence is provided below. >NP_001763.3 Myeloid cell surface antigen CD33 isoform 1 precursor [Hom o sapiens] 1 MPLLLLLPLL WAGALAMDPN FWLQVQESVT VQEGLCVLVP CTFFHPIPYY DKNSPVHGYW 61 FREGAIISRD SPVATNKLDQ EVQEETQGRF RLLGDPSRNN CSLSIVDARR RDNGSYFFRM 121 ERGSTKYSYK SPQLSVHVTD LTHRPKILIP GTLEPGHSKN LTCSVSWACE QGTPPIFSWL 181 SAAPTSLGPR TTHSSVLIIT PRPQDHGTNL TCQVKFAGAG VTTERTIQLN VTYVPQNPTT 241 GIFPGDGSGK QETRAGVVHG AIGGAGVTAL LALCLCLIFF IVKTHRRKAA RTAVGRNDTH 301 PTTGSASPKH QKKSKLHGPT ETSSCSGAAP TVEMDEELHY ASLNFHGMNP SKDTSTEYSE 361 VRTQ

[0129] A "cluster of differentiation 33 (CD33) polynucleotide" refers to a polynucleotide encoding a CD33 polypeptide. An exemplary CD33 nucleic acid sequence is provided below. >NM_001772.4 Homo sapiens CD33 molecule (CD33), translocation transcription variant 1, mRNA 1 ctgctcacac aggaagccct ggaagctgct tcctcagaca tgccgctgct gctactgctg 61 cccctgctgt gggcaggggc cctggctatg gatccaaatt tctggctgca agtgcaggag 121 tcagtgacgg tacaggaggg tttgtgcgtc ctcgtgccct gcactttctt ccatcccata 181 ccctactacg acaagaactc cccagttcat ggttactggt tccgggaagg agccattata 241 tccagggact ctccagtggc cacaaacaag ctagatcaag aagtacagga ggagactcag 301 ggcagattcc gcctccttgg ggatcccagt aggaacaact gctccctgag catcgtagac 361 gccaggagga gggataatgg ttcatacttc tttcggatgg agagaggaag taccaaatac 421 agttacaaat ctccccagct ctctgtgcat gtgacagact tgacccacag gcccaaaatc 481 ctcatccctg gcactctaga acccggccac tccaaaaacc tgacctgctc tgtgtcctgg 541 gcctgtgagc agggacacc cccgatctc tcctgttgt cagctgcccc cacctccctg 601 ggccccagga ctactcactc ctcggtgctc ataatcaccc cacggcccca ggaccacggc 661 accaacctga cctgtcaggt gaagttcgct ggagctgtg tgactacgga gagaaccac 721 cagctcaacg tcacctatgt tccacagac cacaacg gtatctttcc aggagatggc 781 tcagggaac agagaccag agcaggagtg gttcatgggg ccattggagg agctggtgtt 841 acagccctgc tcgctttg tctctgcctc atctcttca tagtgagac ccacaggagg 901 aaagcagcca ggacagcagt gggcaggaat vakacccacc ctaccacagg gtcagcctcc 961 ccgaacacc agagaagtc caagttacat ggcccactg aaacctcag ctgttcaggt 1021 gccgccccta ctgtggagat ggatgaggag ctgcattatg cttccctca cttcatggg 1081 atgaatcctt ccaggacac ctccaccgaa tactcagagg tgaggaccca gtgaggaacc 1141 cacaagagca tcaggctcag ctagaagagatc cacatcctct acaggtcggg gaccaaaggc 1201 tgattctttgg agatttaaca cccacaggc aatggtttta tagacattat gtgagttcc 1261 tgctatatta acatcatctt agactttgca agcagagagt cgtggaatca aatctgtgct 1321 ctttcatttg ctaagtgtat gatgtcacac aagctcctta accttccatg tctccatttt 1381 cttctctgtg aagtaggtat aagaagtcct atctcatagg gatgctgtga gcattaaata 1441 aaggtacaca tggaaaacac ca

[0130] "Cluster of differentiation 52 (CD52) polypeptide" refers to the NCBI reference sequence: NP_00 A protein having at least about 85% amino acid sequence identity with 1794.2 or a fragment thereof. CD52 is also known as CAMPATH-1. The amino acid sequence is provided below. >NP_001794.2 CAMPATH-1 antigen precursor [Homo sapiens ] 1 MKRFLFLLLT ISLLVMVQIQ TGLSGQNDTS QTSSPSASSN ISGGIFLFFV ANAIIHLFCF 61S

[0131] A "cluster of differentiation 52 (CD52) polynucleotide" refers to a polynucleotide encoding a CD52 polypeptide. Exemplary CD52 nucleic acid sequences are provided below. >NM_001803.3 Homo sapiens CD52 molecule (CD52), m RNA 1 agacagccct gagatcacct aaaaagctgc taccaagaca gccacgaaga tcctaccaaa 61 atgaagcgct tcctcttcct cctactcacc atcagcctcc tggttatggt acagatacaa 121 actggactct caggacaaaa cgacaccagc caaaccagca gcccctcagc atccagcaac 181 ataagcggag gcattttcct tttcttcgtg gccaatgcca taatccacct cttctgcttc 241 agttgaggtg acacgtctca gccttagccc tgtgccccct gaaacagctg ccaccatcac 301 tcgcaagaga atcccctcca tctttgggag gggttgatgc cagacatcac caggttgtag 361 aagttgacag gcagtgccat gggggcaaca gccaaaatag gggggtaatg atgtaggggc 421 caagcagtgc ccagctgggg gtcaataaag ttacccttgt acttgca

[0132] "Cluster of differentiation 123 (CD123) polypeptide" refers to the NCBI reference sequence: NP_ A polypeptide having at least about 85% amino acid sequence identity with 002174.1 or a fragment thereof. CD123 is also known as the interleukin-3 receptor. Exemplary amino acid sequences are provided below. >NP_002174.1 Interleukin-3 receptor subunit alpha isoform 1 Precursor [Homo sapiens] 1 MVLLWLTLLL IALPCLLQTK EDPNPPITNL RMKAKAQQLT WDLNRNVTDI ECVKDADYSM 61 PAVNNSYCQF GAISLCEVTN YTVRVANPPF STWILFPENS GKPWAGAENL TCWIHDVDFL 121 SCSWAVGPGA PADVQYDLYL NVANRRQQYE CLHYKTDAQG TRIGCRFDDI SRLSSGSQSS 181 HILVRGRSAA FGIPCTDKFV VFSQIEILTP PNMTAKCNKT HSFMHWKMRS HFNRKFRYEL 241 QIQKRMQPVI TEQVRDRTSF QLLNPGTYTV QIRARERVYE FLSAWSTPQR FECDQEEGAN 301 TRAWRTSLLI ALGTLLALVC VFVICRRYLV MQRLFPRIPH MKDPIGDSFQ NDKLVVWEAG 361 KAGLEECLVT EVQVVQKT

[0133] "Cluster of differentiation 123 (CD123) polynucleotide" means a CD123 polypeptide. Exemplary CD123 nucleic acid sequences are provided below. >NM_002183.4 Homo sapiens interleukin 3 receptor subunit nit α (IL3RA), transcript variant 1, mRNA 1 cttcggtttc tcttcgggga aagctgcttt cagcgcacac gggaagatat cagaaacatc 61 ctaggatcag gacaccccag atcttctcaa ctggaaccac gaaggctgtt tcttccacac 121 agtactttga tctccattta agcaggcacc tctgtcctgc gttccggagc tgcgttcccg 181 atggtcctcc tttggctcac gctgctcctg atcgccctgc cctgtctcct gcaaacgaag 241 gaagatccaa acccaccaat cacgaaccta agatgaaag caaaggctca gcagttgacc 301 tgggacctta acagaaatgt gaccgatatc gagtgtgtta aagacgccga ctattctatg 361 ccggcagtga acaatagcta ttgccagttt ggagcaattt ccttatgtga agtgaccaac 421 tacaccgtcc gagtggccaa cccaccattc tccacgtgga tcctcttccc tgagaacagt 481 gggaagcctt gggcaggtgc ggagaatctg acctgctgga ttcatgacgt ggatttcttg 541 agctgcagct gggcggtagg cccgggggcc cccgcggacg tccagtacga cctgtacttg 601 aacgttgcca acaggcgtca acagtacgag tgtcttcact acaaaacgga tgctcaggga 661 acacgtatcg ggtgtcgttt cgatgacatc tctcgactct ccagcggttc tcaaagttcc 721 cacatcctgg tgcggggcag gagcgcagcc ttcggtatcc cctgcacaga taagtttgtc 781 gtctttcac agattgagat attaactcca cccaacatga ctgcaaagtg tataagaca 841 cattccttta tgcactggaa aatgagaagt catttcaatc gcaaatttcg ctatgagctt 901 cagatacaaa agagaatgca gcctgtaatc acagaacagg tcagagacag aacctccttc 961 cagctactca atcctggaac gtacacagta caaataagag cccgggaaag agtgtatgaa 1021 ttcttgagcg cctggagcac cccccagcgc ttcgagtgcg accaggagga gggcgcaaac 1081 acacgtgcct ggcggacgtc gctgctgatc gcgctgggga cgctgctggc cctggtctgt 1141 gtcttcgtga tctgcagaag gtatctggtg atgcagagac tctttccccg catccctcac 1201 atgaaagacc ccatcggtga cagcttccaa aacgacaagc tggtggtctg ggaggcgggc 1261 aaagccggcc tggaggatg tctggtgact gaagtacagg tcgtgcagaa aacttgagac 1321 tggggttcag ggcttgtggg ggtctgcctc aatctccctg gccgggccag gcgcctgcac 1381 agactggctg ctggacctgc gcacgcagcc caggaatgga cattcctaac gggtggtggg 1441 catgggagat gcctgtgtaa tttcgtccga agctgccagg aagaagaaca gaactttgtg 1501 tgtttatttc atgataaagt gatttttttttttttaaccc a

[0134] "Cluster of differentiation 137 (CD137) polypeptide" refers to the NCBI reference sequence: NP_ A polypeptide having at least about 85% amino acid sequence identity with 001552.2 or a fragment thereof. CD137 is also known as 4-1BB. The acid sequence is provided below. >NP_001552.2 Tumor necrosis factor receptor superfamily member 9 precursor[ Homo sapiens] 1 MGNSCYNIVA TLLLVLNFER TRSLQDPCSN CPAGTFCDNN RNQICSPCPP NSFSSAGGQR 61 TCDICRQCKG VFRTRKECSS TSNAECDCTP GFHCLGAGCS MCEQDCKQGQ ELTKKGCKDC 121 CFGTFNDQKR GICRPWTNCS LDGKSVLVNG TKERDVVCGP SPADLSPGAS SVTPPAPARE 181 PGHSPQIISF FLALTSTALL FLLFFLTLRF SVVKRGRKKL LYIFKQPFMR PVQTTQEEDG 241 CSCRFPEEEE GGCEL

[0135] "Cluster of differentiation 137 (CD137) polynucleotide" means a CD137 polypeptide. Exemplary CD137 nucleic acid sequences are provided below. >NM_001561.6 Homo sapiens TNF receptor superfamily - Member 9 (TNFRSF9), mRNA 1 gcagaagcct gaagaccaag gagtggaaag ttctccggca gccctgagat ctcaagagtg 61 acatttgtga gaccagctaa tttgattaaa attctcttgg aatcagcttt gctagtatca 121 tacctgtgcc agatttcatc atgggaaaca gctgttacaa catagtagcc actctgttgc 181 tggtcctcaa ctttgagagg acaagatcat tgcaggatcc ttgtagtaac tgcccagctg 241 gtacattctg tgataataac aggaatcaga ttgcagtcc ctgtcctcca atagttctct 301 ccagcgcagg tggacaagg acctgtgaca tatgcagca gtgtaaggt gtttcagga 361 ccaggaagga gtgttcctcc accagcaatg cagagtgtga ctgcactcca gggttcact 421 gcctgggggc aggatgcagc atgtgtgaac aggatgta aaaggtcaa gaactgacaa 481 aaaaaggttg tgagactgt tgctttggga catttaacga tcagaacgt gcatctgtc 541 gaccctggac aactgttct tggatggaa agtctgtgct tgtgaatggg acgaaggaga 601 gggacgtggt ctgtggacca tctccagccg accctctcc gggagcatcc tctgtgaccc 661 cgcctgcccc tgcgagagag ccaggacact ctccgcagat catctccttc tttcttgcgc 721 tgacgtcgac tgcgttgctc ttcctgctgt tctcctcac gctccgttc tctgttgtta 781 aacggggcag aaagaaactc ctgtatatat tcaacaacc atttatgaga ccagtacaaa 841 ctactcaaga ggagatggc tgtagctgcc gatttccaga agaagaa ggaggatgtg 901 aactgtgaaa tggagtcaa taggctgtt gggactttct tgaagaag caggaata 961 tgagtcatcc gctatcacag ctttcaaaag caagaacacc atcctacata atacccagga 1021 ttcccccaac acacgttcttt ttctaaatgc caatgagttg gcctttaaaa atgcaccact 1081 tttttttt ttttgacagg gtctcactct gtcacccagg ctggagtgca gtggcaccac 1141 catgctctc tgcagccttg acctctggga gctcaagtga tcctcctgcc tcagctcct 1201 gagtagctgg aactacaagg aagggccacc acacctgact aacttttttg ttttttgttt 1261 ggtaaagatg gcatttcacc atgttgtaca ggctggtctc aaactcctag gttcactttg 1321 gcctcccaaa gtgctgggat tacagacatg aactgccagg cccggccaaa ataatgcacc 1381 acttttaaca gaacagacag atgaggacag agctggtgat aaaaaaaaa aaaaaaagc 1441 atttctaga taccacttaa caggtttgag ctagtttttt tgaaatccaa agaaaattat 1501 agtttaaatt caattacata gtccagtggt ccaactataa ttataatcaa aatcaatgca 1561 ggtttgtttt ttggtgctaa tatgacatat gacaataagc cacgaggtgc agtaagtacc 1621 cgactaaagt ttccgtgggt tctgtcatgt aacacgacat gctccaccgt caggggggag 1681 tatgagcaga gtgcctgagt ttagggtcaa ggacaaaaaa cctcaggcct ggaggaagtt 1741 ttggaaagag ttcaagtgtc tgtatatcct atggtcttct ccatcctcac accttctgcc 1801 tttgtcctgc tcccttttaa gccaggttac attctaaaaa ttcttaactt ttaacataat 1861 attttatacc aaagccaata aatgaactgc atatgatagg tatgaagtac agtgagaaaa 1921 ttaacacctg tgagctcatt gtcctaccac agcactagag tgggggccgc caaactccca 1981 tggccaaacc tggtgcacca tttgcctttg tttgtctgtt ggtttgcttg agacagtctt 2041 gctctgttgc ccaggctgga atggagtggc tattcacagg cacaatcata gcacacttta 2101 gccttaaact cctgggctca agtgatccac ccgcctcagt ctcccaagta gctgggatta 2161 caggtgcaaa cctggcatgc ctgccattgt ttggcttatg atctaaggat agctttttaa 2221 attttattca ttttattttt ttttgagaca gtgtctcact ctgtctccca ggctggagta 2281 cagtggtaca atcttggatc accgcctccc agtttcaagt gatctccctg cctcagcctc 2341 ctaagtagct gggactacag gtatgtgcca ccacgcctgg ctaattttta tatttttagt 2401 agagacgggg tttcaccatg ttgtccaggc tggtctcaaa ctcctgacct caggtgatct 2461 gcccacctct gcctcccaaa gtgctgggat tacaggcatg agccaccatg cctggccatt 2521 tcttacactt ttgtatgaca tgcctattgc aagcttgcgt gcctctgtcc catgttatt 2581 tactctggga tttaggtgga gggagcagct tctatttgga acattggcca tcgcatggca 2641 aatgggtatc tgtcacttct gctcctattt agttggttct actataacct ttagagcaaa 2701 tcctgcagcc aagccaggca tcaatagggc agaaaagtat attctgtaaa taggggtgag 2761 gagaagatat ttctgaacaa tagtctactg fộttccaaa ttgcttttca aagtggctgt 2821 tctaatgtac tcccgtcagt catataagtg tcatgtaagt atcccattga tccacatcct 2881 tgctaccctc tggtactatc aggtgccctt aattttgcca agccagtggg tatagaatga 2941 gatctcactg tggtcttagt ttgcatttgc ttggttactg atgagcacct tgtcaaatat 3001 ttatatacca tttgtgttta tttttttaaa taaaatgctt gctcatgctt ttttgcccat 3061 ttgcaaaaaa acttggggcc gggtgcagtg gctcatgcct gtagtcccag ctctttggga 3121 ggccaaggtg ggcagatcgc ttgagcccag gagttcgaga ccagccttgg caacatggcg 3181 aaaccctgtc tttacaaaaa atacaaaaat tagccgggtg tggtggtgtg cacctgaagt 3241 cccagctact fòaggttc gctttgagcc tgggaggcag aggttgcagt gagctgggac 3301 cgcatcacta cacttcagcc tgggcaacag agaaaaacct tttctcagaa acaaacaaac 3361 ccaaatgtgg ttgtttgtcc tgattcctaa aaggtcttta tgtattctag samaaatct 3421 ttggtcagtt atatgtgtta aaaaatatct tctttgtggc caggcacggt agctcacacc 3481 tgtaatccca gcactttgcg gggctgaggt gggtggatca tctgaggtca agagttcaag 3541 atcagcctgg ccaacacagt gaaaccccat ctctactaaa catgtacaaa acttagctgg 3601 gtatggtggc gggtgcctgt aaccccagct gctccagagg ctgtggcaga agaatcgctt 3661 gaacccagga ggcagaggtt gcagcgagcc aagattgtgc cattgcactc cagactgggt 3721 gacaagagtg aaattctgcc tatctatcta tctatctatc tatatctata tatatatata 3781 tatatatcct ttgtaattta tttttccctt ttaaaaatt tttataaaat tcttttttt 3841 ttttatttt agcagaggtg aggtttctga ggtttcatta tgttgcccag gctggtcttg 3901 aactcctgag ctcaagtgat cctcccacct cagccttcca aagtgctgga attgcagaca 3961 tgagccaccg cgcccctcct gttttctct aattaatggt gtctttcttt gtctttctgg 4021 tataagcaa aaagttcttc atttgatttg gttaaattta taactgtttt ctcatatggt 4081 taacatttt tcttgcctgg ctaaagaaat ccttttctgc ccaatactat aaagaggttt 4141 gcccacattt tattccaaaa gttttaagtt ttgtctttca tcttgaagtc taatgtatca 4201 ggaactggct tttgtgcctg ttgggaggta gtgatccaat tccatgtctt gcatgtaggt 4261 aaccactggt ccctgcgcca tgtattcaat acgtcgtctt tctcctgcgg gtctgcaatc 4321 tcacctacca tccatcaagt ttccataggg ccatgggtct gcttctggc tcctgttct 4381 gttccattgt caatttgtct atcctgtgcc agtatcacac tgtgtttatt acaatagctt 4441 tgtaacagct ctcgatatcc ggtaggacat ctccctccac cttctttttc tacttcagaa 4501 gtgtcttagc taggtcaggc acggtggctc acgcctgtaa tcccagcact ttgggaggcc 4561 gacgcggatg gatcacctga ggtcaggagt tttgagacag cctggccaac atggtgaaac 4621 cccatctcta ctaaaaaata caaaaattag tcaggcatgg tggcatgtgc ctgtaatccc 4681 agctatttgg gaggctgagg ccggagaatt gcttgaaccc ggggggcgga ggttgcagtg 4741 agccgagatc gtaccattgc actccagcct gggtgacaga gcgaaactct gtctcaggaa 4801 aaaaaagaaa agagatgtct tggttattct tggttcttta ttatcaata taaattttag 4861 aagctgaatt tgaaaagatt tggattggaa tttcattaaa tctacaggtc aatttaggga 4921 gagttgataa tttacagaa ttgagtcatc tggtgttcca ataagaataa gagaacaatt 4981 attggctgta caattcttgc caaatagtag gcaaagcaaa gcttaggaag tatactggtg 5041 ccatttcagg aacaaagcta ggtgcgaata tttttgtctt tctgaatcat gatgctgtaa 5101 gttctaaagt gatttctcct cttggctttg gacacatggt gtttaattac ctactgctga 5161 ctatccacaa acagaaagag actggtcatg ccccacaggg ttggggtatc caagataatg 5221 gagcgaggct ctcatgtgtc ctaggttaca caccgaaaat ccacagttta ttctgtgaag 5281 aaaggaggct atgtttatga tacagactgt gatattttta tcatagccta ttctggtatc 5341 atgtgcaaaa gctataaatg aaaaacacag gaacttggca tgtgagtcat tgctccccct 5401 aaatgacaat taataaggaa ggaacattga gacagaataa aatgatcccc ttctgggttt 5461 aatttagaaa gttccataat taggtttaat agaaataaat gtaaatttct atgattaaaa 5521 ataaattagc acatttaggg atacacaaat tataaatcat tttctaaatg ctaaaaacaa 5581 gctcaggttt ttttcagaag aaagttttaa ttttttttct ttagtggaag atatcactct 5641 gacggaaagt tttgatgtga ggggcggatg actataaagt gggcatcttc ccccacagga 5701 agatgtttcc atctgtgggt gagaggtgcc caccgcagct agggcaggtt acatgtgccc 5761 tgtgtgtggt aggacttgga gagtgatctt tatcaacgtt tttatttaaa agactatcta 5821 ataaaacaca aaactatgat gttcacagga aaaaaagaat aagaaaaaaa ga

[0136] "Cluster of differentiation 247 (CD247) polypeptide" refers to the NCBI reference sequence: NP_ A tag having at least about 85% amino acid sequence identity with 932170.1 or a fragment thereof. CD137 is also known as CD3ζ. Exemplary Amino Acids The sequences are provided below. >NP_932170.1 T cell surface glycoprotein CD3 ζ chain isoform 1 precursor [Homo sapiens] 1 MKWKALFTAA ILQAQLPITE AQSFGLLDPK LCYLLDGILF IYGVILTALF LRVKFSRSAD 61 APAYQQGQNQ LYNELNLGRR EEYDVLDKRR GRDPEMGGKP QRRKNPQEGL YNELQKDKMA 121 EAYSEIGMKG ERRRGKGHDG LYQGLSTATK DTYDALHMQA LPPR

[0137] "Cluster of differentiation 247 (CD247) polynucleotide" means a CD247 polypeptide. Exemplary CD247 nucleic acid sequences are provided below. >NM_NM_198053.3 Homo sapiens CD247 molecule (CD2 47), transcript variant 1, mRNA 1 aaccgtccccg gccaccgctg cctcagcctc tgcctcccag cctctttctg agggaaagga 61 caagatgaag tggaaggcgc ttttcaccgc ggccatcctg caggcacagt tgccgattac 121 agaggcacag agctttggcc tgctggatcc caaactctgc tacctgctgg atggaatcct 181 cttcatctat ggtgtcattc tcactgcctt gttcctgaga gtgaagttca gcaggagcgc 241 agacgccccc gcgtaccagc agggccagaa ccagctctat aacgagctca atctaggacg 301 agagaggag tacgatttt tggacagag acgtggccgg gacctgaga tgggggaa 361 gccgcagaga aggaacc ctcaggaagg cctgtacaat gaactgcaga agatagat 421 ggcggaggcc tacagtgaga ttgggatgaa aggcgagcgc cggaggggca aggggcacga 481 tggcctttac cagggtctca gtacagccac CAggacacc tacgacgcc ttcacatgca 541 ggccctgccc cctcgctaac agccaggga ttcaccact aaggccag acctgcagac 601 gcccagatta tgagacacag gatgaagcat ttacaccg gttcactt ctcagccact 661 gaagtattcc cctttatgta caggatgctt tggttatat tagctccaaa ccttcacaca 721 cagactgttg tccctgcact ctttaaggga gtgtactccc agggctcg gccctggcct 781 tgggccctct gtttgccgg tggtgcaggt agactgtct cctggcggtt cctcgttctc 841 cctgggaggc gggcgcactg cctctcacag ctgagttgtt gagtctgtt tgtaagtcc 901 ccagagaag cgcagatgct agcacatgcc ctaatgtctg tatcactg tgtctgagtg 961 gcttcactcc tgctgtaaat tggctctg tgtcacctt cacctccttt caggtact 1021 gtactgggcc atgttgtgcc tccctggtga gagggccggg cagaggggca gatggaaagg 1081 agcctaggcc aggtgcaacc agggagctgc aggggcatgg gaaggtgggc gggcagggga 1141 gggtcagcca gggcctgcga gggcagcggg agcctccctg cctcaggcct ctgtgccgca 1201 ccattgaact gtaccatgtg ctacaggggc cagaagatga acagactgac cttgatgagc 1261 tgtgcacaaa gtggcataaa aaacatgtgg ttacacagtg tgaataaagt gctgcggagc 1321 aagaggaggc cgttgattca cttcacgctt tcagcgaatg acaaaatcat ctttgtgaag 1381 gcctcgcagg aagacccaac acatgggacc tataactgcc cagcggacag tggcaggaca 1441 ggaaaaaccc gtcaatgtac taggatactg ctgcgtcatt acagggcaca ggccatggat 1501 ggaaaacgct ctctactctg ctttttttct actgttttaa tttatactgg catgctaaag 1561 ccttcctatt ttgcataata aatgcttcag tgaaaatgca

[0138] "Co-administration" or "co-administered" means the administration of two or more therapeutic agents or pharmaceutical agents during the course of treatment. Such co-administration may be simultaneous or sequential. The sequential administration of the subsequently administered therapeutic agent or pharmaceutical composition may be in the form of a first pharmaceutical composition or therapeutic agent. This may be done at any time during the course of treatment after administration of the therapeutic agent.

[0139] The term "conservative amino acid substitution" or "conservative mutation" refers to a mutation in which one amino acid is replaced by a common It refers to the substitution of an amino acid with another amino acid that has the same properties as the amino acid. A functional method to define normalized amino acid changes between corresponding proteins of similar organisms is The aim is to analyze the frequency of cases (Schulz, GE and Schirmer, R .H.,Principles of Protein Structure,Spri According to this analysis, Groups of amino acids are preferentially exchanged between amino acids within a group, and thus can be defined when their effects on the overall protein structure are most similar to each other. (Schulz, GE and Schirmer, RH, supra). Non-limiting examples include amino acid substitutions of amino acids (e.g., substitutions of amino acids so that the positive charge can be maintained). Asparagine to lysine, or vice versa, so that a negative charge can be maintained to glutamic acid, or vice versa, to threonine so that the free -OH can be maintained and glutamine to asparagine so that free -NH2 can be maintained. Examples include:

[0140] The terms "coding sequence" or "protein-coding sequence" are used interchangeably herein. It is used to refer to a segment of a polynucleotide that encodes a protein. is bound by a start codon closer to the 5' end and a stop codon closer to the 3' end. A coding sequence may also be referred to as an open reading frame.

[0141] "Codon optimization" refers to the modification of at least one codon (e.g., about 1, 2, 3, 4, 5, 10, 15, 20, 25, 50, or more codons) While maintaining the sequence, it is possible to select the more frequently or most frequently used components in the genes of the host cell. Nucleic acid sequences for improving expression in a host cell of interest by substituting Various species have specific codons for specific amino acids. Codon bias (differences in codon usage between organisms) is often This correlates with the efficiency of translation of messenger RNA (mRNA), which in turn determines, among other things, the efficiency of translation. The properties of the codons involved and the availability of specific transfer RNA (tRNA) molecules The predominance of tRNAs selected in cells is generally dependent on peptides. The codons reflect the most frequently used codons in peptide synthesis. Based on the optimization, the codon usage can be adjusted for optimal gene expression in a given organism. For example, the usage table can be found at www.kazusa.orjp / codon / (July 9, 2002) These are readily available in the "codon usage database" available at http: / / www.ncbi.nlm.nih.gov / pubmed / 2014010100 (see http: / / www.ncbi.nlm.nih.gov / pubmed / 2014010100). The table can be adapted in several ways. don usage tabulated from the internation al DNA sequence databases:status for the See Nucl. Acids Res. 28:292 (2000) Codon selection to optimize a particular sequence for expression in a particular host cell Computer algorithms for this are also available, e.g., Gene Forge ( Aptagen; Jacobus, Pa.) is also available. One or more codons (e.g., 1, 2, 3, 4) in the sequence encoding the engineered nuclease , 5, 10, 15, 20, 25, 50 or more codons, or all codons The codons correspond to the most frequently used codon for a particular amino acid.

[0142] "Cytidine deaminase" is a enzyme that converts amino groups into carbonyl groups through a deamination reaction. In one embodiment, the cytidine deaminase is a polypeptide or fragment thereof that can catalyze a cytidine deaminase (Cd)-dependent catalysis of ... Petase converts cytosine to uracil or 5-methylcytosine to thymine. PmCDA1 derived from Petromyzon marinus (Petromyzon ma rinus cytosine deaminase 1 (“PmCDA1”)), or mammalian (e.g., AID (Activation-Induced Cytidine Deaminase) derived from human, pig, cow, horse, monkey, etc. AICDA and APOBEC are exemplary cytidine deaminases. .

[0143] The nucleotide and amino acid sequences of PmCDA1 and human AID were The amino acid sequence is shown below. >tr|A5H718|A5H718_PETMA Cytosine deaminase OS=Pe Tromyzon marinus OX=7757 PE=2 SV=1 Lamprey MTDAEYVRIHEKLDIYTFKKQFFNNKKSVSHRCYVLFELK RRGERRACFWGYAVNKPQSGTERGIHAEIFSIRKVEEYLR DNPGQFTINWYSSWSPCADCAEKILEWYNQELRGNGHTLK IWACKLYYEKNARNQIGLWNLRDNGVGLNVMVSEHYQCCR KIFIQSSHNQLNENRWLEKTLKRAEKRRSELSIMIQVKIL HTTKSPAV >EF094822.1 Sea lamprey isolate PmCDA. 21 Cytosine deaminase mRNA, full - length CDS TGACACGACACAGCCGTGTATATGAGGAAGGGTAGCTGGA TGGGGGGGGGGGGAATACGTTCAGAGAGGACATTAGCGAG CGTCTTGTTGGTGGCCTTGAGTCTAGACACCTGCAGACAT GACCGACGCTGAGTACGTGAGAATCCATGAGAAGTTGGAC ATCTACACGTTTAAGAAACAGTTTTTCAACAACAAAAAAT CCGTGTCGCATAGATGCTACGTTCTCTTTGAATTAAAACG ACGGGGTGAACGTAGAGCGTGTTTTTGGGGCTATGCTGTG<~ AATAAACCACAGAGCGGGACAGAACGTGGAATTCACGCCG AAATCTTTAGCATTAGAAAAGTCGAAGAATACCTGCGCGA CAACCCCGGACAATTCACGATAAATTGGTACTCATCCTGG AGTCCTTGTGCAGATTGCGCTGAAAAGATCTTAGAATGGT ATAACCAGGAGCTGCGGGGGAACGGCCACACTTTGAAAAT CTGGGCTTGCAAACTCTATTACGAGAAAAATGCGAGGAAT CAAATTGGGCTGTGGAACCTCAGAGATAACGGGGTTGGGT Note: There might be a small error in your original text where is not properly formatted in the original. I've tried to keep it as similar as possible in the translation. Also, the "Petromyzon marinus" in the original was translated as "Sea lamprey" which is a common name for it. If you have any specific preferences regarding these translations, please let me know. TGAATGTAATGGTAAGTGAACACTACCAATGTTGCAGGAA AATATTCATCCAATCGTCGCACAATCAATTGAATGAGAAT AGATGGCTTGAGAAGACTTTGAAGCGAGCTGAAAAACGAC GGAGCGAGTTGTCCATTATGATTCAGGTAAAAATACTCCA CACCACTAAGAGTCCTGCTGTTTAAGAGGCTATGCGGATG GTTTTC >tr|Q6QJ80|Q6QJ80_HUMAN Activation-induced cytidine deaminase OS=Homo sapiens OX=9606 GN=AICDA PE=2 SV =1 MDSLLMNRRKFLYQFKNVRWAKGRRETYLCYVVKRRDSAT SFSLDFGYLRNKNGCHVELLFLRYISDWDLDPGRCYRVTW FTSWSPCYDCARHVADFLRGNPNLSLRIFTARLYFCEDRK AEPEGLRRLHRAGVQIAIMTFKAPV >NG_011588.1:5001-15681 Homo sapiens Activation-induced cytidine deaminase (AICDA), RefSeqGene on chromosome 12 (LR G_17) AGAGAACCATCATTAATTGAAGTGAGATTTTTCTGGCCTG AGACTTGCAGGGAGGCAAGAAGACACTCTGGACACCACTA TGGACAGGTAAAGAGGCAGTCTTCTCGTGGGTGATTGCAC TGGCCTTCCTCTCAGAGCAAATCTGAGTAATGAGACTGGT AGCTATCCCTTTCTCTCATGTAACTGTCTGACTGATAAGA TCAGCTTGATCAATATGCATATATATTTTTTGATCTGTCT CCTTTTCTTCTATTCAGATCTTATACGCTGTCAGCCCAAT TCTTTCTGTTTCAGACTTCTCTTGATTTCCCTCTTTTTCA TGTGGCAAAAGAAGTAGTGCGTACAATGTACTGATTCGTC CTGAGATTTGTACCATGGTTGAAACTAATTTATGGTAATA ATATTAACATAGCAAATCTTTAGAGACTCAAATCATGAAA AGGTAATAGCAGTACTGTACTAAAAACGGTAGTGCTAATT TTCGTAATAATTTTGTAAATATTCAACAGTAAAACAACTT GAAGACACACTTTCCTAGGGAGGCGTTACTGAAATAATTT AGCTATAGTAAGAAAATTTGTAATTTTAGAAATGCCAAGC ATTCTAAATTAATTGCTTGAAAGTCACTATGATTGTGTCC ATTATAAGGAGACAAATTCATTCAAGCAAGTTATTTAATG TTAAAGGCCCAATTGTTAGGCAGTTAATGGCACTTTTACT ATTAACTAATCTTTCCATTTGTTCAGACGTAGCTTAACTT ACCTCTTAGGTGTGAATTTGGTTAAGGTCCTCATAATGTC TTTATGTGCAGTTTTTGATAGGTTATTGTCATAGAACTTA TTCTATTCCTACATTTATGATTACTATGGATGTATGAGAA TAACACCTAATCCTTATACTTTACCTCAATTTAACTCCTT TATAAAGAACTTACATTACAGAATAAAGATTTTTTAAAAA TATATTTTTTTGTAGAGACAGGGTCTTAGCCCAGCCGAGG CTGGTCTCTAAGTCCTGGCCCAAGCGATCTCTCCTGCCTGG GCCTCCTAAAGTGCTGGAATTATAGACATGAGCCATCACA TCCAATATACAGAATAAAGATTTTTAATGGAGGATTTAAT GTTCTTCAGAAAATTTCTTGAGGTCAGACAATGTCAAAT GTTCCCTCAGTTTACACTGAGATTTTGAAAAACAAGTCTGA GCTATAGGTCCTTGTGAAGGGTCCATTGGAAATACTTGTT CAAAGTAAAATGGAAAGCAAAGGTAAAATCAGCAGTTGAA ATTCAGAGAAAGACAGAAAAGGAGAAAAGATGAAATTCAA CAGGACAGAAGGGAAATATATTATCATTAAGGAGGACAGT ATCTGTAGAGCTCATTAGTGATGGCAAAATGACTTGGTCA GGATTATTTTTAACCCGCTTGTTTCTGGTTTGCACGGCTG GGGATGCAGCTAGGGTTCTGCCTCAGGGAGCACAGCTGTC CAGAGCAGCTGTCAGCCTGCAAGCCTGAAACACTCCCTCG GTAAAGTCCTTCCTACTCAGGACAGAAATGACGAGAACAG GGAGCTGGAAACAGGCCCCTAACCAGAGAAGGGAAGTAAT GGATCAACAAAGTTAACTAGCAGGTCAGGATCACGCAATT CATTTCACTCTGACTGGTAACATGTGACAGAAACAGTGTA GGCTTTATTGTATTTTCATGTAGAGTAGGACCCAAAAATCC ACCCAAAGTCCTTTATCTATGCCACATCCTTCTTATCTAT ACTTCCAGGACACTTTTTCTTCCTTATGATAAGGCTCTCT CTCTCTCCACACACACACACACACACACACACACACAC ACACACACCACAAACACACACCCCGCAACCAAGGTGCA TGTAAAAAGATGTAGATTCCTCTGCCTTCTCATCTACAC AGCCCAGGAGGGTAAGTTAAATAAGAGGGATTTATTGGT AAGAGATGATGCTTAATCTGTTTAACACTGGGCCTCAAAG AGAGAATTTCTTTTCTTCTGTACTTATTAAGCACCTATTA TGTGTTGAGCTTATATATACAAAGGGTTATTATATGCTAA TATAGTAATAGTAATGGTGGTTGGTACTATGGTAATTACC ATAAAAATTATTATCCTTTTTAAAATAAAGCTAATTATTAT TGGATCTTTTTTAGTTCATTTTTATGTTTTTTATGTTTT TGATTTTTTAAAAGACAATCTCACCCTGTTACCCAGGCTG GAGTGCAGTGGTGCAATCATAGCTTCTGCAGTCTTGAAC TCCTGGGCTCAAGCAATCCTCCTGCCTTGGCCTCCCAAAG TGTTGGGATACAGTCATGAGCCACTGCATCTGGCCTAGGA TCCATTTAGATTAAAAATATGCATTTTAAATTTTAAAAATA TATGGCTAATTTTTACCTTATGTAATGTGTATACTGGCAA TAAATCTAGTTTGCTGCCTAAAGTTTAAAGTGCTTTCCAG TAAGCTTCATGTACGTGAGGGGAGACATTTAAAGTGAAAC AGACAGCCAGGTGTGGTGGCTCACGCCTGTAATCCCAGCA CTCTGGGAGGCTGAGGTGGGTGGATCGCTTGAGCCCTGGA GTTCAAGACCAGCCTGAGCAACATGGCAAAACGCTGTTTC TATAACAAAAATTAGCCGGGCATGGTGGCATGTGCCTGTG GTCCCAGCTACTAGGGGGCTGAGGCAGGAGAATCGTTGGA GCCCAGGAGGTCAAGGCTGCACTGAGCAGTGCTTGCGCCA CTGCACTCCAGCCTGGGTGACAGGACCAGACCTTGCCTCA AAAAAATAAGAAGAAAAATTAAAAATAAATGGAAACAACT ACAAAGAGCTGTTGTCCTAGATGAGCTACTTAGTTAGGCT GATATTTTGGTATTTAACTTTTAAAGTCAGGGTCTGTCAC CTGCACTACATTATTAAAATATCAATTCTCAATGTATATC CACACAAAGACTGGTACGTGAATGTTCATAGTACCTTTAT TCACAAAACCCCAAAGTAGAGACTATCCAAATATCCATCA ACAAGTGAACAAATAAACAAAATGTGCTATATCCATGCAA TGGAATACCACCCTGCAGTACAAAGAAGCTACTTGGGGAT GAATCCCAAAGTCATGACGCTAAATGAAAGAGTCAGACAT GAAGGAGGAGATAATGTATGCCATACGAAATTCTAGAAAA TGAAAGTAACTTATAGTTACAGAAAGCAAATCAGGGCAGG CATAGAGGCTCACACCTGTAATCCCAGCACTTTGAGAGGC CACGTGGGAAGATTGCTAGAACTCAGGAGTTCAAGACCAG CCTGGGCAACACAGTGAAACTCCATTCTCCACAAAAATGG GAAAAAAAGAAAGCAAATCAGTGGTTGTCCTGTGGGGAGG GGAAGGACTGCAAAGAGGGAAGAAGCTCTGGTGGGGTGAG GGTGGTGATTCAGGTTCTGTATCCTGACTGTGGTAGCAGT TTGGGGTGTTTACATCCAAAAATATTCGTAGAATTATGCA TCTTAAATGGGTGGAGTTTACTGTATGTAAATTATACCTC AATGTAAGAAAAAATAATGTGTAAGAAAACTTTCAATTCT CTTGCCAGCAAACGTTATTCAAATTCCTGAGCCCTTTACT TCGCAAATTCTCTGCACTTCTGCCCCGTACCATTAGGTGA CAGCACTAGCTCCACAAATTGGATAAATGCATTTCTGGAA AAGACTAGGGACAAAATCCAGGCATCACTTGTGCTTTCAT ATCAACCATGCTGTACAGCTTGTGTTGCTGTCTGCAGCTG CAATGGGGACTCTTGATTTCTTTAAGGAAACTTGGGTTAC CAGAGTATTTCCACAAATGCTATTCAAATTAGTGCTTATG ATATGCAAGACACTGTGCTAGGAGCCAGAAAACAAAGAGG AGGAGAAATCAGTCATTATGTGGGAACAACATAGCAAGAT ATTTAGATCATTTTGACTAGTTAAAAAAGCAGCAGAGTAC AAAATCACACATGCAATCAGTATAATCCAAATCATGTAAA TATGTGCCTGTAGAAAGACTAGAGGAATAAACACAAGAAT CTTAACAGTCATTGTCATTAGACACTAAGTCTAATTATTA TTATTAGACACTATGATATTTGAGATTTAAAAAATCTTTA ATATTTTAAAATTTAGAGCTCTTCTATTTTTCCATAGTAT TCAAGTTTGACAATGATCAAGTATTACTCTTTCTTTTTTT TTTTTTTTTTTTTTTTTTGAGATGGAGTTTTGGTCTTGTT GCCCATGCTGGAGTGGAATGGCATGACCATAGCTCACTGC AACCTCCACCTCCTGGGTTCAAGCAAAGCTGTCGCCTCAG CCTCCCGGGTAGATGGGATTACAGGCGCCCACCACCACAC TCGGCTAATGTTTGTATTTTTAGTAGAGATGGGGTTTCAC CATGTTGGCCAGGCTGGTCTCAAACTCCTGACCTCAGAGG ATCCACCTGCCTCAGCCTCCCAAAGTGCTGGGATTACAGA TGTAGGCCACTGCGCCCGGCCAAGTATTGCTCTTATACAT TAAAAAACAGGTGTGAGCCACTGCGCCCAGCCAGGTATTG CTCTTATACATTAAAAAATAGGCCGGTGCAGTGGCTCACG CCTGTAATCCCAGCACTTTGGGAACCAAGGCGGGCAGAA CACCCGAGGTCAGGAGTCCAAGGCCAGCCTGGCCAAGATG GTGAAACCCCGTCTCTTAAAAATACAAACATTACCTGG GCATGATGGTGGGCGCCTGTAATCCCAGCTACTCAGGAGG CTGAGGCAGGAGGATCCGGAGCCTGGCAGATCTGCCTG AGCCTGGGAGGTTGAGGCTACAGTAAGCCAAGATCATGCC AGTATACTTCAGCCTGGGCGACAAAGTGAGACCCGTAACAA AAAAAAAAAAATTTAAAAAAAAAAAAATTGAATTACC AACTGTAAAGTGGCCTAAACTGTAAAGTTTG GAGTTTATTCTGCAGGCAGAAGAGAACCATCAGGGGGTCT TCAGCATGGGAATGGCATGGTGCACCTGGTTTTTGTGAGA TCATGGTGGTGACAGTGTGGGGAATGTTATTTTGGAGGGA C TGGAGGCAGACCGGTTAAAAGGCCAGACACGA AAGGAGGAAGATGAGGGCTTGGACCGAAGCAGAGAAG AGCAAAGGGAAGGTACAAATTCAAGAAATATTGGGGGG TTTGATATTACKTAGNATETATTAAATATTACK CTGAGATAGAAATGAGTCAAGGATGGTTCCAGGCTGC TAGGCTGCTTACCTGAGGTGGCAAAGTCGGGAGGAGTGGC AGTTTAGGACAGGGGGCAGTTGAGGAATATTGTTTTGATC ATTTTGAGTTTGAGGTACAAGTTGGACACTTAGGTAAAGA CTGGAGGGGAAATCTGAATATACAATTATGGGACTGAGGA ACAAGTTTATTTTATTTTTTGTTTCGTTTTCTTGTTGAAG AACAAATTTAATTGTAATCCCAAGTCATCAGCATCTAGAA GACAGTGGCAGGAGGTGACTGTCTTGTGGGTAAGGGTTTG GGGTCCTTGATGAGTATCTCTCAATTGGCCTTAAATATAA GCAGGAAAAGGAGTTTATGATGGATTCCAGGCTCAGCAGG GCTCAGGAGGGCTCAGGCAGCCAGCAGAGGAAGTCAGAGC ATCTTCTTTGGTTTAGCCCAAGTAATGACTTCCTTAAAAA GCTGAAGGAAAATCCAGAGTGACCAGATTATAAACTGTAC TCTTGCATTTTCTCTCCCTCCTCTCACCCACAGCCTCTTG ATGAACCGGAGGAAGTTTCTTTACCAATTCAAAAATGTCC GCTGGGCTAAGGGTCGGCGTGAGACCTACCTGTGCTACGT AGTGAAGAGGCGTGACAGTGCTACATCCTTTTCACTGGAC TTTGGTTATCTTCGCAATAAGGTATCAATTAAAGTCGGCT TTGCAAGCAGTTTAATGGTCAACTGTGAGTGCTTTTAGAG CCACCTGCTGATGGTATTACTTCCATCCTTTTTTGGCATT TGTGTCTCTATCACATTCCTCAAATCCTTTTTTTTATTTC TTTTTCCATGTCCATGCACCCATATTAGACATGGCCCAAA ATATGTGATTTAATTCCTCCCCAGTAATGCTGGGCACCCT AATACCACTCCTTCCTTCAGTGCCAAGAACAACTGCTCCC AAACTGTTTACCAGCTTTCCTCAGCATCTGAATTGCCTTT GAGATTAATTAAGCTAAAAGCATTTTTATATGGGAGAATA TTATCAGCTTGTCCAAGCAAAAATTTTAAATGTGAAAAAC AAATTGTGTCTTAAGCATTTTTGAAAATTAAGGAAGAAGA ATTTGGGAAAAAATTAACGGTGGCTCAATTCTGTCTTCCA AATGATTTCTTTTCCCTCCTACTCACATGGGTCGTAGGCC AGTGAATACATTCAACATGGTGATCCCCAGAAAACTCAGA GAAGCCTCGGCTGATGATTAATTAAATTGATCTTTCGGCT ACCCGAGAGAATTACATTTCCAAGAGACTTCTTCACCAAA ATCCAGATGGGTTTACATAAACTTCTGCCCACGGGTATCT CCTCTCTCCTAACACGCTGTGACGTCTGGGCTTGGTGGAA TCTCAGGGAAGCATCCGTGGGGTGGAAGGTCATCGTCTGG CTCGTTGTTTGATGGTTATATTACCATGCAATTTTCTTTG CCTACATTTGTATTGAATACATCCCAATCTCCTTCCTATT CGGTGACATGACACATTCTATTTCAGAAGGCTTTGATTTT ATCAAGCACTTTCATTTACTTCTCATGGCAGTGCCTATTA CTTCTCTTACAATACCCATCTGTCTGCTTTACCAAAATCT ATTTCCCCTTTTCAGATCCTCCCAAATGGTCCTCATAAAC TGTCCTGCCTCCACCTAGTGGTCCAGGTATATTTCCACAA TGTTACATCAACAGGCACTTCTAGCCATTTTCCTTCTCAA AAGGTGCAAAAAGCAACTTCATAAACACAAATTAAATCTT CGGTGAGGTAGTGTGATGCTGCTTCCTCCCAACTCAGCGC ACTTCGTCTTCCTCATTCCACAAAAACCCATAGCCTTCCT TCACTCTGCAGGACTAGTGCTGCCAAGGGTTCAGCTCTAC CTACTGGTGTGCTCTTTTGAGCAAGTTGCTTAGCCTCTCT GTAACACAAGGACAATAGCTGCAAGCATCCCCAAAGATCA TTGCAGGAGACAATGACTAAGGCTACCAGAGCCGCAATAA AAGTCAGTGAATTTTAGCGTGGTCCTCTCTGTCTCTCCAG AACGGCTGCCACGTGGAATTGCTCTTCCTCCGCTACATCT CGGACTGGGACCTAGACCCTGGCCGCTGCTACCGCGTCAC CTGGTTCACCTCCTGGAGCCCCTGCTACGACTGTGCCCGA CATGTGGCCGACTTTCTGCGAGGGAACCCCAACCTCAGTC TGAGGATCTTCACCGCGCGCCTCTACTTCTGTGAGGACCG CAAGGCTGAGCCCGAGGGGCTGCGGCGGCTGCACCGCGCC GGGGTGCAAATAGCCATCATGACCTTCAAAGGTGCGAAAG GGCCTTCCGCGCAGGCGCAGTGCAGCAGCCCGCATTCGGG ATTGCGATGCGGAATGAATGAGTTAGTGGGGAAGCTCGAG GGGAAGAAGTGGGCGGGGATTCTGGTTCACCTCTGGAGCC GAAATTAAAGATTAGAAGCAGAGAAAAGAGTGAATGGCTC AGAGACAAGGCCCCGAGGAAATGAGAAAATGGGGCCAGGG TTGCTTCTTTCCCCTCGATTTGGAACCTGAACTGTCTTCT ACCCCCATATCCCCGCCTTTTTTTCCTTTTTTTTTTTTTG AAGATTATTTTTACTGCTGGAATACTTTTGTAGAAAACCA CGAAAGAACTTTCAAAGCCTGGGAAGGGCTGCATGAAAAT TCAGTTCGTCTCTCCAGACAGCTTCGGCGCATCCTTTTGG TAAGGGGCTTCCTCGCTTTTTAAATTTTCTTTCTTTCTCT ACAGTCTTTTTTGGAGTTTCGTATATTTCTTATATTTTCT TATTGTTCAATCACTCTCAGTTTTCATCTGATGAAAACTT TATTTCTCCTCCACATCAGCTTTTTCTTCTGCTGTTTCAC CATTCAGAGCCCTCTGCTAAGGTTCCTTTTCCCTCCCTTT TCTTTCTTTTGTTGTTTCACATCTTTAAATTTCTGTCTCT CCCCAGGGTTGCGTTTCCTTCCTGGTCAGAATTCTTTTCT CCTTTTTTTTTTTTTTTTTTTTTTTTTTTAAACAAACAAA CAAAAAACCCAAAAAAACTCTTTCCCAATTTACTTTCTTC CAACATGTTACAAAGCCATCCACTCAGTTTAGAAGACTCT CCGGCCCCACCGACCCCCAACCTCGTTTTGAAGCCATTCA CTCAATTTGCTTCTCTCTTTCTCTACAGCCCCTGTATGAG GTTGATGACTTACGAGACGCATTTCGTACTTTGGGACTTT GATAGCAACTTCCAGGAATGTCACACACGATGAAATATCT CTGCTGAAGACAGTGGATAAAAAACAGTCCTTCAAGTCTT CTCTGTTTTTATTCTTCAACTCTCACTTTCTTAGAGTTTA CAGAAAAAATATTTATATACGACTCTTTAAAAAGATCTAT GTCTTGAAAATAGAGAAGGAACACAGGTCTGGCCAGGGAC GTGCTGCAATTGGTGCAGTTTTGAATGCAACATTGTCCCC TACTGGGAATAACAGAACTGCAGGACCTGGGAGCATCCTA AAGTGTCAACGTTTTTCTATGACTTTTAGGTAGGATGAGA GCAGAAGGTAGATCCTAAAAAGCATGGTGAGAGGATCAAA TGTTTTTATATCAACATCCTTTATTATTTGATTCATTTGA GTTAACAGTGGTGTTAGTGATAGATTTTTCTATTCTTTTC CCTTGACGTTTACTTTCAAGTAACACAAACTCTTCCATCA GGCCATGATCTATAGGACCTCCTAATGAGAGTATCTGGGT GATTGTGACCCCAAACCATCTCTCCAAAGCATTAATATCC AATCATGCGCTGTATGTTTTAATCAGCAGAAGCATGTTTT TATGTTTGTACAAGAAGATTGTTATGGGTGGGGATGGA GGTATAGACCATGCATGGTCACCTTCAAGCTACTTTAATA AAGGATCTTAAAATGGGCAGGAGGACTGTGAACAAGACAC CCTAATAATGGGTTGATGTCTGAAGTAGCAAATCTTCTGG AAACGCAAACTCTTTTAAGGAAGTCCCTAATTTAGAAACA CCCACAAACTTCACATATCATAATTAGCAAACAATTGGAA GGAAGTTGCTTGAATGTTGGGGAGAGGAAAATCTATTGGC TCTCGTGGGTCTCTTCATCTCAGAAATGCCAATCAGGTCA AGGTTTGCTACATTTTGTATGTGTGTGATGCTTCTCCCAA AGGTATATTAACTATATAAGAGAGTTGTGACAAAACAGAA TGATAAAGCTGCGAACCGTGGCACACGCTCATAGTTCTAG CTGCTTGGGAGGTTGAGGAGGGAGGATGGCTTGAACACAG GTGTTCAAGGCCAGCCTGGGCAACATAACAAGATCCTGTC TCTCAAAAAAAAAAAAAAAAGAAGAGAGAGGGCCG GGCGTGGTGGCTCACGCCTGTAATCCCAGCACTTTGGGAG GCCGAGCCGGGCGGATCACCTGTGGTCAGGAGTTTGAGAC CAGCCTGGCCAACATGGCAAAACCCCGTCTGTACTCAAAA TGCAAAAATTAGCCAGGCGTGGTAGCAGGCACCTGTAATC CCAGCTACTTGGGAGGCTGAGGCAGGAGAATCGCTTGAAC CCAGGAGGTGGAGGTTGCAGTAAGCTGAGATCGTGCCGTT GCACTCCAGCCTGGGCGACAAGAGCAAGACTCTGTCTCAG AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA TATTTGGAGAGAAGGATGGGGAAGCATTGCAAGGAAATT GTGCTTTATCCAACAAAATGTAAGGAGCCAATAAGGGATC CCTATTTGTCTCTTTTGGTGTCTATTGTCCCTAACAACT GTCTTTGACAGTGAAAAAATATTCAGAATAACCATATCC CTGTGCCGTTATTACCTAGCAACCCTTGCAATGAAGATGA G CAGATCCACAGGAAAACTTGAATGCACAACTGTCTTATTT TAATCTTTATTGTACATAAGTTTGTAAAAGGTTAAAAAATT GTTACTTCATGTATTCATTTATTTTTATATTATTTTGCG TCTAATGATTTTTTATTAACATGATTTCCTTTTCTGATAT ATTGAAATGGAGTCTCAAAGCTTCATAAATTTATAACTTT AGAAATGATTCTAAAACGTATGTTAATTGTAACATTG CAGTAATGGTGCTACGAAGCCATTCTCTTGATTTTTAGT AAACTTTTATGACAGCAAATTTGCTTCTGGCTCACTTTCA ATCAGTTAAATAAATGATAAATAATTTTGGAAGCTGTGAA GATAAAATACCAAATAAAATAATATAAAAGTGATTTATAT GAAGTTAAAATAAAAAATCAGTATGATGGAATAAACTTG

[0144] Apolipoprotein B mRNA editing enzyme, catalytic polypeptide-like (APOBEC) It is an evolutionarily conserved family of cytidine deaminases. Members of this family The N-terminal domain of APOBEC-like proteins is a catalytic enzyme that edits C to U. The C-terminal domain is the catalytic domain, and the C-terminal domain is the pseudo-catalytic domain. In is a zinc-dependent cytidine deaminase domain that is important for the deamination of cytidine APOBEC family members include APOBEC1, APOBEC2, and AP OBEC3A, APOBEC3B, APOBEC3C, APOBEC3D (currently “AP "APOBEC3E" refers to this), APOBEC3F, APOBEC3G, APOBEC These include 3H, APOBEC4, and activation-induced (cytidine) deaminase. Modified cytidine deaminases are commercially available, including but not limited to SaBE3, S aKKH-BE3, VQR-BE3, EQR-BE3, VRER-BE3, YE1-BE 3, EE-BE3, YE2-BE3, and YEE-BE3, which are Plasmids 85169, 85170, 85171, and 85 172, 85173, 85174, 85175, 85176, 85177).

[0145] Other exemplary deaminases that can be fused to Cas9 according to embodiments of the present disclosure are provided herein. In some embodiments, the active domains of each sequence, e.g., localization signals, are provided. Domains without nuclear localization sequences, nuclear transport signals, and cytoplasmic localization signals are used. It should be understood that this may be possible.

[0146] The terms "deaminase" or "deaminase domain" as used herein In some embodiments, the term "protein" refers to a protein or enzyme that catalyzes a deamination reaction. The deaminase or deaminase domain is a cytidine deaminase and binds to cytidine or or deoxycytidine to uridine or deoxyuridine, respectively. In some embodiments, the deaminase or deaminase domain catalyzes the deaminolysis of Cytosine deaminase is a cytosine deaminase that hydrolytically deaminates cytosine to uracil. In some embodiments, the deaminase is adenosine deaminase, In some embodiments, the deamination of dextran phosphate catalyzes the hydrolytic deamination of dextran to hypoxanthine. Aminase is an adenosine deaminase that converts the ino- tion of adenosine or adenine (A). In some embodiments, the deaminase catalyzes the hydrolytic deamination of hydroxybenzoates to hydroxybenzoates (I). Alternatively, the deaminase domain is an adenosine deaminase, Hydrolytic deamination of hydroxyadenosine to inosine or deoxyinosine, respectively In some embodiments, adenosine deaminase catalyzes the deoxyribonucleic acid The enzymes provided herein catalyze the hydrolytic deamination of adenosine in DNA. adenosine deaminase (e.g., engineered adenosine deaminase, evolved adenosine deaminase) The adendeaminase can be from any organism, such as a bacterium. Nosine deaminase is a ubiquitous enzyme found in bacteria (e.g., Escherichia coli, Staph ylococcus aureus, Salmonella typhimurium, Shewanella putrefaciens, Haemophilus infl uenzae, or Caulobacter crescentus).

[0147] In some embodiments, the deaminase or deaminase domain is selected from the group consisting of human, chimpanzee, Naturally occurring proteins from organisms such as gorilla, monkey, cow, dog, rat, or mouse In some embodiments, the deaminase or deaminase The deaminase domain is not naturally occurring. For example, in some embodiments, The aminase domain has at least 50% similarity to a naturally occurring deaminase, 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 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 less are at least 99.9% identical.

[0148] For example, the deaminase domain is described in International PCT Application No. 2017 / 045381 (WO 2018 / 027078) and PCT / US2016 / 058344 (WO2017 / 070632), each of which is incorporated herein by reference in its entirety. Also, Komor, AC, et al., "Programmable editing of a target base in genomic DNA without double-stranded DNA cleavage”Nat ure 533,420-424(2016), Gaudelli,NM,et a l.,“Programmable base editing of A·T to G·C in genomic DNA without DNA cleavage” Nature 551, 464-471 (2017), Komor, AC, et a l.,“Improved base excision repair inhibition tion and bacteriophage Mu Gam protein yi elds C:G-to-T:A base editors with higher efficiency and product purity”Science A dvances 3:eaao4774(2017), and Rees,HA,et al., “Base editing: precision chemistry o n the genome and transcriptome of living cells.”Nat Rev Genet.2018 Dec;19(12):77 See also 0-788.doi:10.1038 / s41576-018-0059-1 (the entire contents of which are incorporated herein by reference).

[0149] "Detecting" refers to determining the presence, absence, or amount of the analyte being detected.

[0150] A "detectable label" means a label that, when attached to a molecule of interest, is capable of detecting a specific molecule spectroscopically, photochemically, biochemically, or The term "composition" refers to a composition that allows a molecule of interest to be detected through immunological, immunochemical, or chemical means. For example, useful labels include radioisotopes, magnetic beads, metal beads, and colloidal ion particles, fluorescent dyes, electron-dense reagents, enzymes (e.g., enzyme-linked immunosorbent assay (ELISA) Examples include enzymes commonly used in SA (antibody synthesis), biotin, digoxigenin, or haptens. can be done.

[0151] "Disease" means any condition that damages or interferes with the normal function of a cell, tissue, or organ. In one embodiment, the disease is a tumor or cancer. In this case, the disease is a blood cancer. "Blood cancer" refers to a malignant tumor of immune system cells. In some embodiments, the hematological cancer is leukemia, myeloma, and / or lymphoma. Lymphoma and leukemia are examples of "liquid cancers" or cancers that exist in the blood and not in the bone marrow. Leukemia results from the transformation of either hematopoietic precursor cells or mature hematopoietic cells in the blood. It can be lymphoid, myeloid, or acute or chronic. The cells are fully differentiated plasma cells and occur either as dispersed aggregates of malignant cells or in the bone marrow. In lymphoma, transformed lymphocytes in the secondary lymphoid tissues may present as solid masses. Lymphomas are classified as Hodgkin's lymphoma (HL) or non-Hodgkin's lymphoma (NHL). HL).

[0152] In some embodiments, the hematological cancer is a B-cell cancer. The cancer is lymphoma or leukemia. In some cases, leukemia includes preleukemia. In some cases, the leukemia is acute leukemia. Acute leukemias include, for example, acute myeloid leukemia. Acute leukemia includes, for example, acute lymphocytic leukemia or acute myeloid leukemia (AML). This includes lymphocytic leukemia (ALL), which includes B-lineage ALL, T-lineage ALL, and and T-cell acute lymphoblastic leukemia (T-ALL).

[0153] Non-limiting examples of diseases include T-cell acute lymphoblastic leukemia (T-ALL), mycosis MF, Sézary syndrome (SS), peripheral T / NK cell lymphoma, anaplastic large cell lymphoma ALK lymphoma + , primary cutaneous T-cell lymphoma, T-cell large granular lymphocytic leukemia, vascular immunity Blastic T / NK cell lymphoma, hepatosplenic T cell lymphoma, primary cutaneous CD30 + Lymphoproliferative disorder, extranodal NK / T cell lymphoma, adult T cell leukemia / lymphoma, T cell prolymphocytic Leukemia, subcutaneous panniculitis-like T-cell lymphoma, primary cutaneous gamma-delta T-cell lymphoma, aggressive In some embodiments, the disease includes NK-cell leukemia, and enteropathy-associated T-cell lymphoma. In some embodiments, the disease is a liquid tumor. In some embodiments, the disease is T-cell acute lymphoblastic leukemia (T ... In some embodiments, the disease is acute myeloid leukemia (AML). .

[0154] As used herein, the term "effective amount" means an amount sufficient to elicit a desired biological response. In some embodiments, an effective amount refers to an amount of a biologically active agent that is sufficient to treat a patient with a disease compared to an untreated patient. The amount of the compound used in the present invention for the therapeutic treatment of a disease is the amount required to alleviate the symptoms of the disease. The effective amount of active agent used to treat a patient will depend on the method of administration, the age, weight, and general condition of the patient. Varies depending on health status. Ultimately, your doctor or veterinarian will determine the appropriate dosage and dosage regimen. Such an amount is referred to as an "effective" amount. In one embodiment, an effective amount is determined by measuring the cell ( For example, a gene sufficient to introduce a genetic change of interest in a cell (in vitro or in vivo). The base editors of the present invention (e.g., programmable DNA binding proteins, nucleobase In one embodiment, the effective amount of the fusion protein is The amount is determined to achieve a therapeutic effect (e.g., to reduce or eliminate a disease or its symptoms or condition). is the amount of base editor required to control the gene expression level. It is not necessary that the amount of DNA be sufficient to modify the gene of interest in all cells of a tissue or organ. ,Approximately 1%, 5%, 10%, 25%, 50%, 7% of cells present in a subject, tissue, or organ Only 5% or more of the genes of interest need to be modified.

[0155] In some embodiments, the fusion proteins provided herein (e.g., nCas9 domains) adenosine deaminase and deaminase domains (e.g., adenosine deaminase or cytidine deaminase) An effective amount of a nucleobase editor (including a nucleobase editor) described herein is The fusion protein is sufficient to induce editing of a target site that is specifically bound and edited by the As will be understood by those skilled in the art, the amount of a drug (e.g., a fusion protein, Nucleases, hybrid proteins, protein dimers, proteins (or proteins The effective amount of the complex of the protein dimer and the polynucleotide, or the polynucleotide, varies. factors (e.g., the desired biological response, e.g., the specific allele to be edited, the genome, if (e.g., target site, target cells or tissues, and / or drug used) In the context of CAR-T cells, an "effective amount" is an amount that achieves a therapeutic response. This refers to the amount of cells required to administer to a patient for the purpose of

[0156] As used herein, "epitope" means an antigenic determinant. It is part of a protease molecule and, by its structure, has specific functions that recognize and bind to epitopes. The antibody molecule is determined.

[0157] "Fas cell surface death receptor (FAS) polypeptide" refers to the polypeptide identified in NCBI accession number NP having at least about 85% amino acid sequence identity with _000034.1 or a fragment thereof By protein, we mean an exemplary amino acid sequence provided below. >NP_000034.1 Tumor necrosis factor receptor superfamily member 6 isoform Form 1 Precursor [Homo sapiens] 1 MLGIWTLLPL VLTSVARLSS KSVNAQVTDI NSKGLELRKT VTTVETQNLE GLHHDGQFCH 61 KPCPPGERKA RDCTVNGDEP DCVPCQEGKE YTDKAHFSSK CRRCRLCDEG HGLEVEINCT 121 RTQNTKCRCK PNFFCNSTVC EHCDPCTKCE HGIIKECTLT SNTKCKEEGS RSNLGWLCLL 181 LLPIPLIVWV KRKEVQKTCR KHRKENQGSH ESPTLNPETV AINLSDVDLS KYITTIAGVM 241 TLSQVKGFVR KNGVNEAKID EIKNDNVQDT AEQKVQLLRN WHQLHGKKEA YDTLIKDLKK 301 ANLCTLAEKI QTIILKDITS DSENSNFRNE IQSLV

[0158] "Fas cell surface death receptor (FAS) polynucleotide" means a FAS polypeptide. Exemplary FAS nucleic acid sequences are provided below. >NM_000043.6 Homo sapiens Fas cell surface death receptor ( FAS), transcript variant 1, mRNA 1 ctcttctccc gcgggttggt ggacccgctc agtacggagt tggggaagct ctttcacttc 61 ggaggattgc tcaacaacca tgctgggcat ctggaccctc ctacctctgg ttcttacgtc 121 tgttgctaga ttatcgtcca aaagtgttaa tgcccaagtg actgacatca actccaaggg 181 attggaattg aggaagactg ttactacagt tgagactcag aacttggaag gcctgcatca 241 tgatggccaa ttctgccata agccctgtcc tccaggtgaa aggaaagcta gggactgcac 301 agtcaatggg gatgaaccag actgcgtgcc ctgccaagaa gggaaggagt acacagacaa 361 agcccatttt tcttccaaat gcagaagatg tagattgtgt gatgaaggac atggcttaga 421 agtggaaata aactgcaccc ggacccagaa taccaagtgc agatgtaaac caaacttttt 481 ttgtactct actgtagtg aacactgtga cccttgcacc aaatgtgaac atggaatcat 541 caaggaatgc acactcacca gcaacaccaa gtgcaagag gaaggatcca gatctactt 601 ggggtggctt tgtctcttc tttgccaat tccactatt gtttggtga aggaaagga 661 agtacagaaa acatgcagaa agcacagaaa ggaaaaccaa ggttctcatg aatctccaac 721 tttaaatcct gaacagtgg cataaattt atctgatgtt gacttgagta atatatcac 781 cactattgct ggagtcatga cactaagtca agttaaaggc ttgttcgaa agaatggtgt 841 caatgaagcc aaatagatg agatxagaa tgacaatgtc caatgacacag cagacaga 901 agttcaactg cttcgtaatt ggcacact tcatggaag aagaagcgt atgacacatt 961 gattaagat ctcaaaaag ccaatcttg tactctgca gagaaaattc agactacat 1021 cctcaggac attackagtg acctcagaaa ttcaacttc agaatgaaa tccaagctt 1081 gtcttagagt gaaaaacaac aaattcagtt ctgagtatat gcaattagtg tttgaaaga 1141 ttcttaatag ctggctgtaa atactgcttg gtttttact gggtacattt tatcatttat 1201 tagcgctgaa gagccaacat atttgtagat ttttaatc tcatgattct gcctccaagg 1261 atgtttaaaa tctagttggg aaaacaaact tcatcaagag taaatgcagt ggcatgctaa 1321 gtacccaaat aggagtgtat gcagaggatg aaagattag attatgctct ggcatctaac 1381 atatgattct gtagtatgaa tgtaatcagt gtatgttagt acaaatgtct atccacaggc 1441 taaccccact ctatgaatca atcagttact ctatgacctt ttgctgaat atcagttact 1501 gaacaggcag gccactttgc ctctaatta cctctgataa ttctagagat tttaccatat 1561 ttctaaactt tgtttataac tctgagaaga tcatatttat gtaaagtata tgtatttgag 1621 tgcagaattt aaataaggct ctacctcaaa gaccttgca cagtttattg gtgtcatatt 1681 attack tcaattgtga attack aaaacatta attack ttgactatta 1741 fathergtgta tgcatttc tggctcaaaa ctacctactt ctttctcagg catcaaaagc 1801 attttgagca ggagagtatt actagagctt tgccacctct ccattttgc cttggtgctc 1861 atcttaatgg cctaatgcac ccccaaacat ggaaatatca ccaaaaaata cttaatagtc 1921 caccaaaagg caagactgcc cttagaaatt ctagcctggt ttggagatac taactgctct 1981 cagagaaagt agctttgtga catgtcatga acccatgttt gcaatcaaag atgataaaat 2041 agattcttat ttttccccca cccccgaaaa tgttcaataa tgtcccatgt aaaacctgct 2101 acaaatggca gcttatacat agcaatggta aaatcatcat ctggatttag gaattgctct 2161 tgtcataccc ccaagtttct aagatttaag attctcctta ctactatcct acgtttaaat 2221 atctttgaaa gtttgtatta aatgtgaatt ttaagaaata atatttatat ttctgtaaat 2281 gtaaactgtg aagatagtta taaactgaag cagatacctg gaaccaccta aagaacttcc 2341 atttatggag gatttttttg ccccttgtgt ttggaattat aaaatatagg taaaagtacg 2401 taattaaata atgtttttgg tatttctggt tttctctttt ttggtagggg cttgcttttt 2461 ggttttgtct tccttttctc taactgatgc taaatataac ttgtctctttaa tgcttcttgg 2521 atcccttaga aggtacttcc tttttaacct taaccctttt agtagttaaa taattatttc 2581 cataggttgc tattgccaag aagacctctt ccaaacagca catgattatt cgtcaaacag 2641 tttcgtattc cagatactgg aatgtggata agaaagtata catttcaagg ggtaggtttt 2701 attattaaga aagccaaatg aggattttga aatattcttt cctgcatatt atccattcta 2761 gctacatgct ggccagtggg ccacctttct tttctgcaat ttaatgctag tatatattc 2821 tatttaaccc atgagtccca aagttattagc atttcaacat gtaagcatgt cggtaagata 2881 gttgtgcttt gcttagggtt ccctcctgtg ttatggtctg gaaagtgtct ttaggcagaa 2941 agtctgagtg atcacagggt tcactcatta atttctcttt tctgagccat catagtctgt 3001 gctgtctgct ctccagtttt ctatttctag acagaagtag ggcaagttag gtactagtta 3061 ttcttcatgg ccagaagtgc aagttctact ttgcaagaca agattaagtt agagaacacc 3121 ctattccact ttggtgaact cagagcaaga actttgagtt cctttgggag gaagacagtg 3181 gagaagtctt tgtacttggt gatgtggtttt ttttcctcat ggcttcacct agtggcccca 3241 agcatgactt ctcccatgtc aatgagcaca gccacattcc cgagttgagg tgaccccacg 3301 gtccagaatc atcctcattc tggtgaacct ggttctcttt gtggtgggca tactgggtag 3361 gagaatcacc caaaggtcac ccatgagctg cagaaaaaaa ggctatttgc agaaggagct 3421 cacagatcac attgaaagca ttgcatattc aaacatcttg gtcttcttta ttggcatgcc 3481 cacagggtct tctgacctct gattagatca gacacttttt agatattgaa tcatcagttt 3541 ctgtacaact atctgaataa ggtatataat caatgaaatt tagaattttt ttctatgctt 3601 actcctgatt ggtaatttgt ttgggtttag aattctatac aaggccattt gtaattttcc 3661 tcagcacttt aaaaatatta aaccatgttt tcttaa

[0159] By "fragment" is meant a portion of a polypeptide or nucleic acid molecule. at least about 10%, 20%, 30%, 40% of the total length of the nucleic acid molecule or reference polypeptide; Fragments include 50%, 60%, 70%, 80%, or 90%. 0, 50, 60, 70, 80, 90, or 100, 200, 300, 400, 500, Contains 600, 700, 800, 900, or 1000 nucleotides or amino acids It can be seen.

[0160] "Fratricide" refers to the killing of immune cells by other immune cells, and In certain embodiments, the immune cells of the present invention are chimeric. Prevent or reduce the expression of antigens recognized by immune cells expressing antigen receptors (CARs) Genetically modified to reduce the amount of fratricide produced, thereby preventing or reducing fratricide. In various embodiments, the fratricides are administered in vivo (e.g., in a subject) or ex vivo. It may occur in vivo (eg, in immune cell preparations).

[0161] Graft-versus-host disease (GVHD) is an immune response caused by transplanted cells from the donor against the host's cells. Refers to a pathological condition that produces

[0162] A "guide RNA" or "gRNA" is a polynucleotide that can be specific for a target sequence. a programmable nucleotide-binding domain protein (e.g., Cas9 or Cp In one embodiment, the guide polynucleotide is a polynucleotide capable of forming a complex with the guide polynucleotide f1. The nucleotide is a guide RNA (gRNA). gRNA is a compound of two or more RNAs. It can exist as a complex or as a single RNA molecule. The gRNA may be referred to as a single guide RNA (sgRNA), but "gRNA" is It refers to guide RNA that exists as a single molecule or as a complex of two or more molecules. Typically, gRNAs that exist as a single RNA species are expressed as two (1) a domain that shares homology with the target nucleic acid (e.g., also facilitates Cas9 conjugation to the target) (2) a domain that directs binding of the target gene to the target gene, and (3) a domain that binds to the Cas9 protein. In some embodiments, domain (2) is located in a sequence known as tracrRNA. For example, in some embodiments, domain (2) comprises: Presented in Jinek et al., Science 337:816-821 (2012). The tracrRNA is identical to or homologous to the tracrRNA provided herein (the entire contents of which are incorporated by reference). Other examples of gRNAs (e.g., those containing domain 2) are "switchable" US2016 / 0208288 entitled "Cas9 Nuclease and Its Uses" and "Mechanisms No. 9,737,604 entitled "Delivery Systems for Functional Nucleases." (the entire contents of each of which are incorporated herein by reference in their entirety). In one embodiment, the gRNA comprises two or more of domains (1) and (2), and The extended gRNA may be referred to as an "extended gRNA." The extended gRNA may comprise two or more Cas9 transcription factors described herein. The gRNA binds to the target nucleic acid at two or more distinct regions. The nuclease / RNA complex comprises a nucleotide sequence complementary to the target site of the nuclease / RNA complex. It mediates binding to the target site and provides sequence specificity for the nuclease:RNA complex. As will be appreciated, an RNA polynucleotide sequence (e.g., a gRNA sequence) may contain a DNA polynucleotide. The nucleic acid base contained in the nucleotide sequence is not thymine (T), but a pyrimidine derivative. Contains the nucleobase uracil (U). In RNA, uracil forms a base pair with adenine and replaces thymine during DNA transcription.

[0163] A "heterodimer" is a mixture of a wild-type TadA domain and a variant of the TadA domain (e.g., TadA*8), or two variant TadA domains (e.g., TadA*7 containing two domains, such as TadA*8 and TadA*10, or two TadA*8 domains. The term "fusion protein" refers to a fusion protein containing the nucleotide sequence of ... present invention.

[0164] "Host-versus-graft disease" (HVGD) is a condition in which the host's immune system reacts with the transplanted cells of the donor. Refers to a pathological condition that produces a response.

[0165] "Hybridization" means hydrogen bonding between complementary nucleobases, as defined by Watson -Click hydrogen bonding, Hoogsteen hydrogen bonding, or reversed Hoogsteen hydrogen bonding For example, adenine and thymine are complementary amino acids that pair through the formation of hydrogen bonds. It is a nucleic acid base.

[0166] By "immune cell" is meant a cell of the immune system that is capable of producing an immune response.

[0167] "Immune effector cells" are cells that, once activated, initiate an immune response against target cells. In some embodiments, immune effector cells refer to lymphocytes that can induce immune responses. In some embodiments, effector T cells are naive CD8 + T cells, cytotoxic T cells, natural killer T (NKT) cells, natural killer (NK ) cells, or regulatory T (Treg) cells. In some embodiments, immune effector In some embodiments, the effector T cells are effector NK cells. Thymocytes, immature T lymphocytes, mature T lymphocytes, resting T lymphocytes, or activated T lymphocytes In some embodiments, the immune effector cells are CD4 + CD8 + T cells or CD4 - CD8 - In some embodiments, the immune effector cells are T-cells. In some embodiments, the T helper cells are T helper 1 (Th1), T Th2 helper cells, or CD4-expressing helper T cells (CD4+ T cells) is.

[0168] "Immune response regulator gene" or "immune response regulator" refers to a gene involved in the regulation of immune responses. Immune response-regulating genes are genes that encode polypeptides. For example, immune response modulating genes may regulate immune responses at the same or different levels. The immune response-modulating gene may suppress or promote the activation of immune cells (e.g., T cells). The activation threshold of the cell may be increased or decreased. In some embodiments, the immune response modulating gene positively regulates immune cell signaling pathways. In some embodiments, the immune response modulating gene The gene negatively regulates immune cell signaling pathways. In some embodiments, the gene negatively regulates immune response regulation. Nodal genes encode antigens, antibodies, cytokines, or neuroendocrine genes.

[0169] An "immunogenic gene" is a gene that encodes a polypeptide that can elicit an immune response. For example, an immunogenic gene may encode an immunogen that elicits an immune response. In some embodiments, the immunogenic gene encodes a cell surface protein. In embodiments, the immunogenic gene encodes a cell surface antigen or cell surface marker. In some embodiments, the cell surface marker is a T cell marker or a B cell marker. In some embodiments, the immunogenic genes include CD2, CD3e, CD3δ, CD3γ, T RAC, TRBC1, TRBC2, CD4, CD5, CD7, CD8, CD19, CD2 3, CD27, CD28, CD30, CD33, CD52, CD70, CD127, CD 122, CD130, CD132, CD38, CD69, CD11a, CD58, CD9 9, CD103, CCR4, CCR5, CCR6, CCR9, CCR10, CXCR3, Encoding a CXCR4, CLA, CD161, B2M, or CIITA polypeptide .

[0170] The term "inhibitor of base repair" or "IBR" refers to a nucleic acid repair enzyme, e.g., a base It refers to a protein that can inhibit the activity of excision repair (BER) enzymes. BR is an inhibitor of inosine base excision repair. Exemplary inhibitors of base repair include: , APE1, EndoIII, EndoIV, EndoV, EndoVIII, Fpg, hOGG1, hNEIL1, T7 Endo1, T4 PDG, UDG, hSMUG1, and inhibitors of hAAG. In some embodiments, the IBR is an inhibitor of EndoV or or an inhibitor of hAAG. In some embodiments, the IBR is a catalytically inactive EndoV or catalytically inactive hAAG. In some embodiments, the base repair inhibitor is Endo In some embodiments, the base repair inhibitor is an inhibitor of catalytically inactive ATP. active EndoV or catalytically inactive hAAG.

[0171] In some embodiments, the base repair inhibitor is a uracil glycosylase inhibitor (UGI). UGI can inhibit the uracil DNA glycosylase base excision repair enzyme. In some embodiments, the UGI domain refers to a protein that can encode wild-type UGI or a wild-type UGI. In some embodiments, the UGI proteins provided herein include fragments of live UGI. It includes fragments of UGI and proteins homologous to UGI or UGI fragments. In some embodiments, the base repair inhibitor is an inhibitor of inosine base excision repair. In this state, base repair inhibitors act as "catalytically inactive inosine-specific nucleases" or "inactive Without wishing to be bound by any particular theory, Although not required, catalytically inactive inosine glycosylases (e.g., alkyladenine glycosylases) ABA can bind to inosine but creates an abasic site. Neither the inosine can be removed, thereby eliminating the newly formed inosine moiety. In some embodiments, the catalytic molecule can be sterically shielded from DNA damage / repair machinery. The catalytically inactive inosine-specific nuclease can bind to the inosine of nucleic acids, but Non-limiting exemplary catalytically inactive inosine-specific nucleases include: For example, catalytically inactive alkyl adenosine glycosylase (AAG nuclease) from humans ), and, for example, catalytically inactive endonuclease V from E. coli (EndoVnuclease In some embodiments, the catalytically inactive AAG nuclease is E It contains the 125Q mutation or the corresponding mutation in another AAG nuclease.

[0172] An "increase" is a positive change of at least 10%, 25%, 50%, 75%, or 100% means.

[0173] Inteins are used in a process known as protein splicing. A protein that can excise itself and link the remaining fragments (exteins) with peptide bonds. Inteins are also called "protein introns." The process by which the amino acid sequence excises itself and ligates the remainder of the protein is referred to herein as " This is called "protein splicing" or "intein-mediated protein splicing." In some embodiments, the intein of the precursor protein (intein-mediated protein synthesis) The intein-containing protein (pre-spliced ​​protein) is derived from two genes. Such inteins are referred to herein as split inteins (e.g., split inteins). In cyanobacteria, for example, DnaE, ​​the catalytic subunit of DNA polymerase III, is a nucleotide sequence consisting of dnaE-n and dna It is encoded by two separate genes, dnaE and dnan. The intein that is loaded may be referred to herein as "intein-N." The intein encoded by the C gene is referred to herein as "intein-C." It can be done.

[0174] Other intein systems can also be used. For example, dnaE intein, Cfa-N( split intein-N) and Cfa-C (e.g., split intein-C) ) intein pairs have been described (e.g., Stevens et al.,J Am Chem Soc.2016 Feb.24;138(7): 2162-5). Non-limiting examples of intein pairs that may be used in accordance with the present disclosure include C fa DnaE intein, Ssp GyrB intein, Ssp DnaX intein , Ter DnaE3 intein, Ter ThyX intein, Rma DnaB intein Intein, and Cne Prp8 intein (see, e.g., U.S. Patent No. 8,399,439). 4,604, incorporated herein by reference).

[0175] Exemplary nucleotide and amino acid sequences of inteins are provided below. DnaE intein-N DNA: TGCCTGTCATACGAAACCGAGATACTGACAGTAGAATATG GCCTTCTGCCAATCGGGAAGATTGTGGAGAAACGGATAGA ATGCACAGTTTACTCTGTCGATAACAATGGTAACATTTAT ACTCAGCCAGTTGCCCAGTGGCACGACCGGGGAGAGCAGG AAGTATTCGAATACTGTCTGGAGGATGGAAGTCTCATTAG GGCCACTAAGGACCACAAATTTATGACAGTCGATGGCCAG ATGCTGCCTATAGACGAAATCTTTGAGCGAGAGTTGGACC TCATGCGAGTTGACAACCTTCCTAAT DnaE intein-N protein: CLSYETEILTVEYGLLPIGKIVEKRIECTVYSVDNNGNIY TQPVAQWHDRGEQEVFEYCLEDGSLIRATKDHKFMTVDGQ MLPIDEIFERELDLMRVDNLPN DnaE intein-C DNA: ATGATCAAGATAGCTACAAGGAAGTATCTTGGCAAACAAA ACGTTTATGATATTGGAGTCGAAAGAGATCACAACTTTGC TCTGAAGAACGGATTCATAGCTTCTAAT Intein-C: MIKIATRKYLGKQNVYDIGVERDHNFALKNGF IASN Cfa-N DNA: TGCCTGTCTTATGATACCGAGATACTTACCGTTGAATATG GCTTCTTGCCTATTGGAAAGATTGTCGAAGAGAGAATTGA ATGCACAGTATATACTGTAGACAAGAATGGTTTCGTTTAC ACACAGCCCATTGCTCAATGGCACAATCGCGGCGAACAAG AAGTATTTGAGTACTGTCTCGAGGATGGAAGCATCATACG AGCAACTAAAGATCATAAATTCATGACCACTGACGGGCAG ATGTTGCCAATAGATGAGATATTCGAGCGGGGCTTGGATC TCAAACAAGTGGATGGATTGCCA Cfa-N protein: CLSYDTEILTVEYGFLPIGKIVEERIECTVYTVDKNGFVY TQPIAQWHNRGEQEVFEYCLEDGSIIRATKDHKFMTTDGQ MLPIDEIFERGLDLKQVDGLP Cfa-C DNA: ATGAAGAGGACTGCCGATGGATCAGAGTTTGAATCTCCCA AGAAGAAGAGGAAAGTAAAGATAATATCTCGAAAAAGTCT TGGTACCCAAAATGTCTATGATATTGGAGTGGAGAAAGAT CACAACTTCCTTCTCAAGAACGGTCTCGTAGCCAGCAAC Cfa-C protein:MKRTADGSEFESPKKKRKVKIISRKSLGT QNVYDIGVEKDHNFLLKNGLVASN

[0176] For ligation of the N-terminal part of split Cas9 and the C-terminal part of split Cas9 Intein-N and intein-C were inserted into the N-terminal portion of split Cas9, respectively. The fragment can be fused to the C-terminal portion of the split Cas9. For example, In this form, intein-N is fused to the C-terminus of the N-terminal part of split Cas9, That is, the structure of N-[N-terminal part of split Cas9]-[intein-N]-C is formed. In some embodiments, intein-C is located at the N-terminal end of the split Cas9. The C-terminal portion of the split Cas9 is fused to the N-[intein-C]-[C-terminal portion of split Cas9]. The intein is fused to (e.g., split Cas9) to form the [min]-C structure. The mechanism of intein-mediated protein splicing for linking proteins is It is known, for example, Shah et al., Chem Sci. 2014;5(1) :446-461, which is incorporated herein by reference. Methods for designing and using them are known in the art, for example, WO20 14 / 004336, WO2017 / 132580, US2015 / 0344549, and US2018 / 0127780, each of which is incorporated herein by reference. (All of which are incorporated herein by reference in their entirety).

[0177] The terms "isolated," "purified," or "biologically pure" refer to a substance that is in its natural state. " refers to a material that is free, to varying degrees, from components normally associated with it as found in the natural state." "Isolated" refers to the degree of separation from the original source or surroundings. Denotes a degree of separation greater than isolation. "Purified" or "biologically pure" protein The quality is such that it is sufficiently free of other materials so that any impurities do not affect the biological properties of the protein. does not substantially affect or cause other adverse consequences. Nucleic acids or peptides may be derived from cellular material, viruses, or other materials if produced by recombinant DNA technology. Purified or chemically synthesized if it is substantially free of any material or culture medium When prepared, it is considered purified if it is substantially free of chemical precursors or other chemicals. Purity and homogeneity are typically determined by analytical chemistry techniques, e.g., polyacrylamide gel electrophoresis. Purified" as determined using column electrophoresis or high performance liquid chromatography. The term "multiplex" refers to a nucleic acid or protein that forms essentially one band in an electrophoretic gel. For example, it can be shown that a protein undergoes modification (e.g., phosphorylation or glycosylation). In the case of proteins that can be isolated, different modifications can result in different isolated proteins that can be isolated separately. It can be purified.

[0178] An "isolated polynucleotide" is a naturally occurring polynucleotide of the organism from which the nucleic acid molecule of the invention is derived. "gene-free" refers to nucleic acid (e.g., DNA) that does not flank a gene in a genome. Thus, the term includes, for example, vectors, autonomously replicating plasmids or Recombinant DNA integrated into the genomic DNA of viruses, prokaryotes, or eukaryotes or a separate molecule (e.g., cDNA, or PCR or recombinant DNA) independent of other sequences. Exists as genomic or cDNA fragments produced by limited endonuclease digestion In addition, the term includes RNA molecules transcribed from DNA molecules, and recombinant D that is part of a hybrid gene encoding additional polypeptide sequences. Contains NA.

[0179] An "isolated polypeptide" is a polypeptide of the invention that has been separated from components that naturally accompany it. Typically, a polypeptide refers to a protein with which it is naturally associated. and is isolated if it is at least 60%, by weight, free from naturally occurring organic molecules. Preferably, the preparation is at least 75%, by weight, composed of a polypeptide of the invention. Preferably, the content is at least 90% by weight, and most preferably at least 99% by weight. An isolated polypeptide is one obtained by, for example, extracting such polypeptide from a natural source. by expressing a recombinant nucleic acid encoding the peptide, or by producing the protein chemically. The purity can be determined by any suitable method, for example, by by column chromatography, polyacrylamide gel electrophoresis, or HPLC analysis. The "leader peptide" is a peptide that is generated by a newly synthesized secretory protein or or membrane proteins, a short amino acid sequence ( A leader peptide typically refers to a polypeptide having a length of about 16 to 30 amino acids. Located at the N-terminus of a peptide, it is cleaved by a signal peptidase after the polypeptide has crossed the membrane. Leader peptide sequences typically have three general structural features: It contains an N-terminal polar basic region (n region), a hydrophobic core, and a hydrophilic region (c region). In some embodiments, the CARs of the invention comprise a leader peptide sequence (e.g., an antigen-binding domain). An exemplary leader peptide amino acid sequence is METDTLLLW It is VLLLWVPGSTG.

[0180] The term "linker," as used herein, refers to a link between two molecules or moieties, e.g. For example, two components of a protein complex or ribonucleocomplex, or two components of a fusion protein. a domain (e.g., a polynucleotide programmable DNA binding domain (e.g., dCas9) and a deaminase domain (e.g., adenosine deaminase, cytidine deaminase) refers to a bond (e.g., a covalent bond), chemical group, or molecule that links two or more proteins (phosphorylaminolase, phospholipase, or phospholipase). Carr is able to link different components, or different parts of components, of a base editor system. For example, in some embodiments, the linker can be a polynucleotide programmable Nucleotide-binding domain guides polynucleotide-binding domains and deaminase catalysis In some embodiments, the linker can link the CRISPR polypeptide. In some embodiments, the linker can be C In some embodiments, the linker can link as9 to a deaminase. In some embodiments, the linker can link as9 to a deaminase. In some embodiments, the linker can link as9 and the deaminase. In some embodiments, the deaminase can be linked to a phosphorylated polynucleotide. Carr is developing the deamination component of the base editor system and the polynucleotide programmable nucleic acid sequence. In some embodiments, the linker can link the nucleotide binding moieties. RNA-binding moiety of the deaminating component of the Deter system and polynucleotide programmability In some embodiments, the linker can bind a nucleotide binding moiety. The RNA-binding moiety of the deaminating component of the editor system and the polynucleotide programmable The linker can link the RNA-binding portion of the two nucleotide-binding components. Located between or adjacent to groups, molecules, or other moieties and bonded covalently or can be linked to each other through non-covalent interactions, thus linking the two. In some embodiments, the linker can be an organic molecule, group, polymer, or chemical moiety. In some embodiments, the linker can be a polynucleotide. The linker can be a DNA linker. In some embodiments, the linker is an RNA linker. In some embodiments, the linker can be an amino acid that can bind to the ligand. In some embodiments, the ligand may be a carbohydrate, peptide, protein, or In some embodiments, the linker can be derived from a riboswitch. The riboswitch from which the aptamer is derived can include the theophylline riboswitch. switch, thiamine pyrophosphate (TPP) riboswitch, adenosine cobalamin (AdoCb l) Riboswitch, S-adenosylmethionine (SAM) riboswitch, SAH riboswitch flavin mononucleotide (FMN) riboswitch, tetrahydrofolate riboswitch Lysine riboswitch, glycine riboswitch, purine riboswitch, GlmS riboswitch The riboswitch may be selected from a prequeous riboswitch, a ... In this embodiment, the linker comprises an aptamer attached to a polypeptide or protein domain. In some embodiments, the polypeptide may comprise a polypeptide ligand. The tide ligands contain the K homology (KH) domain, the MS2 coat protein domain, and the PP7 Coat protein domain, SfMu Com coat protein domain, steryl α-mono Chief, telomerase Ku binding motif and Ku protein, telomerase Sm7 binding The motif may be an Sm7 protein or an RNA recognition motif. In some embodiments, the polypeptide ligand can be part of a base editor system component. For example, a nucleobase editing component can include a deaminase domain and an RNA recognition motif.

[0181] In some embodiments, the linker is an amino acid or a plurality of amino acids (e.g., a peptide In some embodiments, the linker may be about 5 to 100 amino acids long. Acid length, e.g., about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 1 7, 18, 19, 20, 20-30, 30-40, 40-50, 50-60, 60-70 , 70-80, 80-90, or 90-100 amino acids in length. So, the linker is about 100-150, 150-200, 200-250, 250-30 0, 300-350, 350-400, 400-450, or 450-500 amino acids Longer or shorter linkers are also contemplated.

[0182] In some embodiments, the linker comprises an RNA promoter comprising a Cas9 nuclease domain. The gRNA-binding domain of the gramabru nuclease and a nucleic acid editing protein (e.g., cytidipine) In some embodiments, the catalytic domains of the enzymes (e.g., ATP deaminase or adenosine deaminase) are linked together. In this embodiment, the linker connects the dCas9 and the nucleic acid editing protein. For example, the linker is located between or adjacent to two groups, molecules, or other moieties and is covalently bonded to In some embodiments, the phosphorus A Car is an amino acid or multiple amino acids (eg, a peptide or protein). In some embodiments, the linker is an organic molecule, group, polymer, or chemical moiety. In some embodiments, the linker is 5 to 200 amino acids in length, e.g., 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 35, 4 5, 50, 55, 60, 60, 65, 70, 70, 75, 80, 85, 90, 90, 95 , 100, 101, 102, 103, 104, 105, 110, 120, 130, 140 , 150, 160, 175, 180, 190, or 200 amino acids in length. Longer or shorter linkers are also contemplated. In some embodiments, the linker is an amino It contains the amino acid sequence SGSETPGTSESATPES and may also be referred to as an XTEN linker. In some embodiments, the linker comprises the amino acid sequence SGGS. The winner is (SGGS) n , (GGGS) n , (GGGGS) n , (G) n、 (EAAA K) n , (GGS) n , SGSETPGTSESATPES, or (XP) n motif or any combination thereof, wherein n is independently an integer from 1 to 30. and X is any amino acid. In some embodiments, n is 1, 2, 3, 4, 5 , 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15. In the present study, the linker contains multiple proline residues, with 5-21, 5-14, 5-9, and 5-7 amino acids. Amino acid length, e.g., PAPAP, PAPAPA, PAPAPAP, PAPAPAPA, P (AP)4, P(AP)7, P(AP) 10 Such a proline-rich linker is also referred to as a "rigid" linker.

[0183] In some embodiments, the chimeric antigen receptor comprises at least one linker. One linker is a heavy chain variable (VH) region of the extracellular binding domain of the chimeric antigen receptor. The linker also links or connects the extracellular binding domain to the heavy chain constant (CH) region. The light chain variable (VL) region of the antibody may also be linked to a variable constant (VC) region.

[0184] In some embodiments, the base editor domain comprises a linker comprising the amino acid sequence: are fused via: SGGSSGSETPGTSESATPESSGGS, SGGSSGGSSGSETPGTSESATPESSGGSSGGS, or GGSGGSPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEG SAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPG TSESATPESGPGSEPATSGGSGGS.

[0185] In some embodiments, the base editor domain has the amino acid sequence SGSETPGTS The fusion occurs via a linker containing ESATPES, 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 is 24 amino acids in length. In some embodiments, the link In some embodiments, the linker is 40 amino acids in length. Contains GSSGGSSGSETPGTSESATPESSGGSSGGSSGGSSGGGS In some embodiments, the linker is 64 amino acids in length. The anchor has the amino acid sequence SGGSSGGSSGSETPGTSESATPESSGGSS Contains GGSSGGSSGGSSGSETPGTSESATPESSGGSSGGS. In some embodiments, the linker is 92 amino acids in length. is the amino acid sequence PGSPAGSPTSTEEGTSESATPESGPGTSTEPS EGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSA Contains PGTSESATPESGPGSEPATS.

[0186] As used herein, the term "liquid cancer" refers to cancers that are present in, for example, blood, lymph, and bone marrow. These are cancer cells that exist in bodily fluids, such as leukemia, myeloma, and liquid lymphoma. As used herein, liquid cancers include, but are not limited to, sarcomas, and solid tumors such as carcinomas or solid lymphomas (without cystic or fluid areas) "Liquid cancer" may be recurrent, refractory, or metastatic. The liquid cancer treated by the described methods can be, for example, liquid lymphoma. includes lymphomas that contain cysts or fluid areas.

[0187] "Lymphocyte activation gene 3 (LAG-3) polypeptide" refers to the polypeptide identified under NCBI accession number NP having at least about 85% amino acid sequence identity with _002277.4 or a fragment thereof; It refers to a protein with immunomodulatory activity. Exemplary amino acid sequences are provided below. >NP_002277.4 Lymphocyte activation gene 3 protein precursor [Homo sa piens] 1 MWEAQFLGLL FLQPLWVAPV KPLQPGAEVP VVWAQEGAPA QLPCSPTIPL QDLSLLRRAG 61 VTWQHQPDSG PPAAAPGHPL APGPHPAAPS SWGPRPRRYT VLSVGPGGLR SGRLPLQPRV 121 QLDERGRQRG DFSLWLRPAR RADAGEYRAA VHLRDRALSC RLRLRLGQAS MTASPPGSLR 181 ASDWVILNCS FSRPDRPASV HWFRNRGQGR VPVRESPHHH LAESFLFLPQ VSPMDSGPWG 241 CILTYRDGFN VSIMYNLTVL GLEPPTPLTV YAGAGSRVGL PCRLPAGVGT RSFLTAKWTP 301 PGGGPDLLVT GDNGDFTLRL EDVSQAQAGT YTCHIHLQEQ QLNATVTLAI ITVTPKSFGS 361 PGSLGKLLCE VTPVSGQERF VWSSLDTPSQ RSFSGPWLEA QEAQLLSQPW QCQLYQGERL 421 LGAAVYFTEL SSPGAQRSGR APGALPAGHL LLFLILGVLS LLLLVTGAFG FHLWRRQWRP 481 RRFSALEQGI HPPQAQSKIE ELEQEPEPEP EPEPEPEPEP EPEQL

[0188] "Lymphocyte activation gene 3 (LAG-3) polynucleotide" means a LAG-3 polypeptide. By LAG-3 is meant a nucleic acid encoding a peptide. An exemplary LAG-3 nucleic acid sequence is provided below. >NM_002286.6 Homo sapiens lymphocyte activation 3 (LAG3), mRNA 1 agagaccagc agaacggcat cccagccacg acggccactt tgctctgtct gctctccgcc 61 acggccctgc tctgttccct gggacacccc cgcccccacc tcctcaggct gcctgatctg 121 cccagctttc cagctttcct ctggattccg gcctctggtc atccctcccc accctctctc 181 caaggccctc tcctggtctc ccttcttcta gaaccccttc ctccacctcc ctctctgcag 241 aacttctcct ttacccccca ccccccacca ctgccccctt tccttttctg acctcctttt 301 ggagggctca gcgctgccca gaccatagga gagatgtggg aggctcagtt cctgggcttg 361 ctgtttctgc agccgctttg ggtggctcca gtgaagcctc tccagccagg ggctgaggtc 421 ccggtggtgt gggcccagga gggggctcct gcccagctcc cctgcagccc cacaatcccc 481 ctccaggatc tcagccttct gcgaagagca ggggtcactt ggcagcatca gccagacagt 541 ggcccgcccg ctgccgcccc cggccatccc ctggcccccg gccctcaccc ggcggcgccc 601 tcctcctggg ggcccaggcc ccgccgctac acggtgctga gcgtgggtcc cggaggcctg 661 cgcagcggga ggctgcccct gcagccccgc gtccagctgg atgagcgcgg ccggcagcgc 721 ggggacttct cgctatggct gcgcccagcc cggcgcgcgg acgccggcga gtaccgcgcc 781 gcggtgcacc tcagggaccg cgccctctcc tgccgcctcc gtctgcgcct gggccaggcc 841 tcgatgactg ccagcccccc aggatctctc agagcctccg actgggtcat tttgaactgc 901 tccttcagcc gccctgaccg cccagcctct gtgcattggt tccggaaccg gggccagggc 961 cgagtccctg tccgggagtc cccccatcac cacttagcgg aaagcttcct cttcctgccc 1021 caagtcagcc ccatggactc tgggccctgg ggctgcatcc tcacctacag agatggcttc 1081 aacgtctcca tcatgtataa cctcactgtt ctgggtctgg agcccccaac tcccttgaca 1141 gtgtacgctg gagcaggttc cagggtgggg ctgccctgcc gcctgcctgc tggtgtgggg 1201 acccggtctt tcctcactgc caagtggact cctcctgggg gaggccctga cctcctggtg 1261 actggagaca atggcgactt tacccttcga ctagaggatg tgagccaggc ccaggctggg 1321 acctacacct gccatatcca tctgcaggaa cagcagctca atgccactgt cacattggca 1381 atcatcacag tgactcccaa atcctttggg tcacctggat ccctggggaa gctgctttgt 1441 gaggtgactc cagtatctgg acaagaacgc tttgtgtgga gctctctgga caccccatcc 1501 cagaggagtt tctcaggacc ttggctggag gcacaggagg cccagctcct ttcccagcct 1561 tggcaatgcc agctgtacca gggggagagg cttcttggag cagcagtgta cttcacagag 1621 ctgtctagcc caggtgccca acgctctggg agagccccag gtgccctccc agcaggccac 1681 ctcctgctgt ttctcatcct tggtgtcctt tctctgctcc ttttggtgac tggagccttt 1741 ggctttcacc tttggagaag acagtggcga ccaagacgat tttctgcctt agagcaaggg 1801 attcaccctc cgcaggctca gagcaagata gaggagctgg agcaagaacc ggagccggag 1861 ccggagccgg aaccggagcc cgagcccgag ccccgagccgg agcagctctg acctggagct 1921 gaggcagcca gcagatctca gcagcccagt ccaaataaac tccctgtcag cagcaa

[0189] A "marker" is any molecule that has an altered expression level or activity that is associated with a disease or disorder. The term "protein" refers to any protein or polynucleotide.

[0190] The term "mutation," as used herein, refers to a change in a sequence (e.g., a nucleic acid sequence or The substitution of one residue in an amino acid sequence by another, or the deletion of one or more residues in the sequence, Mutations typically refer to the original residue, followed by the position of the residue in the sequence, and The identity of the newly substituted residues is described herein. Various methods for making amino acid substitutions (mutations) provided herein are known in the art. It is well known, for example, in Green and Sambrook, Molecular C loning:A Laboratory Manual(4th ed.,Cold Spring Harbor Laboratory Press,Cold Spri In some embodiments, the method is provided in the Journal of Microbiology, Vol. 1, No. 1, pp. 111-114, 2012. The base editors of the present disclosure may reduce a significant number of unintended mutations (e.g., unintended point mutations) of nucleic acids (e.g., nucleic acids in a subject's genome) without generating In some embodiments, the intended mutation can be efficiently generated. The mutation is generated by a guide polynucleotide (e.g., a nucleotide sequence) specifically designed to generate the intended mutation. a specific base editor (e.g., a cytidine base editor) bound to a gRNA or adenosine base editors).

[0191] Generally, sequences (e.g., amino acid sequences described herein) are generated or identified. The mutations detected are numbered relative to the reference (or wild-type) sequence, i.e., the sequence that does not contain the mutation. Those skilled in the art will recognize variations in amino acid and nucleic acid sequences relative to the reference sequence. It will be readily apparent how to determine the position of

[0192] "Tumor" refers to cells or tissues that exhibit abnormal growth or proliferation. The term tumor In some embodiments, the tumor is a solid tumor. In other embodiments, the tumor is a liquid tumor. In some embodiments, the tumor is a blood tumor. In some embodiments, the blood cancer is leukemia, myeloma, and / or lymphoma. In some embodiments, the hematological cancer is a B-cell cancer. In some cases, B-cell cancers are lymphomas or leukemias. In some cases, leukemias are pre-leukemias. In some cases, the leukemia is acute leukemia. Acute leukemia includes, for example, acute Acute leukemia includes, for example, acute myeloid leukemia (AML). or acute lymphocytic leukemia (ALL), which includes B-lineage ALL, T-lineage ALL, and LL, and T-cell acute lymphoblastic leukemia (T-ALL).

[0193] Non-limiting examples of tumors include T-cell acute lymphoblastic leukemia (T-ALL), mycosis malignant tumors, and leukemia. MF, Sézary syndrome (SS), peripheral T / NK cell lymphoma, anaplastic large cell lymphoma ALK lymphoma + , primary cutaneous T-cell lymphoma, T-cell large granular lymphocytic leukemia, vascular immunity Blastic T / NK cell lymphoma, hepatosplenic T cell lymphoma, primary cutaneous CD30 + Lymphoproliferative disorder, extranodal NK / T cell lymphoma, adult T cell leukemia / lymphoma, T cell prolymphocytic Leukemia, subcutaneous panniculitis-like T-cell lymphoma, primary cutaneous gamma-delta T-cell lymphoma, aggressive In some embodiments, the tumors include NK-cell leukemia, and enteropathy-associated T-cell lymphoma. In some embodiments, the tumor is T-cell acute lymphoblastic leukemia (T-ALL). is acute myeloid leukemia (AML).

[0194] "Nuclear factor of activated T cells 1 (NFATc1) polypeptide" refers to the polypeptide identified under NCBI accession number N having at least about 85% amino acid sequence identity with M_172390.2 or a fragment thereof refers to a protein that is a component of the activated T cell DNA-binding transcription complex. The amino acid sequence is provided below. >NP_765978.1 Nuclear factor of activated T cells, cytoplasmic 1 isoform A [Homo sapiens] MPSTSFPVPSKFPLGPAAAVFGRGETLGPARAGGTMKSA EEEHYGYASSNVSPALPLPTAHSTLPAPCHNLQTSTPGII PPADHPSGYGAALDGGPAGYFLSSGHTRPDGAPALESPRI EITSCLGLYHNNNQFFHDVEVEDVLPSSSKRSPSTATLSLP SLEAYRDPSCLSPASSLSSRSCNSEASSYESNYSYPYASP QTSPWQSPCVSPKTTDPEEGFPRGLGACTLLGSPRHSPST SPRASVTEESWLGARSSRPASPCNKRKYSLNGRQPPYSPH HSPTPSPHGSPRVSVTDDSWLGNTTQYTSSAIVAAINALT TDSSLDLGDGVPVKSRKTTLEQPPSVALKVEPVGEDLGSP PPPADFAPEDYSSFQHIRKGGFCDQYLAVPQHPYQWAKPK PLSPTSYMSPTLPALDWQLPSHSGPYELRIEVQPKSHHRA HYETEGSRGAVKASAGGHPIVQLHGYLENEPLMLQLFIGT ADDRLLRPHAFYQVHRITGKTVSTTSHEAILSNTKVLEIP LLPENSMRAVIDCAGILKLRNSDIELRKGETDIGRKNTRV RLVFRVHVPQPSGRTLSLQVASNPIECSQRSAQELPLVEK QSTDSYPVVGGKKMVLSGHNFLQDSKVIFVEKAPDGHHVW EMEAKTDRDLCKPNSLVVEIPPFRNQRITSPVHVSFYVCN GKRKRSQYQRFTYLPANGNAIFLTVSREHERVGCFF

[0195] "Nuclear factor of activated T cells 1 (NFATc1) polynucleotide" means a polynucleotide derived from an NFATc1 The NFATc1 gene is a nucleic acid molecule that encodes a polypeptide in T cells. Proteins involved in the inducible expression of cytokine genes (especially IL-2 and IL-4) Exemplary nucleic acids that have been sequenced are provided below. >NM_172390.2 Homo sapiens activated T cell nuclear factor 1 (NFA TC1), transcript variant 1, mRNA GGCGGGCGCTCGGCGACTCGTCCCCGGGGCCCCGCGCGGG CCCGGGCAGCAGGGGCGTGATGTCACGGCAGGGAGGGGGC GCGGGAGCCGCCGGGCCGGCGGGGAGGCGGGGGAGGTGTT TTCCAGCTTTAAAAAGGCAGGAGGCAGAGCGCGGCCCTGC GTCAGAGGCGAGACTCAGAGGCTCCGAACTCGCCGGCGGAG TCGCCGCGCCAGATCCCAGCAGCAGGGCGCGGGCACCGGG GCGCGGGCAGGGCTCGGAGCCACCGCGCAGGTCCTAGGGC CGCGGCCGGGCCCCGCCACGCGCGCACACGCCCCTCGATG ACTTTCCTCCGGGGCGCGCGGCGCTGAGCCCGGGGCGAGG GCTGTCTTCCCGGAGACCCGACCCCGGCAGCGCGGGGGCGG CCGCTTCTCCTGTGCCTCCGCCCGCCGCTCCACTCCCCGC CGCCGCCGCGCGGATGCCAAGCACCAGCTTTCCAGTCCCT TCCAAGTTTCCACTTGGCCCTGCGGCTGCGGTCTTCGGGA GAGGAGAAACTTTGGGGCCCGGCCCGCGCGCCGGCGGCAC CATGAAGTCAGCGGAGGAAGAACACTATGGCTATGCATCC TCCAACGTCAGCCCCGCCCTGCCGCTCCCCACGGCGCACT CCACCCTGCCGGCCCCGTGCCACAACCTTCAGACCTCCAC ACCGGGCATCATCCCGCCGGCGGATCACCCCCTCGGGGTAC GGAGCAGCTTTGGACGGTGGGCCCGCGGGCTACTTCCTCT CCTCCCGGCCACACCAGGCCTGATGGGGCCCCTGCCCTGGA GAGTCCTCGCATCGAGATAACCTCGTGCTTGGGCCTGTAC CACAACAATAACCAGTTTTTCCACGATGTGGAGGTGGAAG ACGTCCTCCCTAGCTCCAAACGGTCCCCCTCCACGCCAC GCTGAGTCTGCCCAGCCTGGAGGCCTACAGAGACCCCTCG TGCCTGAGCCCGGCCAGCAGCCTGTCCTCCCGGAGCTGCA ACTCAGAGGCCTCCTCCTACGAGTCCAACTACTCGTCCC GTACGCGTCCCCCCAGACGTCGCCATGGCAGTCTCCCTGC GTGTCTCCCAAGACCACGGACCCCGAGGAGGGCTTTCCCC GCGGGCTGGGGGCCTGCACACTGCTGGGTTCCCCGCGGCA CTCCCCCTCCACCTCGCCCCGCGCCAGCGTCACTGAGGAG AGCTGGCTGGGTGCCCGCTCCTCCAGACCCGCGTCCCCTT GCAACAAGAGGAAGTACAGCCTCAACGGCCGGCAGCCGCC CTACTCACCCCACCACTCGCCCACGCCGTCCCCGCACGGC TCCCCGCGGGTCAGCGTGACCGACGACTCGTGGTTGGGCA ACACCACCCAGTACACCAGCTCGGCCATCGTGGCCGCCAT CAACGCGCTGACCACCGACAGCAGCCTGGACCTGGGAGAT GGCGTCCCTGTCAAGTCCCGCAAGACCACCCTGGAGCAGC CGCCCTCAGTGGCGCTCAAGGTGGAGCCCGTCGGGGAGGA CCTGGGCAGCCCCCCGCCCCCGGCCGACTTCGCGCCCGAA GACTACTCCTCTTTCCAGCACATCAGGAAGGGCGGCTTCT GCGACCAGTACCTGGCGGTGCCGCAGCACCCCTACCAGTG GGCGAAGCCCAAGCCCCTGTCCCCTACGTCCTACATGAGC CCGACCCTGCCCGCCCTGGACTGGCAGCTGCCGTCCCACT CAGGCCCGTATGAGCTTCGGATTGAGGTGCAGCCCAAGTC CCACCACCGAGCCCACTACGAGACGGAGGGCAGCCGGGGG GCCGTGAAGGCGTCGGCCGGAGGACACCCCATCGTGCAGC TGCATGGCTACTTGGAGAATGAGCCGCTGATGCTGCAGCT TTTCATTGGGACGGCGGACGACCGCCTGCTGCGCCCGCAC GCCTTCTACCAGGTGCACCGCATCACAGGGAAGACCGTGT CCACCACCAGCCACGAGGCCATCCTCTCCAACACCAAAGT CCTGGAGATCCCACTCCTGCCGGAGAACAGCATGCGAGCC GTCATTGACTGTGCCGGAATCCTGAAACTCAGAAACTCCG ACATTGAACTTCGGAAAGGAGAGACGGACATCGGGAGGAA GAACACACGGGTACGGCTGGTGTTCCGCGTTCACGTCCCG CAACCCAGCGGCCGCACGCTGTCCCTGCAGGTGGCCTCCA ACCCCATCGAATGCTCCCAGCGCTCAGCTCAGGAGCTGCC TCTGGTGGAGAAGCAGAGCACGGACAGCTATCCGGTCGTG GGCGGGAAGAAGATGGTCCTGTCTGGCCACAACTTCCTGC AGGACTCCAAGGTCATTTTCGTGGAGAAAGCCCCAGATGG CCACCATGTCTGGGAGATGGAAGCGAAAACTGACCGGGAC CTGTGCAAGCCGAATTCTCTGGTGGTTGAGATCCCGCCAT TTCGGAATCAGAGGATAACCAGCCCCGTTCACGTCAGTTT CTACGTCTGCAACGGGAAGAGAAAGCGAAGCCAGTACCAG CGTTTCACCTACCTTCCCGCCAACGGTAACGCCATCTTTC TAACCGTAAGCCGTGAACATGAGCGCGTGGGGTGCTTTTT CTAAAGACGCAGAAACGACGTCGCCGTAAAGCAGCGTGGC GTGTTGCACATTTAACTGTGTGATGTCCCGTTAGTGAGAC CGAGCCATCGATGCCCTGAAAAGGAAAGGAAAAGGGAAGC TTCGGATGCATTTTCCTTGATCCCTGTTGGGGGTGGGGGG CGGGGGTTGCATACTCAGATAGTCACGGTTATTTTGCTTC TTGCGAATGTATAACAGCCAAGGGGAAAACATGGCTCTTC TGCTCCAAAAAACTGAGGGGGTCCTGGTGTGCATTTGCAC CCTAAAGCTGCTTACGGTGAAAAGGCAAATAGGTATAGCT ATTTTGCAGGCACCTTTAGGAATAAACTTTGCTTTTAAGC CTGTAAAAAAAAAAAAAAA

[0196] The term "non-conservative mutation" refers to amino acid substitutions between different groups (e.g., tryptophan). lysine for phospholipids, or phenylalanine for serine. Non-conservative amino acid substitutions may be made without disrupting or inhibiting the biological activity of the functional variant. Non-conservative amino acid substitutions are preferred because they are intended to ensure that the biological activity of the functional variant is comparable to that of the wild-type variant. The biological activity of the functional variant may be enhanced so that it is increased relative to the protein. can.

[0197] The term "nuclear localization sequence," "nuclear localization signal," or "NLS" refers to a protein The nuclear localization sequence refers to an amino acid sequence that promotes the import of a target protein into the cell nucleus. For example, see the international PCT application filed on November 23, 2000 by Plank et al. PCT / EP2000 / 011690 (as WO / 2001 / 038547, 2001) (Their contents are those of exemplary nuclear localization sequences.) (The disclosure of which is incorporated herein by reference.) In other embodiments, the NLS is, for example, For example, Koblan et al., Nature Biotech. 2018 doi :10.1038 / nbt.4172 is an optimized NLS. In this form, the NLS has the amino acid sequence PKKKRKVEGADKRTADGSEFESPK KKRKV, KRTADGSEFESPKKKRKV, KRPAATKKAGQAKKK K, KKTELQTTNAENKTKKL, KRGINDRNFWRGENGRKTR, RKSGKIAAIVVKRPRK, PKKKRKV, or MDSLLMNRRKFL Contains YQFKNVRWAKGRRETYLC.

[0198] As used herein, the terms "nucleic acid" and "nucleic acid molecule" refer to a nucleic acid molecule having a nucleobase and an acid. a compound containing a moiety (e.g., a nucleoside, a nucleotide, or a polymer of nucleotides) Typically, a polymeric nucleic acid (e.g., a nucleic acid molecule containing three or more nucleotides) is used. A direct linker (a nucleotide) is a chain in which adjacent nucleotides are linked to each other via phosphodiester bonds. In some embodiments, a "nucleic acid" refers to a chain molecule consisting of individual nucleic acid residues (e.g., nucleotides). In some embodiments, a "nucleic acid" refers to a group of three or more Refers to an oligonucleotide chain containing individual nucleotide residues. The terms "oligonucleotide" and "polynucleotide" refer to a polymer of nucleotides. are used interchangeably to refer to a polymer (e.g., a chain of at least three nucleotides) In some embodiments, "nucleic acid" refers to RNA, as well as single-stranded and / or double-stranded nucleic acids. Nucleic acids include naturally occurring nucleic acid molecules (e.g., genomes, transcripts, mRNAs, etc.). NA, tRNA, rRNA, siRNA, snRNA, plasmid, cosmid, chromosome, In contrast, nucleic acid molecules can be , non-naturally occurring molecules (e.g., recombinant DNA or RNA, artificial chromosomes, engineered (a genome or fragment thereof, or synthetic DNA, RNA, or DNA / RNA hybrid) or may include non-naturally occurring nucleotides or nucleosides. The terms "nucleic acid," "DNA," "RNA," and / or similar terms refer to nucleic acids. Nucleic acids may be derived from natural sources. purified from, produced using a recombinant expression system, and optionally purified and chemically Where applicable, e.g., in the case of chemically synthesized molecules, Nucleic acids are nucleotides, such as analogs with chemically modified bases or sugars, and backbone modifications. Nucleic acid sequences are presented in a 5' to 3' direction unless otherwise indicated. In some embodiments, the nucleic acid is composed of natural nucleosides (e.g., adenosine, thymidine, guanosine, cytidine, uridine, deoxyadenosine, deoxythymidine, cytosine, and deoxycytidine), nucleoside analogs (e.g., 2-amino adenosine, 2-thiothymidine, inosine, pyrrolopyrimidine, 3-methyladenosine, 5 -methylcytidine, 2-aminoadenosine, C5-bromouridine, C5-fluorouridine uridine, C5-iodouridine, C5-propynyluridine, C5-propynylcytidine, C5-methylcytidine, 2-aminoadenosine, 7-deazaadenosine, 7-deazagua adenosine, 8-oxoadenosine, 8-oxoguanosine, O(6)-methylguanine, and and 2-thiocytidine), chemically modified bases, biologically modified bases (e.g., methylated bases), insertions bases, modified sugars (e.g., 2'-fluororibose, ribose, 2'-deoxyribose, arabinose, and hexose), and / or modified phosphate groups (e.g., phosphoro thioate and 5'-N-phosphoramidite bridges). The term "nucleic acid programmable DNA binding protein" or "napDNAbp" may be used interchangeably with "polynucleotide programmable nucleotide binding domain" A guide nucleic acid or guide polynucleotide that guides napDNAbp to a specific nucleic acid sequence Proteins that associate with nucleic acids (e.g., DNA or RNA) such as genes (e.g., gRNA) In some embodiments, polynucleotide programmable nucleotide binding The domain is a polynucleotide programmable DNA binding domain. In this embodiment, the polynucleotide programmable nucleotide binding domain is In some embodiments, the polynucleotide protease is a programmable RNA binding domain. The programmable nucleotide binding domain is the Cas9 protein. The protein guides the Cas9 protein to a specific DNA sequence that is complementary to the guide RNA. In some embodiments, the napDNAbp can associate with a guide RNA that is C as9 domain, e.g., nuclease-active Cas9, Cas9 nickase (nCas 9), or nuclease-inactive Cas9 (dCas9). Non-limiting examples of DNA binding proteins include Cas9 (e.g., dCas9 and nCas9). Cas9), Cas12a / Cpfl, Cas12b / C2cl, Cas12c / C2c 3, Cas12d / CasY, Cas12e / CasX, Cas12g, Cas12h, Non-limiting examples of Cas enzymes include Cas12i, Cas12j / CasΦ, and Cas12j / CasΦ. These include Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5, and Cas5 d, Cas5t, Cas5h, Cas5a, Cas6, Cas7, Cas8, Cas8a , Cas8b, Cas8c, Cas9 (also known as Csn1 or Csx12) ), Cas10, Cas10d, Cas12a / Cpfl, Cas12b / C2cl, C as12c / C2c3, Cas12d / CasY, Cas12e / CasX, Cas12 g, Cas12h, Cas12i, Cas12j / CasΦ, Csy1, Csy2, Cs y3, Csy4, Cse1, Cse2, Cse3, Cse4, Cse5e, Csc1, C sc2, Csa5, Csn1, Csn2, Csm1, Csm2, Csm3, Csm4, C sm5, Csm6, Cmr1, Cmr3, Cmr4, Cmr5, Cmr6, Csb1, C sb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Cs x3, Csx1, Csx1S, Csx11, Csf1, Csf2, CsO, Csf4, C sd1, Csd2, Cst1, Cst2, Csh1, Csh2, Csa1, Csa2, C sa3, Csa4, Csa5, type II Cas effector proteins, type V Cas effector proteins effector proteins, type VI Cas effector proteins, CARF, DinG, and Other nucleic acids include homologs of, or modified or engineered versions thereof. Programmable DNA binding proteins are also within the scope of this disclosure; however, they are not For example, Makarova et al. Lassification and Nomenclature of CRISPR -Cas Systems:Where from Here?”CRISPR J.2 018 Oct;1:325-336.doi:10.1089 / crispr.201 8.0033, Yan et al., “Functionally diverse type V CRISPR-Cas systems”Science.2019 J an 4;363(6422):88-91.doi:10.1126 / science See .aav7271 (the entire contents of each of which are incorporated herein by reference). ).

[0199] The terms "nucleobase," "nitrogenous base," or "base" are used interchangeably herein. refers to nitrogen-containing biological compounds that form nucleosides, which are components of nucleotides The ability of nucleic acid bases to base pair and stack with each other is what makes ribonucleic acid (RNA) and It directly connects to long chain helix structures such as ribonucleic acid (DNA). The five nucleobases are tosine (C), guanine (G), thymine (T), and uracil (U) are called primary or canonical. Adenine and guanine are derived from purines, and cytosine , uracil, and thymine are derived from pyrimidines. DNA and RNA also contain modifications Non-limiting exemplary modified nucleobases include: hypoxanthine, xanthine, 7-methylguanine, 5,6-dihydrouracil, 5 -methylcytosine (m5C), and 5-hydromethylcytosine (hydromethyl Hypoxanthine and xanthine are both mutagenic. In the presence of exogenous substances, it is produced through deamination (replacement of amine groups with carbonyl groups). Hypoxanthine can be modified from adenine. Xanthine can be modified from guanine. Uracil can result from the deamination of cytosine. A "nucleoside" is It consists of a nucleic acid base and a five-carbon sugar (either ribose or deoxyribose). Examples of osides are adenosine, guanosine, uridine, cytidine, and 5-methyluridine. (m5U), deoxyadenosine, deoxyguanosine, thymidine, deoxyuridine, and deoxycytidine. Examples of nucleosides with modified nucleobases include , inosine (I), xanthosine (X), 7-methylguanosine (m7G), dihydroguanosine These include lysine (D), 5-methylcytidine (m5C), and pseudouridine (Ψ). A "nucleotide" is a nucleic acid consisting of a nucleic acid base, a pentose sugar (either ribose or deoxyribose), and a and at least one phosphate group.

[0200] The terms "nucleobase editing domain" or "nucleobase editing protein" are used herein. When used in is deamination to thymine (or thymidine), and adenine (or adenosine) Deamination of α to hypoxanthine (or inosine) and non-template nucleotide attachment can catalyze nucleobase modifications in RNA or DNA, such as addition and insertion. In some embodiments, a nucleobase-editing domain refers to a protein or enzyme that a enzyme domain (e.g., adenine deaminase or adenosine deaminase; or cytidine deaminase or cytosine deaminase). In some embodiments, the nucleic acid The base editing domain may comprise two or more deaminase domains (e.g., adenine deaminase or adenosine deaminase and cytidine deaminase or cytosine deaminase) In some embodiments, the nucleobase-editing domain is a naturally occurring nucleobase-editing domain. In some embodiments, the nucleobase-editing domain can be a naturally occurring nucleobase. The nucleobase-editing domain may be an engineered or evolved nucleobase-editing domain derived from a base-editing domain. Acid-base editing domains are found in bacteria, humans, chimpanzees, gorillas, monkeys, cows, dogs, and rats. The antibody may be derived from any organism, such as a mouse or a mammal.

[0201] As used herein, the term "obtaining" in "obtaining a drug" refers to the This includes synthesizing, purchasing, or otherwise obtaining an agent. A "patient" or "subject" as used herein is someone who has been diagnosed with or has a disease or disorder. Being at risk of developing or suspected of having or developing a disease In some embodiments, the term "patient" refers to a mammalian subject or individual having refers to a mammalian subject who has a higher than average likelihood of developing a disease or disorder. has been used in humans, non-human primates, cats, dogs, pigs, cattle, horses, camels, llamas, goats, sheep, rodents (e.g., mice, rabbits, rats, or guinea pigs) and and other mammals that may benefit from the therapies disclosed herein. The patient may be male and / or female.

[0202] A "patient in need thereof" or a "subject in need thereof" is used herein to refer to a person with a disease or Have you been diagnosed with a cancer or disorder (e.g., T-cell or NK-cell malignancies)? , at risk of having it, presumed to have it, or have it This refers to patients suspected of having the disease.

[0203] "Pathogenic mutation," "pathogenic variant," "disease casing" "pathogenic variant," "deleterious mutation," or "predisposing mutation" The term refers to a genetic condition that increases an individual's susceptibility or predisposition to a particular disease or disorder. In some embodiments, a pathogenic mutation refers to a change or mutation in a gene encoded by a gene. At least one pathogenic amino acid has been substituted in the target protein. Both contain one wild-type amino acid.

[0204] The term "pharmaceutically acceptable carrier" refers to a compound that is administered to a site in the body (e.g., a delivery site). ) to another site (e.g., an organ, tissue, or part of the body) Involved liquid or solid fillers, diluents, excipients, manufacturing aids (e.g., lubricants, talc, magnesium, calcium or zinc stearate, or steric acid pharmaceutically acceptable materials, compositions, or bio-based compounds, such as acetaminophen, ... A pharmaceutically acceptable carrier means a substance that is compatible with the other ingredients of the formulation. It is "acceptable" in taste and does not harm the target tissue (e.g., physiologically compatible). (e.g., sterile, physiological pH) "excipient," "carrier," "pharmaceutically acceptable carrier" , "vehicle" and the like terms are used interchangeably herein.

[0205] The term "pharmaceutical composition" means a composition formulated for pharmaceutical use. In some embodiments, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier. In embodiments, the pharmaceutical composition contains an additional agent (e.g., for specific delivery, to increase half-life, or other therapeutic compounds).

[0206] "Programmed cell death 1 (PDCD1 or PD-1) polypeptide" refers to the NCBI-received At least about 85% amino acid sequence identity with accession number AJS10360.1 or a fragment thereof The PD-1 protein is a protein that acts during an immune response and under permissive conditions. It is believed to be involved in regulating T cell function. An exemplary B2M polypeptide sequence is shown below. Provided below. >AJS10360.1 Programmed cell death 1 protein [Homo sapiens] MQIPQAPWPVVWAVLQLGWRPGWFLDSPDRPWNPPTFSPA LLVVTEGDNATFTCSFSNTSESFVLNWYRMSPSNQTDKLA AFPEDRSQPGQDCRFRVTQLPNGRDFHMSVVRARRNDSGT YLCGAISLAPKAQIKESLRAELRVTERRAEVPTAHPSSPSP RPAGQFQTLVVGVVGGLLGSLVLLVWVLAVICSRAARGTI GARRTGQPLKEDPSAVPVFSVDYGELDFQWREKTPEPPVP CVPEQTEYATIVFPSGMGTSPARRGSADGPRSAQPLRPE DGHCSWPL

[0207] "Programmed cell death 1 (PDCD1 or PD-1) polynucleotide" means a PD- The PDCD1 gene refers to a nucleic acid molecule encoding a PDCD1 polypeptide. They encode surface receptors that inhibit T cell effector functions in an antigen-specific manner. A representative PDCD1 nucleic acid sequence is provided below. >AY238517.1 Homo sapiens programmed cell death 1 (PDCD1) mRNA, full-length CDS ATGCAGATCCCACAGGCGCCCTGGCCAGTCGTCTGGGCGG TGCTACAACTGGGCTGGCGGCCAGGATGGTTCTTAGACTC CCCAGACAGGCCCTGGAACCCCCCACCTTCTCCCCAGCC CTGCTCGTGGTGACCGAAGGGGACAACGCCACCTTCACCT GCAGCTTCTCCAACACATCGGAGAGCTTCGTGCTAAACTG GTACCGCATGAGCCCCAGCAACCAGACGGACAAGCTGGCC GCCTTCCCCGAGGACCGCAGCCAGCCCGGCCAGGACTGCC GCTTCCGTGTCACACAACTGCCCAACGGGCGTGACTTCCA CATGAGCGTGGTCAGGGCCCGGCGCAATGACAGCGGCACC TACCTCTGTGGGGCCATCTCCCTGGCCCCCAAGGCGCAGA TCAAAGAGAGCCTGCGGGCAGAGCTCAGGGTGACAGAGAG AAGGGCAGAAGTGCCCACAGCCCACCCCAGCCCCTCACCC AGGCCAGCCGGCCAGTTCCAAACCCTGGTGGTTGGTGTCG TGGGCGGCCTGCTGGGCAGCCTGGTGCTGCTAGTCTGGGT CCTGGCCGTCATCTGCTCCCGGGCCGCACGAGGGACAATA GGAGCCAGGCGCACCGGCCAGCCCCTGAAGGAGGACCCCT CAGCCGTGCCTGTGTTCTCTGTGGACTATGGGGAGCTGGA TTTCCAGTGGCGAGAGAAGACCCCGGAGCCCCCCGTGCCC TGTGTCCCTGAGCAGACGGAGTATGCCACCATTGTCTTTC CTAGCGGAATGGGCACCTCATCCCCCGCCCGCAGGGGCTC AGCTGACGGCCCTCGGAGTGCCCAGCCACTGAGGCCTGAG GATGGACACTGCTCTTGGCCCCTCTGA

[0208] The terms "protein," "peptide," and "polypeptide" and their grammatical meanings Equivalents are used interchangeably herein and are joined together by a peptide (amide) bond. refers to a polymer of linked amino acid residues. The term can be used to refer to any polymer of any size, structure, or function. It refers to a protein, peptide, or polypeptide with a specific function. A protein, peptide, or polypeptide is at least three amino acids in length. Alternatively, polypeptide can refer to an individual protein or a collection of proteins. One or more of the amino acids in a protein, peptide, or polypeptide may be, for example, decarboxylated. hydroxyl group, phosphate group, farnesyl group, isofarnesyl group, fatty acid group, Modification by the addition of chemicals such as anchors (for conjugation, functionalization, or other modifications) The protein, peptide, or polypeptide may be a single molecule. The protein, peptide, or polypeptide may be a molecule or a multi-molecular complex. or simply fragments of naturally occurring proteins or peptides. The peptide, or polypeptide, may be naturally occurring, recombinant, or synthetic, or any of its The term "fusion protein" as used herein may be any combination of these. The term refers to a hybrid polypeptide, which is a polypeptide derived from proteins from at least two different proteins. One protein contains the protein domain. The other protein contains the amino-terminal (N-terminal) portion of the fusion protein. It can be located at the terminal end of the protein, or at the carboxyl terminus (C-terminus) of the protein, and therefore to form an amino-terminal or carboxy-terminal fusion protein, respectively. Proteins can contain distinct domains, e.g., nucleic acid binding domains (e.g., target sites of proteins). a gRNA-binding domain of Cas9 that induces binding to the gRNA) and a nucleic acid cleavage domain, or In some embodiments, the protein may comprise a catalytic domain of a nucleic acid editing protein. The amino acid sequence constituting the protein portion (e.g., nucleic acid binding domain) and the organic compound In some embodiments, the protein may act as a nucleic acid cleaving agent. Proteins are complexed with nucleic acids (e.g., RNA or DNA) or are free-standing. Any of the proteins provided herein are associated with a target protein (e.g., RNA or DNA). They may be produced by any method known in the art, for example, by the methods provided herein. The proteins provided may be produced through recombinant protein expression and purification, and may be peptides. It is particularly suitable for fusion proteins containing a linker. Methods for this are well known and are described in Green and Sambrook, Molecule r Cloning:A Laboratory Manual(4th ed.,Co ld Spring Harbor Laboratory Press,Cold S Spring Harbor, NY (2012) the entire contents of which are incorporated herein by reference).

[0209] The polypeptides and proteins disclosed herein (including functional portions and functional variants thereof) The amino acids (including ants) 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, aminocyclohexane. Carboxylic acid, norleucine, α-amino-n-decanoic acid, homoserine, S-acetylamino monomethyl-cysteine, trans-3- and trans-4-hydroxyproline, 4- Aminophenylalanine, 4-nitrophenylalanine, 4-chlorophenylalanine, 4-carboxyphenylalanine, β-phenylserine, β-hydroxyphenylalanine phenylglycine, α-naphthylalanine, cyclohexylalanine, cyclohexyl Glycine, Indoline-2-carboxylic acid, 1,2,3,4-tetrahydroisoquinoline -3-carboxylic acid, aminomalonic acid monoamide, N'-benzyl-N'-methyl-lysine , N'N'-dibenzyl-lysine, 6-hydroxylysine, ornithine, α-aminocyclohexyl α-aminocyclohexanecarboxylic acid, α-aminocycloheptane carboxylic acid, α-(2-amino-2-norbornane)-carboxylic acid, α,γ-diamino Butyric acid, α,β-diaminopropionic acid, homophenylalanine, and α-tert- Polypeptides and proteins include the polypeptide construct It may be associated with one or more post-translational modifications of amino acids. Non-limiting examples of post-translational modifications include: Phosphorylation, acylation (including acetylation and formylation), glycosylation (N-linked and and O-bonds), amidation, hydroxylation, alkylation (including methylation and ethylation) (including ubiquitination, pyrrolidone carboxylic acid addition, disulfide bridge formation, sulfation) , myristoylation, palmitoylation, isoprenylation, farnesylation, geranylation, These include repiation, lipoylation, and iodination.

[0210] By "promoter" is meant an array of nucleic acid control sequences that direct transcription of a nucleic acid. A promoter contains necessary nucleic acid sequences near the start site of transcription. Optionally, it contains distal enhancer or repressor sequence elements. "-" refers to promoters that are constitutively active and are not regulated by external signals or molecules. In contrast, the activity of an "inducible promoter" is regulated by an external signal or molecule ( For example, the promoter is regulated by a transcription factor (e.g., a CMV promoter). -It can be.

[0211] The term "recombinant" as used herein in the context of a protein or nucleic acid means a protein that is not naturally occurring. refers to a protein or nucleic acid that does not exist and is a man-made product. For example, in some embodiments In other words, a recombinant protein or nucleic acid molecule may have less than 100% repeatability compared to any naturally occurring sequence. At least one, at least two, at least three, at least four, at least five, at least The amino acid sequence or nucleotide sequence contains at least six or at least seven mutations. nothing.

[0212] "Decrease" means a decrease of at least 10%, 25%, 50%, 75%, or 100%. This means a change in

[0213] "Reference" refers to a standard or control condition. In one embodiment, the reference is a wild-type In other embodiments, the reference is an untreated cell and not subjected to the test conditions. or placebo or saline, medium, buffer, and / or A control vector not carrying the polynucleotide is provided.

[0214] A "reference sequence" is a defined sequence used as a basis for sequence comparison. , a subset of a particular sequence or the entire sequence, e.g., a full-length cDNA or gene sequence. It may be a segment of a polypeptide, or the complete cDNA or gene sequence. In this case, the length of the reference polypeptide sequence is generally at least about 16 amino acids, at least about 20 amino acids, at least about 25 amino acids, about 35 amino acids, about 50 amino acids, or about For nucleic acids, the length of a reference nucleic acid sequence is generally at least about 50 amino acids. nucleotides, at least about 60 nucleotides, at least about 75 nucleotides, and About 100 nucleotides or about 300 nucleotides, or thereabouts or therebetween In some embodiments, the reference sequence is the wild-type sequence of the protein of interest. In other embodiments, the reference sequence is a polynucleotide encoding a wild-type protein. It is a code array.

[0215] The terms "RNA-programmable nuclease" and "RNA-guided nuclease" The term refers to a protein that is cleaved with (e.g., bound to or associated with) one or more RNAs that are not targets for cleavage. In some embodiments, the RNA programmable nuclease is used in combination with RNA. When a complex is formed, it can be referred to as a nuclease:RNA complex. The combined RNA is called a guide RNA (gRNA). It can exist as a complex of multiple RNA molecules or as a single RNA molecule. The gRNA present may be referred to as a single guide RNA (sgRNA), but the term "gRNA" is also used. refers to guide RNAs that exist as single molecules or as complexes of two or more molecules. Typically, gRNAs that exist as a single RNA species are Two domains: (1) a domain that shares homology with the target nucleic acid (e.g., a domain that also acts as a Cas9 target) (2) a domain that binds to the Cas9 protein. In some embodiments, domain (2) comprises a sequence known as tracrRNA. The domain (2) corresponds to a sequence of nucleotides and contains a stem-loop structure. For example, in some embodiments, domain (2) Jinek et al., Science 337:816-821(2012) The tracrRNA is identical to or homologous to the tracrRNA provided in Other examples of gRNAs (e.g., those containing domain 2) are "switching" gRNAs. A patent application filed on September 6, 2013, entitled "Possible Cas9 Nucleases and Uses Thereof" was filed in the United States. Provisional Patent Application No. 61 / 874,682, and "Delivery Systems for Functional Nucleases" U.S. Provisional Patent Application No. 61 / 874,746, filed September 6, 2013, entitled "A SYSTEM FOR CARRYING OUT THE INVENTION" (the entire contents of each of which are incorporated herein by reference in their entirety). In some embodiments, the gRNA comprises two or more of domains (1) and (2). For example, an extended gRNA can be any of the extended gRNAs described herein. The target nucleic acid is then bound to two or more Cas9 proteins at two or more distinct regions, such that The gRNA contains a nucleotide sequence that is complementary to the target site, which binds to the nuclease. mediates the binding of the nuclease / RNA complex to the target site and Provides sequence specificity.

[0216] In some embodiments, the RNA programmable nuclease is a CRISPR-associated nuclease. tem) Cas9 endonuclease, e.g., Streptococcus pyoge Cas9 (Csnl) derived from nes (e.g., "Complete genome sequence of an Ml strain of Streptococcus us pyogenes.”Ferretti JJ, et al., Proc.N atl.Acad.Sci.USA98:4658-4663(2001), “C RISPR RNA maturation by trans-encoded sm all RNA and host factor RNase III.”Deltc Heva E., et al., Nature 471:602-607(2011) RNA programmable nucleases (e.g., Cas9) can encode RNA: These targets use DNA hybridization to target DNA cleavage sites. In principle, proteins can target any sequence specified by the guide RNA. For site-specific cleavage (e.g., to modify the genome), Methods for using RNA programmable nucleases are known in the art (e.g., Cong, L. et al., Multiplex genome enginee ring using CRISPR / Cas systems.Science 33 9,819-823(2013), Mali, P. et al., RNA-guided human genome engineering via Cas9.Scien ce 339,823-826(2013), Hwang,WYet al.,Ef ficient genome editing in zebrafish usin ga CRISPR-Cas system.Nature biotechnolo gy 31,227-229(2013), Jinek,M.et al.,RNA-p rogrammed genome editing in human cells. eLife 2,e00471(2013), Dicarlo,JEet al., Genome engineering in Saccharomyces cere visiae using CRISPR-Cas systems.Nucleic acids research(2013), Jiang,W.et al.RNA-g uided editing of bacterial genomes using CRISPR-Cas systems.Nature biotechnology 31, 233-239 (2013), the entire contents of each of which are incorporated herein by reference. (Incorporated herein by reference).

[0217] "Signaling domain" means the intracellular portion of a protein expressed in T cells; This transmits effector function signals (e.g., activation signals) to T cells, leading to their specialization. T cells are activated by several factors. The alloantigen can be induced to bind to a T cell receptor on the surface of the T cell, and the alloantigen can be induced to bind to a T cell receptor on the surface of the T cell. The binding of the T cell co-stimulatory molecule to the T cell surface is , the cognate binding partner on T cells, which specifically binds to costimulatory ligands and thereby Costimulatory molecules mediate costimulatory responses (such as, but not limited to, proliferation) by T cells. In some embodiments, the antigen-binding domains include, but are not limited to, MHC class I molecules. The costimulatory domain is the cytoplasmic domain of CD2. T cell activation is the process by which an immune response (e.g., T cell proliferation and differentiation (e.g., Smith-Garvin et al. See al., Annu. Rev. Immunol., 27:591-619, 2009. Exemplary T cell signaling domains are known in the art. Typical examples include CD2, CD3ζ, CD8, CD28, CD27, CD154, and GI TR (TNFRSF18), CD134 (OX40), and CD137 (4-1BB) Examples include the signal transduction domain of

[0218] "Single chain antibody" or "scFv" refers to a genetically fused single chain molecule. The VH and VL domains of one or more antibodies are linked by a polypeptide linker. (See, e.g., Bird et al., Science ce,242:423-426,1988;Huston et al.,Proc.N atl.Acad.Sci.,85:5879-5883,1988:Ahmad et al. al.,Clin.Dev.Immunol.,2012,doi:10.1155 / 2012 / 980250:Marbry,IDrugs,13:543-549,201 In some embodiments, the VH and VL domains in the scFv are The main intramolecular orientation is VH domain-linker domain-VL domain. In embodiments, the intramolecular orientation of the VH and VL domains in the scFv is domain-linker domain-VH domain.

[0219] The term "single nucleotide polymorphism (SNP)" refers to a single nucleotide polymorphism that occurs at a specific position in the genome. Variations, each of which is present to some degree in a population (e.g., >1 For example, at a particular base position in the human genome, most individuals have a C nucleotide. A oxide may appear, but in a few individuals, the position is occupied by an A. This is because The presence of a SNP at a particular position means there are two possible nucleotide variations. The variant (C or A) at this position is said to be an allele. These differences underlie the differences in susceptibility to illness, the severity of illness, and our body's response to treatment. SNPs are also a manifestation of genetic variation. SNPs can occur in the coding region of a gene, in the non-coding region of a gene, or in the It may be contained in a coding region or an intergenic region (region between genes). However, SNPs within the coding sequence do not necessarily affect the protein produced due to the degeneracy of the genetic code. SNPs in the coding region are classified as synonymous or non-synonymous SNPs. There are two types of SNPs: synonymous SNPs, which do not affect the protein sequence, and non-synonymous SNPs. P changes the amino acid sequence of a protein. Nonsynonymous SNPs are classified as missense and nonsense. SNPs that are not in protein-coding regions affect gene splicing, transcription, and transcription factor binding, messenger RNA degradation, or non-coding RNA sequences Gene expression affected by this type of SNP can be called an eSNP ( Single nucleotide variants (SNVs) are called expressed SNPs and can be located upstream or downstream of a gene. ) is a variation in a single nucleotide with no frequency restriction, which occurs in somatic cells. Somatic single base variations (e.g., associated with cancer) can also occur It can also be called a single base change.

[0220] "Specifically binds" means that the polypeptide and / or nucleic acid molecule of the present invention is recognized and bind to, but do not substantially recognize or bind to, other molecules in a sample (e.g., a biological sample); Nucleic acid molecules, polypeptides, or complexes thereof (e.g., nucleic acid programmable DNA binding proteins) The term "antigen receptor" refers to a specific molecule, compound, or molecule (e.g., a chimeric antigen receptor, a guide nucleic acid, or a chimeric antigen receptor). For example, chimeric antigen receptors specifically bind to particular markers expressed on the surface of cells. but does not bind to other polypeptides, carbohydrates, lipids, or any other compounds on the surface of the cell. Doesn't fit.

[0221] Nucleic acid molecules useful in the methods of the invention include those encoding a polypeptide of the invention or a fragment thereof. Such nucleic acid molecules include any nucleic acid molecule that is 100% identical to an endogenous nucleic acid sequence. Although not necessarily identical, they will typically show substantial identity to endogenous sequences. Polynucleotides that have "qualitative identity" typically have at least one identical sequence to the double-stranded nucleic acid molecule. Nucleic acid molecules useful in the methods of the present invention include those capable of hybridizing to one strand of a nucleic acid molecule. , any nucleic acid molecule that encodes a polypeptide of the invention or a fragment thereof. The nucleic acid molecule need not be 100% identical to the endogenous nucleic acid sequence, but typically will be substantially A polynucleotide having "substantial identity" to an endogenous sequence will be , typically capable of hybridizing to at least one strand of a double-stranded nucleic acid molecule "Hybridize" refers to the ability to hybridize with complementary polynucleotide sequences (e.g., nucleotides) under various conditions of stringency. For example, a gene described herein) or a portion thereof may be paired to form a double-stranded molecule. (See, e.g., Wahl, G.M. and S.L. Berger (1987) Met. hods Enzymol.152:399, Kimmel, AR (1987) Me. (See Methods Enzymol. 152:507).

[0222] For example, the exact salt concentration is typically less than about 750 mM NaCl and less than 75 mM Cl. trisodium enoate, preferably less than about 500 mM NaCl and less than 50 mM more preferably less than about 250 mM NaCl and Less than 25 mM trisodium citrate. Low stringency hybridization is High stringency hybridization can be achieved in the absence of organic solvents, such as formamide. The solution should preferably contain at least about 35% formamide, more preferably at least about 50% The reaction can be carried out in the presence of formamide. Strict temperature conditions usually include at least about 30°C, More preferably, a temperature of at least about 37°C, and most preferably at least about 42°C The hybridization time, detergent (e.g., sodium dodecyl sulfate (S Various additional parameters, such as the concentration of DS) and the inclusion or exclusion of carrier DNA, It is well known to those skilled in the art. By combining these various conditions as needed, Various levels of stringency can be achieved. In one embodiment, hybridization is performed at 75 0 mM NaCl, 75 mM trisodium citrate, and 1% SDS at 30°C. In another embodiment, hybridization is carried out in 500 mM NaCl, 5 0 mM trisodium citrate, 1% SDS, 35% formamide, and 100 μ In another embodiment, the reaction is carried out in 100 μg / mL denatured salmon sperm DNA (ssDNA) at 37°C. Hybridization was performed in 250 mM NaCl, 25 mM trisodium citrate, in 1% SDS, 50% formamide, and 200 μg / mL ssDNA. It is carried out at 42° C. Useful variations on these conditions will be apparent to those skilled in the art.

[0223] In most applications, the washing steps that follow hybridization also require stringency. Wash stringency conditions can be defined by salt concentration and temperature. As mentioned above, wash stringency can be increased by decreasing salt concentration or by increasing temperature. For example, stringency for the wash step can be increased by increasing Suitable salt concentrations are preferably less than about 30 mM NaCl and less than 3 mM trisodium citrate. and most preferably less than about 15 mM NaCl and less than 1.5 mM citrate The exact temperature conditions for the wash step are typically at least about 2 5°C, more preferably at least about 42°C, and even more preferably at least about 68°C In one embodiment, the wash step is performed in 30 mM NaCl, 3 mM chlorine, The preferred method is to perform the cleavage in 0.1% SDS at 25°C. In the form, the wash steps consist of 15 mM NaCl, 1.5 mM trisodium citrate, and 0.1% SDS at 42°C. The solution contains 15 mM NaCl, 1.5 mM trisodium citrate, and 0.1% S The reaction is carried out in DS at 68°C. Further variations in these conditions will be apparent to those skilled in the art. Hybridization techniques are well known to those skilled in the art and are described, for example, in Benton et al. and Davis (Science 196:180, 1977), Grunste in and Hogness(Proc.Natl.Acad.Sci.,USA 7 2:3961,1975), Ausubel et al. ocols in Molecular Biology,Wiley Intersc ience, New York, 2001), Berger and Kimmel (G uide to Molecular Cloning Techniques,198 7, Academic Press, New York), and Sambrook e t al.,Molecular Cloning:A Laboratory Man ual,Cold Spring Harbor Laboratory Press, It is listed in New York.

[0224] "Split" means divided into two or more pieces.

[0225] A "split Cas9 protein" or "split Cas9" is a protein that contains two separate nucleic acids. Ca provided as N- and C-terminal fragments encoded by nucleotide sequences The Cas9 protein is a polypeptide that corresponds to the N-terminal and C-terminal parts of the Cas9 protein. splicing the polypeptide to form the "reconstituted" Cas9 protein In certain embodiments, the Cas9 protein can be, for example, et al.,Cell,Volume 156,Issue 5,pp.935-94 9,2014,or as described in Jiang et al.(2 016)Science 351:867-871.PDB file:5F9R As shown, within the disordered region of the protein, it is split into two fragments, each of which (The sequences are incorporated herein by reference.) In some embodiments, the protein has a length of about amino acid A SpCas between 292 and G364, F445 and K483, or E565 and T637 at any C, T, A, or S within the region of Cas9, or any other Cas9, s9 variants (e.g., nCas9, dCas9) or other napDNAbp In some embodiments, the protein is split into two fragments at corresponding positions in the at T310, T313, A456, S469, or C574 of SpCas9 In some embodiments, a process for splitting a protein into two fragments is used. The process is referred to as "splitting" the protein.

[0226] In other embodiments, the N-terminal portion of the Cas9 protein is selected from the group consisting of S. pyogenes Ca s9 wild type (SpCas9) (NCBI reference sequence: NC_002737.2, Unipr ot reference sequence: Q99ZW2) and amino acids 1 to 573 or 1 to 637 of the Cas The C-terminal part of the protein is located between amino acids 574 and 1368 of the wild-type SpCas9 or 638 to 1368, or their corresponding positions.

[0227] The C-terminal part of the split Cas9 was ligated to the N-terminal part of the split Cas9 to form the complete In some embodiments, the Cas9 protein can be transformed into a complete Cas9 protein. The C-terminal part of the protein begins where the N-terminal part of the Cas9 protein ends. Thus, in some embodiments, the C-terminal portion of the split Cas9 is the amino acid sequence of spCas9. Contains part of the acid (551-651)-1368. The sequence begins at amino acids 551–651 (inclusive) and ends at amino acid 13. For example, the C-terminal part of split Cas9 ends in spCas 9 amino acids 551–1368, 552–1368, 553–1368, 554–136 8, 555~1368, 556~1368, 557~1368, 558~1368, 55 9~1368, 560~1368, 561~1368, 562~1368, 563~13 68, 564~1368, 565~1368, 566~1368, 567~1368, 5 68~1368, 569~1368, 570~1368, 571~1368, 572~1 368, 573~1368, 574~1368, 575~1368, 576~1368, 577~1368, 578~1368, 579~1368, 580~1368, 581~ 1368, 582~1368, 583~1368, 584~1368, 585~1368 , 586-1368, 587-1368, 588-1368, 589-1368, 590 ~1368, 591~1368, 592~1368, 593~1368, 594~136 8, 595~1368, 596~1368, 597~1368, 598~1368, 59 9~1368, 600~1368, 601~1368, 602~1368, 603~13 68, 604~1368, 605~1368, 606~1368, 607~1368, 6 08~1368, 609~1368, 610~1368, 611~1368, 612~1 368, 613~1368, 614~1368, 615~1368, 616~1368, 617~1368, 618~1368, 619~1368, 620~1368, 621~ 1368, 622~1368, 623~1368, 624~1368, 625~1368 , 626~1368, 627~1368, 628~1368, 629~1368, 630 ~1368, 631~1368, 632~1368, 633~1368, 634~136 8, 635~1368, 636~1368, 637~1368, 638~1368, 63 9~1368, 640~1368, 641~1368, 642~1368, 643~13 68, 644~1368, 645~1368, 646~1368, 647~1368, 6 48~1368, 649~1368, 650~1368, or 651~1368 In some embodiments, the split Cas9 protein may comprise any one portion of The C-terminal portion of the SpCas9 gene is located at amino acids 574 to 1368 or 638 to 1368. Includes parts.

[0228] "Subject" means a mammal, whether human or non-human mammal (e.g., bovine, equine, Subjects include, but are not limited to, livestock (dogs, cows, or cats). stock), livestock raised to provide labor and food and other commodities (d omesticated animals), including cows, goats, chickens, horses, and pigs Examples of animals that may be used include, but are not limited to, rabbits, and sheep.

[0229] "Substantially identical" means that the amino acid sequence of a reference amino acid sequence (e.g., an amino acid sequence set forth herein) is any one of the nucleic acid sequences described herein) or a reference nucleic acid sequence (e.g., any one of the nucleic acid sequences described herein) "SEQ ID NO: 1" refers to a polypeptide or nucleic acid molecule that exhibits at least 50% identity to one of the sequences. In one embodiment, such sequences are identical to the sequences used for comparison at the amino acid level or At the nucleic acid level, at least 60%, 80%, or 85%, 90%, 95% or even 9% 9% identical.

[0230] Sequence identity is typically determined using sequence analysis software (e.g., Genetics Co. mputer Group(University of Wisconsin Bio Technology Center, 1710 University Avenue , Madison, Wis. 53705) sequence analysis software package, BLAS T, BESTFIT, COBALT, EMBOSS Needle, GAP, or PI This is measured using the LEUP / PRETTYBOX program. The software assigns degrees of homology to various substitutions, deletions, and / or other modifications. Conservative substitutions are typically made by: Substitutions within the group include: glycine, alanine; valine, isoleucine, leucine; asparta...

Claims

**Claim 1** An ex vivo method for producing CAR-expressing immune cells or a population of CAR-expressing immune cells having reduced immunogenicity, comprising: a) contacting a target polynucleotide in immune cells or a population of immune cells with a base editor comprising a guide polynucleotide and a fusion protein comprising a nucleic acid programmable DNA-binding protein (napDNAbp) and a cytidine deaminase domain, or with a base editor system comprising a guide polynucleotide, a nucleic acid programmable DNA-binding protein (napDNAbp), and a cytidine deaminase domain; b) introducing, by nucleic acid base modification, a mutation that reduces or eliminates the expression of an antigen selected from the group consisting of CD3, CD5, CD7, CD33, and CD123; c) introducing, by nucleic acid base modification, a mutation that reduces or eliminates the expression of at least one polypeptide selected from the group consisting of CD3e, TRAC, LAG-3, FAS, CII TA, TRBC1, TRBC2, CD52, B2M, and PD1 in the immune cells; d) expressing a chimeric antigen receptor targeting a CD3, CD5, CD7, CD33, or CD123 antigen in the immune cells or population of immune cells, thereby producing a CAR-expressing immune cell or population of CAR-expressing immune cells having reduced immunogenicity. **Claim 2** The method according to claim 1, wherein the cytidine deaminase domain comprises an apolipoprotein B mRNA editing complex (APOBEC) selected from ppAPOBEC and rat APOBEC. **Claim 3** The method according to claim 1, wherein the cytidine deaminase domain comprises an amino acid sequence having at least 90% identity to SEQ ID NO: 1380 (ppAPOBEC-1). **Claim 4** The method according to claim 1, wherein the cytidine deaminase domain comprises an amino acid sequence having at least 90% identity to SEQ ID NO: 1378 (rAPOBEC-1). **Claim 5** The method according to claim 1, wherein the napDNAbp is spCas9. **Claim 6** The method according to claim 1, wherein the base editor is a BE4 base editor. **Claim 7** The method according to claim 1, wherein the base editor system further comprises two UGI domains. **Claim 8**: The method according to any one of claims 1 to 7, wherein the CAR comprises, in order from the N-terminus to the C-terminus, an extracellular antigen-binding domain targeting the CD5 or CD7 antigen, a CD8a hinge domain, a CD8 transmembrane domain, and a CD28z signaling domain. **Claim 9** The method according to any one of claims 1 to 8, wherein the CAR is a CD5 CAR. **Claim 10** The method according to claim 9, wherein the CD5 CAR comprises or consists of an amino acid sequence selected from the following: a) 1 MEFGLSWLFLVAILKGVQCIDAMGNIQLVQSGPELKKPGETVKISCKASGYTFTNYGMNW 60 61 VKQAPGKGLRWMGWINTHTGEPTYADDFKGRFAFSLETSASTAYLQINNLKNEDTATYFC 120 121 TRRGYDWYFDVWGAGTTVTVSSGGGGSGGGGSGGGGSDIKMTQSPSSMYASLGERVTITC 180 181 KASQDINSYLSWFHHKPGKSPKTLIYRANRLVDGVPSRFSGSGSGQDYSLTISSLDYEDM 240 241 GIYYCQQYDESPWTFGGGTKLEMKGSGDPAEPKSPDKTHTCPGQPREPQVYTLPPSRDEL 300 301 TKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQ 360 361 QGNVFSCSVMHEALHNHYTQKSLSLSPGKKDPKFWVLVVVGGVLACYSLLVTVAFIIFWV 420 421 RSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSRVKFSRSADAPAYQQGQNQ 480 481 LYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGE 540 541 RRRGKGHDGLYQGLSTATKDTYDALHMQALPPR 573; b) 1 MEFGLSWLFLVAILKGVQCIDAMGNIQLVQSGPELKKPGETVKISCKASGYTFTNYGMNW 60 61 VKQAPGKGLRWMGWINTHTGEPTYADDFKGRFAFSLETSASTAYLQINNLKNEDTATYFC 120 121 TRRGYDWYFDVWGAGTTVTVSSGGGGSGGGGSGGGGSDIKMTQSPSSMYASLGERVTITC 180 181 KASQDINSYLSWFHHKPGKSPKTLIYRANRLVDGVPSRFSGSGSGQDYSLTISSLDYEDM 240 241 GIYYCQQYDESPWTFGGGTKLEMKGSGDPATTTPAPRPPTPAPTIASQPLSLRPEACRPA 300 301 AGGAVHTRGLDFACDFWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPR 360 361 RPGPTRKHYQPYAPPRDFAAYRSRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDK 420 421 RRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTAT 480 481 KDTYDALHMQALPPR 495; c) 1 MEFGLSWLFLVAILKGVQCIDAMGNIQLVQSGPELKKPGETVKISCKASGYTFTNYGMNW 60 61 VKQAPGKGLRWMGWINTHTGEPTYADDFKGRFAFSLETSASTAYLQINNLKNEDTATYFC 120 121 TRRGYDWYFDVWGAGTTVTVSSGGGGSGGGGSGGGGSDIKMTQSPSSMYASLGERVTITC 180 181 KASQDINSYLSWFHHKPGKSPKTLIYRANRLVDGVPSRFSGSGSGQDYSLTISSLDYEDM 240 241 GIYYCQQYDESPWTFGGGTKLEMKGSGDPAIEVMYPPPYLDNEKSNGTIIHVKGKHLCPS 300 301 PLFPGPSKPFWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTR 360 361 KHYQPYAPPRDFAAYRSRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDP 420 421 EMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDA 480 481 LHMQALPPR 489; d) 1 MEFGLSWLFLVAILKGVQCIDAMGNIQLVQSGPELKKPGETVKISCKASGYTFTNYGMNW 60 61 VKQAPGKGLRWMGWINTHTGEPTYADDFKGRFAFSLETSASTAYLQINNLKNEDTATYFC 120 121 TRRGYDWYFDVWGAGTTVTVSSGGGGSGGGGSGGGGSDIKMTQSPSSMYASLGERVTITC 180 181 KASQDINSYLSWFHHKPGKSPKTLIYRANRLVDGVPSRFSGSGSGQDYSLTISSLDYEDM 240 241 GIYYCQQYDESPWTFGGGTKLEMKGSGDPAEPKSPDKTHTCPGQPREPQVYTLPPSRDEL 300 301 TKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQ 360 361 QGNVFSCSVMHEALHNHYTQKSLSLSPGKKDPKPTTTPAPRPPTPAPTIASQPLSLRPEA 420 421 CRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMR 480 481 PVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVL 540 541 DKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLST 600 601 ATKDTYDALHMQALPPR 617; or e) 1 MEFGLSWLFLVAILKGVQCIDAMGNIQLVQSGPELKKPGETVKISCKASGYTFTNYGMNW 60 61 VKQAPGKGLRWMGWINTHTGEPTYADDFKGRFAFSLETSASTAYLQINNLKNEDTATYFC 120 121 TRRGYDWYFDVWGAGTTVTVSSGGGGSGGGGSGGGGSDIKMTQSPSSMYASLGERVTITC 180 181 KASQDINSYLSWFHHKPGKSPKTLIYRANRLVDGVPSRFSGSGSGQDYSLTISSLDYEDM 240 241 GIYYCQQYDESPWTFGGGTKLEMKGSGDPATTTPAPRPPTPAPTIASQPLSLRPEACRPA 300 301 AGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQT 360 361 TQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRR 420 421 DPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKD 480 481 DALHMQALPPRX 494。

11. The method according to any one of claims 1 to 10, wherein the immune cells produced by the method exhibit fratricide resistance and / or increased anti-tumor activity as compared to corresponding control cells.

12. The method according to any one of claims 1 to 11, wherein the immune cells produced by the method contain less than 1% indels.

13. The method according to any one of claims 1 to 12, wherein the mutation reduces the expression of the encoded polypeptide by at least about 50% or more as compared to corresponding control cells lacking the mutation.

14. The method according to any one of claims 1 to 13, wherein the guide polynucleotide comprises a nucleic acid sequence selected from UUCGUAUCUGUAAAACCAAG (SEQ ID NO: 824), UUACCUGUACCAUAACCAGG (SEQ ID NO: 1156), CUCUUACCUGUACCAUAACC (SEQ ID NO: 1155), UGCACCUCUGGGGAGGACCU (SEQ ID NO: 1123), CCUACCUGUCACCAGGACCA (SEQ ID NO: 1128), and CACCUACCUAAGAACCAUCC (SEQ ID NO: 897).

15. The guide polynucleotide is ususcsGUAUCUGUAAAACCAAGGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUsususu (SEQ ID NO: 1452); ususasCCUGUACCAUAACCAGGGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUsususu (SEQ ID NO: 1453); csuscsUUACCUGUACCAUAACCGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUsususu (S SEQ ID NO: 1454); usgscsACCUCUGGGGAGGACCUGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUsususu (S SEQ ID NO: 1455); cscsusACCUGUCACCAGGACCAGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUsususu (SEQ ID NO: 1456); and csascsCUACCUAAGAACCAUCCGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUsususu (SEQ ID NO: 1457) The method according to any one of claims 1 to 14, comprising a nucleic acid sequence selected from the group consisting of, wherein a, c, g, or u represents a 2'-O-methyl analog, and s represents a 3'-phosphorothioate nucleotide internucleoside linkage.

16. The method according to any one of claims 1 to 15, wherein each of the one or more guide nucleic acid sequences targets the napDNAbp to the CD3e, CD5, FAS, LAG-3, CD52, TRAC, B2M, CII TA, TRBC1, TRBC2, and / or PDC1 / PD-1 gene or regulatory element.

17. The method according to any one of claims 1 to 16, wherein the base editor and the one or more guide nucleic acid sequences are introduced into the immune cells via electroporation, nucleofection, cationic lipid-mediated methods, viral transduction, or combinations thereof.

18. The method according to any one of claims 1 to 17, further comprising depleting TCRα / β+ cells from the population of the modified immune cells.

19. The method according to any one of claims 1 to 18, wherein the CD7 CAR comprises or consists of the following amino acid sequence: 7CAR8 1 MALPVTALLLPLALLLHAARPGSDIELTQSPAIMSASLGEEITLTCSASSSVSYMHWYQQ 60 61 KSGTSPKLLIYSTSNLASGVPSRFSGSGSGTFYSLTISSVEAEDAADYYCHQWSSYTFGG 120 121 GTKLEIKRGGGGSGGGGSGGGGSQVKLQESGGGLVKPGGSLKLSCAASGFTFSSYAMSWV 180 181 RQTPEKRLEWVATISSGGSYTYYPDSVKGRFTISRDNAKNTLYLQMSSLRSEDTAMYYCA 240 241 RQDGYYPGWFANWGQGTTVTVSSAAATTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGA 300 301 VHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCRSKRSRLLHSDYMNMTPRRPGPTRK 360 361 HYQPYAPPRDFAAYRSRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPE 420 421 MGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDAL 480 481 HMQALPPR 488.

20. CAR-expressing immune cells having reduced immunogenicity, produced by the method according to any one of claims 1 to 19.

21. A pharmaceutical composition comprising the immune cells according to claim 20 and a pharmaceutically acceptable excipient.