Modified immune cells having enhanced Anti-neoplasia activity and immunosuppression resistance

Abstract

To provide: genetically modified immune cells having enhanced anti-neoplasia activity, enhanced resistance to immune suppression, and decreased risk of eliciting a graft versus host reaction, or a combination thereof; and methods for producing these modified immune cells.SOLUTION: A method for producing modified immune cells with reduced immunogenicity and / or increased anti-neoplasia activity by multiplexed editing comprises: modifying at least four gene sequences or regulatory elements thereof at a single target nucleobase in each thereof in immune cells, thereby generating the modified immune cells with reduced immunogenicity and / or increased anti-neoplasia activity.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] Incorporation by Reference This application is a continuation of U.S. Provisional Patent Application No. 62 / 793,277, filed January 16, 2019, and U.S. Provisional Patent Application No. 62 / 793,277, filed April 16, 2019. This application claims the benefit of U.S. Provisional Patent Application No. 62 / 839,870, filed on January 29, 2014. [Background technology]

[0002] Autologous and allogeneic immunotherapies involve the administration of immune cells expressing chimeric antigen receptors to subjects. A neoplasm treatment approach: generating immune cells expressing chimeric antigen receptors (CARs) To do this, immune cells are first isolated from the subject (autologous) or from a donor different from the subject receiving treatment (allogeneic). ) and genetically modified to express a chimeric antigen receptor. The resulting cells are expressing a chimeric antigen receptor on its cell surface (e.g., CAR T cells) and administering them to a subject. Thus, the chimeric antigen receptor binds to a marker expressed by the neoplastic cell. Interaction with the CAR activates the CAR-T cells, which then kill the neoplastic cells. However, for autologous or allogeneic cell therapy to be effective and efficient, T cell signaling must be regulated. Significant conditions and cellular responses, such as inhibition of cellular signaling, must be overcome or avoided. For allogeneic cell therapy, graft-versus-host disease and host rejection of CAR-T cells pose additional challenges. Editing genes involved in these processes could improve CAR-T cell function and immune suppression. or enhance resistance to inhibition, but current methods for performing such editing are This methodology induces large genomic rearrangements in CAR-T cells, thereby impacting their efficacy. Therefore, it is necessary to make immune cells, especially CAR-T cells, more positive. There is a significant need in the art for accurately modifying the phenotype of phenotype. This application addresses this and other important needs. It is directed towards the needs. Summary of the Invention

[0003] As described below, the present invention provides anti-neoplastic activity, enhanced resistance to immunosuppression, and and graft-versus-host reaction or host CD8 + T cells recognize the graft as non-self (e.g., Inducing a host-versus-graft reaction (in which the transplant recipient mounts an immune response against the transplanted organ) The present invention is characterized by genetically modified immune cells having a reduced risk of developing a disease or a combination thereof. In an embodiment, a subject having or prone to developing graft-versus-host disease (GVHD) is Elephants are administered CAR-T cells that lack or have reduced levels of functional TRAC. In one embodiment, the subject has or is prone to develop host-versus-graft disease (HVGD). The subject lacks or has reduced levels of functional beta-2 microglobulin (B2M). The present invention also provides a method for producing and administering these modified immune cells to a patient. and methods for using the same.

[0004] In one embodiment, modified antibodies have reduced immunogenicity and / or increased antineoplastic activity. A method for producing modified immune cells by multiplex editing, comprising: At least four gene sequences or their regulatory elements are identified using a single target nucleic acid sequence in each of the sequences. modified with a group, thereby reducing immunogenicity and / or increasing antineoplastic activity Provided herein are methods comprising generating modified immune cells.

[0005] In another embodiment, modified antibodies have reduced immunogenicity and / or increased antineoplastic activity. A method for producing a population of modified immune cells by multiplex editing, comprising: At least four gene sequences or their regulatory elements in the population are identified as single and modifying the target nucleobase of the target nucleic acid, thereby providing reduced immunogenicity and / or anti-neoplastic activity. Provided herein are methods for generating a population of modified immune cells with increased activity. can be.

[0006] In some embodiments, at least one of the at least four gene sequences is a checker. a cross-linking factor gene sequence, an immune response modulating gene sequence, or an immunogenic gene sequence be.

[0007] In some embodiments, modifying comprises modifying at least four of the gene sequences. Both reduce the expression of one.

[0008] In some embodiments, the expression of at least one of the at least four genes is altered. The expression level is reduced by at least 80% compared to control cells that do not have the gene.

[0009] In some embodiments, the expression of each of the at least four genes is unaltered. The expression level is reduced by at least 80% compared to control cells.

[0010] In some embodiments, the expression of at least one of the at least four genes is is reduced in at least 50% of the population of cells.

[0011] In some embodiments, the expression of each of the at least four genes is associated with immune cell recruitment. Reduced by at least 50% of the group.

[0012] In some embodiments, the at least four gene sequences include a TRAC gene sequence. .

[0013] In some embodiments, the at least four gene sequences are Contains factor gene sequences.

[0014] In some embodiments, the at least four gene sequences include a PDCD1 gene sequence. .

[0015] In some embodiments, the at least four gene sequences are T cell marker gene sequences. Contains columns.

[0016] In some embodiments, the at least four gene sequences include a CD52 gene sequence. .

[0017] In some embodiments, the at least four gene sequences include a CD7 gene sequence.

[0018] In some embodiments, the at least four gene sequences include a TRAC gene sequence, a PDCD1 gene sequence, CD52 gene sequence, or CD7 gene sequence.

[0019] In some embodiments, the at least four sequences are a TCR complex gene sequence, a CD7 gene sequence, gene sequence, CD52 gene sequence, and CIITA, CD2 gene sequence, CD4 gene sequence, CD5 gene sequence sequence, CD7 gene sequence, CD30 gene sequence, CD33 gene sequence, CD52 gene sequence, CD70 gene a gene sequence selected from the group consisting of a B2M gene sequence, a CIITA gene sequence, and a B2M gene sequence. nothing.

[0020] In some embodiments, the at least four gene sequences include a CD2 gene sequence, a TRAC gene sequence, and a nucleotide sequence. Gene sequence, CD3 epsilon gene sequence, CD3 gamma gene sequence, CD3 delta gene sequence, TRB C1 gene sequence, TRBC2 gene sequence, CD4 gene sequence, CD5 gene sequence, CD7 gene sequence, CD30 gene sequence, CD33 gene sequence, CD52 gene sequence, CD70 gene sequence, B2M gene sequence, and and CIITA gene sequences.

[0021] The methods of some embodiments described herein involve detecting five or more gene sequences in immune cells. or the regulatory elements at a single target nucleobase in each thereof.

[0022] The methods of some embodiments described herein involve detecting up to six gene sequences in immune cells. or the regulatory elements at a single target nucleobase in each thereof.

[0023] The methods of some embodiments described herein involve detecting up to seven gene sequences in immune cells. or the regulatory elements at a single target nucleobase in each thereof.

[0024] The methods of some embodiments described herein involve detecting up to eight gene sequences in immune cells. or the regulatory elements at a single target nucleobase in each thereof.

[0025] The methods of some embodiments described herein involve detecting five genes in a population of immune cells. modifying a sequence or its regulatory elements at a single target nucleic acid base in each thereof include.

[0026] The methods of some embodiments described herein involve detecting six genes in a population of immune cells. modifying a sequence or its regulatory elements at a single target nucleic acid base in each thereof include.

[0027] The methods of some embodiments described herein involve detecting seven genes in a population of immune cells. modifying a sequence or its regulatory elements at a single target nucleic acid base in each thereof include.

[0028] The methods of some embodiments described herein involve detecting eight genes in a population of immune cells. modifying a sequence or its regulatory elements at a single target nucleic acid base in each thereof include.

[0029] In some embodiments, five, six, seven, or eight gene sequences or sequences thereof The nodal elements are the CD2 gene sequence, the TRAC gene sequence, the CD3 epsilon gene sequence, the CD3 cancer sequence, CD3 delta gene sequence, TRBC1 gene sequence, TRBC2 gene sequence, CD4 gene sequence sequence, CD5 gene sequence, CD7 gene sequence, CD30 gene sequence, CD33 gene sequence, CD52 gene sequence , CD70 gene sequence, B2M gene sequence, and CIITA gene sequence.

[0030] In some embodiments, five, six, seven, or eight gene sequences or sequences thereof The nodal elements are the CD3 gene sequence, the CD7 gene sequence, the CD2 gene sequence, the CD5 gene sequence, and and CD52 gene sequences.

[0031] In some embodiments, the modifying deaminates a single target nucleobase. This includes:

[0032] In some embodiments, the deaminating step comprises cleaving a polypeptide comprising a deaminase. This is carried out by the government.

[0033] In some embodiments, the deaminase is a nucleic acid programmable DNA binding protein. It associates with the substrate (napDNAbp) to form a base editor.

[0034] In some embodiments, the deaminase is a nucleic acid programmable DNA binding protein. It is fused to the protein (napDNAbp).

[0035] In some embodiments, the napDNAbp comprises a Cas9 polypeptide or a portion thereof. .

[0036] In some embodiments, the napDNAbp is a Cas9 nickase or nuclease-inactive Ca Includes s9.

[0037] In some embodiments, the deaminase is a cytidine deaminase.

[0038] In some embodiments, the single target nucleobase is cytosine (C), wherein: The modification includes converting C to thymine (T).

[0039] In some embodiments, the base editor comprises a uracil glycosylase inhibitor. Further includes:

[0040] In some embodiments, the deaminase is adenosine deaminase.

[0041] In some embodiments, the single target nucleobase is adenosine (A), wherein , the modification involves converting A to guanine (G).

[0042] In some embodiments, modifying comprises contacting an immune cell with a guide nucleic acid sequence. This includes:

[0043] In some embodiments, the modifying comprises modifying the immune cell to at least four guide nuclei. and contacting the napDNAbp with a guide nucleic acid sequence, wherein each guide nucleic acid sequence binds the napDNAbp to at least four genes. sequence or one of its regulatory elements.

[0044] In some embodiments, the guide nucleic acid sequence is a guide RNase A sequence of Table 8A, Table 8B, or Table 8C. A sequence selected from the group consisting of:

[0045] In some embodiments, the guide nucleic acid sequence is UUCGUAUCUGUAAAACCAAG, CCUACCUGUC ACCAGGACCA, CUCUUACCUGUACCAUAACC, CACCUACCUAAGAACCAUCC, ACUCACGCUGGAUAGCCUCC, AC UCACCCAGCAUCCCCAGC, CACUCACCUUAGCCUGAGCA, and CACGCACCUGGACAGCUGAC The sequence includes a sequence selected from the group consisting of:

[0046] In some embodiments, modifying comprises a reverse transcriptase and an extended guide nucleic acid. Substitution of a single target nucleobase with a different nucleobase by target-primed reverse transcription using a base sequence. This includes replacing.

[0047] In some embodiments, the extended guide nucleic acid sequence is a reverse transcription template sequence. , a reverse transcription primer binding site, or a combination thereof.

[0048] In some embodiments, the single target nucleobase is in an exon.

[0049] In some embodiments, the modifying generates a premature stop codon in the exon. do.

[0050] In some embodiments, the single target nucleobase is in exon 1 of the TRAC gene sequence, Located in exon 2 or exon 3.

[0051] In some embodiments, the single target nucleobase is in exon 1 of the PCDC1 gene sequence, Located in exon 2 or exon 5.

[0052] In some embodiments, the single target nucleobase is located in exon 1 or exon 2 of the CD52 gene sequence. or in exon 2.

[0053] In some embodiments, the single target nucleobase is located in exon 1, exon 2, exon 3, exon 4, exon 5, exon 6, exon 7, exon 8, exon 9, exon 10, exon 11, exon 12, exon 13, exon 14, exon 15, exon 16, exon 17, exon 18, exon 19, exon 20, ex It is located in exon 2 or exon 3.

[0054] In some embodiments, the single target nucleobase is located in exon 1 or exon 2 of the B2M gene sequence. is in exon 2.

[0055] In some embodiments, the single target nucleobase is located in exon 2, exon 3, of the CD5 gene sequence. within exon 3, exon 4, exon 5, exon 6, exon 7, or exon 8.

[0056] In some embodiments, the single target nucleobase is located in exon 2, exon 3, of the CD2 gene sequence. within exon 3, exon 4, or exon 5.

[0057] In some embodiments, the single target nucleobase is in exon 1 of the CIITA gene sequence, Exon 2, exon 4, exon 7, exon 8, exon 9, exon 10, exon 11, Within exon 12, exon 14, exon 15, exon 18, or exon 19.

[0058] In some embodiments, the single target nucleobase is a splice donor site or splice donor site. Located in the price acceptor site.

[0059] In some embodiments, the single target nucleobase is located in exon 1 sequence of the TRAC gene sequence. Splice acceptor site, exon 1 splice donor site, or exon 3 splice It is located in the acceptor site.

[0060] In some embodiments, the single target nucleobase is located in exon 1 sequence of the PDCD1 gene sequence. Splice acceptor site, exon 1 splice donor site, exon 2 splice acceptor site Sceptor site, exon 3 splice donor site, exon 4 splice acceptor site , at the exon 4 splice donor site, or at the exon 5 splice acceptor site .

[0061] In some embodiments, the single target nucleobase is located in exon 1 sequence of the CD52 gene sequence. Located in the splice donor site or exon 2 splice acceptor site.

[0062] In some embodiments, the single target nucleobase is located in exon 1 splice of the CD7 gene sequence. Rice donor site, exon 2 splice donor site, exon 2 splice acceptor site, or at the exon 3 splice acceptor site.

[0063] In some embodiments, the single target nucleobase is located in exon 1 splice of the B2M gene sequence. Rice donor site, exon 2 splice donor site, exon 2 splice acceptor site, or at the exon 3 splice acceptor site.

[0064] In some embodiments, the single target nucleobase is located in exon 3 splice of the CD2 gene sequence. It is located in the rice donor area.

[0065] In some embodiments, the single target nucleobase is located in exon 1 splice of the CD5 gene sequence. Rice donor site, exon 1 splice acceptor site, exon 3 splice acceptor site exon 3 splice donor site, exon 4 splice acceptor site, Exon 5 splice donor site, exon 6 splice acceptor site, exon 9 splice splice donor site, in the exon 10 splice acceptor site.

[0066] In some embodiments, the single target nucleobase is an exon of the CIITACIITA gene sequence. Exon 1 splice donor site, exon 7 splice donor site, exon 8 splice accession site Sceptor site, exon 9 splice donor site, exon 10 splice acceptor site position, exon 11 splice acceptor site, exon 14 splice acceptor site, Exon 14 splice donor site, exon 15 splice donor site, exon 16 splice donor site splice acceptor site, exon 16 splice donor site, exon 17 splice accession site splice donor site, exon 17 splice donor site, or exon 19 splice acceptor site - Located in the area.

[0067] In some embodiments, the immune cells are human cells. Immune cells include cytotoxic T cells, regulatory T cells, helper T cells, dendritic cells, B cells, and or NK cells.

[0068] In some embodiments, the population of immune cells is human cells.

[0069] In some embodiments, the population of immune cells includes cytotoxic T cells, regulatory T cells, hemoglobin-binding proteins, and the like. These cells are T cells, dendritic cells, B cells, or NK cells.

[0070] In some embodiments, the modifying is ex vivo.

[0071] In some embodiments, the immune cells or population of immune cells are derived from a single human donor. It originates from.

[0072] In some embodiments, the method comprises: and a polynucleotide encoding a target chimeric antigen receptor (CAR) or a functional fragment thereof. It further includes causing

[0073] In some embodiments, an immune cell or population of immune cells is treated with a polypeptide encoding a CAR. contacting the lentivirus containing the oligonucleotide;

[0074] In some embodiments, the immune cell or population of immune cells is treated with napDNAbp, and CA contacting a donor DNA sequence comprising a polynucleotide encoding R;

[0075] In some embodiments, the napDNAbp is Cas12b.

[0076] In some embodiments, the CAR specifically binds to a marker associated with a neoplasm .

[0077] In some embodiments, the neoplasm is a T-cell cancer, a B-cell cancer, a lymphoma, a leukemia, Or multiple myeloma.

[0078] In some embodiments, the CAR specifically binds to CD7.

[0079] In some embodiments, the CAR specifically binds to BCMA.

[0080] In some embodiments, the immune cell or population of immune cells contains a detectable translocation. In some embodiments, at least 50% of the population of immune cells expresses a CAR. In some embodiments, at least 50% of the population of immune cells is viable. In some embodiments, at least 50% of the population of immune cells are of the same type without the modification. expand at a rate that is at least 80% of the expansion rate of a population of control cells.

[0081] In some embodiments of the methods described herein, the modifying comprises modifying immune cells. In some embodiments, the modifying generates less than 1% indels in In some embodiments, the modified gene produces less than 5% non-targeted editing in immune cells. This produces less than 5% off-target editing in immune cells.

[0082] In one aspect, the present invention provides a method for producing a medicament for the treatment of ... Modified immune cells are provided herein.

[0083] In one aspect, the present invention provides a method for producing a medicament for the treatment of ... Provided herein are populations of modified immune cells.

[0084] In another embodiment, each of at least four gene sequences or their regulatory elements is independently Reduced immunogenicity or increased antineoplastic activity, including modification of one targeted nucleobase, Modified immune cells are provided herein. In some embodiments, the modified In the isolated immune cells, each of the at least four gene sequences was found to inhibit checkpoint inhibition. factor gene sequence, immune response modulating gene sequence, or immunogenic gene sequence.

[0085] In the modified immune cell of the previous embodiment, the at least four gene sequences are TCR complexes. Contains gene sequences.

[0086] In some embodiments, the at least four gene sequences include the TRAC gene. In some embodiments, the at least four gene sequences are checkpoint inhibitors. In some embodiments, the at least four gene sequences include PDCD1 Contains gene sequences.

[0087] In some embodiments, the at least four gene sequences are T cell marker gene sequences. Contains columns.

[0088] In some embodiments, the at least four gene sequences include a CD52 gene sequence. .

[0089] In some embodiments, the at least four gene sequences include a CD7 gene sequence.

[0090] In some embodiments, the expression of one of the at least four genes is unaltered. At least 80% reduction compared to control cells.

[0091] In some embodiments, the expression of each of the at least four genes is unaltered. The expression level is reduced by at least 90% compared to control cells.

[0092] In some embodiments, the immune cells express five gene sequences or regulatory elements thereof. Each of the five gene sequences contains an alteration at a single target nucleic acid base in each of the five gene sequences. a cross-linking factor gene sequence, an immune response modulating gene sequence, or an immunogenic gene sequence be.

[0093] In some embodiments, the immune cells express six gene sequences or their regulatory elements. Each of the six gene sequences contains an alteration at a single target nucleic acid base in each of the six gene sequences. a cross-linking factor gene sequence, an immune response modulating gene sequence, or an immunogenic gene sequence be.

[0094] In some embodiments, the immune cells express seven gene sequences or regulatory elements thereof. Each of the seven gene sequences contains an alteration at a single target nucleic acid base in each of the seven gene sequences. a cross-linking factor gene sequence, an immune response modulating gene sequence, or an immunogenic gene sequence be.

[0095] In some embodiments, the immune cells express eight gene sequences or regulatory elements thereof. Each of the eight gene sequences contains an alteration at a single target nucleic acid base in each of the eight gene sequences. a cross-linking factor gene sequence, an immune response modulating gene sequence, or an immunogenic gene sequence be.

[0096] In some embodiments, at least one of the five, six, seven, or eight genes Expression is reduced by at least 90% compared to control cells without the modification.

[0097] In some embodiments, the expression of each of five, six, seven, or eight genes is altered. The mutation is reduced by at least 90% compared to control cells without the mutation.

[0098] In some embodiments, five, six, seven, or eight gene sequences or their regulation The elements are the CD2 gene sequence, the TRAC gene sequence, the CD3 epsilon gene sequence, and the CD3 gamma gene sequence. gene sequence, CD3 delta gene sequence, TRBC1 gene sequence, TRBC2 gene sequence, CD4 gene sequence , CD5 gene sequence, CD7 gene sequence, CD30 gene sequence, CD33 gene sequence, CD52 gene sequence, A sequence selected from the group consisting of a CD70 gene sequence, a B2M gene sequence, and a CIITA gene sequence. Includes.

[0099] In one embodiment, a CD3 gene sequence, a CD5 gene sequence, a CD52 gene sequence, and a CD7 gene sequence and modified immune cells containing a single target nucleic acid base modification in each of the child sequences, exhibiting reduced immunogenicity or increased antineoplastic activity compared to control cells of the same type that do not express the antibody; Modified immune cells are provided herein.

[0100] In some embodiments, the modified immune cells contain a CD2 gene sequence, CIITA, or Each of the regulatory elements further comprises a single targeted nucleobase modification.

[0101] In some embodiments, the modified immune cells contain a TRAC gene sequence, CD3 epsilon, the CD3 gamma gene sequence, the CD3 delta gene sequence, the TRBC1 gene sequence, or the T A single targeted nucleic acid base alteration in the RBC2 gene sequence, a CD4 gene sequence, a CD30 gene sequence The sequence consists of the CD33 gene sequence, the CD70 gene sequence, the B2M gene sequence, and the CIITA gene sequence. a single target nucleic acid base in a gene sequence selected from the group consisting of: Further modifications are included.

[0102] In some embodiments, the modified immune cells contain a TRAC gene sequence, a PDCD1 gene sequence, or sequence, CD52 gene sequence, CD7 gene sequence, CD2 gene sequence, CD5 gene sequence, CIITA gene sequence sequence, and the B2M gene sequence each contain a single targeted nucleobase modification.

[0103] In some embodiments, the modified immune cells do not contain detectable translocations.

[0104] In some embodiments, the modified immune cells contain less than 1% indels.

[0105] In some embodiments, the engineered immune cells contain less than 5% non-targeted editing.

[0106] In some embodiments, the engineered immune cells contain less than 5% off-target editing. nothing.

[0107] In some embodiments, the modified immune cells exhibit growth or In some embodiments, the reference cells have increased viability. These are immune cells that have been modified using

[0108] In some embodiments, the modified immune cells are mammalian cells.

[0109] In some embodiments, the modified immune cells are human cells.

[0110] In some embodiments, the engineered immune cells are cytotoxic T cells, regulatory T cells, , helper T cells, dendritic cells, B cells, or NK cells.

[0111] In some embodiments, the modified immune cells are in ex vivo culture.

[0112] In some embodiments, the modified immune cells are derived from a single human donor.

[0113] In some embodiments, the engineered immune cells contain an exogenous functional chimeric antigen receptor. (CAR) or a functional fragment thereof.

[0114] In some embodiments, the polynucleotide encoding the CAR is It is built into the system.

[0115] In some embodiments, the CAR specifically binds to a marker associated with a neoplasm .

[0116] In some embodiments, the neoplasm is a T-cell cancer, a B-cell cancer, a lymphoma, a leukemia, Or multiple myeloma.

[0117] In some embodiments, the CAR specifically binds to CD7.

[0118] In some embodiments, the CAR specifically binds to BCMA.

[0119] In some embodiments, the single target nucleobase is in an exon.

[0120] In some embodiments, the single target nucleobase is in exon 1 of the TRAC gene sequence, Located in exon 2 or exon 3.

[0121] In some embodiments, the single target nucleobase is in exon 1 of the PCDC1 gene sequence, Located in exon 2 or exon 5.

[0122] In some embodiments, the single target nucleobase is located in exon 1 or exon 2 of the CD52 gene sequence. or in exon 2.

[0123] In some embodiments, the single target nucleobase is located in exon 1, exon 2, exon 3, exon 4, exon 5, exon 6, exon 7, exon 8, exon 9, exon 10, exon 11, exon 12, exon 13, exon 14, exon 15, exon 16, exon 17, exon 18, exon 19, exon 20, ex It is located in exon 2 or exon 3.

[0124] In some embodiments, the single target nucleobase is a splice donor site or splice donor site. Located in the price acceptor site.

[0125] In some embodiments, the single target nucleobase is located in exon 1 sequence of the TRAC gene sequence. Splice acceptor site, exon 1 splice donor site, or exon 3 splice It is located in the acceptor site.

[0126] In some embodiments, the single target nucleobase is located in exon 1 sequence of the PDCD1 gene sequence. Splice acceptor site, exon 1 splice donor site, exon 2 splice acceptor site Sceptor site, exon 3 splice donor site, exon 4 splice acceptor site , at the exon 4 splice donor site, or at the exon 5 splice acceptor site .

[0127] In some embodiments, the single target nucleobase is located in exon 1 sequence of the CD52 gene sequence. Located in the splice donor site or exon 2 splice acceptor site.

[0128] In some embodiments, the single target nucleobase is located in exon 1 splice of the CD7 gene sequence. Rice donor site, exon 2 splice donor site, exon 2 splice acceptor site, or at the exon 3 splice acceptor site.

[0129] In one embodiment, the population of modified immune cells comprises a plurality of cells. A single target nucleic acid base modification is introduced into each of at least four gene sequences or their regulatory elements. and comparing a population of cells having the alteration with a population of control cells of the same type that do not have the alteration. The present invention provides a population of modified immune cells that exhibit reduced immunogenicity or increased antineoplastic activity in response to a given antibody. The details are provided in the specification.

[0130] In some embodiments, the plurality of cells comprises at least 50% of the population.

[0131] In some embodiments, each of the at least four gene sequences is a checkpoint The gene sequence may be an immune inhibitor gene sequence, an immune response modulating gene sequence, or an immunogenic gene sequence.

[0132] In some embodiments, the at least four gene sequences include TCR component gene sequences, It contains a checkpoint inhibitor gene sequence or a T cell marker gene sequence.

[0133] In some embodiments, the at least four gene sequences include a TRAC gene sequence. .

[0134] In some embodiments, the at least four gene sequences include a PDCD1 gene sequence. .

[0135] In some embodiments, the at least four gene sequences include a CD52 gene sequence. .

[0136] In some embodiments, the at least four gene sequences include a CD7 gene sequence.

[0137] In some embodiments, the expression of at least one of the at least four genes is , at least 80% in a plurality of cells having the modification compared to control cells not having the modification is reduced.

[0138] In some embodiments, the expression of each of the at least four genes is altered. is reduced by at least 80% in a plurality of cells having the modification compared to control cells not having the modification. do.

[0139] In some embodiments, the plurality of populations comprises five gene sequences or regulatory elements thereof. Each of the five gene sequences contains an alteration at a single target nucleic acid base in each of the five genes. checkpoint inhibitor gene sequence, immune response modulating gene sequence, or immunogenic gene sequence is.

[0140] In some embodiments, the plurality of populations comprises six gene sequences or regulatory elements thereof. Each of the six sequences contains an alteration at a single target nucleobase in each of the checkpoints. an immune inhibitor gene sequence, an immune response modulating gene sequence, or an immunogenic gene sequence. .

[0141] In some embodiments, the plurality of populations comprises seven gene sequences or regulatory elements thereof. Each of the seven gene sequences contains an alteration at a single target nucleic acid base in each of the seven genes. checkpoint inhibitor gene sequence, immune response modulating gene sequence, or immunogenic gene sequence is.

[0142] In some embodiments, the plurality of populations comprises eight gene sequences or regulatory elements thereof. Each of the eight gene sequences contains an alteration at a single target nucleic acid base in each of the eight gene sequences. checkpoint inhibitor gene sequence, immune response modulating gene sequence, or immunogenic gene sequence is.

[0143] In some embodiments, at least five, six, seven, or eight genes are present. One expression is less pronounced in cells with the modification compared to control cells without the modification. At least 90% reduction.

[0144] In some embodiments, the expression of each of the five, six, seven, or eight genes is is at least 90% in the plurality of cells having the modification compared to the control cells not having the modification. % reduction.

[0145] In some embodiments, at least five, six, seven, or eight genes are present. One expression is less pronounced in cells with the modification compared to control cells without the modification. At least 90% reduction.

[0146] In some embodiments, the expression of each of five, six, seven, or eight genes is altered. At least a 90% reduction in cells having the alteration compared to control cells not having the alteration. will be done.

[0147] In some embodiments, five, six, seven, or eight gene sequences or sequences thereof The nodal elements are the CD2 gene sequence, the TRAC gene sequence, the CD3 epsilon gene sequence, the CD3 cancer sequence, CD3 delta gene sequence, TRBC1 gene sequence, TRBC2 gene sequence, CD4 gene sequence sequence, CD5 gene sequence, CD7 gene sequence, CD30 gene sequence, CD33 gene sequence, CD52 gene sequence , CD70 gene sequence, B2M gene sequence, and CIITA gene sequence.

[0148] In one embodiment, the population of modified immune cells, wherein a plurality of the populations are mutated using the TRAC gene A single target nucleic acid for each of the sequences, the PDCD1 gene sequence, the CD52 gene sequence, and the CD7 gene sequence. The cells contain a base alteration, and multiple populations are immunoreactive compared to multiple control cells of the same type that do not have the alteration. Modified immune cell populations that exhibit reduced immunogenicity or increased anti-neoplastic activity are described herein. Provided.

[0149] In some embodiments, the plurality of populations comprises CD2 gene sequences, CD5 gene sequences, CIITA a single target nucleic acid base in the gene sequence, the B2M gene sequence, or their respective regulatory elements; In some embodiments, the plurality of populations further comprises a modification of the CD2 gene sequence, TRAC gene sequence, CD3 epsilon gene sequence, CD3 gamma gene sequence, CD3 delta gene sequence, T RBC1 gene sequence, TRBC2 gene sequence, CD4 gene sequence, CD5 gene sequence, CD7 gene sequence, CD 30 gene sequence, CD33 gene sequence, CD52 gene sequence, CD70 gene sequence, B2M gene sequence, and and CIITA gene sequences or the respective sequences of the genes selected from the group consisting of In some embodiments, the node element further comprises a single target nucleobase modification. Multiple populations have been identified using TRAC gene sequences, PDCD1 gene sequences, CD52 gene sequences, CD7 gene sequences, and CD 2 gene sequence, CD5 gene sequence, CIITA gene sequence, and B2M gene sequence each had a single nucleus. Includes acid-base modifications.

[0150] In some embodiments, the population of modified immune cells includes multiple populations with detectable translocation. Does not include seat.

[0151] In some embodiments of the modified population of immune cells, at least one of the population of immune cells In some embodiments, the population of modified immune cells is At least 60% of the population of immune cells exhibits an expansion rate faster than a population of control cells of the same type that do not have the modification. In some embodiments, the population of modified immune cells is expanded by at least 80%. The population of immune cells is human. In some embodiments, the population of modified immune cells is human. In this study, immune cell populations were classified into cytotoxic T cells, regulatory T cells, helper T cells, dendritic cells, B In some embodiments, the population of engineered immune cells is a human NK cell, or a human NK cell. The population of immune cells is derived from a single human donor. In the population of cells, multiple cells bearing the modification are identified as expressing an exogenous functional chimeric antigen receptor (CAR). ) or a functional fragment thereof.

[0152] In some embodiments, at least 50% of the population of immune cells express the CAR.

[0153] In some embodiments, the CAR specifically binds to a marker associated with a neoplasm .

[0154] In some embodiments, the neoplasm is a T-cell cancer, a B-cell cancer, a lymphoma, a leukemia, Or multiple myeloma.

[0155] In some embodiments, the CAR specifically binds to CD7.

[0156] In some embodiments, the CAR specifically binds to BCMA.

[0157] In some embodiments, the single target nucleobase is in an exon.

[0158] In some embodiments, the single target nucleobase is in exon 1 of the TRAC gene sequence, Located in exon 2 or exon 3.

[0159] In some embodiments, the single target nucleobase is in exon 1 of the PCDC1 gene sequence, Located in exon 2 or exon 5.

[0160] In some embodiments, the single target nucleobase is located in exon 1 or exon 2 of the CD52 gene sequence. or in exon 2.

[0161] In some embodiments, the single target nucleobase is located in exon 1, exon 2, exon 3, exon 4, exon 5, exon 6, exon 7, exon 8, exon 9, exon 10, exon 11, exon 12, exon 13, exon 14, exon 15, exon 16, exon 17, exon 18, exon 19, exon 20, ex It is located in exon 2 or exon 3.

[0162] In some embodiments of the population of engineered immune cells, the single target nucleobase is Located at the splice donor site or splice acceptor site.

[0163] In some embodiments, the single target nucleobase is located in exon 1 sequence of the TRAC gene sequence. Splice acceptor site, exon 1 splice donor site, or exon 3 splice It is located in the acceptor site.

[0164] In some embodiments, the single target nucleobase is located in exon 1 sequence of the PDCD1 gene sequence. Splice acceptor site, exon 1 splice donor site, exon 2 splice acceptor site Sceptor site, exon 3 splice donor site, exon 4 splice acceptor site , at the exon 4 splice donor site, or at the exon 5 splice acceptor site .

[0165] In some embodiments, the single target nucleobase is located in exon 1 sequence of the CD52 gene sequence. Located in the splice donor site or exon 2 splice acceptor site.

[0166] In some embodiments, the single target nucleobase is located in exon 1 splice of the CD7 gene sequence. Rice donor site, exon 2 splice donor site, exon 2 splice acceptor site, or at the exon 3 splice acceptor site.

[0167] In one embodiment, a composition comprising a deaminase and a nucleic acid sequence, The array is UUCGUAUCUGUAAAACCAAG, CCUACCUGUCACCAGGACCA, CUCUUACCUGUACCAUAACC, CACCUA CCUAAGAACCAUCC, ACUCACGCUGGAUAGCCUCC, ACUCACCCAGCAUCCCCAGC, CACUCACCUUAGCCUGAGCA and CACGCACCUGGACAGCUGAC. will be provided to.

[0168] In some embodiments, the deaminase is a nucleic acid programmable DNA binding protein. It associates with the substrate (napDNAbp) to form a base editor.

[0169] In some embodiments, the napDNAbp is a Cas9 nickase or nuclease-inactive Ca s9, wherein the deaminase is a cytidine deaminase.

[0170] In some embodiments, the base editor comprises a uracil glycosylase inhibitor. Further includes:

[0171] In some embodiments, the napDNAbp is a Cas9 nickase or nuclease-inactive Ca s9, wherein the deaminase is adenosine deaminase.

[0172] In one embodiment, a composition comprising a polymerase and a guide nucleic acid sequence, The nucleic acid sequence is UUCGUAUCUGUAAAACCAAG, CCUACCUGUCACCAGGACCA, CUCUUACCUGUACCAUAACC, CACCUACCUAAGAACCAUCC, ACUCACGCUGGAUAGCCUCC, ACUCACCCAGCAUCCCCAGC, CACUCACCUUAGCC The composition of the present invention comprises a sequence selected from the group consisting of UGAGCA, and CACGCACCUGGACAGCUGAC. Provided in the specification.

[0173] In some embodiments, the polymerase is a reverse transcriptase, wherein the guide nucleus The nucleic acid sequence is a reverse transcription template sequence, a reverse transcription primer binding site, or a combination thereof. and an extended guide nucleic acid sequence comprising:

[0174] In one embodiment, modified antibodies have reduced immunogenicity and / or increased antineoplastic activity. A method for producing an altered immune cell, comprising: a) introducing a first gene sequence or b) modifying a single target nucleic acid base in the regulatory element; and c) modifying a Cas12 polypeptide. The peptide is used to modify a second gene sequence or its regulatory element in the immune cell. wherein the Cas12 polypeptide generates a site-specific cleavage in the second gene sequence. wherein each of the first gene and the second gene is an immunogenic gene, a checkpoint inhibitor, factor genes, or immune response-modulating genes, thereby reducing immunogenicity and Provided herein are methods for generating modified immune cells with increased anti-neoplastic and / or anti-cancer activity. It is served.

[0175] In some embodiments, the method comprises expressing an exogenous functional chimeric antigen receptor (CAR) in an immune cell. CAR) or a functional fragment thereof.

[0176] In some embodiments, a polynucleotide encoding a CAR or a functional fragment thereof The gene is inserted into the site-specific cleavage site generated by the Cas12 polypeptide.

[0177] In some embodiments, the Cas12 polypeptide is a Cas12b polypeptide.

[0178] In one embodiment, a method for producing a reduced immunogenic and / or antineoplastic antibody comprising the steps of: Provided herein are methods for producing modified immune cells with increased activity:

[0179] a) a single target nucleic acid salt in a first gene sequence or its regulatory element in an immune cell; and b) modifying the second gene to include an exogenous functional chimeric antigen receptor (CAR) or or a functional fragment thereof, or an exogenous functional T cell receptor or a functional fragment thereof. modifying the second gene sequence or its regulatory element in the immune cell by wherein each of the first gene and the second gene is an immunogenic gene, a checkpoint inhibitor, or immune response-modulating genes, thereby reducing immunogenicity and The present invention generates modified immune cells with increased immune and / or anti-neoplastic activity.

[0180] In some embodiments, step b) comprises using a nucleic acid programmable DNA binding protein (na pDNAbp) to generate a site-specific cleavage in the second gene sequence.

[0181] In some embodiments, the napDNAbp is Cas12b.

[0182] In some embodiments, the first gene is expressed as a nucleotide sequence similar to that of a control cell of the same type that does not have the modification. The expression of a gene is reduced by at least 60%, or the expression of a second gene is reduced by at least 60%. can be.

[0183] In some embodiments, the first gene is CD3 epsilon, CD3 gamma, CD3 delta , CD4, TRAC, TRBC1, TRBC2, PDCD1, CD30, CD33, CD7, CD52, B2M, CD70, CIITA, CD2, and CD5.

[0184] In some embodiments, the first gene or the second gene is TRAC, CIITA, CD2, It is selected from the group consisting of CD5, CD7, and CD52.

[0185] In some embodiments, the second gene is TRAC.

[0186] In some embodiments, step a) comprises comparing two other gene sequences or their regulatory elements. The method further comprises modifying a single target nucleobase in the target nucleic acid.

[0187] In some embodiments, step a) comprises detecting three other gene sequences or their regulatory elements. The method further comprises modifying a single target nucleobase in the target nucleic acid.

[0188] In some embodiments, step a) comprises detecting four other gene sequences or their regulatory elements. The method further comprises modifying a single target nucleobase in the target nucleic acid.

[0189] In some embodiments, step a) comprises detecting five other gene sequences or their regulatory elements. The method further comprises modifying a single target nucleobase in the target nucleic acid.

[0190] In some embodiments, step a) comprises the step of selecting six other gene sequences or their regulatory elements. The method further comprises modifying a single target nucleobase in the target nucleic acid.

[0191] In some embodiments, step a) comprises detecting seven other gene sequences or their regulatory elements. The method further comprises modifying a single target nucleobase in the target nucleic acid.

[0192] In some embodiments, the modifying in step a) comprises a deaminase and Single base editor containing nucleic acid programmable DNA binding protein (napDNAbp) The method includes deaminating the target nucleobase of

[0193] In some embodiments, the napDNAbp is a Cas9 nickase or nuclease-inactive Cas9 Includes.

[0194] In some embodiments, the deaminase is a cytidine deaminase, wherein: The modification includes converting a cytidine (C) to a thymine (T).

[0195] In some embodiments, the deaminase is adenosine deaminase, , the modification involves converting an adenine (A) to a guanine (G).

[0196] In some embodiments, the modifying in a) comprises inducing immune cells to a guide nucleic acid sequence. contacting the array.

[0197] In some embodiments, the guide nucleic acid sequence is UUCGUAUCUGUAAAACCAAG, CCUACCUGUC ACCAGGACCA, CUCUUACCUGUACCAUAACC, CACCUACCUAAGAACCAUCC, ACUCACGCUGGAUAGCCUCC, AC UCACCCAGCAUCCCCAGC, CACUCACCUUAGCCUGAGCA, and CACGCACCUGGACAGCUGAC The sequence includes a sequence selected from the group consisting of:

[0198] In some embodiments, the modifying in b) comprises inducing immune cells to a guide nucleic acid sequence. contacting the array.

[0199] In some embodiments, the guide nucleic acid sequence is a sequence selected from the sequences in Table 1. Contains columns.

[0200] In some embodiments, the modifying in a) comprises a reverse transcriptase and an elongation A single target nucleobase is synthesized by target-primed reverse transcription using a guide nucleic acid sequence that has been selected. the extended guide nucleic acid sequence is a reverse transcription template sequence, , a reverse transcription primer binding site, or a combination thereof.

[0201] In some embodiments, the modifying in a) and b) is Generates less than 1% indels.

[0202] In some embodiments, the modifying in a) and b) is generate less than 5% off-target modifications.

[0203] In some embodiments, the modifying in a) and b) is to generate less than 5% non-target modifications.

[0204] In some embodiments, the immune cells are human cells.

[0205] In some embodiments, the immune cells are cytotoxic T cells, regulatory T cells, helper T cells, or the like. T cells, dendritic cells, B cells, or NK cells.

[0206] In some embodiments, the CAR specifically binds to a marker associated with a neoplasm .

[0207] In some embodiments, the CAR specifically binds to CD7.

[0208] In one embodiment, a compound having reduced immunogenicity and / or antineoplastic activity, including Provided herein are increased, modified immune cells:

[0209] a) modifying a single target nucleic acid base in a first gene sequence or a regulatory element thereof and and b) a modification in the second gene sequence or its regulatory element, which is The first gene and the second gene are modified by site-specific cleavage generated by the cleavage gene. Each of the children expresses an immunogenic gene, a checkpoint inhibitor gene, or an immune response regulator gene. In one embodiment, the immune cells are carrying an exogenous functional chimeric antigen receptor (CAR) or or a functional fragment thereof.

[0210] In some embodiments, a polynucleotide encoding a CAR or a functional fragment thereof The gene is inserted into the site-specific cleavage site generated by the Cas12 polypeptide.

[0211] In one embodiment, modified antibodies have reduced immunogenicity and / or increased antineoplastic activity. a) a first gene sequence or a regulatory element thereof in the immune cell; and b) a single targeted nucleic acid base modification in a second gene sequence or a regulatory element thereof. an exogenous chimeric antigen receptor (CAR) or a functional fragment thereof, or a first gene and a second gene, comprising a modification that is the insertion of an exogenous T cell receptor or a functional fragment thereof; Each of the second genes is an immunogenic gene, a checkpoint inhibitor gene, or an immunosuppressant gene. Provided herein are modified immune cells that are response regulator genes.

[0212] In some embodiments, the modification in b) is performed by site-specific cleavage using Cas12b. Therefore,

[0213] In some embodiments, the first gene is expressed as a nucleotide sequence similar to that of a control cell of the same type that does not have the modification. The expression of a gene is reduced by at least 60%, or the expression of a second gene is reduced by at least 60%. can be.

[0214] In some embodiments, the first gene or the second gene is CD3 epsilon, CD3 Gamma, CD3 delta, CD4, TRAC, TRBC1, TRBC2, PDCD1, CD30, CD33, CD7, CD52, B2M, C D70, CIITA, CD2, and CD5.

[0215] In some embodiments, the first gene or the second gene is selected from the group consisting of TRAC, CD2, CD5, CD 7, and CD52.

[0216] In some embodiments, the second gene is TRAC.

[0217] In some embodiments, the immune cells express two other gene sequences or their regulatory elements. The invention further comprises a modification to a single targeted nucleobase in the cleavage region.

[0218] In some embodiments, the immune cells express three other gene sequences or their regulatory elements. The invention further comprises a modification to a single targeted nucleobase in the cleavage region.

[0219] In some embodiments, the immune cells express four other gene sequences or their regulatory elements. The invention further comprises a modification to a single targeted nucleobase in the cleavage region.

[0220] In some embodiments, the immune cells express five other gene sequences or their regulatory elements. The invention further comprises a modification to a single targeted nucleobase in the cleavage region.

[0221] In some embodiments, the immune cells express six other gene sequences or their regulatory elements. The invention further comprises a modification to a single targeted nucleobase in the cleavage region.

[0222] In some embodiments, the immune cells express seven other gene sequences or their regulatory elements. The invention further comprises a modification to a single targeted nucleobase in the cleavage region.

[0223] In some embodiments, the modification in a) is carried out by a deaminase and a nucleic acid program. It is generated by a base editor containing a tunable DNA-binding protein (napDNAbp).

[0224] In some embodiments, the deaminase is a cytidine deaminase and the modified involves the conversion of a cytidine (C) to a thymine (T).

[0225] In some embodiments, the deaminase is adenosine deaminase, , the modification involves converting an adenine (A) to a guanine (G).

[0226] In some embodiments, the immune cells contain less than 1% indels in their genome.

[0227] In some embodiments, the immune cells are human cells.

[0228] In some embodiments, the immune cells are cytotoxic T cells, regulatory T cells, helper T cells, or the like. T cells, dendritic cells, B cells, or NK cells.

[0229] In some embodiments, the CAR specifically binds to a marker associated with a neoplasm .

[0230] In some embodiments, the CAR specifically binds to CD7.

[0231] In some embodiments, the modification in b) is in exon 1 of the TRAC gene sequence. It is an insertion.

[0232] In one embodiment, the population of modified immune cells comprises a plurality of immune cells selected from a) of a single target nucleic acid base in a first gene sequence or its regulatory element in an immune cell. and b) a modification in a second gene sequence or its regulatory element, a first gene and a second gene, including a modification that is a site-specific cleavage produced by the polypeptide; Each of the second genes is an immunogenic gene, a checkpoint inhibitor gene, or an immunosuppressant gene. Response regulator genes, multiple populations of which are expressed in exogenous chimeric antigen receptors (CARs) or their functional Provided herein are populations of modified immune cells that include the fragments.

[0233] In some embodiments, a polynucleotide encoding a CAR or a functional fragment thereof The gene is inserted into the site-specific cleavage site generated by the Cas12 polypeptide.

[0234] In one embodiment, the population of modified immune cells comprises a plurality of immune cells selected from a) a) modifying a single target nucleic acid base in a first gene sequence or a regulatory element thereof; and b) Modifications in the second gene sequence or its regulatory elements to enhance the expression of exogenous chimeric antigen receptors. receptor (CAR) or a functional fragment thereof, or an exogenous T cell receptor or a functional fragment thereof The first gene and the second gene each contain an immunogenic gene, a) or b) A plurality of cells having the modification exhibit reduced immunogenicity and / or increased antineoplastic activity. Provided herein are populations of altered immune cells. In some embodiments, b) The modifications in are generated by site-specific cleavage using Cas12b. the degree of the modification in a) or b) compared to multiple control cells of the same type that do not have the modification. In a plurality of cells having the mutation, expression of the first gene is reduced by at least 60%, or The expression of 2 genes is reduced by at least 60%.

[0235] In some embodiments, the first gene or the second gene is CD3 epsilon, CD3 Gamma, CD3 delta, CD4, TRAC, TRBC1, TRBC2, PDCD1, CD30, CD33, CD7, CD52, B2M, C D70, CIITA, CD2, and CD5.

[0236] In some embodiments, the first gene or the second gene is TRAC, CIITA, CD2, It is selected from the group consisting of CD5, CD7, and CD52.

[0237] In some embodiments, the first gene is TRAC, CD7, or CD52.

[0238] In some embodiments, the second gene is TRAC.

[0239] In some embodiments, the plurality of cells having the modification in a) or b) comprises two Further modifications to single target nucleic acid bases in other gene sequences or their regulatory elements include.

[0240] In some embodiments, the plurality of cells having the modification in a) or b) comprises three a single target in four, five, or six other gene sequences or their regulatory elements The nucleic acid further comprises a target nucleobase.

[0241] In some embodiments, the modification in a) is to form a base editor. Deaminase and nucleic acid programmable DNA binding protein (napDNAbp) containing base editor generated by the

[0242] In some embodiments, the deaminase is a cytidine deaminase, wherein: The modification includes converting a cytidine (C) to a thymine (T).

[0243] In some embodiments, the deaminase is adenosine deaminase, , the modification involves converting an adenine (A) to a guanine (G).

[0244] In some embodiments, the base editor further comprises a uracil glycosylase inhibitor. This includes:

[0245] In some embodiments, at least 60% of the population of immune cells is viable.

[0246] In some embodiments, at least 60% of the population of immune cells are identical to those that do not have the modification. expand at a rate that is at least 80% of the expansion rate of a population of control cells of the same type.

[0247] In some embodiments, the population of modified immune cells is compared to a reference population of cells. In some embodiments, the reference population is , a population of immune cells modified using Cas9 nuclease.

[0248] In some embodiments, the immune cells are human cells.

[0249] In some embodiments, the immune cells are cytotoxic T cells, regulatory T cells, helper T cells, or the like. T cells, dendritic cells, B cells, or NK cells.

[0250] In some embodiments, the CAR specifically binds to a marker associated with a neoplasm .

[0251] In some embodiments, the CAR specifically binds to CD7.

[0252] In some embodiments, the modification in b) is in exon 1 in the TRAC gene sequence. It is an insertion that can be done.

[0253] In one embodiment, a method for producing modified immune cells with increased anti-neoplastic activity. The method comprises the step of detecting the Cbl proto-oncogene B (CBLB) gene sequence or its regulatory element in an immune cell. The method includes modifying a single target nucleic acid base in an immune cell, wherein the modification differentiates the immune cell from an immune cell lacking the modification. In comparison, the improved antibody reduced the activation threshold of immune cells, thereby increasing anti-neoplastic activity. Methods for generating altered immune cells are provided herein.

[0254] In one embodiment, the composition comprises modified immune cells having increased anti-neoplastic activity. Therefore, the modified immune cells express the Cbl proto-oncogene B (CBLB) gene sequence or its regulatory elements. The modified immune cells contain a modification at a single target nucleobase in the target nucleobase, and the modified immune cells contain a modification at a single target nucleobase in the target nucleobase. Provided herein are compositions that exhibit a reduced activation threshold compared to control immune cells of the same type. can be.

[0255] In one embodiment, the population of immune cells comprises a plurality of immune cells expressing the CBLB gene. A single target nucleic acid base in the sequence or its regulatory element may be modified, and multiple nucleic acid bases containing the modification may be modified. A population of immune cells of a certain number exceeds the activation threshold compared to a control population of immune cells of the same type that does not have the modification. Provided herein are populations of immune cells that exhibit a reduced value of IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17, IL-18, IL-19, IL-20,

[0256] In one embodiment, the method produces a population of modified immune cells with increased anti-neoplastic activity. The method comprises detecting Cbl proto-oncogene B (CBLB) gene sequences or sequences thereof in a population of immune cells. wherein the target nucleic acid sequence is a sequence encoding a single target nucleic acid in a regulatory element of the immune cell. The method is described herein, wherein at least 50% of a population of nucleic acids is modified to contain a single target nucleic acid base modification. Provided in the specification.

[0257] In one embodiment, a combination comprising at least four different guide nucleic acid sequences for base editing. In some embodiments, the composition comprises a base editor. and a polynucleotide encoding the polypeptide, These include nucleic acid programmable DNA binding proteins (napDNAbp) and deaminases. In some embodiments, the polynucleotide encoding the base editor is be.

[0258] In some embodiments, the deaminase is a cytidine deaminase or an adenosine It is a deaminase.

[0259] In some embodiments, the composition further comprises a base editor polypeptide, The base editor polypeptide is a nucleic acid programmable DNA binding protein (napDNAbp) and Contains deaminase.

[0260] In some embodiments, the deaminase is a cytidine deaminase or an adenosine It is a deaminase.

[0261] In some embodiments, the composition further comprises lipid nanoparticles.

[0262] In some embodiments, the at least four guide nucleic acid sequences are each a CD2, CD3 epsilon, CD3 gamma, CD3 delta, CD4, CD5, CD7, CD30, CD33, CD52, CD70, and C In some embodiments, the nucleic acid sequence hybridizes to a gene sequence selected from the group consisting of: IITA. At least one, two, three, four, five, six, seven, eight, or more genes in The children or their regulatory elements are CD2, CD3 epsilon, CD3 gamma, CD3 delta, CD4, CD5 , CD7, CD30, CD33, CD52, CD70, and CIITA.

[0263] In some embodiments, at least 1, 2, 3, 4, 5, 6, 7, 8, or more genes or their regulatory elements, such as CD2, CD3 epsilon, CD3 gamma, 1 selected from MA, CD3 delta, CD4, CD5, CD7, CD30, CD33, CD52, CD70, and CIITA In some embodiments, the gene comprises at least one, two, three, or more genes. , 4, 5, 6, 7, 8 or more genes or their regulatory elements are CAT1, ACLY, ADORA2A, AXL, B2M, BATF, BCL2L11, BTLA, CAMK2D, cAMP, CASP8, Cblb, C CR5, CD2, CD3D, CD3E, CD3G, CD4, CD5, CD7, CD8A, CD33, CD38, CD52, CD70, CD82, C D86, CD96, CD123, CD160, CD244, CD276, CDK8, CDKN1B, Chi3l1, CIITA, CISH, CSF2CS K, CTLA-4, CUL3, Cyp11a1, DCK, DGKA, DGKZ, DHX37, ELOB(TCEB2), ENTPD1(CD39), FAD D, FAS, GATA3, IL6, IL6R, IL10, IL10RA, IRF4, IRF8, JUNB, Lag3, LAIR-1(CD305), L DHA, LIF, LYN, MAP4K4, MAPK14, MCJ, MEF2D, MGAT5, NR4A1, NR4A2, NR4A3, NT5E(CD73 ), ODC1, OTULINL(FAM105A), PAG1, PDCD1, PDIA3, PHD1(EGLN2), PHD2(EGLN1), PHD3(EG LN3), PIK3CD, PIKFYVE, PPARa, PPARd, PRDMI1, PRKACA, PTEN, PTPN2, PTPN6, PTPN11 , PVRIG(CD112R), RASA2, RFXANK, SELPG / PSGL1, SIGLEC15, SLA, SLAMF7, SOCS1, Spry1 , Spry2, STK4, SUV39, H1TET2, TGFbRII, TIGIT, Tim-3, TMEM222, TNFAIP3, TNFRSF8(C D30), TNFRSF10B, TOX, TOX2, TRAC, TRBC1, TRBC2, UBASH3A, VHL, and VISTA In some embodiments, each of the at least four guide nucleic acid sequences is a CD3-type Chiron, CD3 delta, CD3 gamma, TRAC, TRBC1 and TRBC2, CD2, CD5, CD7, CD52, CD70 and CIITA.

[0264] In some embodiments, the at least four guide nucleic acid sequences are: UUCGUAUCUGUAAAACC AAG, CCUACCUGUCACCAGGACCA, CUCUUACCUGUACCAUAACC, CACCUACCUAAGAACCAUCC, ACUCACGCU GGAUAGCCUCC, ACUCACCCAGCAUCCCCAGC, CACUCACCUUAGCCUGAGCA, and CACGCACCUGGACAGCU GAC.

[0265] In one aspect, an immune cell comprising the composition of any of the above embodiments, The composition is introduced into the immune cells using electroporation, the immune cells being Provided.

[0266] In one aspect, an immune cell comprising the composition of any of the above embodiments, The composition may be introduced by electroporation, nucleofection, viral transduction, or other methods. Provided herein are immune cells that are introduced into the immune system using these combinations.

[0267] Other features and advantages of the invention will become apparent from the detailed description and claims. There will be.

[0268] [Definition] Unless otherwise defined, all technical and scientific terms used herein are It has the meaning commonly understood by a person skilled in the art to which the present invention pertains. The reference provides those skilled in the art with general definitions of many of the terms used in this invention: Single ton et al., Dictionary of Microbiology and Molecular Biology (2nd ed. 1994); Cambridge Dictionary of Science and Technology (Walker ed., 1988); The Glossary of Genetics, 5th Ed., R. Rieger et al. (eds.), Springer Verlag (1991); and H ale & Marham, The Harper Collins Dictionary of Biology (1991). used herein where applicable, the following terms are applied hereinafter unless otherwise specified: It has meaning.

[0269] "Adenosine deaminase" refers to the enzyme that hydrolyzes the deamination of adenine or adenosine. In one embodiment, the term "de" refers to a polypeptide or fragment thereof that is capable of catalyzing the deactivation of a protein. The aminase or deaminase domain hydrolyzes adenosine to inosine. Catalyzes the hydrolytic deamination of deoxyadenosine or deoxyadenosine to deoxyinosine In some embodiments, the adenosine deaminase is a deoxyadenosine deaminase. It catalyzes the hydrolytic deamination of adenine or adenosine in nucleic acid (DNA). The adenosine deaminases provided herein (e.g., engineered adenosine deaminases) aminase, evolved adenosine deaminase) can be derived from any organism, e.g., bacteria. In some embodiments, the deaminase or deaminase domain may be In some embodiments, the deaminase is a variant of a naturally occurring deaminase derived from a plant. Alternatively, the deaminase domain may be non-naturally occurring. In a similar manner, the deaminase or deaminase domain is a naturally occurring deaminase At least 50%, at least 55%, at least 60%, at least 65%, at least 70% %, at least 75% at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% In some embodiments, the adenosine deaminase is derived from a gene encoding adenosine deaminase from a plant, e.g., E. coli, S. aureus, S. typhi, S. putrefaciens, H. influenzae, or C. crescentus. In some embodiments, the adenosine deaminase is TadA deaminase. In some embodiments, the TadA deaminase is E. coli TadA (ecTadA) deaminase or This is a fragment of it.

[0270] For example, truncated ecTadA may have one or more N-terminal amino acids compared to full-length ecTadA. In some embodiments, the truncated ecTadA is a truncated ecTadA fragment of the full-length ecTadA. Compared to TadA, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 6, 17, 18, 19, Alternatively, the 20 N-terminal amino acid residues may be deleted. The packed ecTadA has 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 1 compared to the full-length ecTadA. The C-terminal amino acid residues of 4, 15, 6, 17, 18, 19, or 20 may be deleted. In some embodiments, the ecTadA deaminase does not contain an N-terminal methionine. In embodiments, the TadA deaminase is an N-terminal truncated TadA. In PCT / US2017 / 045381, the entire contents of which are incorporated herein by reference, TadA is There is one of the listed TadAs.

[0271] In certain embodiments, the adenosine deaminase comprises the following amino acid sequence: MSEVEFSHEYWMRHALTLAKRAWDEREVPVGAVLVHNNRVIGEGWNRPIGRHDPT AHAEIMALRQGGLVMQNYRLIDAT LYVTLEPCVMCAGAMIHSRIGRVVFGARDAKT GAAGSLMDVLHHPGMNHRVEITEGILADECAALLSDFFRMRRQEIKA QKKAQSSTD This is called the "TadA reference sequence."

[0272] In some embodiments, the TadA deaminase is a full-length E. coli TadA deaminase. For example, In certain embodiments, the adenosine deaminase comprises the following amino acid sequence: MRRAFITGVFFLSEVEFSHEYWMRHALTLAKRAWDEREVPVGAVLVHNNRVIGEG WNRPIGRHDPTAHAEIMALRQGGL VMQNYRLIDATLYVTLEPCVMCAGAMIHSRIG RVVFGARDAKTGAAGSLMDVLHHPGMNHRVEITEGILADECAALLSD FFRMRRQEI KAQKKAQSSTD

[0273] However, additional adenosine deaminases useful herein will be apparent to those skilled in the art. It should be understood that these are obvious and within the scope of the present disclosure. The aminase may be a homolog of adenosine deaminase (AD AT) that acts on tRNA Exemplary AD AT homologs include, but are not limited to, the following:

[0274] Staphylococcus aureus TadA: MGSHMTNDIYFMTLAIEEAKKAAQLGEVPIGAIITKDDEVIARAHNLRETLQQPTAH AEHIAIERAAKVLGSWRLEGCT LYVTLEPCVMCAGTIVMSRIPRVVYGADDPKGGCS GS LMNLLQQS NFNHRAIVDKG VLKE AC S TLLTTFFKNL RANKKS TN

[0275] Bacillus subtilis TadA: MTQDELYMKEAIKEAKKAEEKGEVPIGAVLVINGEIIARAHNLRETEQRSIAHAEML VIDEACKALGTWRLEGATLYVT LEPCPMCAGAVVLSRVEKVVFGAFDPKGGC S GTLMN LLQEERFNHQAEVVSGVLEEECGGMLSAFFRELRKKKKAAR KNLSE

[0276] MPPAFITGVTSLSDVELDHEYWMRHALTLAKRAWDEREVPVGAVLVHNHRVIGEG WNRPIGRHDPTAHAEIMALRQGGL VLQNYRLLDTTLYVTLEPCVMCAGAMVHSRIG RVVFGARDAKTGAAGSLIDVLHHPGMNHRVEIIEGVLRDECATLLSD FFRMRRQEIK ALKKADRAEGAGPAV

[0277] Shewanella putrefaciens (S. putrefaciens) TadA: MDE YWMQVAMQM AEKAEAAGE VPVGA VLVKDGQQIATGYNLS IS QHDPT AHAEI LCLRSAGKKLENYRLLDA TLYITLEPCAMCAGAMVHSRIARVVYGARDEKTGAAGT VVNLLQHPAFNHQVEVTSGVLAEACSAQLSRFKRRRDEKK ALKLAQRAQQGIE

[0278] Haemophilus influenzae F3031 (H. influenzae) TadA: MDAAKVRSEFDEKMMRYALELADKAEALGEIPVGAVLVDDARNIIGGWNLSIVQS DPT ΑΗAEIIALRNG AKNIQ N YRLLNS TLY VTLEPCTMC AG AILHS RIKRLVFG AS D YK TGAIGSRFHFFDDYKMNHTLEITSGVLAEEC SQKLSTFFQKRREEKKIEKALLKSLSD K

[0279] Caulobacter crescentus (C. crescentus) TadA: MRTDESEDQDHRMMRLALDAARAAAEAGETPVGAVILDPSTGEVIATAGNGPIAAH DPTAHAEIAAMRAAAAKLGNYRL TDLTLVVTLEPCAMCAGAISHARIGRVVFGADD PKGGAVVHGPKFFAQPTCHWRPEVTGGVLADESADLLRGFFRARRK COST

[0280] Geobacter sulfurreducens (G. sulfurreducens) TadA: MSSLKKTPIRDDAYWMGKAIREAAKAAAARDEVPIGAVIVRDGAVIGRGHNLREGSN DPSAHAEMIAIRQAARRSANWRL TGATLYVTLEPCLMCMGAIILARLERVVFGCYDP KGGAAGSLYDLSADPRLNHQVRLSPGVCQEECGTMLSDFFRDLRR RKKAKATPALF IDERKVPPEP

[0281] TadA7.10 MSEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWNRAIGLHDPTAHEIMALRQGGLVMQNYRLIDATL YVTFEPCVMCAGAMIHSRIGRVVFGVRNAKTGAAGSLMDVLHYPGMNHRVEITEGILADECAALLCYFFRMPRQVFNAQK KAQSSTD

[0282] An "agent" is any small molecule compound, antibody, nucleic acid molecule, or polypeptide. , or fragments thereof.

[0283] "Alteration" refers to any alteration that can be detected by standard art known methods such as those described herein. This refers to a change in the structure, expression level, or activity of a gene or polypeptide. As used herein, an alteration (e.g., an increase or decrease) is defined as a 10% change in expression level, a 2% change in expression level, or a 10% change in expression level. These include a 5% change, a 40% change, and a 50% or greater change in expression level.

[0284] As used herein, "allogeneic" refers to cells of the same species that are genetically distinct from the cells being compared. refers to the cells of the

[0285] "Analog" means a molecule that is not identical but has similar functional or structural characteristics. For example, a polypeptide analog may possess the biological activity of the corresponding naturally occurring polypeptide. specific sequences that enhance the function of the analog compared to the native polypeptide Such modifications may be made without, for example, altering polynucleotide binding activity. It can increase the protease resistance, membrane permeability, or half-life of the analogue. In another example, a polynucleotide analog may have an analogous structure compared to a naturally occurring polynucleotide. The biological properties of the corresponding naturally occurring polynucleotides, with certain modifications that enhance their function. Such modifications may affect the affinity of the polynucleotide for DNA, its half-life, and / or may increase nuclease resistance, and the analogs may be unnatural nucleotides or may comprise amino acids.

[0286] "Antineoplastic activity" means preventing or inhibiting the maturation and / or growth of neoplasms. means.

[0287] As used herein, "autologous" refers to cells derived from the same subject.

[0288] "B cell maturation antigen or tumor necrosis factor receptor superfamily member 17 polypeptide" BCMA is a marker expressed on mature B lymphocytes, NCBI accession number NP_001183 or its equivalent. An exemplary B protein is a protein having at least about 85% amino acid sequence identity with a fragment of B. The CMA polypeptide sequence is provided below.

[0289] >NP_001183.2 Tumor necrosis factor receptor superfamily member 17 [Homo sapiens] MLQMAGQCSQNEYFDSLLHACIPCQLRCSSNTPPLTCQRYCNASVTNSVKGTNAILWTCLGLSLIISLAVFVLMFLLRKI NSEPLKDEFKNTGSGLLGMANIDLEKSRTGDEIILPRGLEYTVEECTCEDCIKSKPKVDSDHCFPLPAMEEGATILVTTK TNDYCKSLPAALSATEIEKSISAR

[0290] This antigen is used in the treatment of relapsed or refractory multiple myeloma and other hematological neoplasms. It can be targeted.

[0291] "B-cell maturation antigen or tumor necrosis factor receptor superfamily member 17, (BCMA) "Polynucleotide" means a nucleic acid molecule that encodes a BCMA polypeptide. The gene encodes a cell surface receptor that recognizes a B cell activating factor. The octide sequences are provided below.

[0292] >NM_001192.2 Homo sapiens TNF receptor superfamily member 17 (TNFRSF17), m RNA AAGACTCAAACTTAGAAACTTGAATTAGATGTGGTATTCAAATCCTTAGCTGCCGCGAAGACACAGACAGCCCCCGTAAG AACCCACGAAGCAGGCGAAGTTCATTGTTCTCAACATTCTAGCTGCTCTTGCTGCATTTGCTCTGGAATTCTTGTAGAGA TATTACTTGTCCTTCCAGGCTGTTCTTTCTGTAGCTCCCTTGTTTTCTTTTTGTGATCATGTTGCAGATGGCTGGGCAGT GCTCCCAAAATGAATATTTTGACAGTTTGTTGCATGCTTGCATACCTTGTCAACTTCGATGTTCTTCTAATACTCCTCCT CTAACATGTCAGCGTTATTGTAATGCAAGTGTGACCAATTCAGTGAAAGGAACGAATGCGATTCTCTGGACCTGTTTGGG ACTGAGCTTAATAATTTCTTTGGCAGTTTTCGTGCTAATGTTTTTGCTAAGGAAGATAAACTCTGAACCATTAAAGGACG AGTTTAAAAACACAGGATCAGGTCTCCTGGGCATGGCTAACATTGACCTGGAAAAGAGCAGGACTGGTGATGAAATTATT CTTCCGAGAGGCCTCGAGTACACGGTGGAAGAATGCACCTGTGAAGACTGCATCAAGAGCAAACCGAAGGTCGACTCTGA CCATTGCTTTCCACTCCCAGCTATGGAGGAAGGCGCAACCATTCTTGTCACCACGAAAACGAATGACTATTGCAAGAGCC TGCCAGCTGCTTTGAGTGCTACGGAGATAGAGAAATCAATTTCTGCTAGGTAATTAACCATTTCGACTCGAGCAGTGCCA CTTTAAAAAATCTTTTGTCAGAATAGATGATGTGTCAGATCTCTTTAGGATGACTGTATTTTTCAGTTGCCGATACAGCTT TTTGTCCTCTAACTGTGGAAACTCTTTATGTTAGATATATTTCTCTAGGTTACTGTTGGGAGCTTAATGGTAGAAACTTC CTTGGTTTCATGATTAAACTCTTTTTTTTCCTGA

[0293] "Base editor (BE)" or "nucleobase editor (NBE)" refers to a polynucleobase In one embodiment, the term "agent" refers to an agent that binds a nucleotide and has nucleobase modifying activity. The drug uses nucleic acid programmable DNA binding proteins to specifically target polynucleotides. In another embodiment, the base editor binds to a nucleic acid molecule (e.g., In some embodiments, the enzyme is capable of modifying cytidine bases in DNA. Thus, base editors can deaminate bases within nucleic acid molecules. In embodiments, the base editor can deaminate bases within a DNA molecule. In some embodiments, the base editor deaminates cytidines in DNA In some embodiments, the base editor is a cytidine deaminase or a fusion protein comprising adenosine deaminase. The base editor is fused to a cytidine deaminase or an adenosine deaminase. In some embodiments, the base editor is a cytidine deoxynucleotide. The Cas9 nickase (nCas9) is fused to adenosine deaminase or adenosine deaminase. In some embodiments, the base editor is an inhibitor of base excision repair, such as a UGI domain. In some embodiments, the fusion protein is fused to a deaminase and Cas9 nickase fused to an inhibitor of base excision repair, such as a UGI domain. In some embodiments, the cytidine deaminase or adenosine deaminase comprises the following domains: AB Nosine deaminase nucleobase editor polypeptide: NH2-[AB]-COOH

[0294] wherein A is a cytidine deaminase domain, an adenosine deaminase domain, or and B comprises one or more domains having nucleic acid sequence-specific binding activity. In one embodiment, the cytidine or adenosine deaminase nucleobase edta of the previous aspect The interpolypeptide contains:

[0295] NH2-[A n -B o ]-COOH, where A is a cytidine deaminase domain, adenosine deaminase domain, or an active fragment thereof, wherein n is an integer: 1, 2, 3, 4, or 5; and B is a nucleic acid. It contains a domain having sequence-specific binding activity, and o is an integer: 1, 2, 3, 4, or 5. In embodiments, the polypeptide contains one or more nuclear localization sequences. In an embodiment, the polypeptide comprises at least one of the nuclear localization sequences at the N-terminus or C-terminus. In one embodiment, the polypeptide contains a bipartite nuclear localization signal. In one embodiment, the polypeptide contains a nuclear localization signal that is phosphorylated. It contains one or more domains linked by a carrier.

[0296] 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 this embodiment, the base editors are adenosine base editors (ABEs) and citrates. In some embodiments, the base editor is adenosine. A nuclease-inactive Cas9 (dCas9) fused to a deaminase. In the present invention, Cas9 is a circular permutant Cas9 (e.g., spCas9 or saCas9). Circularly permuted Cas9 is known in the art and is described, for example, in Oakes et al., Cell 176 , 254-267, 2019. In some embodiments, the base editor is It is fused to an inhibitor of excision repair, e.g., a UGI domain or a dISN domain. In this case, the fusion protein contains a deaminase and a base-removing domain, such as a UGI or dISN domain. In another embodiment, the base editor comprises a Cas9 nickase fused to an inhibitor of repair. is an abasic base editor.

[0297] In some embodiments, the adenosine deaminase is evolved from TadA. In some embodiments, the polynucleotide programmable DNA binding domain is a CRISPR-associated In some embodiments, the base editor is a cleavage enzyme (e.g., Cas or Cpf1). The target gene is a catalytically inactive Cas9 (dCas9) fused to a deaminase domain. In embodiments, the base editor comprises a Cas9 nickase fused to a deaminase domain ( In some embodiments, the base editor is a base excision repair (BER) In some embodiments, the inhibitor of base excision repair is fused to an inhibitor of base excision repair. In some embodiments, the base excision is a uracil DNA glycosylase inhibitor (UGI). Inhibitors of excision repair are inosine base excision repair inhibitors. International PCT application numbers PCT / 2017 / 045381 (WO2018 / 027078) and PCT / US2016 / 058344 (WO2017 / 070 632), each of which is incorporated herein by reference in its entirety. , AC, et al., "Programmable editing of a target base in genomic DNA without do "Uble-stranded DNA cleavage" Nature 533, 420-424 (2016); Gaudelli, NM, et al., "Programmable base editing of A·T to G·C in genomic DNA without DNA cleavage" Nature 551, 464-471 (2017); Komor, AC, et al., "Improved base excision repair inhibition and bacteriophage Mu Gam protein yields C: G-to-T: A editor bases wi th higher efficiency and product purity" Science Advances 3: eaao4774 (2017), and Rees, HA, et al., "Base editing: precision chemistry on the genome and nscriptome of living cells." Nat Rev Genet. 2018 Dec;19(12): 770-788. doi: 10.1 See also 038 / s41576-018-0059-1, the entire contents of which are incorporated herein by reference. (This is the case.)

[0298] In some embodiments, the base editor is an adenosine deaminase variant (e.g., TadA*7.10) in combination with a circularly permuted Cas9 (e.g., spCAS9) and a bipartite nuclear localization sequence. Circularly permuted Cas9 is generated by cloning into a scaffold containing These methods are known in the art and are described, for example, in Oakes et al., Cell 176, 254-267, 2019. Exemplary circularly permuted sequences are shown below, where the bolded sequence is Ca The sequences derived from s9 are shown, the italicized sequences indicate linker sequences, and the underlined sequences The columns represent bipartite nuclear localization sequences.

[0299] CP5 (MSP) - "NGC = Pam variant with mutation; conventional Cas9 prefers NGG." PID = protein interaction domain, and "D10A" nickase):

[0300] JPEG2024174899000002.jpg197169

[0301] Nucleobase components of base editor systems and polynucleotide programmable nucleases The nucleotide binding moieties can be covalently or non-covalently bound to one another. For example, In some embodiments, the deaminase domain is a polynucleotide programming Targeted to a target nucleotide sequence by a nucleotide-binding domain that can be In some embodiments, the polynucleotide has a programmable nucleotide binding domain. In some embodiments, the deaminase domain may be fused or linked to the deaminase domain. The polynucleotide programmable nucleotide binding domain is a deaminase The deaminase domain binds to the ATPase domain by non-covalently interacting with or binding to the ATPase domain. It can be targeted to a target nucleotide sequence. For example, in some embodiments In the method, a nucleobase editing component, e.g., a deaminase component, is used to program polynucleotides. and a further heterologous moiety or domain that is part of the nucleotide-binding domain that can be bound. Additional heterologous moieties or domains that can interact, associate, or form complexes. In some embodiments, the additional heterologous moiety can include a polypeptide. Some examples are capable of binding, interacting, associating, or forming complexes with In embodiments, the additional heterologous moiety binds to, interacts with, or associates with the polynucleotide, or can form a complex. In some embodiments, the additional heterologous moiety In some embodiments, additional The heterologous moiety can be attached to a polypeptide linker. In this case, the additional heterologous moiety can be attached to the polynucleotide linker. The species portion may be a protein domain. In some embodiments, additional heterologous sequences may be present. The seed portion contains the K homology (KH) domain, the MS2 coat protein domain, and the PP7 coat protein. domain, SfMu Com coat protein domain, steryl α motif, telomerase Ku binding binding motif and Ku protein, telomerase Sm7 binding motif and Sm7 protein, or an RNA recognition motif.

[0302] The base editor system can further comprise a guide polynucleotide component. The components of the base editor system may be linked by covalent bonds, non-covalent interactions, or both. It should be understood that the compounds may be coupled to one another via any combination of bonds and interactions. In some embodiments, the deaminase domain is selected from the group consisting of: It can be targeted to a target nucleotide sequence. For example, in some embodiments The nucleobase editing component of the base editor system, e.g., the deaminase component, interacts with and binds to a portion or segment of a nucleotide (e.g., a polynucleotide motif) or further heterologous moieties or domains (e.g. RNA or DNA binding) that can form complexes. In some embodiments, the polypeptide may comprise a polynucleotide binding domain, such as a ligation protein. In the present invention, an additional heterologous moiety or domain (e.g., a polypeptide such as an RNA or DNA binding protein) may be used. The nucleotide-binding domain can be fused or linked to the deaminase domain. In some embodiments, the additional heterologous moiety binds to, interacts with, or associates with the polypeptide. In some embodiments, the polypeptide may be bound to or complexed with the polypeptide. Thus, the additional heterologous moiety binds to, interacts with, associates with, or binds to the polynucleotide. In some embodiments, the additional heterologous nucleotide can be conjugated to a heterologous nucleotide. The moiety is capable of binding to a guide polynucleotide. In some embodiments, the additional heterologous moiety can be attached to the polypeptide linker. In some embodiments, the additional heterologous moiety can be attached to the polynucleotide linker. The additional heterologous moiety may be a protein domain. The additional heterologous moieties are the K homology (KH) domain, the MS2 coat protein domain, and the PP7 coat protein domain. Protein domain, SfMu Com coat protein domain, sterile alpha motif, telomerase 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.

[0303] In one embodiment, the base editor system includes a base excision repair (BER) component. The components of the base editor system can further comprise an inhibitor of a shared base. through binding, non-covalent interactions, or any combination of such binding and interactions. It should be understood that inhibitors of BER components can be linked to each other by base excision repair. In some embodiments, the inhibitor of base excision repair is a uracil DNA group inhibitor. In some embodiments, the inhibitors of base excision repair may be glycosylase inhibitors (UGIs). can be an inosine base excision repair inhibitor. In some embodiments, the inhibitor of base excision repair The factor binds to the target nucleotide via a polynucleotide-programmable nucleotide-binding domain. In some embodiments, the polynucleotide sequence can be targeted to a nucleotide sequence. Ramming-competent nucleotide-binding domains fused or linked to inhibitors of base excision repair In some embodiments, polynucleotide programmable nucleotide binding can be performed. The domain is fused or linked to a deaminase domain and an inhibitor of base excision repair. In some embodiments, the polynucleotide programmable nucleotide The base-binding domain interacts non-covalently with inhibitors of base excision repair or binds to salts. By associating with the inhibitor of base excision repair, the inhibitor of base excision repair is directed to the target nucleoside. For example, in some embodiments, the targeting can be to a nucleotide sequence such as Inhibitors of base excision repair components inhibit programmable nucleotide bonds in polynucleotides. interacting, associating, or complexing with further heterologous moieties or domains that are part of the binding domain In some embodiments, the base removal may comprise additional heterologous moieties or domains that can form complexes. The inhibitor of repair is targeted to the target nucleotide sequence by the guide polynucleotide. For example, in some embodiments, an inhibitor of base excision repair can be Interacts with a portion or segment of a polynucleotide (e.g., a polynucleotide motif) Further heterologous moieties or domains (e.g., RNA or or a polynucleotide-binding domain such as a DNA-binding protein). In this embodiment, additional heterologous moieties or domains of the guide polynucleotide (e.g. For example, polynucleotide-binding domains such as RNA or DNA binding proteins are involved in base excision. In some embodiments, the additional heterologous moiety may be fused or linked to an inhibitor of repair. The molecule can bind, interact, associate, or complex with the polynucleotide. In some embodiments, the additional heterologous moiety is capable of binding to the guide polynucleotide. In some embodiments, the additional heterologous moiety can be added to the polypeptide linker. In some embodiments, the additional heterologous moiety can be linked to the polynucleoside. The additional heterologous moiety can be a protein domain that can be linked to a tid linker. In some embodiments, the additional heterologous moiety may be a K homology (KH) domain, an MS2 domain, or a coat protein domain, PP7 coat protein domain, SfMu Com coat protein domain Main, sterile alpha motif, telomerase Ku binding motif and Ku protein, telomerase The motif may be a nucleotide sequence encoding the Sm7 nucleotide sequence and the Sm7 protein, or an RNA recognition motif. "Base editing activity" refers to the ability to chemically change bases within a polynucleotide. In one embodiment, the first base is converted to the second base. The base editing activity is a cytidine deaminase activity, for example, converting the target C·G to T·A. In another embodiment, the base editing activity is adenosine deaminase activity. For example, it is the activity of converting A·T to G·C.

[0304] "Beta-2 microglobulin (B2M) polypeptide" is a reference to UniProt accession number P61769. or a fragment thereof, and has immunomodulatory activity. By B2M is meant a protein. Exemplary B2M polypeptide sequences are provided below. >sp|P61769|B2MG__HUMAN Beta-2-Microglobulin OS=Homo sapiens OX=9606 G N=B2M PE=1 SV=1 MSRSVALAVLALLSLSGLEAIQRTPKIQVYSRHPAENGKSNFLNCYVSGFHPSDIEVDLL KNGERIEKVEHSDLSFSKDWSFYLLYYTEFTPTEKDEYACRVNHVTLSQPKIVKWDRDM

[0305] "Beta-2 microglobulin (B2M) polynucleotide" refers to a polynucleotide encoding a B2M polypeptide. The beta-2 microglobulin gene is a nucleic acid molecule that encodes the major histocompatibility complex (MHC). B2M encodes a serum protein associated with B2M. B2M is involved in non-self recognition by host CD8+ T cells. Exemplary B2M polynucleotide sequences are provided below. >DQ217933.1 Homo sapiens beta-2-microglobulin (B2M) gene, complete cds CATGTCATAAATGGTAAGTCCAAGAAAAATACAGGTATTCCCCCCCAAAGAAAACTGTAAAATCGACTTTTTTCTATCTG TACTGTTTTTTATTGGTTTTTAAATTGGTTTTCCAAGTGAGTAAATCAGAATCTATCTGTAATGGATTTTAAATTTAGTG TTTCTCTGTGATGTAGTAAACAAGAAACTAGAGGCAAAAATAGCCCTGTCCCTTGCTAAACTTCTAAGGCACTTTTCTAG TACAACTCAACACTAACATTTCAGGCCTTTAGTGCCTTATATGAGTTTTTAAAAGGGGGAAAAGGGAGGGAGCAAGAGTG TCTTAACTCATACATTTAGGCATAACAATTATTCTCATATTTTAGTTATTGAGAGGGCTGGTAGAAAAACTAGGTAAATA ATATTAATAATTATAGCGCTTATTAAACACTACAGAACACTTACTATGTACCAGGCATTGTGGGAGGCTCTCTCTTGTGC ATTATCTCATTTCATTAGGTCCATGGAGAGTATTGCATTTTCTTAGTTTAGGCATGGCCTCCACAATAAAGATTATCAAA AGCCTAAAAATATGTAAAAGAAACCTAGAAGTTATTTGTTGTGCTCCTTGGGGAAGCTAGGCAAATCCTTTCAACTGAAA ACCATGGTGACTTCCAAGATCTCTGCCCCTCCCCATCGCCATGGTCCACTTCCTCTTCTCACTGTTCCTCTTAGAAAAGA TCTGTGGACTCCACCACCACGAAATGGCGGCACCTTATTTATGGTCACTTTAGAGGGTAGGTTTTCTTAATGGGTCTGCC TGTCATGTTTAACGTCCTTGGCTGGGTCCAAGGCAGATGCAGTCCAAACTCTCACTAAAATTGCCGAGCCCTTTGTCTTC CAGTGTCTAAAATATTAATGTCAATGGAATCAGGCCAGAGTTTGAATTCTAGTCTCTTAGCCTTTGTTTCCCCTGTCCAT AAAATGAATGGGGGTAATTCTTTCCTCCTACAGTTTATTTATATATTCACTAATTCATTCATTCATCCATCCATTCGTTC ATTCGGTTTACTGAGTACCTACTATGTGCCAGCCCCTGTTCTAGGGTGGAAACTAAGAGAATGATGTACCTAGAGGGCGC TGGAAGCTCTAAAGCCCTAGCAGTTACTGCTTTTACTATTAGTGGTCGTTTTTTTCTCCCCCCCGCCCCCCGACAAATCA ACAGAACAAAGAAAATTACCTAAACAGCAAGGACATAGGGAGGAACTTCTTGGCACAGAACTTTCCAAACACTTTTTCCT GAAGGGATACAAGAAGCAAGAAAGGTACTCTTTCACTAGGACCTTCTCTGAGCTGTCCTCAGGATGCTTTTGGGACTATT TTTCTTACCCAGAGAATGGAGAAACCCTGCAGGGAATTCCCAAGCTGTAGTTATAAACAGAAGTTCTCCTTCTGCTAGGT AGCATTCAAAGATCTTAATCTTCTGGGTTTCCGTTTTCTCGAATGAAAAATGCAGGTCCGAGCAGTTAACTGGCTGGGGC ACCATTAGCAAGTCACTTAGCATCTCTGGGGCCAGTCTGCAAAGCGAGGGGGCAGCCTTAATGTGCCTCCAGCCTGAAGT CCTAGAATGAGCGCCCGGTGTCCCAAGCTGGGGCGCGCACCCCAGATCGGAGGGCGCCGATGTACAGACAGCAAACTCAC CCAGTCTAGTGCATGCCTTCTTAAACATCACGAGACTCTAAGAAAAGGAAACTGAAAACGGGAAAGTCCCTCTCTCTAAC CTGGCACTGCGTCGCTGGCTTGGAGACAGGTGACGGTCCCTGCGGGCCTTGTCCTGATTGGCTGGGCACGCGTTTAATAT AAGTGGAGGCGTCGCGCTGGCGGGCATTCCTGAAGCTGACAGCATTCGGGCCGAGATGTCTCGCTCCGTGGCCTTAGCTG TGCTCGCGCTACTCTCTCTTTCTGGCCTGGAGGCTATCCAGCGTGAGTCTCTCCTACCCTCCCGCTCTGGTCCTTCCTCT CCCGCTCTGCACCCTCTGTGGCCCTCGCTGTGCTCTCTCGCTCCGTGACTTCCCTTCTCCAAGTTCTCCTTGGTGGCCCG CCGTGGGGCTAGTCCAGGGCTGGATCTCGGGGAAGCGGCGGGGTGGCCTGGGAGTGGGGAAGGGGGTGCGCACCCGGGAC GCGCGCTACTTGCCCCTTTCGGCGGGGAGCAGGGGAGACCTTTGGCCTACGGCGACGGGAGGGTCGGGACAAAGTTTAGG GCGTCGATAAGCGTCAGAGCGCCGAGGTTGGGGGAGGGTTTCTCTTCCGCTCTTTCGCGGGGCCTCTGGCTCCCCCAGCG CAGCTGGAGTGGGGGACGGGTAGGCTCGTCCCAAAGGCGCGGCGCTGAGGTTTGTGAACGCGTGGAGGGGCGCTTGGGGT CTGGGGGAGGCGTCGCCCGGGTAAGCCTGTCTGCTGCGGCTCTGCTTCCCTTAGACTGGAGAGCTGTGGACTTCGTCTAG GCGCCCGCTAAGTTCGCATGTCCTAGCACCTCTGGGTCTATGTGGGGCCACACCGTGGGGAGGAAACAGCACGCGACGTT TGTAGAATGCTTGGCTGTGATACAAAGCGGTTTCGAATAATTAACTTATTTGTTCCCATCACATGTCACTTTTAAAAAAT TATAAGAACTACCCGTTATTGACATCTTTCTGTGTGCCAAGGACTTTATGTGCTTTGCGTCATTTAATTTTGAAAACAGT TATCTTCCGCCATAGATAACTACTATGGTTATCTTCTGCCTCTCACAGATGAAGAAACTAAGGCACCGAGATTTTAAGAA ACTTAATTACACAGGGGATAAATGGCAGCAATCGAGATTGAAGTCAAGCCTAACCAGGGCTTTTGCGGGAGCGCATGCCT TTTGGCTGTAATTCGTGCATTTTTTTTTAAGAAAAACGCCTGCCTTCTGCGTGAGATTCTCCAGAGCAAACTGGGCGGCA TGGGCCCTGTGGTCTTTTCGTACAGAGGGCTTCCTCTTTGGCTCTTTGCCTGGTTGTTTCCAAGATGTACTGTGCCTCTT ACTTTCGGTTTTGAAAACATGAGGGGGTTGGGCGTGGTAGCTTACGCCTGTAATCCCAGCACTTAGGGAGGCCGAGGCGG GAGGATGGCTTGAGGTCCGTAGTTGAGACCAGCCTGGCCAACATGGTGAAGCCTGGTCTCTACAAAAAATAATAACAAAA ATTAGCCGGGTGTGGTGGCTCGTGCCTGTGGTCCCAGCTGCTCCGGTGGCTGAGGCGGGAGGATCTCTTGAGCTTAGGCT TTTGAGCTATCATGGCGCCAGTGCACTCCAGCGTGGGCAACAGAGCGAGAGACCCTGTCTCTCTCAAAAGAAAAAAAAAAAA AAAGAAAGAGAAAAGAAAAGAAAGAAAGAAGTGAAGGTTTGTCAGTCAGGGGAGCTGTAAAACCATTAATAAAGATAATC CAAGATGGTTACCAAGACTGTTGAGGACGCCAGAGATCTTGAGCACTTTCTAAGTACCTGGCAATACACTAAGCGCGCTC ACCTTTTCCTCTGGCAAAACATGATCGAAAGCAGAATGTTTTGATCATGAGAAAATTGCATTTAATTTGAATACAATTTA TTTACAACATAAAGGATAATGTATATATCACCACCATTACTGGTATTTGCTGGTTATGTTAGATGTCATTTTTAAAAAATA ACAATCTGATATTTAAAAAAAAATCTTATTTTGAAAATTTCCAAAGTAATACATGCCATGCATAGACCATTTCTGGAAGA TACCACAAGAAACATGTAATGATGATTGCCTCTGAAGGTCTATTTTCCTCCTCTGACCTGTGTGTGGGTTTTGTTTTTGT TTTACTGTGGGCATAAATTAATTTTTCAGTTAAGTTTTGGAAGCTTAAATAACTCTCCAAAAGTCATAAAGCCAGTAACT GGTTGAGCCCAAATTCAAACCCAGCCTGTCTGATACTTGTCCTCTTCTTAGAAAAGATTACAGTGATGCTCTCACAAAAT CTTGCCGCCTTCCCTCAAACAGAGAGTTCCAGGCAGGATGAATCTGTGCTCTGATCCCTGAGGCATTTAATATGTTCTTA TTATTAGAAGCTCAGATGCAAAGAGCTCTCTTAGCTTTTAATGTTATGAAAAAAATCAGGTCTTCATTAGATTCCCCAAT CCACCTCTTGATGGGGCTAGTAGCCTTTCCTTAATGATAGGGTGTTTCTAGAGAGATATATCTGGTCAAGGTGGCCTGGT ACTCCTCCTTCTCCCCACAGCCTCCCAGACAAGGAGGAGTAGCTGCCTTTTAGTGATCATGTACCCTGAATATAAGTGTA TTTAAAAGAATTTTATACACATATATTTAGTGTCAATCTGTATATTTAGTAGCACTAACACTTCTCTTCATTTTCAATGA AAAATATAGAGTTTATAATATTTTCTTCCCACTTCCCCATGGATGGTCTAGTCATGCCTCTCATTTTGGAAAGTACTGTT TCTGAAACATTAGGCAATATATTCCCAACCTGGCTAGTTTACAGCAATCACCTGTGGATGCTAATTAAAACGCAAATCCC ACTGTCACATGCATTACTCCATTTGATCATAATGGAAAGTATGTTCTGTCCCATTTGCCATAGTCCTCACCTATCCCTGT TGTATTTTATCGGGTCCAACTCAACCATTTAAGGTATTTGCCAGCTCTTGTATGCATTTAGGTTTTGTTTCTTTGTTTTT TAGCTCATGAAATTAGGTACAAAGTCAGAGAGGGGTCTGGCATATAAAACCTCAGCAGAAATAAAGAGGTTTTGTTGTTT GGTAAGAACATACCTTGGGTTGGTTGGGCACGGTGGCTCGTGCCTGTAATCCCAACACTTTGGGAGGCCAAGGCAGGCTG ATCACTTGAAGTTGGGAGTTCAAGACCAGCCTGGCCAACATGGTGAAATCCCGTCTCTACTGAAAATACAAAAATTAACC AGGCATGGTGGTGTGTGCCTGTAGTCCCAGGAATCACTTGAACCCAGGAGGCGGAGGTTGCAGTGAGCTGAGATCTCACC ACTGCACACTGCACTCCAGCCTGGGCAATGGAATGAGATTCCATCCCAAAAAATAAAAAAATAAAAAAATAAAGAACATA CCTTGGGTTGATCCACTTAGGAACCTCAGATAATAACATCTGCCACGTATAGAGCAATTGCTATGTCCCAGGCACTCTAC TAGACACTTCATACAGTTTAGAAAATCAGATGGGTGTAGATCAAGGCAGGAGCAGGAACCAAAAAGAAAGGCATAAACAT AAGAAAAAAAATGGAAGGGGTGGAAACAGAGTACAATAACATGAGTAATTTGATGGGGGCTATTATGAACTGAGAAATGA ACTTTGAAAAGTATCTTGGGGCCAAATCATGTAGACTCTTGAGTGATGTGTTAAGGAATGCTATGAGTGCTGAGAGGGCA TCAGAAGTCCTTGAGAGCCTCCAGAGAAAGGCTCTTAAAAATGCAGCGCAATCTCCAGTGACAGAAGATACTGCTAGAAA TCTGCTAGAAAAAAAACAAAAAAGGCATGTATAGAGGAATTATGAGGGAAAGATACCAAGTCACGGTTTATTCTTCAAAA TGGAGGTGGCTTGTTGGGAAGGTGGAAGCTCATTTGGCCAGAGTGGAAATGGAATTGGGAGAAATCGATGACCAAATGTA AACACTTGGTGCCTGATATAGCTTGACACCAAGTTAGCCCCAAGTGAAATACCCTGGCAATATTAATGTGTCTTTTCCCG ATATTCCTCAGGTACTCCAAAGATTCAGGTTTACTCACGTCATCCAGCAGAGAATGGAAAGTCAAATTTCCTGAATTGCT ATGTGTCTGGGTTTCATCCATCCGACATTGAAGTTGACTTACTGAAGAATGGAGAGAGAATTGAAAAAGTGGAGCATTCA GACTTGTCTTTCAGCAAGGACTGGTCTTTCTATCTCTTGTACTACACTGAATTCACCCCCACTGAAAAAGATGAGTATGC CTGCCGTGTGAACCATGTGACTTTGTCACAGCCCAAGATAGTTAAGTGGGGTAAGTCTTACATTCTTTTGTAAGCTGCTG AAAGTTGTGTATGAGTAGTCATATCATAAAGCTGCTTTGATATAAAAAAGGTCTATGGCCATACTACCCTGAATGAGTCC CATCCCATCTGATATAAACAATCTGCATATTGGGATTGTCAGGGAATGTTCTTAAAGATCAGATTAGTGGCACCTGCTGA GATACTGATGCACAGCATGGTTTCTGAACCAGTAGTTTCCCTGCAGTTGAGCAGGGAGCAGCAGCAGCACTTGCACAAAT ACATATACACTCTTAACACTTCTTACCTACTGGCTTCCTCTAGCTTTTGTGGCAGCTTCAGGTATATTTAGCACTGAACG AACATCTCAAGAAGGTATAGGCCTTTGTTTGTAAGTCCTGCTGTCCTAGCATCCTATAATCCTGGACTTCTCCAGTACTT TCTGGCTGGATTGGTATCTGAGGCTAGTAGGAAGGGCTTGTTCCTGCTGGGGTAGCTCTAAAACAATGTATTCATGGGGTAGG AACAGCAGCCTATTCTGCCAGCCTATTTCTAACCATTTTAGACATTTGTTAGTACATGGTATTTAAAAGTAAACTTA ATGTCTTCCTTTTTTTTCTCCACTGTCTTTTTCATAGATCGGAGACATGTAAGCAGCATCATGGAGGTAAGTTTTTGACCT TGAGAAAATGTTTTTTGTTTCACTGTCCTGAGGACTATTTATAGACAGCTCTAACATGATAACCCTCACTATTGTGGAGAAC ATTGACAGAGTAACATTTTAGCAGGGAAAGAAGAATCCTACGGGTCATGTTCCCTTCTCCTGTGGAGTGGCATGAAGAA GGTGTATGGCCCACAGTATGGCCATATTACTGACCCTCTACAGAGAGGGCAAAGGAACTGCCAGTATGGTATTGCAGGAT AAAGGCAGGTGGTTACCCACATTACCTGCAAGGCTTTGATCTTTCTTCTGCCATTTCCACATTGGACATCTCTGCTGAGGG AGAGAAAATGAACCACTCTTTTCCTTTGTATAATGTTGTTTTATTCTTCAGACAGAGAGAGGAGTTATACAGCTCTGCA GACATCCCATTCCTGTATGGGGACTGTGTTTGCCTCTTAGAGGTTCCCCAGGCCACTAGAGGAGATAAAGGGAAAACAGATT GTTATAACTTGATATAATGATACTATAATAGATGTAACTACAAGGAGCTCCAGAAGCAAGAGAGAGGGAGGAACTTGGAC TTCTCTGCATCTTTAGTTGGAGTCCAAAGGCTTTTCAATGAAATTCTACTGCCCAGGGTACATTGATGCTGAAACCCCAT TCAAATCTCCTGTTATATTCTAGAACAGGGAATTGATTTGGGAGAGCATCAGGAAGGTGGATGATCTGCCCAGTCACACT GTTAGTAAATTGTAGAGCCAGGACCTGAACTCTAATATAGTCATGTGTTACTTAATGACGGGGACATGTTCTGAGAAATG CTTACACAAACCTAGGTGTTGTAGCCTACTACACGCATAGGCTACATGGTATAGCCTATTGCTCCTAGACTACAAACCTG TACAGCCTGTTACTGTACTGAATACTGTGGGCAGTTGTAACACAATGGTAAGTATTTGTGTATCTAAACATAGAAGTTGC AGTAAAAATATGCTATTTTAATCTTATGAGACCACTGTCATATATACAGTCCATCATTGACCAAAACATCATATCAGCAT TTTTTCTTCTAAGATTTTGGGAGCACCAAAGGGATACACTAACAGGATATACTCTTTATAATGGGTTTGGAGAACTGTCT GCAGCTACTTCTTTTAAAAAGGTGATCTACACAGTAGAAATTAGACAAGTTTGGTAATGAGATCTGCAATCCAAATAAAA TAAATTCATTGCTAACCTTTTTCTTTTCTTTTCAGGTTTGAAGATGCCGCATTTGGATTGGATGAATTCCAAATTCTGCT TGCTTGCTTTTTAATATTGATATGCTTATACACTTACACTTTATGCACAAAATGTAGGGTTATAATAATGTTAACATGGA CATGATCTTCTTTATAATTCTACTTTGAGTGCTGTCTCCATGTTTGATGTATCTGAGCAGGTTGCTCCACAGGTAGCTCT AGGAGGGCTGGCACTTAGAGGTGGGGAGCAGAGAATTCTCTTATCCAACATCAACATCTTGGTCAGATTTGAACTCTTC AATCTCTTGCACTCAAAGCTTGTTAAGATAGTTAAGCGTGCATAAGTTAACTTCCATTTACATACTCTGCTTAGAATTT GGGGGAAAATTTAGAAATATAATTGACAGGATTATTGGAAATTTGTTATAATGAATGAACATTTTGTCATATAAGATTC ATATTTACTTCTTATACATTTGATAAAGTAAGGCATGGTTGTGGTTAATCTGGTTTATTTTGTTCCACAAGTTAAATAA ATCATAAAACTTGATGTGTTATCTCTTATATCTCACTCCCACTATTACCCCTTTATTTTCAAACAGGGAAACAGTCTTCA AGTTCCACTTGGTAAAAATGTGAACCCCTTGTATAGAGTTTGGCTCACAGTGTAAAGGGCCTCAGTGATTCACATTT TCCAGATTAGGAATCTGATGCTCAAAGAAGTTAAATGGCATAGTTGGGGTGACACAGCTGTCTAGTGGGAGGCCAGCCTT CTATATTTTAGCCAGCGTTCTTTCCTGCGGGCCAGGTCATGAGGAGTATGCAGACTCTAAGAGGGAGCAAAAGTATCTGA AGGATTTAATATTTTAGCAAGGAATAGATATACAATCATCCCTTGGTCTCCCTGGGGGATTGGTTTCAGGACCCCTTCTT GGACACCAAATCTATGGATATTTAAGTCCCTTCTATAAAATGGTATAGTATTTGCATATAACCTATCCACATCCTCCTGT ATACTTTAAATCATTTCTAGATTACTTGTAATACCTAATACAATGTAAATGCTATGCAAATAGTTGTTATTGTTTAAGGA ATAATGACAAGAAAAAAAAGTCTGTACATGCTCAGTAAAGACACAACCATCCCTTTTTTCCCCAGTGTTTTTGATCCAT GGTTTGCTGAATCCACAGATGTGGAGCCCCTGGATACGGAAGGCCCGCTGTACTTTGAATGACAAATAACAGATTTAAA

[0306] The term "Cas9" or "Cas9 domain" refers to a Cas9 protein or a fragment thereof (e.g., Cas9 Active, inactive, or partially active DNA cleavage domains of Cas9 and / or gRNA binding of Cas9 Cas9 nuclease refers to an RNA-guided nuclease containing a c domain. asn1 nuclease or CRISPR (clustered regularly interspaced short palindrome) CRISPR is sometimes called a mobile genetic element (viral repeat)-associated nuclease. CRISPR is an adaptive immune system that provides defense against pathogens (e.g., viruses, transposable elements, and conjugative plasmids). The star comprises a spacer, a sequence complementary to the preceding mobile element, and a target invading nucleic acid. CRISPR clusters are transcribed and processed into CRISPR RNA (crRNA). Type II CRISPR In our system, the correct processing of pre-crRNA is mediated by a transcoding small RNA (tracrRNA), tracrRNA requires the ribonuclease 3 (rnc) and Cas9 protein. This guides the processing of pre-crRNA by Cas9 / crRNA / tracrRNA. The RNA endonuclease cleaves a linear or circular dsDNA target complementary to the spacer. The target strand that is not complementary to the crRNA is first endonucleolytically cleaved and then exonucleolytically cleaved. In nature, DNA binding and cleavage typically occurs via transcription. However, single guide RNA ("sgRNA" or The gene encoding the gene for the gene encoding the tracrRNA (gNRA for short) is engineered to incorporate aspects of both the crRNA and tracrRNA into a single RNA species. For example, Jinek M., Chylinski K., Fonfara I., Hauer M., Doudna J. See Charpentier E. Science 337: 816-821 (2012) (the entire contents of which are incorporated herein by reference). Cas9 targets short motifs (PAMs) in the CRISPR repeats. or protospacer adjacent motif) to help distinguish self from non-self. The sequence and structure of Cas9 nuclease are well known to those skilled in the art (see, for example, "Complete ete genome sequence of an Ml strain of Streptococcus pyogenes." Ferretti et al., JJ, McShan WM, Ajdic DJ, Savic DJ, Savic G., Lyon K., Primeaux C, Sezat e S., Suvorov AN, Kenton S., Lai HS, Lin SP, Qian Y., Jia HG, Najar FZ , Ren Q., Zhu H., Song L., White J., Yuan X., Clifton SW, Roe BA, McLaughlin RE, Proc. Natl. Acad. Sci. USA 98: 4658-4663(2001); "CRISPR RNA maturation by trans-encoded small RNA and host factor RNase III. " Deltcheva E., Chylinski K., Sharma CM., Gonzales K., Chao Y., Pirzada ZA, Eckert MR, Vogel J., Char Pentier E., Nature 471: 602-607(2011); and "A programmable dual-RNA-guided DNA A endonuclease in adaptive bacterial immunity." Jinek M., Chylinski K., Fonfara I., Hauer M., Doudna JA, Charpentier E. Science 337: 816-821(2012). The entire contents of which are incorporated herein by reference.) Cas9 orthologs include, but are not limited to: Although not exclusively S. pyogenes, S. thermophilus have been described in various species, including S. pyogenes and S. thermophilus. Additional suitable Cas9 nucleases and sequences will be apparent to those of skill in the art based on the present disclosure. Such Cas9 nucleases and sequences are described in Chylinski, Rhun, and Charpentier, "The tracrRNA and Cas9 families of type II CRISPR-Cas immunity systems" (2013) R NA Biology 10: 5, 726-737. , the entire contents of which are incorporated herein by reference. In some embodiments, Cas9 Nucleases have an inactive (e.g., inactivated) DNA cleavage domain, i.e., their C as9 is a nickase.

[0307] Nuclease-inactivated Cas9 proteins are interchangeably referred to as "dCas9" proteins (nuclease-"d The Cas9 protein (or Cas9 protein) has an inactive DNA cleavage domain. Methods for generating the ribosomal protein (or fragments thereof) are known (e.g., Jinek et al., Science. 337: 816-8 21(2012); Qi et al, “Repurposing CRISPR as an RNA-Guided Platform for Sequence-S pecific Control of Gene Expression" (2013) Cell. 28; 152(5): 1173-83 (each content (The text is incorporated herein by reference.) For example, the DNA cleavage domain of Cas9 can be expressed as a HNH nucleic acid. It is known to contain two subdomains: the leucine subdomain and the RuvC1 subdomain. The HNH subdomain cleaves the strand complementary to the gRNA, while the RuvC1 subdomain cleaves the non-complementary strand. Mutations within these subdomains can suppress the nuclease activity of Cas9. For example, mutations D10A and H840A completely abolish the nuclease activity of S. pyogenes Cas9. Activates (Jinek et al, Science. 337: 816-821(2012); Qi et al, Cell. 28; 152(5) : 1173-83 (2013)). In some embodiments, proteins comprising fragments of Cas9 are provided. For example, in some embodiments, the protein contains one of the following two Cas9 domains: (1) a gRNA binding domain of Cas9; and (2) a DNA cleavage domain of Cas9. In this context, proteins containing Cas9 or fragments thereof are referred to as "Cas9 variants." The variants share homology with Cas9 or a fragment thereof. For example, the Cas9 variants are At least about 70% identical, at least about 80% identical, at least about 90% identical, at least about 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% identical, at least about 99.5% identical, or at least about 99.9% identical. In some embodiments, the Cas9 mutant has 1, 2, 3, 4, 5, 6, 7, or more functional groups compared to wild-type Cas9. 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 21, 24, 25, 26, 27, 2 8, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 4 In some embodiments, the Cas gene may have 8, 49, 50 or more amino acid changes. 9 variants include a fragment of Cas9 (e.g., the gRNA binding domain or the DNA cleavage domain), The fragment is at least about 70% identical to the corresponding fragment of wild-type Cas9, and at least about 80% Identical, at least about 90% identical, at least about 95% identical, at least about 96% identical % identical, at least about 97% identical, at least about 98% identical, at least about 9 9% identical, at least about 99.5% identical, or at least about 99.9% identical. In some embodiments, the fragments are at least 30% of the amino acid length of the corresponding wild-type Cas9. At least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60% , at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% identical, at least 96%, at least 97%, at least 98%, At least 99%, or at least 99.5%.

[0308] In some embodiments, the fragment is at least 100 amino acids in length. In the above, the fragment is at least 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 60 0, 650, 700, 750, 800, 850, 900, 950, 1000, 1050, 1100, 1150, 1200, 1250, or In some embodiments, the wild-type Cas9 is at least 1300 amino acids in length. Cas9 from Escherichia coli pyogenes (NCBI reference sequence: NC_17053.1, nucleotide sequence and The amino acid sequence is as follows: ATGGATAAGAAATACTCAATGCTTAGATATCGGCACAAATAGCGTCGGATGGGCGGTGATCACTGATGATTATAAGGT TCCGTCTAAAAAGTTCAAGGTTCTGGGAAATACAGACCGCCACAGTATCAAAAAAAATCTTATAGGGGCTCTTTTATTTG GCAGTGGAGAGACAGCGGAAGCGACTCGTCTCAAACGGACAGCTCGTAGAAGGTATACACGTCGGAAGAATCGTATTTGT TATCTACAGGAGATTTTTTCAAATGAGATGGCGAAAGTAGATGATAGTTTCTTTCATCGACTTGAAGAGTCTTTTTTGGT GGAAGAAGACAAGAAGCATGAACGTCATCCTATTTTTGGAAATATAGTAGATGAAGTTGCTTATCATGAGAAATATCCAA CTATCTATCATCTGCGAAAAAAATTGGCAGATTCTACTGATAAAGCGGATTTGCGCTTAATCTATTTGGCCTTAGCGCAT ATGATTAAGTTTCGTGGTCATTTTTTGATTGAGGGAGATTTAAATCCTGATAATAGTGATGTGGACAAACTATTTATCCA GTTGGTACAAATCTACAATCAATTATTTGAAGAAAACCCTATTAACGCAAGTAGAGTAGATGCTAAAGCGATTCTTTCTG CACGATTGAGTAAATCAAGACGATTAGAAAATCTCATTGCTCAGCTCCCCGGTGAGAAGAGAAATGGCTTGTTTGGGAAT CTCATTGCTTTGTCATTGGGATTGACCCCTAATTTTAAATCAAATTTTGATTTGGCAGAAGATGCTAAATTACAGCTTTC AAAAGATACTTACGATGATGATTTAGATAATTTATTGGCGCAAATTGGAGATCAATATGCTGATTTGTTTTTGGCAGCTA AGAATTTATCAGATGCTATTTTACTTTCAGATATCCTAAGAGTAAATAGTGAAATAACTAAGGCTCCCCTATCAGCTTCA ATGATTAAGCGCTACGATGAACATCATCAAGACTTGACTCTTTTAAAAGCTTTAGTTCGACAACAACTTCCAGAAAAGTA TAAAGAAATCTTTTTTGATCAATCAAAAAACGGATATGCAGGTTATATTGATGGGGGAGCTAGCCAAGAAGAATTTTATA AATTTATCAAACCAATTTTAGAAAAAATGGATGGTACTGAGGAATTATTGGTGAAACTAAATCGTGAAGATTTGCTGCGC AAGCAACGACCTTTGACAACGGCTCTATTCCCCATCAAATTCACTTGGGTGAGCTGCATGCTATTTTGAGAAGACAAGA AGACTTTTATCCATTTTTAAAAGACAATCGTGAGAAGATTGAAAAAATCTTGACTTTTCGAATTCCTTATTATGTTGGTC CATTGGCGCGTGGCAATAGTCGTTTTGCATGGATGACTCGGAAGTCTGAAGAAACAATTACCCCATGGAATTTTGAAGAA GTTGTCGATAAAGGTGCTTCAGCTCAATCATTTATTGAACGCATGACAAACTTTGATAAAAATCTTCCAAATGAAAAAGT ACTACCAAAACATAGTTTGCTTATGAGTATTTTACGGTTTATAACGAATTGACAAAGGTCAAATATGTTACTGAGGGAA TGCGAAAACCAGCATTTCTTTCAGGTGAACAGAAGAAAGCCATTGTTGATTTACTCTTCAAAACAAATCGAAAAGTAACC GTTAAGCAATTAAAAAGAAGATTATTTCAAAAAAATAGAATGTTTTGATAGTGTTGAAATTTCAGGAGTTGAAGATAGATT TAATGCTTCATTAGGCGCCTACCATGATTTGCTAAAAATTATTAAAGATAAAGATTTTTTGGATAATGAAGAAAATGAAG ATATCTTAGAGGATATTGTTTTAACATTGACCTTATTTGAAGATAGGGGGATGATTGAGGAAAGACTTAAAACATATGCT CACCTCTTTGATGATAAGGTGATGAAACAGCTTAAACGTCGCCGTTATACTGGTTGGGGACGTTTGTCTCGAAAATTGAT TAATGGTATTAGGGATAAGCAATCTGGCAAAACAATATTAGATTTTTTGAAATCAGATGGTTTTGCCAATCGCAATTTTA TGCAGCTGATCCATGATGATAGTTTGACATTTAAAGAAGATATTCAAAAAGCACAGGTGTCTGGACAAGGCCATAGTTTA CATGAACAGATTGCTAACTTAGCTGGCAGTCCTGCTATTAAAAAAGGTATTTTACAGACTGTAAAAATTGTTGATGAACT GGTCAAAGTAATGGGGCATAAGCCAGAAAATATCGTTATTGAAATGGCACGTGAAAATCAGACAACTCAAAAGGGCCAGA AAAATTCGCGAGAGCGTATGAAACGAATCGAAGAAGGTATCAAAGAATTAGGAAGTCAGATTCTTAAAGAGCATCCTGTT GAAAATACTCAATTGCAAAATGAAAAGCTCTATCTCTATTATCTACAAAATGGAAGAGACATGTATGTGGACCAAGAATT AGATATTAATCGTTTAAGTGATTATGATGTCGATCACATTGTTCCACAAAGTTTCATTAAAGACGATTCAATAGACAATA AGGTACTAACGCGTTCTGATAAAAATCGTGGTAAATCGGATAACGTTCCAAGTGAAGAAGTAGTCAAAAAGATGAAAAAC TATTGGAGACAACTTCTAAACGCCAAGTTAATCACTCAACGTAAGTTTGATAATTTAACGAAAGCTGAACGTGGAGGTTT GAGTGAACTTGATAAAGCTGGTTTTATCAAACGCCAATTGGTTGAAACTCGCCAAATCACTAAGCATGTGGCACAAATTT TGGATAGTCGCATGAATACTAAATACGATGAAAATGATAAACTTATTCGAGAGGTTAAAGTGATTACCTTAAAATCTAAA TTAGTTTCTGACTTCCGAAAAGATTTCCAATTCTATAAAGTACGTGAGATTAACAATTACCATCATGCCCATGATGCGTA TCTAAATGCCGTCGTTGGAACTGCTTTGATTAAGAAATATCCAAAACTTGAATCGGAGTTTGTCTATGGTGATTATAAAG TTTATGATGTTCGTAAAATGATTGCTAAGTCTGAGCAAGAAATAGGCAAAGCAACCGCAAAATATTTCTTTTACTCTAAT ATCATGAACTTCTTCAAAACAGAAATTACACTTGCAAATGGAGAGATTCGCAAACGCCCTCTAATCGAAACTAATGGGGA AACTGGAGAAATTGTCTGGGATAAAGGGCGAGATTTTGCCACAGTGCGCAAAGTATTGTCCATGCCCCAAGTCAATATTG TCAAGAAAACAGAAGTACAGACAGGCGGATTCTCCAAGGAGTCAATTTTACCAAAAAGAAATTCGGACAAGCTTATTGCT CGTAAAAAGACTGGGATCCAAAAAAAATATGGTGGTTTTGATAGTCCAACGGTAGCTTATTCAGTCCTAGTGGTTGCTAA GGTGGAAAAAGGGAAATCGAAGAAGTTAAAATCCGTTAAAGAGTTACTAGGGATCACAATTATGGAAAGAAGTTCCTTTG AAAAAATCCGATTGACTTTTTAGAAGCTAAAGGATAAGGAAGTTAAAAGACTTAATCATTAAACTACCTAATAT AGTCTTTTTGAGTTAGAAAACGGTCGTAAACGGATGCTGGCTAGTGCCGGAATTACAAAAAGGAAATGAGCTGGCTCT GCCAAGCAAATGTGAATTTTTTATTTAGCTAGTCATTATGAAAAGTTGAAGGGTAGTCCAGAAGATAACGAACAAA AACAATTGTTTGTGGAGCAGCATAAGCATTATTTGAGATTATTGAGCAAATCAGTGAATTTTCTAAGCGTGTTATT TTAGCAGATGCCAATTTAGATAAAGTTCTTAGTGCATATAACAAACATAGAGACAAACCAATACGTGAACAAGCAGAAAA TATTATTCATTTATTTACGTTGACGAATCTTGGAGCTCCCGCTGCTTTTAAATATTTTGATACAACAATTGATCGTAAAC GATATACGTCTACAAAGAAGTTTTAGATGCCACTCTTATCCATCCATCCATGGTCTTTGAAACACGCATTGAT TTGAGTCAGCTAGGAGGTGACTGA

[0309] JPEG2024174899000003.jpg172166

[0310] (Single underline: HNH domain; double underline: RuvC domain)

[0311] In one embodiment, the wild-type Cas9 has the following nucleotide and / or amino acid sequence: Corresponding to or containing the following sequence: ATGGATAAAAAGTATTCTATTGGTTTAGACATCGGCACTAATTCCGTTGGATGGGCTGTCATAACCGATGAATACAAAGT ACCTTCAAAGAAATTTAAGGTGTTGGGGAACACAGACCGTCATTCGATTAAAAAAGAATCTTATCGGTGCCCTCCTATTCG ATAGTGGCGAAACGGCAGAGGCGACTCGCCTGAAACGAACCGCTCGGAGAAGGTATACACGTCGCAAGAACCGAATATGT TACTTACAAGAAATTTTTAGCAATGAGATGGCCAAAGTTGACGATTCTTTCTTTCACCGTTTGGAAGAGTCCTTCCTTGT CGAAGAGGACAAGAAACATGAACGGCACCCCATCTTTGGAAACATAGTAGATGAGGTGGCATATCATGAAAAGTACCCAA CGATTTATCACCTCAGAAAAAAGCTAGTTGACTCAACTGATAAAGCGGACCTGAGGTTAATCTACTTGGCTCTTGCCCAT ATGATAAAGTTCCGTGGGCACTTTCTCATTGAGGGTGATCTAAATCCGGACAACTCGGATGTCGACAAACTGTTCATCCA GTTAGTACAAACCTATAATCAGTTGTTTGAAGAGAACCCTATAAAATGCAAGTGGCGTGGATGCGAAGGCTATTCTTAGCG CCCGCCTCTCTAAATCCCGACGGCTAGAAAAACCTGATCGCACAATTACCCGGAGAGAAAAAAATGGTTGTTCGGTAAC CTTATAGCGCTCTCACTAGGCCTGACACCAAATTTAAGTCGAACTTCGACTTAGCTGAAGATGCCAAATTGCAGCTTAG TAAGGACACGTACGATGACGATCTCGCAATCTACTGGCACAAATTGGAGATCAGTATGCGGACTTATTTTTGGCTGCCA AAAACCTTAGCGATGCAATCCTCCTATCTGACATACTGAGAGTTAATACTGAGATTACCAAGGCGCCGTTATCCCGCTTCA ATGATCAAAAGGTACGATGAACATCACCAAGACTTGACACTTCTCAAGGCCCTAGTCCGTCAGCAACTGCCTGAGAAATA TAAGGAAATATTCTTTGATCAGTCGAAAACGGGTACGCAGGTTATATTGACGGGCGGAGCGAGTCAAGAGGAATTCTACA AGTTTATCAAACCCATATTAGAGAATGGATGGGACGGAAGAGTTGCTTGTAAAACTCAATCGCGAAGATCTACTGCGA AAGCAGCGGACTTTCGACAACGGTAGCATTCCACATCAAATCCACTTAGGCGAATTGCATGCTATACTTAGAAGGCAGGA GGATTTTTATCCGTTCCTCAAAGACAATCGTGAAAAGATTGAGAAAATCCTAACCTTTCGCATACCTACTATGTGGGAC CCCTGGCCGAGGGAACTCTCGGTTCGCATGGATGACAAGAAAGTCCGAAGAAACGATTACTCCATGGAATTTTGAGGAA GTTGTCGATAAAGGTGCGTCAGCTCAATCGTTCATCGAGAGGATGACCAACTTTGACAAGAATTTACCGAACGAAAAAGT ATTGCCTAAGCACAGTTTACTTTACGAGTATTTCACAGTGTACAATGAACTCACGAAAGTTAAGTATGTCACTGAGGGCA TGCGTAAACCCGCCTTTCTAAGCGGAGAACAGAAGAAAGCAATAGTAGATCTGTTATTCAAGACCAACCGCAAAGTGACA GTTAAGCAATTGAAAGAGGACTACTTTAAGAAAATTGAATGCTTCGATTCTGTCGAGATCTCCGGGGTAGAAGATCGATT TAATGCGTCACTTGGTACGTATCATGACCTCCTAAAGATAATTAAAGATAAGGACTTCCTGGATAACGAAGAGAATGAAG ATATCTTAGAAGATATAGTGTTGACTCTTACCCTCTTTGAAGATCGGGAAATGATTGAGGAAAGACTAAAAACATACGCT CACCTGTTCGACGATAAGGTTATGAAACAGTTAAAGAGGCGTCGCTATACGGGCTGGGGACGATTGTCGCGGAAACTTAT CAACGGGATAAGAGACAAGCAAAGTGGTAAAACTATTCTCGATTTTCTAAAGAGCGACGGCTTCGCCATAGGAACTTTA TGCAGCTGATCCATGATGACTCTTTAACCTTCAAAGAGGATATACAAAAGGCACAGGTTTCCGGACAAGGGGACTCATTG CACGAACATATTGCGAATCTTGCTGGGTTCGCCAGCCATCAAAGGGCATACTCCAGACAGTCAAAGTAGTGGATGAGCT AGTTAAGGTCATGGGACGTCACAAACCGGAAAACATTGTAATCGAGATGGCACGCGAAAATCAAACGACTCAGAAGGGGC AAAAAAACAGTCGAGAGCGGATGAAGAGAATAGAAGAGGGTATTAAAGAACTGGGCAGCCAGATCTTAAAGGAGCATCCT GTGGAAAATACCCAATTGCAGAACGAGAAACTTTACCTCTATTACCTACAAAATGGAAGGGACATGTATGTTGATCAGGA ACTGGACATAAACCGTTTATCTGATTACGACGTCGATCACATTGTACCCCAATCCTTTTTGAAGGACGATTCAATCGACA ATAAAGTGCTTACACGCTCGGATAAGAACCGAGGGAAAAGTGACAATGTTCCAAGCGAGGAAGTCGTAAAGAAAATGAAG AACTATTGGCGGCAGCTCCTAAATGCGAAACTGATAACGCAAAGAAAGTTCGATAACTTAACTAAAGCTGAGAGGGGTGG CTTGTCTGAACTTGACAAGGCCGGATTTATTAAACGTCAGCTCGTGGAAACCCGCCAAATCACAAAGCATGTTGCACAGA TACTAGATTCCCGAATGAATACGAAATACGACGAGAACGATAAGCTGATTCGGGAAGTCAAAGTAATCACTTTAAAGTCA AAATTGGTGTCGGACTTCAGAAAGGATTTTCAATTCTATAAAGTTAGGGAGATAAATAACTACCACCATGCGCACGACGC TTATCTTAATGCCGTCGTAGGGACCGCACTCATTAAGAAATACCCGAAGCTAGAAAGTGAGTTTGTGTATGGTGATTACA AAGTTTATGACGTCCGTAAGATGATCGCGAAAGCGAACAGGAGATAGGCAAGGCTACAGCCAAATACTTCTTTTATTCT AACATTATGAATTTCTTTAAGACGGAAATCACTCTGGCAAACGGAGAGATACGCAAACGACCTTTAATTGAAACCAATGG GGAGACAGGTGAAATCGTATGGGATAAGGGCCGGGACTTCGCGACGGTGAGAAAAGTTTTGTCCATGCCCCAAGTCAACA TAGTAAAGAAAACTGAGGTGCAGACCGGAGGGTTTTCAAAGGAATCGATTCTTCCAAAAAGGAATAGTGATAAGCTCATC GCTCGTAAAAAGGACTGGGACCCGAAAAAGTACGGTGGCTTCGATAGCCCTACAGTTGCCTATTCTGTCCTAGTAGTGGC AAAAGTTGAGAAGGGAAATCCAAGAAACTGAAGTCAGTCAAAGAATTATTGGGGATAACGATTATGGAGCGCTCGTCTT TTGAAAAGACCCCATCGACTTCCTTGAGGCGAAAGGTTACAAGGAAGTAAAAAAGGATCTCATAATTAAACTACCAAAG TATAGTCTGTTTGAGTTAGAAATGGCCGAAAACGGATGTTGGCTAGCGCCGGAGAGCTTCCAAAGGGGAACGAACTCGC ACTACCGTCTAAATACGTGAATTTCCTGTATTTAGCGTCCCATTACGAGAAGTTGAAAGGTTCACCTGAAGATAACGAAC AGAAGCAACTTTTTGTTGAGCAGCACAAACATTATCTCGACGAAATCATAGAGCAAATTTCGGAATTCAGTAAGAGAGTC ATCCTAGCTGATGCCAATCTGGACAAAGTATTAAGCGCATACAACAAGCACAGGGATAAACCCATACGTGAGCAGGCGGA AAATATTATCCATTTGTTTACTCTTACCAACCTCGGCGCTCCAGCCGCATTCAAGTATTTTGACACAACGATAGATCGCA AACGATACACTTCTACCAAGGAGGTGCTAGACGCGACACTGATTCACCAATCCATCACGGGATTATATGAAACTCGGATA GATTTGTCACAGCTTGGGGGTGACGGATCCCCAAGAAGAAGAGGAAAGTCTCGAGCGACTACAAAGACCATGACGGTGA TTATAAAGATCATGACATCGATTACAAGGATGACGATGACAAGGCTGCAGGA JPEG2024174899000004.jpg172165

[0312] (Single underline: HNH domain; double underline: RuvC domain)

[0313] In some embodiments, wild-type Cas9 is directed against Cas9 from Streptococcus pyogenes. (NCBI reference sequence: NC_002737.2 (nucleotide sequence below); and Uniprot reference sequence: String: Q99ZW2 (amino acid sequence below). ATGGATAAGAAATACTCAATGCTTAGATATCGGCACAAATAGCGTCGGATGGGCGGTGATCACTGATGAATATAAGGT TCCGTCTAAAAAGTTCAAGGTTCTGGGAAATACAGACCGCCACAGTATCAAAAAAAATCTTATAGGGGCTCTTTTATTTG ACAGTGGAGAGACAGCGGAAGCGACTCGTCTCAAACGGACAGCTCGTAGAAGGTATACACGTCGGAAGAATCGTATTTGT TATCTACAGGAGATTTTTTCAAATGAGATGGCGAAAGTAGATGATAGTTTCTTTCATCGACTTGAAGAGTCTTTTTTGGT GGAAGAAGACAAGAAGCATGAACGTCATCCTATTTTTGGAAATATAGTAGATGAAGTTGCTTATCATGAGAAATATCCAA CTATCTATCATCTGCGAAAAAAATTGGTAGATTCTACTGATAAAGCGGATTTGCGCTTAATCTATTTGGCCTTAGCGCAT ATGATTAAGTTTCGTGGTCATTTTTTGATTGAGGGAGATTTAAATCCTGATAATAGTGATGTGGACAAACTATTTATCCA GTTGGTACAAACCTACAATCAATTATTTGAAGAAAACCCTATTAACGCAAGTGGAGTAGATGCTAAAGCGATTCTTTCTG CACGATTGAGTAAATCAAGACGATTAGAAAATCTCATTGCTCAGCTCCCCGGTGAGAAGAAAAATGGCTTATTTGGGAAT CTCATTGCTTTGTCATTGGGTTTGACCCCTAATTTTAAATCAAATTTTGATTTGGCAGAAGATGCTAAATTACAGCTTTC AAAAGATACTTACGATGATGATTTAGATAATTTATTGGCGCAAATTGGAGATCAATATGCTGATTTGTTTTTGGCAGCTA AGAATTTATCAGATGCTATTTTACTTTCAGATATCCTAAGAGTAAATACTGAAATAACTAAGGCTCCCCTATCAGCTTCA ATGATTAAACGCTACGATGAACATCATCAAGACTTGACTCTTTTAAAAGCTTTAGTTCGACAACAACTTCCAGAAAAGTA TAAAGAAATCTTTTTTGATCAATCAAAAAACGGATATGCAGGTTATATTGATGGGGGAGCTAGCCAAGAAGAATTTTATA AATTTATCAAACCAATTTTAGAAAAAATGGATGGTACTGAGGAATTATTGGTGAAACTAAATCGTGAAGATTTGCTGCGC AAGCAACGACCTTTGACAACGGCTCTATTCCCCATCAAATTCACTTGGGTGAGCTGCATGCTATTTTGAGAAGACAAGA AGACTTTTATCCATTTTTAAAAGACAATCGTGAGAAGATTGAAAAAATCTTGACTTTTCGAATTCCTTATTATGTTGGTC CATTGGCGCGTGGCAATAGTCGTTTTGCATGGATGACTCGGAAGTCTGAAGAAACAATTACCCCATGGAATTTTGAAGAA GTTGTCGATAAAGGTGCTTCAGCTCAATCATTTATTGAACGCATGACAAACTTTGATAAAAATCTTCCAAATGAAAAAGT ACTACCAAAACATAGTTTGCTTATGAGTATTTTACGGTTTATAACGAATTGACAAAGGTCAAATATGTTACTGAAGGAA TGCGAAAACCAGCATTTCTTTCAGGTGAACAGAAGAAAGCCATTGTTGATTTACTCTTCAAAACAAATCGAAAAGTAACC GTTAAGCAATTAAAAAGAAGATTATTTCAAAAAAATAGAATGTTTTGATAGTGTTGAAATTTCAGGAGTTGAAGATAGATT TAATGCTTCATTAGGTACCTACCATGATTTGCTAAAAATTATTAAAGATAAAGATTTTTTGGATAATGAAGAAATGAAG ATATCTTAGAGGATATTGTTTTAACATTGACCTTATTTGAAGATAGGGAGATGATTGAGGAAAGACTTAAAACATATGCT CACCTCTTTGATGATAAGGTGATGAAACAGCTTAAACGTCGCCGTTATACTGGTTGGGGACGTTTGTCTCGAAAATTGAT TAATGGTATTAGGGATAAGCAATCTGGCAAAACAATATTAGATTTTTTGAAATCAGATGGTTTTGCCAATCGCAATTTTA TGCAGCTGATCCATGATGATAGTTTGACATTTAAAGAAGACATTCAAAAAGCACAAGTGTCTGGACAAGGCGATAGTTTA CATGAACATATTGCAAATTTAGCTGGTAGCCCTGCTATTAAAAAGGTATTTTACAGACTGTAAAAGTTGTTGATGAATT GGTCAAAGTAATGGGGCGGCATAAGCCAGAAATATCGTTATTGAAATGGCACGTGAAAATCAGACAACTCAAAAGGGCC AGAAAAATTCGCGAGAGCGTATGAAACGAATCGAAGAAGGTATCAAAGAATTAGGAAGTCAGATTCTTAAAAGAGCATCCT GTTGAAAATACTCAAATTGCAAATGAAAAGCTCTATCTCTATTATCTCCAAAATGGAAGAGACATGTATGTGGACCAAGA ATTAGATATTAATCGTTTAAGTGATTATGATGTCGATCACATTGTTCCACAAAGTTTCCTTAAAGACGATTCAATAGACA ATAAGGTCTTAACGCGTTCTGATAAAAATCGTGGTAAATCGGATAACGTTCCAAGTGAAGAAGTAGTCAAAAAGATGAAA AACTATTGGAGACAACTTCTAAACGCCAAGTTAATCACTCAACGTAAGTTTGATAATTTAACGAAAGCTGAACGTGGAGG TTTGAGTGAACTTGATAAAGCTGGTTTTATCAAACGCCAATTGGTTGAAACTCGCCAAATCACTAAGCATGTGGCACAAA TTTTGGATAGTCGCATGAATACTAAATACGATGAAAATGATAAACTTATTCGAGAGGTTAAAGTGATTACCTTAAAATCT AATTAGTTTCTGACTTCCGAAGATTTCCAATTCTATAAAGTACGTGAGATTAACAATTACCATCATGCCCATGATGC GTATCTAAATGCCGTCGTTGGAACTGCTTTGATTAAGAAATATCCAAAACTTGAATCGGAGTTTGTCTATGGTGATTATA AAGTTTATGATGTTCGTAAAATGATTGCTAAGTCTGAGCAAGAAATAGGCAAAGCAACCGCAAAATATTTCTTTTACTCT AATATCATGAACTTCTTCAAAACAGAAATTACACTTGCAAATGGAGAGATTCGCAAACGCCCTCTAATCGAAACTAATGG GGAAACTGGAGAAATTGTCTGGGATAAAGGGCGAGATTTTGCCACAGTGCGCAAAGTATTGTCCATGCCCCAAGTCAATA TTGTCAAGAAAACAGAAGTACAGACAGGCGGATTCTCCAAGGAGTCAATTTTTACCAAAAGAAATTCGGACAAGCTTATT GCTCGTAAAAAAGACTGGGATCCAAAAAAATATGGTGGTTTTGATAGTCCAACGGTAGCTTATTCAGTCCTAGTGGTTGC TAAGGTGGAAAAAGGGAAATCGAAGAAGTTAAAATCCGTTAAAGAGTTACTAGGGATCACAATTATGGAAAGAAGTTCCT TTGAAAAAAATCCGATTGACTTTTTAGAAGCTAAAGGATATAAGGAAGTTAAAAAAGACTTAATCATTAAACTACCTAAA TATAGTCTTTTTGAGTTAGAAAACGGTCGTAAACGGATGCTGGCTAGTGCCGGAGAATTACAAAAAGGAAATGAGCTGGC TCTGCCAAGCAAATATGTGAATTTTTTATATTTAGCTAGTCATTATGAAAAGTTGAAGGGTAGTCCAGAAGATAACGAAC AAAAACAATTGTTTGTGGAGCAGCATAAGCATTATTTAGATGAGATTATTGAGCAAATCAGTGAATTTTCTAAGCGTGTT ATTTTAGCAGATGCCAATTTAGATAAAGTTCTTAGTGCATATAACAAACATAGAGACAAACCAATACGTGAACAAGCAGA AAATATTATTCATTTATTTACGTTGACGAATCTTGGAGCTCCCGCTGCTTTTAAATATTTTGATACAACAATTGATCGTA AACGATATACGTCTACAAAAGAAGTTTTAGATGCCACTCTTATCCATCAATCCATCACTGGTCTTTATGAAACACGCATT GATTTGAGTCAGCTAGGAGGTGACTGA

[0314] JPEG2024174899000005.jpg168163(Underlined once: HNH domain; Underlined twice: RuvC domain)

[0315] In some embodiments, Cas9 is derived from Corynebacterium ulcerans (NCBI Refs: NC_01 5683.1, NC_017317.1); Corynebacterium diphtheria (NCBI Refs: NC_016782.1, NC_016 786.1); Spiroplasma syrphidicola (NCBI Ref: NC_021284.1); Prevotella intermedia (NCBI Ref: NC_017861.1); Spiroplasma taiwanense (NCBI Ref: NC_021846.1); Strept ococcus iniae (NCBI Ref: NC_021314.1); Belliella baltica (NCBI Ref: NC_018010.1) ; Psychroflexus torquisI (NCBI Ref: NC_018721.1); Streptococcus thermophilus (NC BI Ref: YP_820832.1), Listeria innocua (NCBI Ref: NP_472073.1), Campylobacter je juni (NCBI Ref: YP_002344900.1) or Neisseria meningitidis (NCBI Ref: YP_0 02342100.1), or Cas9 from any other organism.

[0316] In some embodiments, dCas9 contains one or more nucleotides that inactivate Cas9 nuclease activity. It corresponds to or comprises part or all of a mutated Cas9 amino acid sequence. For example, in some embodiments, the dCas9 domain contains the D10A and H840A mutations or contains a corresponding mutation in another Cas9. In some embodiments, dCas9 is The amino acid sequence of dCas9 (D10A and H840A) is as follows:

[0317] JPEG2024174899000006.jpg171165 (single underline: HNH domain; double underline: RuvC domain)

[0318] In some embodiments, the Cas9 domain comprises a D10A mutation, while the the residue at position 840 in the amino acid sequence provided herein, or The residue at the corresponding position in either sequence remains a histidine.

[0319] In other embodiments, D10A, e.g., resulting in nuclease-inactivated Cas9 (dCas9), and dCas9 variants having mutations other than H840A. For example, other amino acid substitutions at D10 and H840, or the nuclease domain of Cas9 may be used. Other substitutions within the domain (e.g., HNH nuclease subdomain and / or RuvC1 subdomain) (including substitutions in

[0320] In some embodiments, a variant or homolog of dCas9, which is at least about 70% identical , at least about 80% identical, at least about 90% identical, at least about 95% identical, at least about 98% Identical, at least about 99% identical, at least about 99.5% identical, or at least about 99.9% identical In some embodiments, the amino acid sequence is about 5 amino acids, about 10 amino acids, about 15 amino acids, or the like. amino acids, about 20 amino acids, about 25 amino acids, about 30 amino acids, about 40 amino acids, about 50 amino acids, about dCa having an amino acid sequence shorter or longer by 75 amino acids, about 100 amino acids or more The s9 variant will be offered.

[0321] In some embodiments, the Cas9 fusion proteins as provided herein are The full-length amino acid sequence of the s9 protein, e.g., comprising one of the Cas9 sequences provided herein. However, in other embodiments, the fusion proteins as provided herein , does not contain the full-length Cas9 sequence, but only a fragment thereof. For example, in some embodiments, The Cas9 fusion proteins provided herein comprise a Cas9 fragment, wherein the fragment binds to crRNA and tracrRN. A, or sgRNA that binds but contains a truncated version of the nuclease domain, e.g., functional nucleases in that they contain only the nuclease domain or no nuclease domain at all. It does not contain a lease domain.

[0322] Exemplary amino acid sequences of suitable Cas9 domains and Cas9 fragments are provided herein, and Cas9 Further suitable sequences of domains and fragments will be apparent to those skilled in the art.

[0323] In some embodiments, Cas9 is isolated from Corynebacterium ulcerans (NCBI Refs: NC_01 5683.1, NC_017317.1); Corynebacterium diphtheria (NCBI Refs: NC_016782.1, NC_016 786.1); Spiroplasma syrphidicola (NCBI Ref: NC_021284.1); Prevotella intermedia (NCBI Ref: NC_017861.1); Spiroplasma taiwanense (NCBI Ref: NC_021846.1); Strept ococcus iniae (NCBI Ref: NC_021314.1); Belliella baltica (NCBI Ref: NC_018010.1) ; Psychroflexus torquisI (NCBI Ref: NC_018721.1); Streptococcus thermophilus (NC BI Ref: YP_820832.1); Listeria innocua (NCBI Ref: NP_472073.1); Campylobacter je juni (NCBI Ref: YP_002344900.1); or Neisseria meningitidis (NCBI Ref: YP_002 This refers to Cas9 derived from 342100.1).

[0324] Additional Cas9 proteins (e.g., nuclease-dead Cas9 (dCas9), Cas9 nCas9, or nuclease-active Cas9, its variants and homologs It should be understood that within the scope of this disclosure are any and all Cas9 proteins, including: In some embodiments, Cas9 includes, but is not limited to, those provided below. The protein is a nuclease-inactive Cas9 (dCas9). In some embodiments, the Cas9 protein In some embodiments, the Cas9 protein is a Cas9 nickase (nCas9). The quality is nuclease-active Cas9.

[0325] Exemplary catalytically inactive Cas9 (dCas9): DKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGETEATRLKRTARRRRYTRRKNRICY LQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKLVDSTDKADLRIYLALAHM IKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNL IALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASM IKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRK QRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEEV VDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTV KQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAH LFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLH EHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEGIGELGSQILKEHPV ENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDAIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKN YWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKSK LVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSN IMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIA RKKDWDPKKYGGFDSPTVAYSVLVVAKVEGKKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKY SLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVI LADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRID LSQLGGD

[0326] Exemplary catalyst of Cas9ニッカーゼ(nCas9): DKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGETAEATRLKRTARRRYTRRKNRICY LQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHM IKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNL IALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASM IKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRK QRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEEETITPWNFEEV VDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTV KQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDRIEMIEERLKTYAH LFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLH EHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEGIGELGSQILKEHPV ENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKN YWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKSK LVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSN IMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIA RKKDWDPKKYGGFDSPTVAYSVLVVAKVEGKKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKY SLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVI LADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRID LSQLGGD

[0327] Exemplary catalyst activityCas9: DKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGETAEATRLKRTARRRYTRRKNRICY LQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHM IKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNL IALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASM IKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRK QRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEEETITPWNFEEV VDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTV KQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDRIEMIEERLKTYAH LFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLH EHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEGIGELGSQILKEHPV ENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKN YWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKSK LVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSN IMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIA RKKDWDPKKYGGFDSPTVAYSVLVVAKVEGKKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKY SLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVI LADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRID LSQLGGD

[0328] In some embodiments, Cas9 is expressed in the Archaea (Archaea), which comprise the domain and kingdom of unicellular prokaryotic microorganisms. In some embodiments, Cas9 refers to a Cas9 derived from, for example, Burstein et al., "New CRISPR-Cas systems from uncultivated microbes." Cell Res. 2017 Feb 21. doi: 10.1038 / cr.2017.21 refers to CasX or CasY, and the entire contents thereof. The text is incorporated herein by reference. Genome-resolved metagenomics can be used to study the ancient history of life. Many CRISPR-Cas systems have been identified, including Cas9, which was first reported in the fungal domain. Branched Cas9 proteins from the lab are able to activate CRISPR in little-studied nanoarchaea In bacteria, two previously unknown systems, CRIS and CRIS, were discovered as part of the CRIS system. The discovery of PR-CasX and CRISPR-CasY, which are the most compact proteins discovered to date, In some embodiments, Cas9 expresses CasX or a variant of CasX. In some embodiments, Cas9 represents CasY or a variant of CasY. Programmable DNA binding protein (napDNAbp) Other RNA-guided DNA binding proteins may also be used as the ng protein and are within the scope of this disclosure. It should be understood that:

[0329] In some embodiments, nucleic acid programmable DNA binding proteins (napDNAbp) or or any of the fusion proteins provided herein is a CasX or CasY protein. In some embodiments, the napDNAbp is a CasX protein. In some embodiments, the napDNAbp is a CasY protein. NAbps are at least 85% identical to naturally occurring CasX or CasY proteins and at least 9 0%, 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% In some embodiments, napDNAbp comprises an amino acid sequence having at least 100% identity to the napDNAbp. In some embodiments, the napDNAbp is a naturally occurring CasX or CasY protein. is at least 85% for any CasX or CasY protein described herein , 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 Contains amino acid sequences with at least 99.5% identity. CasX and Cas from other bacterial species It is to be understood that Y may also be used in accordance with the present disclosure.

[0330] CasX (uniprot.org / uniprot / F0NN87; uniprot.org / uniprot / F0NH53)

[0331] >tr|F0NN87|F0NN87_SULIH CRISPR-associated Casx protein OS = Icelandic Sulfolobus s (strain HVE10 / 4) GN = SiH_0402 PE=4 SV=1 MEVPLYNIFGDNYIQVATEAENSTIYNNKVEIDDEELRNVLNLAYKIAKNNEDAAAERRGKAKKKGEEGETTTSNIIL PLSGNDKNPWTETLKCYNFPTTVALSEVFKNFSQVKECEEVSAPSFVKPEFYEFGRSPGMVERTRRVKLEVEPHYLIIAA AGWVLTRLGKAKVSEGDYVGVNVFTRPTRGILYSLIQNVNGIVPGIKPETAFGLWIARKVSSVTNPNVSVVRIYTISDAV GQNPTTINGGFSIDLTKLLEKRYLLSERLEAIARNALSSNMRERYIVLANIIYEYLTG SKRLEDLLYFANRDLIMNL NSDDGKVRDLKLISAIVNGELIRGEG

[0332] >tr|F0NH53|F0NH53_SULIR CRISPR associated protein, Casx OS = Sulfolobus islandic us (strain REY15A) GN=SiRe_0771 PE=4 SV=1 MEVPLYNIFGDNYIQVATEAENSTIYNNKVEIDDEELRNVLNLAYKIAKNNEDAAAERRGKAKKKGEEGETTTSNIIL PLSGNDKNPWTETLKCYNFPTTVALSEVFKNFSQVKECEEVSAPSFVKPEFYKFGRSPGMVERTRRVKLEVEPHYLIMAA AGWVLTRLGKAKVSEGDYVGVNVFTRPTRGILYSLIQNVNGIVPGIKPETAFGLWIARKVSSVTNPNVSVVSIYTISDAV GQNPTTINGGFSIDLTKLLEKRDLLSERLEAIARNALSISSNMRERYIVLANYIYEYLTGSKRLEDLLYFANRDLIMNLN SDDGKVRDLKLISAYVNGELIRGEG

[0333] CasY (ncbi.nlm.nih.gov / protein / APG80656.1)

[0334] >APG80656.1 CRISPR-associated protein CasY [uncultured Parcubacteria group bacte rium] MSKRHPRISGVKGYRLHAQRLEYTGKSGAMRTIKYPLYSSPSGGRTVPREIVSAINDDYVGLYGLSNFDDLYNAEKRNEE KVYSVLDFWYDCVQYGAVFSYTAPGLLKNVAEVRGGSYELTKTLKGSHLYDELQIDKVIKFLNKKEISRANGSLDKLKKD IIDCFKAEYRERHKDQCNKLADDIKNAKKDAGASLGERQKKLFRDFFGISEQSENDKPSFTNPLNLTCCLLPFDTVNNNR NRGEVLFNKLKEYAQKLDKNEGSLEMWEYIGIGNSGTAFSNFLGEGFLGRLRENKITELKKAMMDITDAWRGQEQEEELE KRLRILAALTIKLREPKFDNHWGGYRSDINGKLSSWLQNYINQTVKIKEDLKGHKKDLKKAKEMINRFGESDTKEEAVVS SLLESIEKIVPDDSADDEKPDIPAIAIYRRFLSDGRLTLNRFVQREDVQEALIKERLEAEKKKKPKKRKKKSDAEDEKET IDFKELFPHLAKPLKLVPNFYGDSKRELYKKYKNAAIYTDALWKAVEKIYKSAFSSSLKNSFFDTDFDKDFFIKRLQKIF SVYRRFNTDKWKPIVKNSFAPYCDIVSLAENEVLYKPKQSRSRKSAAIDKNRVRLPSTENIAKAGIALARELSVAGFDWK DLLKKEEHEEYIDLIELHKTALALLLAVTETQLDISALDFVENGTVKDFMKTRDGNLVLEGRFLEMFSQSIVFSELRGLA GLMSRKEFITRSAIQTMNGKQAELLYIPHEFQSAKITTPKEMSRAFLDLAPAEFATSLEPESLSEKSLLKLKQMRYYPHY FGYELTRTGQGIDGGVAENALRLEKSPVKKREIKCKQYKTLGRGQNKIVLYVRSSYYQTQFLEWFLHRPKNVQTDVAVSG SFLIDEKKVKTRWNYDALTVALEPVSGSERVFVSQPFTIFPEKSAEEEGQRYLGIDIGEYGIAYTALEITGDSAKILDQN FISDPQLKTLREEVKGLKLDQRRGTFAMPSTKIARIRESLVHSLRNRIHHLALKHKAKIVYELEVSRFEEGKQKIKKVYA TLKKADVYSEIDADKNLQTTVWGKLAVASEISASYTSQFCGACKKLWRAEMQVDETITTQELIGTVRVIKGGTLIDAIKD FMRPPIFDENDTPFPKYRDFCDKHHISKKMRGNSCLFICPFCRANADADIQASQTIALLRYVKEEKKVEDYFERFRKLKN IKVLGQMKKI

[0335] The term "Cas12b" or "Cas12b domain" refers to the Cas12b / C2c1 protein or a fragment thereof. (e.g., an active, inactive, or partially active DNA cleavage domain of Cas12b and / or Ca refers to RNA-guided nucleases containing the gRNA-binding domain of s12b (those The contents of each of these are incorporated herein by reference. Cas12b orthologs include various It has been described in various species, including Alicyclobacillus acidoterrestris, Alicyclobacillus lobacillus acidophilus (Teng et al., Cell Discov. 2018 Nov 27; 4: 63), Bacillus hisashi, and Bacillus sp. V3-13. Additional suitable Ca The s12b nuclease and sequences will be apparent to those skilled in the art based on the present disclosure.

[0336] In some embodiments, a protein comprising Cas12b or a fragment thereof is referred to as a "Cas12b protein." Cas12b variants are called "variants." Cas12b variants share homology with Cas12b or fragments thereof. For example, a Cas12b variant may be at least about 70% identical to wild-type Cas12b, at least about 80% identical to wild-type Cas12b, or 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 In some embodiments, the Cas12b variant is at least about 99.9% identical to Compared to wild-type Cas12b, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12 pieces, 13 pieces, 14 pieces, 15 pieces, 16 pieces, 17 pieces, 18 pieces, 19 pieces, 20 pieces, 21 pieces, 22 pieces, 21 pieces, 24 pieces, 25 pieces, 26 pieces, 27 pieces, 28 pieces, 29 pieces, 30 pieces, 31 pieces, 32 pieces, 33 pieces, 34 pieces, 35 pieces, 36 pieces, 37 pieces, 38 pieces , 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, and In some embodiments, the Cas12b variant may have more amino acid changes. wherein the fragment is at least about 70% identical, at least about 80% identical to the corresponding fragment of wild-type Cas12b; 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 A fragment of Cas12b (e.g., a gRNA binding domain or a DNA cleavage domain) that is at least about 99.9% identical to the Cas12b fragment. In some embodiments, the fragment comprises a corresponding wild-type Cas12b fragment. At least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, At least 80%, at least 85%, at least 90%, at least 95% identical, at least 96%, At least 97%, at least 98%, at least 99%, or at least 99.5%. Suitable Cas12b polypeptides are listed below.

[0337] Cas12b / C2c1 (uniprot.org / uniprot / T0D7A2#2)

[0338] sp|T0D7A2|C2C1_ALIAG CRISPR-associated endonuclease C2c1 OS=Alicyclobacillus a cido-terrestris (strain ATCC 49025 / DSM 3922 / CIP 106132 / NCIMB 13137 / GD3B)GN=c2c1 PE=1 SV=1 MAVKSIKVKLRLDDMPEIRAGLWKLHKEVNAGVRYYTEWLSLLRQENLYRRSPNGDGEQECDKTAEECKAELLERLRARQ VENGHRGPAGSDDELLQLARQLYELLVPQAIGAKGDAQQIARKFLSPLADKDAVGGLGIAKAGNKPRWVRMREAGEPGWE EEKEKAETRKSADRTADVLRALADFGLKPLMRVYTDSEMSSVEWKPLRKGQAVRTWDRDMFQQAIERMMSWESWNQRVGQ EYAKLVEQKNRFEQKNFVGQEHLVHLVNQLQQDMKEASPGLESKEQTAHYVTGRALRGSDKVFEKWGKLAPDAPFDLYDA EIKNVQRRNTRRFGSHDLFAKLAEPEYQALWREDASFLTRYAVYNSILRKLNHAKMFATFTLPDATAHPIWTRFDKLGGN LHQYTFLFNEFGERRHAIRFHKLLKVENGVAREVDDVTVPISMSEQLDNLLPRDPNEPIALYFRDYGAEQHFTGEFGGAK IQCRRDQLAHMHRRRGARDVYLNVSVRVQSQSEARGERRPPYAAVFRLVGDNHRAFVHFDKLSDYLAEHPDDGKLGSEGL LSGLRVMSVDLGLRTSASISVFRVARKDELKPNSKGRVPFFFPIKGNDNLVAVHERSQLLKLPGETESKDLRAIREERQR TLRQLRTQLAYLRLLVRCGSEDVGRRERSWAKLIEQPVDAANHMTPDWREAFENELQKLKSLHGICSDKEWMDAVYESVR RVWRHMGKQVRDWRKDVRSGERPKIRGYAKDVVGGNSIEQIEYLERQYKFLKSWSFFGKVSGQVIRAEKGSRFAITLREH IDHAKEDRLKKLADRIIMEALGYVYALDERGKGKWVAKYPPCQLILLEELSEYQFNNDRPPSENNQLMQWSHRGVFQELI NQAQVHDLLVGTMYAAFSSRFDARTGAPGIRCRRVPARCTQEHNPEPFPWWLNKFVVEHTLDACPLRADDLIPTGEIF VSPFSAEEGDFHQIHADLNAAQNLQQRLWSDFDISQIRRLCDWGEVDGELVLIPRLTGKRTADSYSNKVFYTNTGVTYYE RERGKKRRKVFAQEKLSEEEAELLVEADEAREKSVVLMRDPSGIINRGNWTRQKEFWSMV NQRIEGYLVKQIRSRVPLQ DSACENTGDI

[0339] AacCas12b (Alicyclobacillus acidiphilus) - WP_067623834 MAVKSMKVKLRLDNMPEIRAGLWKLHTEVNAGVRYYTEWLSLLRQENLYRRSPNGDGEQECYKTAEECKAELLERLRARQ VENGHCGPAGSDELLQLARQLYELLVPQAIGAKGDAQQIARKFLSPLADKDAVGGGLGIAKAGNKPRWVRMREAGEPGWE EEKAKAEARKSTDTRDAVLRALADFGLKPLMRVYTDSDMSSVQWKPLRKGQAVRTWDRDMFQQAIERMMSWESWNQRVGE AYAKLVEQKSRFEQKNFVGQEHLVQLVNQLQQDMKEASGHLESKEQTAHYLTGRALRGSDKVFEKWEKLDPDAPFDLYDT EIKNVQRRNTRRFGHSHDLFAKLAEPKYQALWREDASFLTRYAVYNSIVRKLNHAKMFATFTLPDATAHPIWTRFDKLGGN LHQYTFLFNEFGEGRHAIRFQKLLTVEDGVAKEVDDVTVPIMSAQLDDLLPRDPHELVALYFQDYGAEQHLAGEFGGAK IQYRRDQLNHLHARRGARDVYLNLSVRVQSQSEARGERRPPYAAVFRLVGDNHRAFVHFDKLSDYLAEHPDDGKLGSEGL LSGLRVMSVDLGLRTSASISVFRVARKDELKPNSEGRVPFCFPIEGNENLVAVHERSQLLKLPGETESKDLRAIREERQR TLRQLRTQLAYLRLLVRCGSEDVGRRERSWAKLIEQPMDANQMTPDWREAFEDELQKLKSLYGICGDREWTEAVYESVRR VWRHMGKQVRDWRKDVRSGERPKIRGYQKDVVGGNSIEQIEYLERQYKFLKSWSFFGKVSGQVIRAEKGSRFAITLREHI DHAKEDRLKKLADRIIMEALGYVYALDDERGKGKWVAKYPPCQLILLEELSEYQFNNDRPPSENNQLMQWSHRGVFQELL NQAQVHDLLVGTMYAAFSSRFDARTGAPGIRCRRVPARCAREQNPEPFPWWLNKFVAEHKLDGCPLRADDLIPTGEGEFF VSPFSAEEGDFHQIHADLNAAQNLQRRLWSDFDISQIRRLCDWGEVDGEPVLIPRTTGKRTADSYGNKVFYTKTGVTYYE RERGKKRRKVFAQEELSEEEAELLVEADEAREKSVVLMRDPSGIINRGDWTRQKEFWSMVNQRIEGYLVKQIRSRVRLQE SACENTGDI

[0340] BhCas12b (Bacillus hisashii) NCBI reference sequence: WP_095142515 MAPKKKRKVGIHGVPAAATRSFILKIEPNEEVKKGLWKTHEVLNHGIAYYMNILKLIRQEAIYEHHEQDPKNPKKVSKAE IQAELWDFVLKMQKCNSFTHEVDKDEVFNILRELYEELVPSSVEKKGEANQLSNKFLYPLVDPNSQSGKGTASSGRKPRW YNLKIAGDPSWEEEKKKWEEDKKKDPLAKILGKLAEYGLIPLFIPYTDSNEPIVKEIKWMEKSRNQSVRRLDKDMFIQAL ERFLSWESWNLKVKEEYEKVEKEYKTLEERIKEDIQALKALEQYEKERQEQLLRDTLNTNEYRLSKRGLRGWREIIQKWL KMDENEPSEKYLEVFKDYQRKHPREAGDYSVYEFLSKKENHFIWRNHPEYPYLYATFCEIDKKKKDAKQQATFTLADPIN HPLWVRFEERSGSNLNKYRILTEQLHTEKLKKKLTVQLDRLIYPTESGGWEEKGKVDIVLLPSRQFYNQIFLDIEEKGKH AFTYKDESIKFPLKGTLGGARVQFDRDHLRRYPHKVESGNVGRIYFNMTVNIEPTESPVSKSLKIHRDDFPKVVNFKPKE LTEWIKDSKGKKLKSGIESLEIGLRVMSIDLGQRQAAAASIFEVVDQKPDIEGKLFFPIKGTELYAVHRASFNIKLPGET LVKSREVLRKAREDNLKLMNQKLNFLRNVLHFQQFEDITEREKRVTKWISRQENSDVPLVYQDELIQIRELMYKPYKDWV AFLKQLHKRLEVEIGKEVKHWRKSLSDGRKGLYGISLKNIDEIDRTRKFLLRWSLRPTEPGEVRRLEPGQRFAIDQLNHL NALKEDRLKKMANTIIMHALGYCYDVRKKKWQAKNPACQIILFEDLSNYNPY EERSRFENSKLM K WSRREIPRQVALQGE IYGLQVGEVGAQFSSRFHAKTGSPGIRCSVVTKEKLQDNRFFKNLQREGRLTLDKIAVLKEGDLYPDKGGEKFISLSKDR KCVTTHADINAAQNLQKRFWTRTHGFYKVYCKAYQVDGQTVYIPESKDQKQKIIEEFGEGYFILKDGVYEWVNAGKLKIK KGSSKQSSSELVDSDILKDSFDLASELKGEKLMLYRDPSGNVFPSDKWMAAGVFFGKLERILISKLTNQYSISTIEDDSS KQSMKRPAATKKAGQAKKKK It contains a variant called BvCas12b V4 (S893R / K846R / E837G changes compared to the wild type mentioned above). nothing

[0341] BvCas12b (Bacillus sp. V3-13) NCBI reference sequence: WP_101661451.1 MAIRSIKLKMKTNSGTDSIYLRKALWRTHQLINEGIAYYMNLLTLYRQEAIGDKTKEAYQAELINIIRNQQRNNGSSEEH GSDQEILALLRQLYELIIPSSIGESGDANQLGNKFLYPLVDPNSQSGKGTSNAGRKPRWKRLKEEGNPDWELEKKKDEER KAKDPTVKIFDNLNKYGLLPLFPLFTNIQKDIEWLPLGKRQSVRKWDKDMFIQAIERLLSWESWNRRVADEYKQLKEKTE SYYKEHLTGGEEWIEKIRKFEKERNMELEKNAFAPNDGYFITSRQIRGWDRVYEKWSKLPESAPEELWKVVAEQQNKMS EGFGDPKVFSFLANRENRDIWRGHSERIYHIAAYNGLQKKLSRTKEQATFTLPDAIEHPLWIRYESPGGTNLNLFKLEEK QKKNYYVTLSKIIWPSEEKWIEKENIEIPLAPSIQFNRQIKLKQHVKGKQEISFSDYSSRISLDGVLGGSRIQFNRKYIK NHKELLGEGDIGPVFFNLVVDVAPLQETRNGRLQSPIGKALKVISSDFSKVIDYKPKELMDWMNTGSASNSFGVASLLEG MRVMSIDMGQRTSASVSIFEVVKELPKDQEQKLFYSINDTELFAIHKRSFLLNLPGEVVTKNNKQQRQERRKKRQFVRSQ IRMLANVLRLETKKTPDERKKAIHKLMEIVQSYDSWTASQKEVWEKELNLLTNMAAFNDEIWKESLVELHHRIEPYVGQI VSKWRKGLSEGRKNLAGISMWNIDELEDTRRLLISWSKRSRTPGEANRIETDEPFGSSLLQHIQNVKDDRLKQMANLIIM TALGFKYDKEEKDRYKRWKETYPACQIILFENLNRYLFNLDRSRRRENSRLMKWAHRSIPRTVSMQGEMFGLQVGDVRSEY SSRFHAKTGAPGIRCHALTEEDLKAGSNTLKRLIEDGFINESELAYLKKGDIIPSQGGELFVTLSKRYKKDSDNNELTVI HADINAAQNLQKRFWQQNSEVYRVPCQLARMGEDKLYIPKSQTETIKKYFGKGSFVKNNTEQEVYKWEKSEKMKIKTDTT FDLQDLDGFEDISKTIELAQEQQKKYLTMFRDPSGYFFNNETWRPQKEYWSIVNNIIKSCLKKKILSNKVEL

[0342] "Cbl proto-oncogene B (CBLB) polypeptide" is a GenBank having at least about 85% amino acid sequence identity with Accession No. ABC86700.1 or a fragment thereof By "CBLB" is meant a protein. Exemplary CBLB polypeptide sequences are provided below.

[0343] >ABC86700.1 CBL-B [Homo sapiens] MANSMNGRNPGGRGGNPRKGRILGIIDAIQDAVGPPKQAAADRRTVEKTWKLMDKVVRLCQNPKLQLKNSPPYILDILPD TYQHLRLILSKYDDNQKLAQLSENEYFKIYIDSLMKKSKRAIRLFKEGKERMYEEQSQDRRNLTKLSLIFSHMLAEIKAI FPNGQFQGDNFRITKADAAEFWRKFFGDKTIVPWKVFRQCLHEVHQISSGLEAMALKSTIDLTCNDYISVFEFDIFTRLF QPWGSILRNWNFLAVTHPGYMAFLTYDEVKARLQKYSTKPGSYIFRLSCTRLGQWAIGYVTGDGNILQTIPHNKPLFQAL IDGSREGFYLYPDGRSYNPDLTGLCEPTPHDHIKVTQEQYELYCEMGSTFQLCKICAENDKDVKIEPCGHLMCTSCLTAW QESDGQGCPFCRCEIKGTEPIIVDPFDPRDEGSRCCSIIDPFGMPMLDLDDDDDREESLMMNRLANVRKCTDRQNSPVTS PGSSPLAQRRKPQPDPLQIPHLSLPPVPPRLDLIQKGIVRSPCGSPTGSPKSSPCMVRKQDKPLPAPPPPPLRDPPPPPPE RPPPIPPDNRLSRHIHHVESVPSRDPPMPLEAWCPRDVFGTNQLVGCRLLGEGSPKPGITASSNVNGRHSRVGSDPVLMR KHRRHDLPLEGAKVFSNGHLGSEEYDVPPRLSPPPPVTTLLPSIKCTGPLANSLSEKTRDPVEEDDDEYKIPSSHPVSLN SQPSHCHNVKPPVRSCDNGHCMLNGTHGPSSEKKSNIPDLSIYLKGDVFDSASDPVPLPPARPPTRDNPKHGSSLNRTPS DYDLLIPPLGEDAFDALPPSLPPPPPPPARHSLIEHSKPPGSSSRPSSGQDLFLLPSDPFVDLASGQVPLPPARRLPGENV KTNRTSQDYDQLPSCSDGSQAPARPPKPRPRRTAPEIHHRKPHGPEAALENVDAKIAKLMGEGYAFEEVKRALEIAQNNV EVARSILREFAFPPPVSPRLNL "Cbl proto-oncogene B (CBLB) polynucleotide" means a nucleic acid sequence encoding a CBLB polypeptide. The CBLB gene encodes an E3 ubiquitin ligase. Exemplary CBLB Nucleic Acids The sequence is provided below. Additional exemplary CBLB genome sequences are available in the NCBI reference sequence: NC_000003. 12, or as shown in transcript reference NM_001321813.1. >DQ349203.1 Homo sapiens CBL-B mRNA, complete cds ATGGCAAACTCAATGAATGGCAGAAACCCTGGTGGTCGAGGAGGAAATCCCGAAAGGTCGAATTTTGGGTATTATTGA TGCTATTCAGGATGCAGTTGGACCCCCTAAGCAAGCTGCCGCAGATCGCAGGACCGTGGAGAAGACTTGGAAGCTCATGG ACAAAGTGGTAAGACTGTGCCAAAATCCCAAACTTCAGTTGAAAAATAGCCCACCATATATACTTGATATTTTGCCTGAT ACATATCAGCATTTACGACTTATATTGAGTAAATATGATGACAACCAGAAACTTGCCCAACTCAGTGAGAATGAGTACTT TAAAATCTACATTGATAGCCTTATGAAAAAGTCAAAACGGGCAATAAGACTCTTTAAAGAAGGCAAGGAGAGAATGTATG AAGAACAGTCACAGGACAGAACGAATCTCACAAAACTGTCCCTTATCTTCAGTCACATGCTGGCAGAAATCAAAGCAATC TTTCCCAATGGTCAATTCCAGGGAGATAACTTTCGTATCCAAAAGCAGATGCTGCTGAATTCTGGAGAAAGTTTTTTGG AGACAAAACTATCGTACCATGGAAAGTATTCAGACAGTGCCTTCATGAGGTCCACCAGATTAGCTCTGGCCTGGAAGCAA TGGCTCTAAAATCAACAATTGATTTAACTTGCAATGATTACATTTCAGTTTTTGAATTTGATATTTTTACCAGGCTGTTTT CAGCCTTGGGGCTCTATTTTGCGGAATTGGAATTTCTTAGCTGTGACACATCCAGGTTACATGGCATTTCTCACATATGA TGAAGTTAAAGCACGACTACAGAAATATAGCACCAAACCCGGGAAGCTATATTTCCGGTTAAGTTGCACTCGATTGGGAC AGTGGGCCATTGGCTATGTGACTGGGGATGGGAATATCTTACAGACCATACCTCATAACAAGCCCTTATTTCAAGCCCTG ATTGATGGCAGCAGGGAAGGATTTTATCTTTATCCTGATGGGAGGAGTTATAATCCTGATTTAACTGGATTATGTGAACC TACACCTCATGACCATATAAAAGTTACACAGGAACAAATATGAATTATATTGTGAAATGGGCTCCACTTTTCAGCTCTGTA AGATTTGTGCAGAGAATGACAAAGATGTCAAGATTGAGCCTTGTGGGCATTTGATGTGCACCTCTTGCCTTACGGCATGG CAGGAGTCGGATGGTCAGGGCTGCCCTTTCTGTCGTTGTGAAATAAAAGGAACTGAGCCCATAATCGTGGACCCCTTTGA TCCAAGAGATGAAGGCTCCAGGTGTTGCAGCATCATTGACCCCTTTGGCATGCCGATGCTAGACTTGGACGACGATGATG ATCGTGAGGAGTCCTTGATGATGAATCGGTTGGCAAACGTCCGAAAGTGCACTGACAGGCAGAACTCACCAGTCACATCA CCAGGATCCTCTCCCCTTGCCCAGAGAAGAAAGCCACAGCCTGACCCACTCCAGATCCCACATCTAAGCCTGCCACCCGT GCCTCCTCGCCTGGATCTAATTCAGAAAGGCATAGTTAGATCTCCCTGTGGCAGCCCAACGGGTTCACCAAAGTCTTCTC CTTGCATGGTGAGAAAACAAGATAAACCACTCCCAGCACCACCTCCTCCCTTAAGAGATCCTCCTCCACCGCCACCTGAA AGACCTCCACCAATCCCACCAGACAATAGACTGAGTAGACACATCCATCATGTGGAAAGCGTGCCTTCCAGAGACCCGCC AATGCCTCTTGAAGCATGGTGCCCTCGGGATGTGTTTGGGACTAATCAGCTTGTGGGATGTCGACTCCTAGGGGAGGGCT CTCCAAAACCTGGAATCACAGCGAGTTCAAATGTCAATGGAAGGCACAGTAGAGTGGGCTCTGACCCAGTGCTTATGCGG AAACACAGACGCCATGATTTGCCTTTAGAAGGAGCTAAGGTCTTTTCCAATGGTCACCTTGGAAGTGAAGAATATGATGT TCCTCCCCGGCTTTCTCCTCCTCCTCCAGTTACCACCCTCCTCCCTAGCATAAAGTGTACTGGTCCGTTAGCAAATTCTC TTTCAGAGAAAACAAGAGACCCAGTAGAGGAAGATGATGATGAATACAAGATTCCTTCATCCCACCCTGTTTCCCTGAAT TCACAACCATCTCATTGTCATAATGTAAAACCTCCTGTTCGGTCTTGTGATAATGGTCACTGTATGCTGAATGGAACACA TGGTCCATCTTCAGAGAAGAAATCAAACATCCCTGACTTAAGCATATATTTAAAGGGAGATGTTTTTGATTCAGCCTCTG ATCCCGTGCCATTACCACCTGCCAGGCCTCCAACTCGGGACAATCCAAAGCATGGTTCTTCACTCAACAGGACGCCCTCT GATTATGATCTTCTCATCCCTCCATTAGGTGAAGATGCTTTTGATGCCCTCCCTCCATCTCTCCCACCTCCCCCACCTCC TGCAAGGCATAGTCTCATTGAACATTCAAAACCTCCTGGCTCCAGTAGCCGGCCATCCTCAGGACAGGATCTTTTTCTTC TTCCTTCAGATCCCTTTGTTGATCTAGCAAGTGGCCAAGTTCCTTTGCCTCCTGCTAGAAGGTTACCAGGTGAAAATGTC AAAACTAACAGAACATCACAGGACTATGATCAGCTTCCTTCATGTTCAGATGGTTCACAGGCACCAGCCAGACCCCCTAA ACCACGACCGCGCAGGACTGCACCAGAAATTCACCACAGAAACCCCATGGGCCTGAGGCGGCATTGGAAAATGTCGATG CAAAAATTGCAAAACTCATGGGAGAGGGTTATGCCTTTGAAGAGGTGAAGAGAGCCTTAGAGATAGCCCAGAATAATGTC GAAGTTGCCCGGAGCATCCTCGGAGAATTTGCCTTCCCTCCTCCAGTATCCCCACGTCTAAATCTATAG

[0344] "Chimeric antigen receptor" refers to an extracellular antigen-binding protein that confers specificity to immune cells for an antigen. refers to a synthetic receptor containing a domain, a transmembrane domain, and an intracellular signaling domain. do.

[0345] In this disclosure, the terms "comprises," "comprising," and "contain" are used interchangeably. "container," "having," and the like shall have the meanings ascribed to them in U.S. patent law. It can mean "includes," "including," etc., and "essentially from "consisting essentially of" or "consists essentially of" "y")" also has the meaning given to it in U.S. patent law, and such term is open-ended. The basic or novel properties of the described thing are not present in other things than the described thing. Unless otherwise varied, other than those described are permitted, except that the prior art implementation Exclude aspects.

[0346] "Cluster of differentiation 2 (CD2)" refers to NCBI accession number NP_001315538.1 or a fragment thereof. It means a protein having at least about 85% amino acid sequence identity with the target gene and having immunomodulatory activity. Exemplary amino acid sequences are provided below. >NP_001315538.1 T cell surface antigen CD2 isoform 1 precursor [Homo sapiens] MSFPCKFVASFLLIFFINVSSKGAVSKEITNALETWGALGQDINLDIPSFQMSDDIDDIKWEKTSDKKKIAQFRKEKETFKE KDTYKLFKNGTLKIKHLKTDDQDIYKVSIYDTKGKNVLEKIFDLKIQERVSKPKISWTCINTTLTCEVMNGTDPELNLYQ DGKHLKLSQRVITHKWTTSLSAKFKCTAGNKVSKESSVEPVSCPGGSILGQSNGLSAWTPPSHPTSLPFAEKGLDIYLII GICGGGSLLMVFVALLVFYITKRKKQRSRRNDEELETRAHRVATEERGRKPHQIPASTPQNPATSQHPPPPPGHRSQAPS HRPPPPGHRVQHQPQKRPPAPSGTQVHQQKGPPLPRPRVQPKPPHGAAENSLSPSSN

[0347] "Cluster of Differentiation 2 (CD2)" refers to a nucleic acid encoding a CD2 polypeptide. The D2 nucleic acid sequence is provided below. >NM_001328609.2 Homo sapiens CD2 molecule (CD2), transcript variant 1, mRNA AGTCTCACTTCAGTTCCTTTTGCATGAAGAGCTCAGAATCAAAAGAGGAAACCAACCCCTAAGATGAGCTTTCCATGTAA ATTTGTAGCCAGCTTCCTTCTGATTTTCAATGTTTCTTCCAAAGGTGCAGTCTCCAAAGAGATTACGAATGCCTTGGAAA CCTGGGGTGCCTTGGGTCAGGACATCAACTTGGACATTCCTAGTTTTCAAATGAGTGATGATATTGACGATAAAAAATGG GAAAAAACTTCAGACAAGAAAAGATTGCACAATTCAGAAAAGAGAGACTTTCAAGGAAAAAGATACATATAAGCT ATTTAAAAAATGGAACTCTGAAAATTAAGCATCTGAAGACCGATGATCAGGATATCTACAAGGTATCAATATATGATACAA AAGGAAAAAATGTGTTGGAAAAAATTTGATTTGAAGATTCAAGAGAGGGTCTCAAAACCAAAGATCTCCTGGACTTGT ATCAACACAACCCTGACCTGTGAGGGTAATGAATGGAACTGACCCCGAATTAAACCTGTATCAAGATGGGAAACATCTAAA ACTTTCTCAGAGGGTCATCACACACAAGTGGACCACCAGCCTGAGTGCAAAATTCAAGTGCACAGCAGGGAACAAAGTCA GCAAGGAATCCAGTGTCGAGCCTGTCAGCTGTCCAGGAGGCAGCATCCTTGGCCAGAGTAATGGGCTCTCTGCCTGGACC CCTCCCAGCCATCCACTTCTCTTCCTTTGCAGAGAAAGGTCTGGACATCTATCTCATCATTGGCATATGTGGAGGAGG CAGCCTCTTGATGGTCTTTGTGGCACTGCTCGTTTTCTATATCACCAAAAGGAAAAACAGAGGAGTCGGAGAAATGATG AGGAGCTGGAGACAAGAGCCCACAGAGTAGCTACTGAAGAAAGGGGCCGGAAGCCCCACCAAATTCCAGCTTCAACCCCT CAGAATCCAGCAACTTCCCAACATCCTCCTCCACCACCTGGTCATCGTCCCAGGCACCTAGTCATCGTCCCCCGCCTCC TGGACACCGTGTTCAGCACCAGCCTCAGAAGAGGCCTCCTGCTCCGTCGGGCACACAAGTTCACCAGCAGAAAGGCCCGC CCCTCCCCAGACCTCGAGTTCAGCCAAAACCTCCCCATGGGGCAGCAGAAAACTCATTGTCCCCTTCCTCTAATTAAAAAA AGATAGAAACTGTCTTTTTCAAATAAAAAGCACTGTGGATTTCTGCCTCCTGATGTGCATATCCGTACTTCCATGAGGTG TTTTCTGTGTGCAGAACATTGTCACCTCCTGAGGCTGTGGCCACAGCCACCTCTGCATCTTCGAACTCAGCCATGTGGT CAACATCTGGAGTTTTTGGTCTCCTCAGAGAGCTCCATCACACCAGTAAGGAGAAGCAATATAAGTGTGATTGCAAGAAT GGTAGAGGACCGAGCACAGAAATCTTAGAGATTTCTTGTCCCCTCTCAGGTCATGTGTAGATGCGATAAATCAAGTGATT GGTGTGCCTGGGTCTCACTACAAGCAGCCTATCTGCTTAAGAGACTCTGGAGTTTCTTATGTGCCCTGGTGGACACTTGC CCACCATCCTGTGAGTAAAAGTGAAATAAAAGCTTTGACTAGA

[0348] "Cluster of differentiation 3 epsilon (CD3e or CD3 epsilon)" is NCBI accession number NP_0 00724.1 or a fragment thereof, and having at least about 85% amino acid sequence identity with 00724.1 or a fragment thereof, and having immunomodulatory activity. Exemplary amino acid sequences are provided below. >NP_000724.1 T cell surface glycoprotein CD3 epsilon chain precursor [Homo sapiens] MQSGTHWRVLGLCLLSVGVWGQDGNEEMGGITQTPYKVSISGTTVILTCPQYPGSEILWQHNDKNIGGDEDDKNIGSDED HLSLKEFSELEQSGYYVCYPRGSKPEDANFYLYLRARVCENCMEMDVMSVATIVIVDICITGGLLLLVYYWSKNRKAKAK PVTRGAGAGGRQRGQNKERPPPVPNPDYEPIRKGQRDLYSGLNQRRI

[0349] "Cluster of differentiation 3 epsilon (CD3e or CD3 epsilon)" refers to a molecule that contains the CD3e polypeptide. Exemplary CD3e nucleic acid sequences are provided below. >NM_000733.4 Homo sapiens CD3e molecule (CD3E), mRNA AGAAACCCTCCTCCCCTCCCAGCCTCAGGTGCCTGCTTCAGAAAATGAAGTAGTAAGTCTGCTGGCCTCCGCCATCTTAG TAAAGTAACAGTCCCATGAAACAAAGATGCAGTCGGGCACTCACTGGAGAGTTCTGGGCCTCTGCCTCTTATCAGTTGGC GTTTGGGGGCAAGATGGTAATGAAGAAATGGGTGGTATTACACAGACACCATATAAAGTCTCCATCTCTGGAACCACAGT AATATTGACATGCCCTCAGTATCCTGGATCTGAAATACTATGGCAACACAATGATAAAAACATAGGCGGTGATGAGGATG ATAAAACATAGGCAGTGATGAGGATCACCTGTCACTGAAGGAATTTTCAGAATTGGAGCAAAGTGGTTATTATGTCTGC TACCCCAGAGGAAGCAAACCAGAAGATGCGAACTTTTATCTCTACCTGAGGGCAAGAGTGTGTGAGAACTGCATGGAGAT GGATGTGATGTCGGTGGCCACAATTGTCATAGTGGACATCTGCATCACTGGGGGCTTGCTGCTGCTGGTTTACTACTGGA GCAAGAATAGAAAGGCCAAGGCCAAGCCTGTGACACGAGGAGCGGGTGCTGGCGGCAGGCAAAGGGGACAAAACAAGGAG AGGCCACCACCTGTTCCCAACCCAGACTATGAGCCCATCCGGAAAGGCCAGCGGGACCTGTATTCTGGCCTGAATCAGAG ACGCATCTGACCCTCTGGAGAACACTGCCTCCCGCTGGCCCAGGTCTCCTCTCCAGTCCCCCTGCGACTCCCTGTTTCCT GGGCTAGTCTTGGACCCCACGAGAGAGAATCGTTCCTCAGCCTCATGGTGAACTCGCGCCCTCCAGCCTGATCCCCCGCT CCCTCCTCCCTGCCTTCTCTGCTGGTACCCAGTCCTAAAATATTGCTGCTTCCTCTTCCTTTGAAGCATCATCAGTAGTC ACACCCTCACAGCTGGCCTGCCCTCTTGCCAGGATATTTATTTGTGCTATTCACTCCCTTCCCTTTGGATGTAACTTCTC CGTTCAGTTCCCTCCTTTTCTTGCATGTAAGTTGTCCCCCATCCCAAAGTATTCCATCTACTTTTCTATCGCCGTCCCCT TTTGCAGCCCTCTCTGGGGATGGACTGGGTAAATGTTGACAGAGGCCCTGCCCCGTTCACAGATCCTGGCCCTGAGCCAG CCCTGTGCTCCTCCCTCCCCCAACACTCCCTACCAACCCCCTAATCCCCTACTCCCTCCACCCCCCCTCCACTGTAGGCC ACTGGATGGTCATTTGCATCTCCGTAAATGTGCTCTGCTCCTCAGCTGAGAGAGAAAAAAATAAACTGTATTTGGCTGCA A

[0350] "Cluster of differentiation 3 gamma (CD3g or CD3 gamma)" refers to NCBI accession number NP_000064.1 or a fragment thereof, and having at least about 85% amino acid sequence identity therewith and having immunomodulatory activity. Exemplary amino acid sequences are provided below. >NP_000064.1 T cell surface glycoprotein CD3 gamma chain precursor [Homo sapiens] MEQGKGLAVLILAIILLQGTLAQSIKGNHLVKVYDYQEDGSVLLTCDAEAKNITWFKDGKMIGFLTEDKKKWNLGSNAKD PRGMYQCKGSQNKSKPLQVYYRMCQNCIELNAATISGFLFAEIVSIFVLAVGVYFIAGQDGVRQSRASDKQTLLPNDQLY QPLKDREDDQYSHLQGNQLRRN

[0351] "Cluster of differentiation 3 gamma (CD3g or CD3 gamma)" refers to a molecule encoding the CD3g polypeptide. By nucleic acid is meant an exemplary CD3g nucleic acid sequence, as provided below: >NM_000073.3 Homo sapiens CD3g molecule (CD3G), mRNA AGTCTAGCTGCTGCACAGGCTGGCTGGCTGGCTGGCTGCTAAGGGCTGCTCCACGCTTTTGCCGGAGGACAGAGACTGAC ATGGAACAGGGGAAGGGCCTGGCTGTCCTCATCCTGGCTATCATTCTTCTTCAAGGTACTTTGGCCCAGTCAATCAAAGG AAACCACTTGGTTAAGGTGTATGACTATCAAGAAGATGGTTCGGTACTTCTGACTTGTGATGCAGAAGCCAAAAATATCA CATGGTTTAAAGATGGGAAGATGATCGGCTTCCTAACTGAAGATAAAAAAAAATGGAATCTGGGAAGTAATGCCAAGGAC CCTCGAGGGATGTATCAGTGTAAAGGATCACAGAACAAGTCAAAACCACTCCAAGTGTATTACAGAATGTGTCAGAACTG CATTGAACTAAATGCAGCCACCCATATCTGGCTTTCTCTTTGCTGAAATCGTCAGCATTTTCGTCCTTGCTGTTGGGGTCT ACTTCATTGCTGGACAGGATGGAGTTCGCCAGTCGAGAGCTTCAGACAAGCAGACTCTGTTGCCCAATGACCAGCTCTAC CAGCCCCTCAAGGATCGAGAAGATGACCAGTACAGCCACCTTCAAGGAAACCAGTTGAGGAGGAATTGAACTCAGGACTC AGAGTAGTCCAGGTGTTCTCCTCCTATTCAGTTCCCAGAATCAAAGCAATGCATTTTGGAAAGCTCCTAGCAGAGAGACT TTCAGCCTAAATCTAGACTCAAGGTTCCCAGAGATGACAAATGGAGAAGAAAGGCCATCAGAGCAAATTTGGGGGTTTC TCAAATAAAATAAAAATAAAACAAATACTGTGTTTCAGAAGCGCCACCTATTGGGGAAAATTGTAAAGGAAAAATGAAA AGATCAAATAACCCCCTGGATTTGAATATAATTTTTTGTGTTGTAATTTTTATTTCGTTTTTGTATAGGTTATAATTCAC ATGGCTCAAATATTCAGTGAAAGCTCTCCCTCCACCGCCATCCCCTGCTACCCAGTGACCCTGTTGCCCTCTTCAGAGAC AAATTAGTTTCTCTTTTTTTTTTTTTTTTTTTTTTTTTTGAGACAGTCTGGCTCTGTCACCCAGGCTGAAATGCAGTGGC ACCATCTCGGCTCACTGCAACCTCTGCCTCCTGGGTTCAAGCGATTCTCCTGCCTCAGCCTCCCGGGCAGCTGGGATTAC AGGCACACACTACCACACCTGGCTAATTTTTGTATTTTTAGTAGAGACAGGGTTTTGCTCTGTTGGCCAAGCTGGTCTCG AACTCCTGACCTCAAGTGATCCGCCCGCCTCAGCCTCCCAAAGTGCTGGGATTACAGGTGTGAGCCACCATGCCTGGTCT TAAAACCAGTTTCTTATATATCTCTCTGGAGGTATTCTAGGCATATATGAGCACATTCTCAAGTACATATTATCCTCCCT TCCCCTATCTTTTAGACAAATGATATCAAACTATACATCTTGTGAGATTATTGCATACCATTATATGAAGATACCATTAT ATCCTTTTTAATGCAACCATATTGTACAAATAGACTATGATTTATTTAACCTGTTATCTATCAGTGGATATTTAAGTTGG TAGTTGGTTCCAATCTTTTGCTCTTACAACAATTCTGCAATGACTAACATTGTATAAATATCATTTTTAAAAATAATTGC ATTGAAGCATAATGTACATGCCATAAAATCCACCCATCTTAAGTGATTTCACCTGTTCTCAGAAATTTTTAGTAAATTTA ACTAATTGTACAGCCATTACCATAATCCAGCTTTAGGACATTTTCTTTTTTTTCTTTTCTTTTCTTTTTTTTCTTTTTTT TTTTTTTTTGAAGTGGAATCTTGCTCTGTGGCCCAGGCTGGAGTGCAGTGGCGCGATCTCAGCTCACTGCAACCTCCACC TCCTGGGTTCAAGCGATTCTCTTGCCTTGGCCTCCCGAGTAGCTGAGACTACAGGCACATGCCACCACGCCCAGCTCATT TTTTGTGTATTTAGTATTTGTGTATCTAGTATTTGTGTACTTAGTAGAGACAGGGTTTCACCATGTTGGCCAGGCTGGTC TCCAATTCCTGACCTCAGGCGATCCACCCGCCTTGACCTCCCAAAGTGCTGGGATTACAGGTGTGAGCCACCGCGCCAGG CCCGTAACTGTATTTTAATATAGCCATTCTATGGATTTAATATGGTATTTTATTATGGCCTTAATTTGCATTTCCCTAGA TACTAACCATGCTGAGTGTCCTGTCTTGTGTTTATTAACCATTCATATATTTTTAGTGAAATGTGTATCAAATCTTTTGC CCATTTTTAAGTTGACTTATTTGTTTGTCTTCTTACTATTGGGTTGCATATGTTTTTGATATAAGTCCTTTATCAGATAT ATGATTTGGAAATATTTTCTACCAATCTGTGGTTTGTTTTTCTTAATGGTGTCTTTTGAAGTGCAAAAGGTTTGAATTTT GAAGTACATTTTATTGATTTTTTCTTCTATATATTGTGCTTTTGGTATCATGTCTAATAAATCTTTACCAAACCCACAGT TACAAAGATTTTCTCCTGTCTTCTTTTTATACTTTTTACAGCTTTATGGTTTTAGCTCTAACAATAAATGTGATTTTGAA CATACATAAGACTATTTGTAACAAACACAAATAAATTGAATTGTTGGGCA

[0352] "Cluster of differentiation 3 delta (CD3d or CD3 delta)" refers to NCBI accession number NP_000723.1 or a fragment thereof, and having at least about 85% amino acid sequence identity therewith and having immunomodulatory activity. Exemplary amino acid sequences are provided below. >NP_000723.1 T cell surface glycoprotein CD3 delta chain isoform A precursor [Homo sapien vinegar] MEHSTFLSGLVLATLLSQVSPFKIPIEELEDRVFVNCNTSITWVEGTVGTLLSDITRLDLGKRILDPRGIYRCNGTDIYK DKESTVQVHYRMCQSCVELDPATVAGIIVTDVIATLLLALGVFCFAGHETGRLSGAADTQALLRNDQVYQPLRDRDDAQY SHLGGNWARNK

[0353] "Cluster of differentiation 3 delta (CD3d or CD3 delta)" refers to a molecule encoding the CD3d polypeptide. By nucleic acid is meant an exemplary CD3d nucleic acid sequence, as provided below: >NM_000732.4 Homo sapiens CD3d molecule (CD3D), transcript variant 1, mRNA AGAGAAGCAGACATCTTCTAGTTCCTCCCCCACTCTCCTCTTTCCGGTACCTGTGAGTCAGCTAGGGGAGGGCAGCTCTC ACCCAGGCTGATAGTTCGGTGACCTGGCTTTATCTACTGGATGAGTTCCGCTGGGAGATGGAACATAGCACGTTTCTCTC TGGCCTGGTACTGGCTACCCTTCTCTCGCAAGTGAGCCCCTTCAAGATACCTATAGAGGAACTTGAGGACAGAGTGTTTG TGAATTGCAATACCAGCATCACATGGGTAGAGGGAACGGTGGGAACACTGCTCTCAGACATTACAAGACTGGACCTGGGA AAACGCATCCTGGACCCACGAGGAATATATAGGTGTAATGGGACAGATATATACAAGGACAAAGAATCTACCGTGCAAGT TCATTATCGAATGTGCCAGAGCTGTGTGGAGCTGGATCCAGCCACCGTGGCTGGCATCATTGTCACTGATGTCATTGCCA CTCTGCTCCTTGCTTTGGGAGTCTTCTGCTTTGCTGGACATGAGACTGGAAGGCTGTCTGGGGCTGCCGACACACAAGCT CTGTTGAGGAATGACCAGGTCTATCAGCCCCTCCGAGATCGAGATGATGCTCAGTACAGCCACCTTGGAGGAAACTGGGC TCGGAACAAGTGAACCTGAGACTGGTGGCTTCTAGAAGCAGCCATTACCAACTGTACCTTCCCTTCTTGCTCAGCCAATA AATATATCCTCTTTTCACTCAGAAAAAAAAAAAAAAAAAAAAAAAAAAA

[0354] "Cluster of differentiation 4 (CD4)" refers to NCBI accession number NP_000607.1 or at least a fragment thereof. Both refer to proteins that have approximately 85% amino acid sequence identity and have immunomodulatory activity. Exemplary amino acid sequences are provided below. >NP_000607.1 T cell surface glycoprotein CD4 isoform 1 precursor [Homo sapiens] MNRGVPFRHLLLVLQLALLPAATQGKKVVLGKKGDTVELTCTASQKKSIQFHWKNSNQIKILGNQGSFLTKGPSKLNDRA DSRRSLWDQGNFPLIIKNLKIEDSDTYICEVEDQKEEVQLLVFGLTANSSDTHLLQGQSLTLTLESPPGSSPSVQCRSPRG KNIQGGKTLSVSQLELQDSGTWTCTVLQNQKKVEFKIDIVVLAFQKASSIVYKKEGEQVEFSFPLAFTVEKLTGSGELWW QAERASSSKSWITFDLKNKEVSVKRVTQDPKLQMGKKLPLHLTLPQALPQYAGSGNLTLALEAKTGKLHQEVNLVVMRAT QLQKNLTCEVWGPTSPKLMLSLKLENKEAKVSKREKAVWVLNPEAGMWQCLLSDSGQVLLESNIKVLPTWSTPVQPMALI VLGGVAGLLLFIGLGIFFCVRCRHRRRQAERMSQIKRLLSEKKTCQCPHRFQKTCSPI

[0355] "Cluster of Differentiation 4 (CD4)" refers to a nucleic acid encoding a CD4 polypeptide. A suitable CD4 nucleic acid sequence is provided below. >NM_000616.5 Homo sapiens CD4 molecule (CD4), transcript variant 1, mRNA CTCTCTTCATTTAAGCACGACTCTGCAGAAGGAACAAAGCACCCTCCCCACTGGGCTCCTGGTTGCAGAGCTCCAAGTCC TCACACAGATACGCCTGTTTGAGAAGCAGCGGGCAAGAAAGACGCAAGCCCAGAGGCCCTGCCATTTCTGTGGGCTCAGG TCCCTACTGGCTCAGGCCCCTGCCTCCCTCGGCAAGGCCACAATGAACCGGGGAGTCCCTTTTAGGCACTTGCTTCTGGT GCTGCAACTGGCGCTCCTCCCAGCAGCCACTCAGGGAAAGAAAGTGGTGCTGGGCAAAAAAGGGGATACAGTGGAACTGA CCTGTACAGCTTCCCAGAAGAAGAGCATACAATTCCACTGGAAAAACTCCAACCAGATAAAGATTCTGGGAAATCAGGGC TCCTTCTTAACTAAAGGTCCATCCAAGCTGAATGATCGCGCTGACTCAAGAAGAAGCCTTTGGGACCAAGGAAACTTTCC CCTGATCATCAAGAATCTTAAGATAGAAGACTCAGATACTTACATCTGTGAAGTGGAGGACCAGAAGGAGGAGGTGCAAT TGCTAGTGTTCGGATTGACTGCCAACTCTGACACCCACCTGCTTCAGGGGCAGAGCCTGACCCTGACCTTGGAGAGCCCC CCTGGTAGTAGCCCCTCAGTGCAATGTAGGAGTCCAAGGGGTAAAAACATACAGGGGGGGAAGACCCTCTCCGTGTCTCA GCTGGAGCTCCAGGATAGTGGCACCTGGACATGCACTGTCTTGCAGAACCAGAAGAAGGTGGAGTTCAAAATAGACATCG TGGTGCTAGCTTTCCAGAAGGCCTCCAGCATAGTCTATAAGAAAGAGGGGGAACAGGTGGAGTTCTCCTTCCCACTCGCC TTTACAGTTGAAAAGCTGACGGGCAGTGGCGAGCTGTGGTGGCAGGCGGAGAGGGCTTCCTCCTCCAAGTCTTGGATCAC CTTTGACCTGAAGAACAAGGAAGTGTCTGTAAAACGGGTTACCCAGGACCCTAAGCTCCAGATGGGCAAGAAGCTCCCGC TCCACCTCACCCTGCCCCAGGCCTTGCCTCAGTATGCTGGCTCTGGAAACCTCACCCTGGCCCTTGAAGCGAAAACAGGA AAGTTGCATCAGGAAGTGAACCTGGTGGTGATGAGAGCCACTCAGCTCCAGAAAAATTTGACCTGTGAGGTGTGGGGACC CACCTCCCCTAAGCTGATGCTGAGTTTGAAACTGGAGAACAAGGAGGCAAAGGTCTCGAAGCGGGAGAAGGCGGTGTGGG TGCTGAACCCTGAGGCGGGGATGTGGCAGTGTCTGCTGAGTGACTCGGGACAGGTCCTGCTGGAATCCAACATCAAGGTT CTGCCCACATGGTCCACCCCGGTGCAGCCAATGGCCCTGATTGTGCTGGGGGGCGTCGCCGGCCTCCTGCTTTTCATTGG GCTAGGCATCTTCTTCTGTGTCAGGTGCCGGCACCGAAGGCGCCAAGCAGAGCGGATGTCTCAGATCAAGAGACTCCTCA GTGAGAAGAAGACCTGCCAGTGTCCTCACCGGTTTCAGAAGACATGTAGCCCCATTTGAGGCACGAGGCCAGGCAGATCC CACTTGCAGCCTCCCCAGGTGTCTGCCCCGCGTTTCCTGCCTGCGGACCAGATGAATGTAGCAGATCCCCAGCCTCTGGC CTCCTGTTCGCCTCCTCTACAATTTGCCATTGTTTCTCCTGGGTTAGGCCCCGGCTTCACTGGTTGAGTGTTGCTCTCTA GTTTCCAGAGGCTTAATCACACCGTCCTCCACGCCATTTCCTTTTCCTTCAAGCCTAGCCCTTCTCTCATTATTTCTCTC TGACCCTCTCCCCACTGCTCATTTGGATCCCAGGGGAGTGTTCAGGGCCAGCCCTGGCTGGCATGGAGGGTGAGGCTGGG TGTCTGGAAGCATGGAGCATGGGACTGTTCTTTTACAAGACAGGACCCTGGGACCACAGAGGGCAGGAACTTGCACAAAA TCACACAGCCAAGCCAGTCAAGGATGGATGCAGATCCAGAGGTTTCTGGCAGCCAGTACCTCCTGCCCCATGCTGCCCGC TTCTCACCCTATGTGGGTGGGACCACAGACTCACATCCTGACCTTGCACAAACAGCCCCTCTGGACACAGCCCCATGTAC ACGGCCTCAAGGGATGTCTCACATCCTCTGTCTATTTGAGACTTAGAAAAATCCTACAAGGCTGGCAGTGACAGAACTAA GATGATCATCTCCAGTTTATAGACCAGAACCAGAGCTCAGAGAGGCTAGATGATTGATTACCAAGTGCCGGACTAGCAAG TGCTGGAGTCGGGACTAACCCAGGTCCCTTGTCCCAAGTTCCACTGCTGCCTCTTGAATGCAGGGACAAATGCCACACGG CTCTCACCAGTGGCTAGTGGTGGGTACTCAATGTGTACTTTTGGGTTCACAGAAGCACAGCACCCATGGGAAGGGTCCAT CTCAGAGAATTTACGAGCAGGGATGAAGGCCTCCCTGTCTAAAATCCCTCCTTCATCCCCCGCTGGTGGCAGAATCTGTT ACCAGAGGACAAAGCCTTTGGCTCTTCTAATCAGAGCGCAAGCTGGGAGCACAGGCACTGCAGGAGAGAATGCCCAGTGA CCAGTCACTGACCCTGTGCAGAACCTCCTGGAAGCGAGCTTTGCTGGGAGAGGGGGTAGCTAGCCTGAGAGGGAACCCTC TAAGGGACCTCAAAGGTGATTGTGCCAGGCTCTGCGCCTGCCCCACACCCTCCCTTACCCTCCTCCAGACCATTCAGGAC ACAGGGAAATCAGGGTTACAAATCTTCTTGATCCACTTCTCTCAGGATCCCCTCTTCCTACCCTTCCTCACCACTTCC CTCAGTCCCAACTCCTTTTCCCTATTTCCTTCTCCTCCTGTCTTTAAAGCCTGCCTCTTCCAGGAAGACCCCCCTATTGC TGCTGGGGCTCCCCATTTGCTTACTTTGCATTTGTGCCCACTCTCCACCCCTGCTCCCCTGAGCTGAAATAAAAATACAA TAAACTTAC

[0356] "Cluster of differentiation 5 (CD5)" refers to NCBI accession number NP_001333385.1 or a fragment thereof. It means a protein having at least about 85% amino acid sequence identity with the target gene and having immunomodulatory activity. Exemplary amino acid sequences are provided below. >NP_001333385.1 T cell surface glycoprotein CD5 isoform 2 [Homo sapiens] MVCSQSWGRSSKQWEDPSQASKVCQRLNCGVPLSLGPFLVTYTPQSSIICYGQLGSFSNCSHSRNDMCHSLGLTCLEPQK TTPPTTRPPPTTTPEPTAPPRLQLVAQSGGQHCAGVVEFYSSGSLGGTISYEAQDKTQDLENFLCNNLQCGSFLKHLPETE AGRAQDPGEPREHQPLPIQWKIQNSSCTSLEHCFRKIKPQKSGRVLALLCSGFQPKVQSRLVGGSSICEGTVEVRQGAQW AALCDSSSARSSLRWEEVCREQQCGSVNSYRVLDAGDPTSRGLFCPHQKLSQCHELWERNSYCKKVFVTCQDPNPAGLAA GTVASIILALVLLVVLLVVCGPLAYKKLVKKFRQKKQRQWIGPTGMNQNMSFHRNHTATVRSHAENPTASHVDNEYSQPP RNSHLSAYPALEGALHRSSMQPDNSSDSDYDLHGAQRL

[0357] "Cluster of Differentiation 5 (CD5)" refers to a nucleic acid encoding a CD5 polypeptide. A suitable CD5 nucleic acid sequence is provided below. >NM_001346456.1 Homo sapiens CD5 molecule (CD5), transcript variant 2, mRNA GAGTCTTGCTGATGCTCCCGGCTGAATAAACCCCTTCCTTCTTTAACTTGGTGTCTGAGGGGTTTTGTCTGTGGCTTGTC CTGCTACATTTCTTGGTTCCCTGACCAGGAAGCAAAGTGATTAACGGACAGTTGAGGCAGCCCCTTAGGCAGCTTAGGCC TGCCTTGTGGAGCATCCCCGCGGGGAACTCTGGCCAGCTTGAGCGACACGGATCCTCAGAGCGCTCCCAGGTAGGCAATT GCCCCAGTGGAATGCCTCGTCAGAGCAGTGCATGGCAGGCCCCTGTGGAGGATCAACGCAGTGGCTGAACACAGGGAAGG AACTGGCACTTGGAGTCCGGACAACTGAAACTTGTCGCTTCCTGCCTCGGACGGCTCAGCTGGTATGACCCAGATTTCCA GGCAAGGCTCACCCGTTCCAACTCGAAGTGCCAGGGCCAGCTGGAGGTCTACCTCAAGGACGGATGGCACATGGTTTGCA GCCAGAGCTGGGGCCGGAGCTCCAAGCAGTGGGAGGACCCCAGTCAAGCGTCAAAAGTCTGCCAGCGGCTGAACTGTGGG GTGCCCTTAAGCCTTGGCCCCTTCCTTGTCACCTACACACCTCAGAGCTCAATCATCTGCTACGGACAACTGGGCTCCTT CTCCAACTGCAGCCACAGCAGAAATGACATGTGTCACTCTCTGGGCCTGACCTGCTTAGAACCCCAGAAGACAACACCTC CAACGACAAGGCCCCCGCCCACCACAACTCCAGAGCCCACAGCTCCTCCCAGGCTGCAGCTGGTGGCACAGTCTGGCGGC CAGCACTGTGCCGGCGTGGTGGAGTTCTACAGCGGCAGCCTGGGGGGTACCATCAGCTATGAGGCCCAGGACAAGACCCA GGACCTGGAGAACTTCCTCTGCAACAACCTCCAGTGTGGCTCCTTCTTGAAGCATCTGCCAGAGACTGAGGCAGGCAGAG CCCAAGACCCAGGGGAGCCACGGGAACACCAGCCCTTGCCAATCCAATGGAAGATCCAGAACTCAAGCTGTACCTCCCTG GAGCATTGCTTCAGGAAAATCAAGCCCCAGAAAAGTGGCCGAGTTCTTGCCCTCCTTTGCTCAGGTTTCCAGCCCAAGGT GCAGAGCCGTCTGGTGGGGGGCAGCAGCATCTGTGAAGGCACCGTGGAGGTGCGCCAGGGGGCTCAGTGGGCAGCCCTGT GTGACAGCTCTTCAGCCAGGAGCTCGCTGCGGTGGGAGGAGGTGTGCCGGGAGCAGCAGTGTGGCAGCGTCAACTCCTAT CGAGTGCTGGACGCTGGTGACCCAACATCCCGGGGGCTCTTCTGTCCCCATCAGAAGCTGTCCCAGTGCCACGAACTTTG GGAGAGAAATTCCTACTGCAAGAAGGTGTTTGTCACATGCCAGGATCCAAACCCCGCAGGCCTGGCCGCAGGCACGGTGG CAAGCATCATCCTGGCCCTGGTGCTCCTGGTGGTGCTGCTGGTCGTGTGCGGCCCCCTTGCCTACAAGAAGCTAGTGAAG AAATTCCGCCAGAAGAAGCAGCGCCAGTGGATTGGCCCAACGGGAATGAACCAAAACATGTCTTTCCATCGCAACCACAC GGCAACCGTCCGATCCCATGCTGAGAACCCCACAGCCTCCCACGTGGATAACGAATACAGCCAACCTCCCAGGAACTCCC ACCTGTCAGCTTATCCAGCTCTGGAAGGGGCTCTGCATCGCTCCTCCATGCAGCCTGACAACTCCTCCGACAGTGACTAT GATCTGCATGGGGCTCAGAGGCTGTAAAGAACTGGGATCCATGAGCAAAAAGCCGAGAGCCAGACCTGTTTGTCCTGAGA AAACTGTCCGCTCTTCACTTGAAATCATGTCCCTATTTCTACCCCGGCCAGAACATGGACAGAGGCCAGAAGCCTTCCGG ACAGGCGCTGCTGCCCCGAGTGGCAGGCCAGCTCACACTCTGCTGCACAACAGCTCGGCCGCCCCTCCACTTGTGGAAGC TGTGGTGGGCAGAGCCCCAAAACAAGCAGCCTTCCAACTAGAGACTCGGGGGTGTCTGAAGGGGGCCCCCTTTCCCTGCC CGCTGGGGAGCGGCGTCTCAGTGAAATCGGCTTTCTCCTCAGACTCTGTCCCTGGTAAGGAGTGACAAGGAAGCTCACAG CTGGGCGAGTGCATTTTGAATAGTTTTTTGTAAGTAGTGCTTTTCCTCCTTCCTGACAAATCGAGCGCTTTGGCCTCTTC TGTGCAGCATCCACCCCTGCGGATCCCTCTGGGGAGGACAGGAAGGGGACTCCCGGAGACCTCTGCAGCCGTGGTGGTCA GAGGCTGCTCACCTGAGCACAAAGACAGCTCTGCACATTCACCGCAGCTGCCAGCCAGGGGTCTGGGTGGGCACCACCCT GACCCACAGCGTCACCCCACTCCCTCTGTCTTATGACTCCCCTCCCCAACCCCCTCATCTAAAGACACCTTCCTTTCCAC TGGCTGTCAAGCCCACAGGGCACCAGTGCCACCCAGGGCCCGGCACAAAGGGGCGCCTAGTAAACCTTAACCAACTTGGT TTTTTGCTTCACCCAGCAATTAAAAGTCCCAAGCTGAGGTAGTTTCAGTCCATCACAGTTCATCTTCTAACCCAAGAGTC AGAGATGGGGCTGGTCATGTTCCTTTGGTTTGAATAACTCCCTTGACGAAAACAGACTCCTCTAGTACTTGGAGATCTTG GACGTACACCTAATCCCATGGGGCCTCGGCTTCCTTAACTGCAAGTGAGAAGAGGAGGTCTACCCAGGAGCCTCGGGTCT GATCAAGGGAGAGGCCAGGCGCAGCTCACTGCGGCGGCTCCCTAAGAAGGTGAAGCAACATGGGAACACATCCTAAGACA GGTCCTTTCTCCACGCCATTTGATGCTGTATCTCCTGGGAGCACAGGCATCAATGGTCCAAGCCGCATAATAAGTCTGGA AGAGCAAAAGGGAGTTACTAGGATATGGGGTGGGCTGCTCCCAGAATCTGCTCAGCTTTCTGCCCCCACCAACACCCTCC AACCAGGCCTTGCCTTCTGAGAGCCCCCGTGGCCAAGCCCAGGTCACAGATCTTCCCCCGACCATGCTGGGAATCCAGAA ACAGGGACCCCATTTGTCTTCCCATATCTGGTGGAGGTGAGGGGGCTCCTCAAAAAGGGAACTGAGAGGCTGCTCTTAGGG AGGGCAAAGGTTCGGGGGCAGCCAGTGTCTCCCATCAGTGCCTTTTTTAATAAAAGCTCTTTCATCTATAGTTTGGCCAC CATACAGTGGCCTCAAAGCAACCATGGCCTACTTAAAAACCAAACAAAAAAATAAAGAGTTTAGTTGAGGAGAAAAAAAAA AAAAAAAAAAAAAAAA

[0358] "Cluster of differentiation 7 (CD7)" refers to NCBI accession number NP_006128.1 or at least a fragment thereof. Both refer to proteins that have approximately 85% amino acid sequence identity and have immunomodulatory activity. Exemplary amino acid sequences are provided below. >NP_006128.1 T cell antigen CD7 precursor [Homo sapiens] MAGPPRLLLLPLLLLALARGLPGALAAQEVQQSPHCTTVPVGASVNITCSTSGGLRGIYLRQLGPQPQDIIYYEDGVVPTT DRRFRGRIDFSGSQDNLTITMHRLQLSDTGTYTCQAITEVNVYGSGTLVLVTEEQSQGWHRCSDAPPRASALPAPPTGSA LPDPQTASALPDPPAASALPAALAVISFLLGLGVACVLARTQIKKLCSWRDKNSAACVVYEDMSHSRCNTLSSPNQYQ

[0359] "Cluster of Differentiation 7 (CD7)" refers to a nucleic acid encoding a CD7 polypeptide. A suitable CD7 nucleic acid sequence is provided below. >NM_006137.7 Homo sapiens CD7 molecule (CD7), mRNA CTCTCTGAGCTCTGAGCGCCTGCGGTCTCCTGTGTGCTGCTCTCTGTGGGGTCCTGTAGACCCAGAGAGGCTCAGCTGCA CTCGCCCGGCTGGGAGAGCTGGGTGTGGGGAACATGGCCGGGCCTCCGAGGCTCCTGCTGCTGCCCCTGCTTCTGGCGCT GGCTCGCGGCCTGCCTGGGGCCCTGGCTGCCCAAGAGGTGCAGCAGTCTCCCACTGCACGACTGTCCCCGTGGGAGCCT CCGTCAACATCACCTGCTCCACCAGCGGGGGCCTGCGTGGGATCTACCTGAGGCAGCTCGGGCCACAGCCCCAAGACATC ATTTACTACGAGGACGGGGTGGTGCCCACTACGGACAGACGGTTCCGGGGCCGCATCGACTTCTCAGGGTCCCAGGACAA CCTGACTATCACCATGCACCGCCTGCAGCTGTCGGACACTGGCACCTACACCTGCCAGGCCATCACGGAGGTCAATGTCT ACGGCTCCGGCACCCTGGTCCTGGTGACAGAGGAACAGTCCCAAGGATGGCACAGATGCTCGGACGCCCCACCAAGGGCC TCTGCCCTCCCTGCCCCACCGACAGGCTCCGCCCTCCCTGACCCGCAGACAGCCTCTGCCCTCCCTGACCCGCCAGCAGC CTCTGCCCTCCCTGCGGCCCTGGCGGTGATCTCCTTCCTCCTCGGGCTGGGCCTGGGGGTGGCGTGTGTGCTGGCGAGGA CACAGATAAAGAAACTGTGCTCGTGGCGGGATAAGAATTCGGCGGCATGTGTGGTGTACGAGGACATGTCGCACAGCCGC TGCAACACGCTGTCCTCCCCCAACCAGTACCAGTGACCCAGTGGGCCCCTGCACGTCCCGCCTGTGGTCCCCCCAGCACC TTCCCTGCCCCACCATGCCCCCCACCCTGCCACACCCCTCACCCTGCTGTCCTCCCACGGCTGCAGCAGAGTTTGAAGGG CCCAGCCGTGCCCAGCTCCAAGCAGACACACAGGCAGTGGCCAGGCCCCACGGTGCTTCTCAGTGGACAATGATGCCTCC TCCGGGAAGCCTTCCCTGCCCAGCCCACGCCGCCACCGGGAGGAAGCCTGACTGTCCTTTGGCTGCATCTCCCGACCATG GCCAAGGAGGGCTTTTCTGTGGGATGGGCCTGGGCACGCGGCCCTCTCCTGTCAGTGCCGGCCCACCCACCAGCAGGCCC CCAACCCCCAGGCAGCCCGGCAGAGGACGGGAGGAGACCAGTCCCCCACCCAGCCGTACCAGAAATAAAGGCTTCTGTGC TTCC

[0360] "Cluster of Differentiation 30 (CD30)" refers to a protein having at least about 85% amino acid sequence identity with NCBI accession number NP_001234.3 or a fragment thereof and having immunomodulatory activity. ​Exemplary amino acid sequences are provided below. >NP_001234.3 Tumor necrosis factor receptor superfamily member 8 isoform 1 precursor [Homo sapiens] MRVLLAALGLLFLGALRAFPQDRPFEDTCHGNPSHYYDKAVRRCCYRCPMGLFPTQQCPQRPTDCRKQCEPDYYLDEADR CTACVTCSRDDLVEKTPCAWNSSRVCECRPGMFCSTSAVNSCARCFFHSVCPAGMIVKFPGTAQKNTVCEPASPGVSPAC ASPENCKEPSSGTIPQAKPTPVSPATSSASTMPVRGGTRLAQEAASKLTRAPDSPSSVGRPSSDPGLSPTQPCPEGSGDC RKQCEPDYYLDEAGRCTACVSCSRDDLVEKTPCAWNSSRTCECRPGMICATSATNSCARCVPYPICAAETVTKPQDMAEK DTTFEAPPLGTQPDCNPTPENGEAPASTSPTQSLLVDSQASKTLPIPTSAPVALSSTGKPVLDAGPVLFWVILVLVVVVG SSAFLLCHRRACRKRIRQKLHLCYPVQTSQPKLELVDSRPRRSSTQLRSGASVTEPVAEERGLMSQPLMETCHSVGAAYL ESLPLQDASPAGGPSSPRDLPEPRVSTEHTNNKIEKIYIMKADTVIVGTVKAELPEGRGLAGPAEPELEEELEADHTPHY PEQETEPPLGSCSDVMLSVEEEGKEDPLPTAASGK

[0361] "Cluster of Differentiation 30 (CD30)" refers to a nucleic acid encoding a CD30 polypeptide. A representative CD30 nucleic acid sequence is provided below. >NM_001243.5 Homo sapiens TNF receptor superfamily member 8 (TNFRSF8), translocation Writing variant 1, mRNA CTGAGTCATCTCTGCACGTGTTTGCCCCCTTTTTTCTTCGCTGCTTGTAGCTAAGTGTTCCTGGAACCAATTTGATACGG GAGAACTAAGGCTGAAACCTCGGAGGAACAACCACTTTTGAAGTGACTTCGCGGCGTGCGTTGGGTGCGGACTAGGTGGC CGCGGCGGGAGTGTGCTGGAGCCTGAAGTCCACGCGCGCGGCTGAGAACCGCCGGGACCGCACGTGGGCGCCGCGCGCTT CCCCCGCTTCCCAGGTGGGCGCCGGCCGCCAGGCCACCTCACGTCCGGCCCCGGGGATGCGCGTCCTCCTCGCCGCGCTG GGACTGCTGTTCCTGGGGGCGCTACGAGCCTTCCCACAGGATCGACCCTTCGAGGACACCTGTCATGGAAACCCCAGCCA CTACTATGACAAGGCTGTCAGGAGGTGCTGTTACCGCTGCCCCATGGGGCTGTTCCCGACACAGCAGTGCCCACAGAGGC CTACTGACTGCAGGAAGCAGTGTGAGCCTGACTACTACCTGGATGAGGCCGACCGCTGTACAGCCTGCGTGACTTGTTCT CGAGACGACCTCGTGGAGAAGACGCCGTGTGCATGGAACTCCTCCCGTGTCTGCGAATGTCGACCCGGCATGTTCTGTTC CACGTCTGCCGTCAACTCCTGTGCCCGCTGCTTCTTCCATTCTGTCTGTCCGGCAGGGATGATTGTCAAGTTCCCAGGCA CGGCGCAGAAGAACACGGTCTGTGAGCCGGCTTCCCCAGGGGTCAGCCCTGCCTGTGCCAGCCCAGAGAACTGCAAGGAA CCCTCCAGTGGCACCATCCCCCAGGCCAAGCCCACCCCGGTGTCCCCAGCAACCTCCAGTGCCAGCACCATGCCTGTAAG AGGGGGCACCCGCCTCGCCCAGGAAGCTGCTTCTAAACTGACGAGGGCTCCCGACTCTCCCTCCTCTGTGGGAAGGCCTA GTTCAGATCCAGGTCTGTCCCCAACACAGCCATGCCCAGAGGGGTCTGGTGATTGCAGAAAGCAGTGTGAGCCCGACTAC TACCTGGACGAGGCCGGCCGCTGCACGGCCTGCGTGAGCTGTTCTCGAGATGACCTTGTGGAGAAGACGCCATGTGCATG GAACTCCTCCCGCACCTGCGAATGTCGACCTGGCATGATCTGTGCCACATCAGCCACCAACTCCTGTGCCCGCTGTGTCC CCTACCCAATCTGTGCAGCAGAGACGGTCACCAAGCCCCAGGATATGGCTGAGAAGGACACCACCTTTGAGGCGCCACCC CTGGGGACCCAGCCGGACTGCAACCCCACCCCAGAGAATGGCGAGGCGCCTGCCAGCACCAGCCCCACTCAGAGCTTGCT GGTGGACTCCCAGGCCAGTAAGACGCTGCCCATCCCAACCAGCGCTCCCGTCGCTCTCTCCTCCACGGGGAAGCCCGTTC TGGATGCAGGGCCAGTGCTCTTCTGGGTGATCCTGGTGTTGGTTGTGGTGGTCGGCTCCAGCGCCTTCCTCCTGTGCCAC CGGAGGGCCTGCAGGAAGCGAATTCGGCAGAAGCTCCACCTGTGCTACCCGGTCCAGACCTCCCAGCCCAAGCTAGAGCT TGTGGATTCCAGACCCAGGAGGAGCTCAACGCAGCTGAGGAGTGGTGCGTCGGTGACAGAACCCGTCGCGGAAGAGCGAG GGTTAATGAGCCAGCCACTGATGGAGACCTGCCACAGCGTGGGGGCAGCCTACCTGGAGAGCCTGCCGCTGCAGGATGCC AGCCCGGCCGGGGGCCCCTCGTCCCCCAGGGACCTTCCTGAGCCCCGGGTGTCCACGGAGCACACCAATAACAAGATTGA GAAAATCTACATCATGAAGGCTGACACCGTGATCGTGGGGACCGTGAAGGCTGAGCTGCCGGAGGGCCGGGGCCTGGCGG GGCCAGCAGAGCCCGAGTTGGAGGAGGAGCTGGAGGCGGACCATACCCCCCACTACCCCGAGCAGGAGACAGAACCGCCT CTGGGCAGCTGCAGCGATGTCATGCTCTCAGTGGAAGAGGAAGGGAAAGAAGACCCCTTGCCCACAGCTGCCTCTGGAAA GTGAGGCCTGGGCTGGGCTGGGGCTAGGAGGGCAGCAGGGTGGCCTCTGGGAGGCCAGGATGGCACTGTTGGCACCGAGG TTGGGGGCAGAGGCCCATCTGGCCTGAACTGAGGCTCCAGCATCTAGTGGTGGACCGGCCGGTCACTGCAGGGGTCTGGT GGTCTCTGCTTGCATCCCCAACTTAGCTGTCCCCTGACCCAGAGCCTAGGGGATCCGGGGCTTGTACAGAAGAGACAGTC CAAGGGGACTGGATCCCAGCAGTGATGTTGGTTGAGGCAGCAAACAGATGGCAGGATGGGCACTGCCGAGAACAGCATTG GTCCCAGAGCCCTGGGCATCAGACCTTAACCACCAGGCCCACAGCCCAGCGAGGGAGAGGTCGTGAGGCCAGCTCCCGGG GCCCCTGTAACCCTACTCTCCTCTCTCCCTGGACCTCAGAGGTGACACCCATTGGGCCCTTCCGGCATGCCCCCAGTTAC TGTAAATGTGGCCCCCAGTGGGCATGGAGCCAGTGCCTGTGGTTGTTTCTCCAGAGTCAAAAGGGAAGTCGAGGGATGGG GCGTCGTCAGCTGGCACTGTCTCTGCTGCAGCGGCCACACTGTACTCTGCACTGGTGTGAGGGCCCCTGCCTGGACTGTG GGACCCTCCTGGTGCTGCCCACCTTCCCTGTCCTGTAGCCCCCTCGGTGGGCCCAGGGCCTAGGGCCCAGGATCAAGTCA CTCATCTCAGAATGTCCCCACCAATCCCCGCCACAGCAGGCGCCTCGGGTCCCAGATGTCTGCAGCCCTCAGCAGCTGCA GACCGCCCCTCACCAACCCAGAGAACCTGCTTTACTTTGCCCAGGGACTTCCTCCCCATGTGAACATGGGGAACTTCGGG CCCTGCCTGGAGTCCTTGACCGCTCTCTGTGGGCCCCACCCACTCTGTCCTGGGAAATGAAGAAGCATCTTCCTTAGGTC TGCCCTGCTTGCAAATCCACTAGCACCGACCCCACCACCTGGTTCCGGCTCTGCACGCTTTGGGGTGTGGATGTCGAGAG GCACCACGGCCTCACCCAGGCATCTGCTTTACTCTGGACCATAGGAAACAAGACCGTTTGGAGGTTTCATCAGGATTTTG GGTTTTTCACATTTCACGCTAAGGAGTAGTGGCCCTGACTTCCGGTCGGCTGGCCAGCTGACTCCCTAGGGCCTTCAGAC GTGTATGCAAATGAGTGATGGATAAGGATGAGTCTTGGAGTTGCGGGCAGCCTGGAGACTCGTGGACTTACCGCCTGGAG GCAGGCCCGGGAAGGCTGCTGTTTACTCATCGGGCAGCCACGTGCTCTCTGGAGGAAGTGATAGTTTCTGAAACCGCTCA GATGTTTTGGGGAAAGTTGGAGAAGCCGTGGCCTTGCGAGAGGTGGTTACACCAGAACCTGGACATTGGCCAGAAGAAGC TTAAGTGGGCAGACACTGTTTGCCCAGTGTTTGTGCAAGGATGGAGTGGGTGTCTCTGCATCACCCACAGCCGCAGCTGT AAGGCACGCTGGAAGGCACACGCCTGCCAGGCAGGGCAGTCTGGCGCCCATGATGGGAGGGATTGACATGTTTCAACAAA ATAATGCACTTCCTTACCTAGTGGCCCTTCACACAACTTTTGAATCTCTAAAAATCCATAAAATCCTTAAAGAACTGTAA

[0362] "Cluster of differentiation 33 (CD33)" refers to NCBI accession number NP_001763.3 or a fragment thereof. It means a protein having at least about 85% amino acid sequence identity with the target gene and having immunomodulatory activity. Exemplary amino acid sequences are provided below. >NP_001763.3 Myeloid cell surface antigen CD33 isoform 1 precursor [Homo sapiens] MPLLLLLPLLWAGALAMDPNFWLQVQESVTVQEGLCVLVPCTFFHPIPYYDKNSPVHGYWFREGAIISRDSPVATNKLDQ EVQEETQGRFRLLGDPSRNNCSLSIVDARRRDNGSYFFRMERGSTKYSYKSPQLSVHVTDLTHRPKILIPGTLEPGHSKN LTCSVSWACEQGTPPIFSWLSAAPTSLGPRTTHSSVLIITPRPQDHGTNLTCQVKFAGAGVTTERTIQLNVTYVPQNPTT GIFPGDGSGKQETRAGVVHGAIGGAGVTALLALCLCLIFFIVKTHRRKAARTAVGRNDTHPTTGSASPKHQKKSKLHGPT ETSSCSGAAPTVEMDEELHYASLNFHGMNPSKDTSTEYSEVRTQ

[0363] "Cluster of Differentiation 33 (CD33)" refers to a nucleic acid encoding a CD33 polypeptide. A representative CD33 nucleic acid sequence is provided below. >NM_001772.4 Homo sapiens CD33 molecule (CD33), transcript variant 1, mRNA CTGCTCACACAGGAAGCCCTGGAAGCTGCTTCCTCAGACATGCCGCTGCTGCTACTGCTGCCCCTGCTGTGGGCAGGGGC CCTGGCTATGGATCCAAATTTCTGGCTGCAAGTGCAGGAGTCAGTGACGGTACAGGAGGGTTTGTGCGTCCTCGTGCCCT GCACTTTCTTCCATCCCATACCCTACTACGACAAGAACTCCCCAGTTCATGGTTACTGGTTCCGGGAAGGAGCCATTATA TCCAGGGACTCTCCAGTGGCCACAAACAAGCTAGATCAAGAAGTACAGGAGGAGACTCAGGGCAGATTCCGCCTCCTTGG GGATCCCAGTAGGAACAACTGCTCCCTGAGCATCGTAGACGCCAGGAGGAGGGATAATGGTTCATACTTCTTTCGGATGG AGAGAGGAAGTACCAAATACAGTTACAAATCTCCCCAGCTCTCTGTGCATGTGACAGACTTGACCCACAGGCCCAAAATC CTCATCCCTGGCACTCTAGAACCCGGCCACTCCAAAAACCTGACCTGCTCTGTGTCCTGGGCCTGTGAGCAGGGAACACC CCCGATCTTCTCCTGGTTGTCAGCTGCCCCCACCTCCCTGGGCCCCAGGACTACTCACTCCTCGGTGCTCATAATCACCC CACGGCCCCAGGACCACGGCACCAACCTGACCTGTCAGGTGAAGTTCGCTGGAGCTGGTGTGACTACGGAGAGAACCATC CAGCTCAACGTCACCTATGTTCCACAGAACCCAACAACTGGTATCTTTCCAGGAGATGGCTCAGGGAAACAAGAGACCAG AGCAGGAGTGGTTCATGGGGCCATTGGAGGAGCTGGTGTTACAGCCCTGCTCGCTCTTTGTCTCTGCCTCATCTTCTTCA TAGTGAAGACCCACAGGAGGAAAGCAGCCAGGACAGCAGTGGGCAGGAATGACACCCACCCTACCACAGGGTCAGCCTCC CCGAAACACCAGAAGAAGTCCAAGTTACATGGCCCCACTGAAACCTCAAGCTGTTCAGGTGCCGCCCCTACTGTGGAGAT GGATGAGGAGCTGCATTATGCTTCCCTCAACTTTCATGGGATGAATCCTTCCAAGGACACCTCCACCGAATACTCAGAGG TCAGGACCCAGTGAGGAACCCACAAGAGCATCAGGCTCAGCTAGAAGATCCACATCCTCTACAGGTCGGGGACCAAAGGC TGATTCTTGGAGATTTAACACCCCACAGGCAATGGGTTTATAGACATTATGTGAGTTTCCTGCTATATTAACATCATCTT AGACTTTGCAAGCAGAGAGTCGTGGAATCAAATCTGTGCTCTTTCATTTGCTAAGTGTATGATGTCACACAAGCTCCTTA ACCTTCCATGTCTCCATTTTCTTCTCTGTGAAGTAGGTATAAGAAGTCCTATCTCATAGGGATGCTGTGAGCATTAAATA AAGGTACACATGGAAAACACCA

[0364] "Cluster of differentiation 52 (CD52)" refers to NCBI accession number NP_001794.2 or a fragment thereof. It means a protein having at least about 85% amino acid sequence identity with the target gene and having immunomodulatory activity. Exemplary amino acid sequences are provided below. >NP_001794.2 CAMPATH-1 antigen precursor [Homo sapiens]

[0365] MKRFLFLLLTISLLVMVQIQTGLSGQNDTSQTSSPSASSNISGGIFLFFVANAIIHLFCFS

[0366] "Cluster of Differentiation 52 (CD52)" refers to a nucleic acid encoding a CD52 polypeptide. A representative CD52 nucleic acid sequence is provided below. >NM_001803.3 Homo sapiens CD52 molecule (CD52), mRNA AGACAGCCCTGAGATCACCTAAAAAGCTGCTACCAAGACAGCCACGAAGATCCTACCAAAATGAAGCGCTTCCTCTTCCT CCTACTCACCATCAGCCTCCTGGTTATGGTACAGATACAAACTGGACTCTCAGGACAAAACGACACCAGCCAAACCAGCA GCCCCTCAGCATCCAGCAACATAAGCGGAGGCATTTTCCTTTTCTTCGTGGCCAATGCCATAATCCACCTCTTCTGCTTC AGTTGAGGTGACACGTCTCAGCCTTAGCCCTGTGCCCCTGAAAACAGCTGCCACCATCACTCGCAAGAGAATCCCCTCCA TCTTTGGGAGGGGTTGATGCCAGACATCACCAGGTTGTAGAAGTTGACAGGCAGTGCCATGGGGGCAACAGCCAAAATAG GGGGGTAATGATGTAGGGGCCAAGCAGTGCCCAGCTGGGGGTCAATAAAGTTACCCTTGTACTTGCA

[0367] "Cluster of differentiation 70 (CD70)" refers to NCBI accession number NP_001243.1 or a fragment thereof. It means a protein having at least about 85% amino acid sequence identity with the target gene and having immunomodulatory activity. Exemplary amino acid sequences are provided below. >NP_001243.1 CD70 antigen isoform 1 [Homo sapiens] MPEEGSGCSVRRRPYGCVLRAALVPLVAGLVICLVVCIQRFAQAQQQLPLESLGWDVAELQLNHTGPQQDPRLYWQGGPA LGRSFLHGPELDKGQLRIHRDGIYMVHIQVTLAICSSTTASRHHPTTLAVGICSPASRSISLLRLSFHQGCTIASQRLTP LARGDTLCTNLTGTLLPSRNTDETFFGVQWVRP

[0368] "Cluster of Differentiation 70 (CD70)" refers to a nucleic acid encoding a CD70 polypeptide. Typical CD70 nucleic acid sequences are provided below. >NM_001252.5 Homo sapiens CD70 molecule (CD70), transcript variant 1, mRNA AGAGAGGGGCAGGCTGGTCCCCTGACAGGTTGAAGCAAGTAGACGCCCAGGAGCCCCGGGAGGGGGCTGCAGTTTCCTTC CTTCCTTCTCGGCAGCGCTCCGCGCCCCCATCGCCCCTCCTGCGCTAGCGGAGGTGATCGCCGCGGCGATGCCGGAGGAG GGTTCGGGCTGCTCGGTGCGGCGCAGGCCCTATGGGTGCGTCCTGCGGGCTGCTTTGGTCCCATTGGTCGCGGGCTTGGT GATCTGCCTCGTGGTGTGCATCCAGCGCTTCGCACAGGCTCAGCAGCAGCTGCCGCTCGAGTCACTTGGGTGGGACGTAG CTGAGCTGCAGCTGAATCACACAGGACCTCAGCAGGACCCCAGGCTATACTGGCAGGGGGGCCCAGCACTGGGCCGCTCC TTCCTGCATGGACCAGAGCTGGACAAGGGGCAGCTACGTATCCATCGTGATGGCATCTACATGGTACACATCCAGGTGAC GCTGGCCATCTGCTCCTCCACGACGGCCTCCAGGCACCACCCCACCACCCTGGCCGTGGGAATCTGCTCTCCCGCCTCCC GTAGCATCAGCCTGCTGCGTCTCAGCTTCCACCAAGGTTGTACCATTGCCTCCCAGCGCCTGACGCCCCTGGCCCGAGGG GACACACTCTGCACCAACCTCACTGGGACACTTTTGCCTTCCCGAAACACTGATGAGACCTTCTTTGGAGTGCAGTGGGT GCGCCCCTGACCACTGCTGCTGATTAGGGTTTTTTAAATTTTATTTTATTTTATTTAAGTTCAAGAGAAAAAGTGTACAC ACAGGGGCCACCCGGGGTTGGGGTGGGAGTGTGGTGGGGGGTAGTGGTGGCAGGACAAGAGAAGGCATTGAGCTTTTTCT TTCATTTTCCTATTAAAAAATACAAAAATCA

[0369] "Class II, major histocompatibility complex, transactivator tibility complex, transactivator (CIITA)” is NCBI accession number NP_001273331. 1 or a fragment thereof and having at least about 85% amino acid sequence identity with the Exemplary amino acid sequences are provided below. >NP_001273331.1 MHC class II transactivator isoform 1 [Homo sapiens] MRCLAPRPAGSYLSEPQGSSQCATMELGPLEGGYLELLNSDADPLCLYHFYDQMDLAGEEEIELYSEPDTDTINCDQFSR LLCDMEGDEETREAYANIAELDQYVFQDSQLEGLSKDIFIEHIGPDEVIGESMEMPAEVGQKSQKRPFPEELPADLKHWK PAEPTVVTGSLVGPVSDCSTLPCLPLPALFNQEPASGQMRLEKTDQIPMPFSSSSLSCLNLPEGPIQFVPTISTLPHG LWQISEAGTGVSSIFIYHGEVPQASQVPPPSGFTVHGLPTSPDRPGSTSPFAPSATDLPSMPEPALTSRANMTEHKTSPT QCPAAGEVSNKLPKWPEPVEQFYRSLQDTYGAEPAGPDGILVEVDLVQARLERSSSKSLERELATPDWAERQLAQGGLAE VLLAAKEHRRPRETRVIAVLGKAGQGKSYWAGAVSRAWACGRLPQYDFVFSVPCHCLNRPGDAYGLQDLLFSLGPQPLVA ADEVFSHILKRPDRVLLILDGFEELEAQDGFLHSTCGPAPAEPCSLRGLLAGLFQKKLLRGCTLLLTARPRGRLVQSLSK ADALFELSGFSMEQAQAYVMRYFESSGMTEHQDRALTLLRDRPLLLSHSHSPTLCRAVCQLSEALLELGEDAKLPSTLTG LYVGLLGRAALDSPPGALAELAKLAWELGRRHQSTLQEDQFPSADVRTWAMAKGLVQHPPRAAESELAFPSFLLQCFLGA LWLALSGEIKDKELPQYLALTPRKKRPYDNWLEGVPRFLAGLIFQPPARCLGALLGPSAAASVDRKQKVLARYLKRLQPG TLRARQLLELLHCAHEAEEAGIWQHVVQELPGRLSFLGTRLTPPDAHVLGKALEAAGQDFSLDLRSTGICPSGLGSLVGL SCVTRFRAALSDTVALWESLQQHGETKLLQAAEEKFTIEPFKAKSLKDVEDLGKLVQTQRTRSSSEDTAGELPAVRDLKK LEFALGPVSGPQAFPKLVRILTAFSSLQHLDLDALSENKIGDEGVSQLSATFPQLKSLETLNLSQNNITDLGAYKLAEAL PSLAASLLRLSLYNNCICDVGAESLARVLPDMVSLRVMDVQYNKFTAAGAQQLAASLRRCPHVETLAMWTPTIPFSVQEH LQQQDSRISLR [[ID=2o]]

[0370] "class II, major histocompatibility complex, transactivator (class II, major histocompa It should be noted that there seems to be an error in the original text where "2o" is likely a typo and should be "20". Also, the text "クラスII、主要組織適合複合体、トランス活性化因子(class II, major histocompa" seems incomplete."complex, transactivator (CIITA)" refers to a gene encoding a CIITA polypeptide. By "nucleic acid" is meant an exemplary CIITA nucleic acid sequence, as provided below: >NM_001286402.1 Homo sapiens class II major histocompatibility complex transactivator (CII TA), transcript variant 1, mRNA GGTTAGTGATGAGGCTAGTGATGAGGCTGTGTGCTTCTGAGCTGGGCATCCGAAGGCATCCTTGGGGAAGCTGAGGGCAC GAGGAGGGGCTGCCAGACTCCGGGAGCTGCTGCCTGGCTGGGATTCCTACACAATGCGTTGCCTGGCTCCACGCCCTGCT GGGTCCTACCTGTCAGAGCCCCAAGGCAGCTCACAGTGTGCCACCATGGAGTTGGGGCCCCTAGAAGGTGGCTACCTGGA GCTTCTTAACAGCGATGCTGACCCCCTGTGCCTCTACCACTTCTATGACCAGATGGACCTGGCTGGAGAAGAAGAGATTG AGCTCTACTCAGAACCCGACACAGACACCATCAACTGCGACCAGTTCAGCAGGCTGTTGTGTGACATGGAAGGTGATGAA GAGACCAGGGAGGCTTATGCCAATATCGCGGAACTGGACCAGTATGTCTTCCAGGACTCCCAGCTGGAGGGCCTGAGCAA GGACATTTTCATAGAGCACATAGGACCAGATGAAGTGATCGGTGAGAGTATGGAGATGCCAGCAGAAGTTGGGCAGAAAA GTCAGAAAGACCCTTCCCAGAGGAGCTTCCGGCAGACCTGAAGCACTGGAAGCCAGCTGAGCCCCCCACTGTGGTGACT GGCAGTCTCCTAGTGGGACCAGTGAGCGACTGCTCCACCCTGCCCTGCCTGCCACTGCCTGCGCTGTTCAACCAGGAGCC AGCCTCCGGCCAGATGCGCCTGGAGAAAACCGACCAGATTCCCATGCCTTTCTCCAGTTCCTCGTTGAGCTGCCTGAATC TCCCTGAGGGACCCATCCAGTTTGTCCCCACCATCTCCACTCTGCCCCATGGGCTCTGGCAAATCTCTGAGGCTGGAACA GGGGTCTCCAGTATATTCATCTACCATGGTGAGGTGCCCCAGGCCAGCCAAGTACCCCCTCCCAGTGGATTCACTGTCCA CGGCCTCCCAACATCTCCAGACCGGCCAGGCTCCACCAGCCCCTTCGCTCCATCAGCCACTGACCTGCCCAGCATGCCTG AACCTGCCCTGACCTCCCGAGCAAACATGACAGAGCACAAGACGTCCCCCACCCAATGCCCGGCAGCTGGAGAGGTCTCC AACAAGCTTCCAAAATGGCCTGAGCCGGTGGAGCAGTTCTACCGCTCACTGCAGGACACGTATGGTGCCGAGCCCGCAGG CCCGGATGGCATCCTAGTGGAGGTGGATCTGGTGCAGGCCAGGCTGGAGAGGAGCAGCAGCAAGAGCCTGGAGCGGGAAC TGGCCACCCCGGACTGGGCAGAACGGCAGCTGGCCCAAGGAGGCCTGGCTGAGGTGCTGTTGGCTGCCAAGGAGCACCGG CGGCCGCGTGAGACACGAGTGATTGCTGTGCTGGGCAAAGCTGGTCAGGGCAAGAGCTATTGGGCTGGGGCAGTGAGCCG GGCCTGGGCTTGTGGCCGGCTTCCCCAGTACGACTTTGTCTTCTCTGTCCCCTGCCATTGCTTGAACCGTCCGGGGGATG CCTATGGCCTGCAGGATCTGCTCTTCTCCCTGGGCCCACAGCCACTCGTGGCGGCCGATGAGGTTTTCAGCCACATCTTG AAGAGACCTGACCGCGTTCTGCTCATCCTAGACGGCTTCGAGGAGCTGGAAGCGCAAGATGGCTTCCTGCACAGCACGTG CGGACCGGCACCGGCGGAGCCCTGCTCCCTCCGGGGGCTGCTGGCCGGCCTTTTCCAGAAGAAGCTGCTCCGAGGTTGCA CCCTCCTCCTCACAGCCCGGCCCCGGGGCCGCCTGGTCCAGAGCCTGAGCAAGGCCGACGCCCTATTTGAGCTGTCCGGC TTCTCCATGGAGCAGGCCCAGGCATACGTGATGCGCTACTTTGAGAGCTCAGGGATGACAGAGCACCAAGACAGAGCCCT GACGCTCCTCCGGGACCGGCCACTTCTTCTCAGTCACAGCCACAGCCCTACTTTGTGCCGGGCAGTGTGCCAGCTCTCAG AGGCCCTGCTGGAGCTTGGGGAGGACGCCAAGCTGCCCTCCACGCTCACGGGACTCTATGTCGGCCTGCTGGGCCGTGCA GCCCTCGACAGCCCCCCCGGGGCCCTGGCAGAGCTGGCCAAGCTGGCCTGGGAGCTGGGCCGCAGACATCAAAGTACCCT ACAGGAGGACCAGTTCCCATCCGCAGACGTGAGGACCTGGGCGATGGCCAAAGGCTTAGTCCAACACCCACCGCGGGCCG CAGAGTCCGAGCTGGCCTTCCCCAGCTTCCTCCTGCAATGCTTCCTGGGGGCCCTGTGGCTGGCTCTGAGTGGCGAAATC AAGGACAAGGAGCTCCCGCAGTACCTAGCATTGACCCCAAGGAAGAAGAGGCCCTATGACAACTGGCTGGAGGGCGTGCC ACGCTTTCTGGCTGGGCTGATCTTCCAGCCTCCCGCCCGCTGCCTGGGAGCCCTACTCGGGCCATCGGCGGCTGCCTCGG TGGACAGGAAGCAGAAGGTGCTTGCGAGGTACCTGAAGCGGCTGCAGCCGGGGACACTGCGGGCGCGGCAGCTGCTGGAG CTGCTGCACTGCGCCCACGAGGCCGAGGAGGCTGGAATTTGGCAGCACGTGGTACAGGAGCTCCCCGGCCGCCTCTCTTT TCTGGGCACCCGCCTCACGCCTCCTGATGCACATGTACTGGGCAAGGCCTTGGAGGCGGCGGGCCAAGACTTCTCCCTGG ACCTCCGCAGCACTGGCATTTGCCCCTCTGGATTGGGGAGCCTCGTGGGACTCAGCTGTGTCACCCGTTTCAGGGCTGCC TTGAGCGACACGGTGGCGCTGTGGGAGTCCCTGCAGCAGCATGGGGAGACCAAGCTACTTCAGGCAGCAGAGGAGAAGTT CACCATCGAGCCTTTCAAAGCCAAGTCCCTGAAGGATGTGGAAGACCTGGGAAAGCTTGTGCAGACTCAGAGGACGAGAA GTTCCTCGGAAGACACAGCTGGGGAGCTCCCTGCTGTTCGGGACCTAAAGAAACTGGAGTTTGCGCTGGGCCCTGTCTCA GGCCCCCAGGCTTTCCCCAAACTGGTGCGGATCCTCACGGCCTTTTCCTCCCTGCAGCATCTGGACCTGGATGCGCTGAG TGAGAACAAGATCGGGGACGAGGGTGTCTCGCAGCTCTCAGCCACCTTCCCCCAGCTGAAGTCCTTGGAAACCCTCAATC TGTCCCAGAACAACATCACTGACCTGGGTGCCTACAAACTCGCCGAGGCCCTGCCTTCGCTCGCTGCATCCCTGCTCAGG CTAAGCTTGTACAATAACTGCATCTGCGACGTGGGAGCCGAGAGCTTGGCTCGTGTGCTTCCGGACATGGTGTCCCTCCG GGTGATGGACGTCCAGTACAACAAGTTCACGGCTGCCGGGGCCCAGCAGCTCGCTGCCAGCCTTCGGAGGTGTCCTCATG TGGAGACGCTGGCGATGTGGACGCCCACCATCCCATTCAGTGTCCAGGAACACCTGCAACAACAGGATTCACGGATCAGC CTGAGATGATCCCAGCTGTGCTCTGGACAGGCATGTTCTCTGAGGACACTAACCACGCTGGACCTTGAACTGGGTACTTG TGGACACAGCTCTTCTCCAGGCTGTATCCCATGAGCCTCAGCATCCTGGCACCCGGCCCCTGCTGGTTCAGGGTTGGCCC CTGCCCGGCTGCGGAATGAACCACATCTTGCTCTGCTGACAGACACAGGCCCGGCTCCAGGCTCCTTTAGCGCCCAGTTG GGTGGATGCCTGGTGGCAGCTGCGGTCCACCCAGGAGCCCCGAGGCCTTCTCTGAAGGACATTGCGGACAGCCACGGCCA GGCCAGAGGGAGTGACAGAGGCAGCCCCATTCTGCCTGCCCAGGCCCCTGCCACCCTGGGGAGAAAGTACTTCTTTTTTT TTATTTTTAGACAGAGTCTCACTGTTGCCCAGGCTGGCGTGCAGTGGTGCGATCTGGGTTCACTGCAACCTCCGCCTCTT GGGTTCAAGCGATTCTTCTGCTTCAGCCTCCCGAGTAGCTGGGACTACAGGCACCCACCATCATGTCTGGCTAATTTTTC ATTTTTAGTAGAGACAGGGTTTTGCCATGTTGGCCAGGCTGGTCTCAAACTCTTGACCTCAGGTGATCCACCCACCTCAG CCTCCCAAAGTGCTGGGATTACAAGCGTGAGCCACTGCACCGGGCCACAGAGAAAGTACTTCTCCACCCTGCTCTCCGAC CAGACACCTTGACAGGGCACACCGGGCACTCAGAAGACACTGATGGGCAACCCCCAGCCTGCTAATTCCCCAGATTGCAA CAGGCTGGGCTTCAGTGGCAGCTGCTTTTGTCTATGGGACTCAATGCACTGACATTGTTGGCCAAAGCCAAAGCTAGGCC TGGCCAGATGCACCAGCCCTTAGCAGGGAAACAGCTAATGGGACACTAATGGGGCGGTGAGAGGGGAACAGACTGGAAGC ACAGCTTCATTTCCTGTGTCTTTTTTCACTACATTATAAATGTCTCTTTAATGTCACAGGCAGGTCCAGGGTTTGAGTTC ATACCCTGTTACCATTTTGGGGTACCCACTGCTCTGGTTATCTAATATGTAACAAGCCACCCCAAATCATAGTGGCTTAA AACAACACTCACATTTA

[0371] "Cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) polypeptide" refers to the NCBI Access

[0023] The present invention also refers to a protein having at least about 85% sequence identity with the sequence number EAW70354.1 or a fragment thereof. Exemplary amino acid sequences are provided below. >EAW70354.1 Cytotoxic T-lymphocyte-associated protein 4 [Homo sapiens] MACLGFQRHKAQLNLATRTWPCTLLFFLLFIPVFCKAMHVAQPAVVLASSRGIASFVCEYASPGKATEVRVTVLRQADSQ VTEVCAATYMMGNELTFLDDSICTGTSSGNQVNLTIQGLRAMDTGLYICKVELMYPPPYYLGIGNGTQIYVIDPEPCPDS DFLLWILAAVSSGLFFYSFLLTAVSLSKMLKKRSPLTTGVYVKMPPTEPECEKQFQPYFIPIN

[0372] "Cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) polynucleotide" means a CTLA-4 polynucleotide. The CTLA-4 gene refers to a nucleic acid molecule encoding an immunoglobulin superfamily polypeptide. family and encode proteins that transmit inhibitory signals to T cells Exemplary CTLA-4 nucleic acid sequences are provided below. >BC074842.2 Homo sapiens cytotoxic T-lymphocyte-associated protein 4, mRNA (cDNA clone) MGC: 104099 IMAGE: 30915552), complete CDs GACCTGAACACCGCTCCCATAAAGCCATGGCTTGCCTTGGATTTCAGCGGCACAAGGCTCAGCTGAACCTGGCTACCAGG ACCTGGCCCTGCACTCTCCTGTTTTTTCTTCTCTTCATCCCTGTCTTCTGCAAAGCAATGCACGTGGCCCAGCCTGCTGT GGTACTGGCCAGCAGCCGAGGCATCGCCAGCTTTGTGTTGAGTATGCATCTCCAGGCAAAGCCACTGAGGTCCGGGTGA CAGTGCTTCGGCAGGCTGACAGCCAGGTGACTGAAGTCTGTGCGGCAACCTACATGATGGGGAATGAGTTGACCTTCCTA GATGATTCCATCTGCACGGGCACCTCCAGTGGAAATCAAGTGAACCTCACTATCCAAGGACTGAGGGCCATGGACACGGG ACTCTACATCTGCAAGGTGGAGCTCATGTACCCACCGCCATACTACCTGGGCATAGGCAACGGAACCCAGATTTATGTAA TTGATCCAGAACCGTGCCCAGATTCTGACTTCCTCCTCTGGATCCTTGCAGCAGTAGTTCGGGGTTGTTTTTTTATAGC TTTCTCCTCACAGCTGTTTCTTTGAGCAAAATGCTAAAGAAAAGAAGCCCTCTTACAACAGGGGTCTATGTGAAAATGCC CCCAACAGAGCCAGAATGTGAAAAGCAATTTCAGCCTTATTTTATTCCCATCAATTGAGAAACCATTATGAAGAAGAGAG TCCATATTTCAATTTCCAAGAGCTGAGG

[0373] "Cytidine deaminase" is a enzyme that converts amino groups into carbonyl groups through a deamination reaction. In one embodiment, the term "catalyzed polypeptide" refers to a polypeptide or fragment thereof that is capable of catalyzing , cytidine deaminase converts cytosine to uracil or 5-methylcytosine to thymine PmCDA1 (Petromyzon marinus cytosine deaminase) from Petromyzon marinus 1), or AID (activating inducer inhibitor) derived from mammals (e.g., humans, pigs, cows, horses, monkeys, etc.) AICDA (anti-inducible cytidine deaminase), and APOBEC are exemplary cytidine deaminases. do.

[0374] The nucleotide and amino acid sequences of PmCDA1 and human AID The columns are shown below. >tr|A5H718|A5H718_PETMA Cytosine deaminase OS=Petromyzon marinus OX=7757 PE=2 SV =1 MTDAEYVRIHEKLDIYTFKKQFFNNKKSVSHRCYVLFELKRRGERRACFWGYAVNKPQSGTERGIHAEIFSIRKVEEYLR DNPGQFTINWYSSWSPCADCAEKILEWYNQELRGNGHTLKIWACKLYYEKNARNQIGLWNLRDNGVGLNVMVSEHYQCCR KIFIQSSHNQLNENRWLEKTLKRAEKRRSELSIMIQVKILHTTKSPAV >EF094822.1 Petromyzon marinus isolate PmCDA.21 cytosine deaminase mRNA, complete e cds TGACACGACACAGCCGTGTATATGAGGAAGGGTAGCTGGATGGGGGGGGGGGGAATACGTTCAGAGAGGACATTAGCGAG CGTCTTGTTGGTGGCCTTGAGTCTAGACACCTGCAGACATGACCGACGCTGAGTACGTGAGAATCCATGAGAAGTTGGAC ATCTACACGTTTAAGAAACAGTTTTTCAACAACAAAAAATCCGTGTCGCATAGATGCTACGTTCTCTTTGAATTAAAACG ACGGGGTGAACGTAGAGCGTGTTTTTGGGGCTATGCTGTGAATAAACCACAGAGCGGGACAGAACGTGGAATTCACGCCG AAATCTTTAGCATTAGAAAAGTCGAAGAATACCTGCGCGACAACCCCGGACAATTCACGATAAATTGGTACTCATCCTGG AGTCCTTGTGCAGATTGCGCTGAAAAGATCTTAGAATGGTATAACCAGGAGCTGCGGGGGAACGGCCACACTTTGAAAAT CTGGGCTTGCAAACTCTATTACGAGAAAAATGCGAGGAATCAAATTGGGCTGTGGAACCTCAGAGATAACGGGGTTGGGT TGAATGTAATGGTAAGTGAACACTACCAATGTTGCAGGAAAATATTCATCCAATCGTCGCACAATCAATTGAATGAGAAT AGATGGCTTGAGAAGACTTTGAAGCGAGCTGAAAAACGACGGAGCGAGTTGTCCATTATGATTCAGGTAAAAATACTCCA CACCACTAAGAGTCCTGCTGTTTAAGAGGCTATGCGGATGGTTTTC >tr|Q6QJ80|Q6QJ80_HUMAN Activation-induced cytidine deaminase OS=Homo sapiens OX =9606 GN=AICDA PE=2 SV=1 MDSLLMNRRKFLYQFKNVRWAKGRRETYLCYVVKRRDSATSFSLDFGYLRNKNGCHVELLFLRYISDWDLDPGRCYRVTW FTSWSPCYDCARHVADFLRGNPNLSLRIFTARLYFCEDRKAEPEGLRRLHRAGVQIAIMTFKAPV >NG_011588.1: 5001-15681 Homo sapiens activation induced cytidine deaminase (AIC DA), RefSeqGene (LRG_17) on chromosome 12 AGAGAACCATCATTTGAAGTGAGATTTTTCTGGCCTGAGACTTGCAGGGAGGCAAGAAGACACTCTGGACACCACTA TGGACAGGTAAAGAGGCAGTCTTCTCGTGGGTGATTGCACTGGCCTTCCTCTCAGAGCAAATCTGAGTAATGAGACTGGT AGCTATCCCTTTCTCTCATGTAACTGTCTGACTGATAAGATCAGCTTGATCAATATGCATATATTTTTTGATCTGTCT CCTTTTCTTCTATTCAGATCTTATACGCTGTCAGCCCAATTCTTTCTGTTTCAGACTTCTCTTGATTTCCCTCTTTTTCA TGTGGCAAAAGAAGTAGTGCGTACAATGTACTGATTCGTCCTGAGATTTGTACCATGGTTGAAACTAATTTATGGTAATA ATATTAACATAGCAAATCTTTTAGAGACTCAAATCATGAAAAGGTAATAGCAGTACTGTACTAAAAACGGTAGTGCTAATT TTCGTAATAATTTGTAAATATTCAACAGTAAAACAACTTGAAGACACCTTCCTAGGGAGGCGTTACTGAAATAATTT AGCTATAGTAAGAAAATTTGTAATTTTGAATGCCAAGCATTCTAAATTAATTGCTTGAAAGTCACTATGATTGTGTCC ATTATAAGGAGACAAATTCATTCAAGCAAGTTATTTAATGTTAAAGGCCCAATTGTTAGGCAGTTAATGGCACTTTTACT ATTAACTAATCTTTCCATTTGTTCAGACGTAGCTTAACTTACCTCTTAGGTGTGAATTTGGTTAAGGTCCTCATAATGTC TTTATGTGCAGTTTTTGATAGGTTATTGTCATAGAACTTATTCTATTCCTACATTTATGATTACTATGGATGTATGAGAA TAACACCTAATCCTTAACTTTACCTCAATTTAACTCCTTTATAAAGAACTTACATTACAGAATAAAGATTTTTTAAAAA TATATTTTTTTGTAGAGACAGGGTCTTAGCCCAGCCCGAGGCTGGTCTCTAAGTCCTGGCCCAAGCGATCCTCCTCGCCTGG GCCTCCTAAAGTGCTGGAATTATAGACATGAGCCATCACATCCAATATACAGAATAAAGATTTTTAATGGAGGATTTAAT GTTCTTCAGAAAATTTCTTGAGGTCAGACAATGTCAAATGTCTCCTCAGTTTCACACTGAGATTTTGAAAAACAAGTCTGA GCTATAGGTCCTTGTGAAGGGTCCATTGGAAATACTTGTTCAAAGTAAAATGGAAAGCAAAGGTAAAATCAGCAGTTGAA ATTCAGAGAAAGACAGAAAAGGAGAAAAGATGAAAATTCAACAGGACAGAAGGGAAATATATTATCATTAAGGAGGACAGT ATCTGTAGAGCTCATTAGTGATGGCAAAATGACTTGGTCAGGATTATTTTTAACCCGCTTGTTTCTGGTTTGCACGGCTG GGGATGCAGCTAGGGTTCTGCCTCAGGGAGCACAGCTGTCCAGAGCAGCTGTCAGCCTGCAAGCCTGAAACACTCCCTCG GTAAAGTCCTTCCTACTCAGGACAGAAATGACGAGAACAGGGAGCTGGAACAGGCCCCTAACCAGAGAAGGGAAGTAAT GGATCAACAAAGTTAACTAGCAGGTCAGGATCACGCAATTCATTTCACTCTGACTGGTAACATGTGACAGAAACAGTGTA GGCTTTATTGTATTTTCATGTAGAGTAGGACCCAAAAATCCACCCAAAGTCCTTTATCTATGCCACATCCTTCTTATCTAT ACTTCCAGGACACTTTTTCTTCCTTATGATAAGGCTCTCTCTCTCCCACACACACACACACACACACACACACACAC ACACACACACACAACACACACCCCGCCAACCAAGGTGCATGTAAAAAGATGTAGATTCCTCTGCCTTCTCTCATCTACAC AGCCCAGGAGGGTAAGTTAAATAAGAGGGATTTATTGGTAAGAGATGATGCTTAATCTGTTTAACACTGGGCCTCAAAG AGAGAATTTCTTTTCTTCTGTACTTATTAAGCACCTATTATGTGTTGAGCTTATATATACAAAGGGTTATTATATGCTAA TATAGTAATAGTAATGGTGGTTGGTACTATGGTAATTACCATAAAAATTATTATCCTTTAAAATAAAGCTAATTATTAT TGGATCTTTTTAGTATTCATTTATGTTTTTTATGTTTTTGATTTTTTAAAGACAATCTCACCCTGTTACCCAGGCTG GAGTGCAGTGGTGCAATCATAGCTTTCTGCAGTCTTGAACTCCTGGGCTCAAGCAATCCTCCTGCCTTGGCCTCCCAAAG TGTTGGGATACAGTCATGAGCCACTGCATCTGGCCTAGGATCCATTTAGATTAAAATATGCATTTTAAATTTTAAATAA TATGGCTAATTTTTACCTTATGTAATGTGTATACTGGCAATAAATCTAGTTTGCTGCCTAAAGTTTAAAGTGCTTTCCAG TAAGCTTCATGTACGTGAGGGGAGACATTTAAAGTGAAACAGACAGCCAGGTGTGGTGGCTCACGCCTGTAATCCCAGCA CTCTGGGAGGCTGAGGTGGGTGGATCGCTTGAGCCCTGGAGTTCAAGACCAGCCTGAGCAACATGGCAAAACGCTGTTTC TATAACAAAAATTAGCCGGGCATGGTGGCATGTGCCTGTGGTCCCAGCTACTAGGGGGCTGAGGCAGGAGAATCGTTGGA GCCCAGGAGGTCAAGGCTGCACTGAGCAGTGCTTGCGCCACTGCACTCCAGCCTGGGTGACAGGACCAGACCTTGCCTCA AAAAAATAAGAAGAAAAATTAAAAATAAATGGAAACAACTACAAAGAGCTGTTGTCCTAGATGAGCTACTTAGTTAGGCT GATATTTTGGTATTTAACTTTTAAAGTCAGGGTCTGTCACCTGCACTACATTATTAAAATATCAATTCTCAATGTATATC CACACAAAGACTGGTACGTGAATGTTCATAGTACCTTTATTCACAAAACCCCAAAGTAGAGACTATCCAAATATCCATCA ACAAGTGAACAAATAAACAAAATGTGCTATATCCATGCAATGGAATACCACCCTGCAGTACAAAGAAGCTACTTGGGGAT GAATCCCAAAGTCATGACGCTAAATGAAAGAGTCAGACATGAAGGAGGAGATAATGTATGCCATACGAAATTCTAGAAAA TGAAGTAACTTATAGTTACAGAAAGCAAATCAGGGCAGGCATAGAGGCTCACACCTGTAATCCCAGCACTTTGAGAGGC CACGTGGGAAGATTGCTAGAACTCAGGAGTTCAAGACCAGCCTGGGCAACACAGTGAAACTCCATTCTCCACAAAAATGG GAAAAAAGAAAGCAAATCAGTGGTTGTCCTGTGGGGAGGGAAGGACTGCAAAGAGGGAAGAAGCTCTGGTGGGTGAG GGTGGTGATTCAGGTTCTGTATCCTGACTGTGGTAGCAGTTTGGGGTGTTTACATCCAAAAAATTCGTAGAATTATGCA TCTTAAATGGGTGGAGTTTACTGTATGTAAATTATACCTCAATGTAAGAAAAAAATAATGTGTAAGAAAACTTTCAATTCT CTTGCCAGCAAACGTTATTCAAATTCCTGAGCCCTTTACTTCGCAAATTCTCTGCACTTCTGCCCCGTACCATTAGGTGA CAGCACTAGCTCCACAAATTGGATAAATGCATTTCTGGAAAAGACTAGGGACAAAATCCAGGCATCACTTGTGCTTTCAT ATCAACCATGCTGTACAGCTTGTGTTGCTGTCTGCAGCTGCAATGGGACTCTTGATTTCTTTAAGGAAACTTGGGTTAC CAGAGTATTTCCACAAATGCTATTCAAATTAGTGCTTATGATATGCAAGACACTGTGCTAGGAGCCAGAAAACAAAGAGG AGGAGAAATCAGTCATTATGTGGGAACAACATAGCAAGATATTTAGATCATTTTGACTAGTTAAAAAGCAGCAGGAGTAC AAAATCACACATGCAATCAGTATAATCCAAATCATGTAAATATGTGCCTGTAGAAAGACTAGAGGAATAAACACAAGAAT CTTAACAGTCATTGTCATTAGACACTAAGTCTAATTATTATTATTAGACACTATGATATTTGAGATTTAAAAAATTCTTTA ATATTTTAAATTTAGAGCTCTTCTATTTTTCCATAGTATTCAAGTTTGACAATGATCAAGTATTACTCTTTCTTTTTTT TTTTTTTTTTTTTTTTTTGAGATGGAGTTTTGGTCTTGTTGCCCATGCTGGAGTGGAATGGCATGACCATAGCTCACTGC AACCTCCACCTCCTGGGTTCAAGCAAAGCTGTCGCCTCAGCCTCCCGGGTAGATGGGATTACAGGCGCCCACCACCACAC TCGGCTAATGTTTGTATTTTTAGTAGAGATGGGGTTTCACCATGTTGGCCAGGCTGGTCTCAAACTCCTGACCTCAGAGG ATCCACCTGCCTCAGCCTCCCAAAGTGCTGGGATTACAGATGTAGGCCACTGCGCCCGGCCAAGTATTGCTCTTATACAT TAAAAAACAGGTGTGAGCCACTGCGCCCAGCCAGGTATTGCTCTTATACATTAAAAAATAGGCCGGTGCAGTGGCTCACG CCTGTAATCCCAGCACTTTGGGAAGCCAAGGCGGGCAGAACACCCGAGGTCAGGAGTCCAAGGCCAGCCTGGCCAAGATG GTGAAACCCCGTCTCTATTAAAAATACAAACATTACCTGGGCATGATGGTGGGCGCCTGTAATCCCAGCTACTCAGGAGG CTGAGGCAGGAGGATCGCGGAGCCTGGCAGATCTGCCTGAGCCTGGGAGGTTGAGGCTACAGTAAGCCAAGATCATGCC AGTATACTTCAGCCTGGGCGACAAAGTGAGACCGTAACAAAAAAAAAAAAATTTAAAAAAGAAATTTAGATCAAGATCC AACTGTAAAAAGTGGCCTAAACACCACATTAAAGAGTTTGGAGTTTATTCTGCAGGCAGAAGAGAACCATCAGGGGGTCT TCAGCATGGGAATGGCATGGTGCACCTGGTTTTGTGAGATCATGGTGGTGACAGTGTGGGGAATGTTATTTTGGAGGGGA CTGGAGGCAGACAGACCGGTTAAAAGGCCAGCACAACAGATAAGGAGGAAAAGATGAGGGCTTGGACCGAAGCAGAGAA GAGCAAACAGGGAAGGTACAAATTCAAGAAATATTGGGGGGTTGAATCAAACACATTTAGATGATTAATTAAATATGAGG ACTGAGGAATAAGAAATGAGTCAAGGATGGTTCCAGGCTGCTAGGCTGCTTACCTGAGGTGGCAAAGTCGGGAGGAGTGGG CAGTTTAGGACAGGGGGCAGTTGAGGAATATTGTTTTGATCATTTTGAGTTTGAGGTACAAGTTGGACACTTAGGTAAAG ACTGGAGGGGAAATCTGAATATACAATTATGGGACTGAGGAACAAGTTTATTTTATTTTTTGTTTCGTTTTCTTGTTGAA GAACAAATTTAATTGTAATCCCAAGTCATCAGCATCTAGAAGACAGTGGCAGGAGGTGACTGTCTTGTGGGTAAGGGTT GGGGTCCTTGATGAGTATCTCTCAATTGGCCTTAAATATAAGCAGGAAAAGGAGTTTATGATGGATTCCAGGCTCAGCAG GGCTCAGGAGGGCTCAGGCAGCCAGCAGAGGAAGTCAGAGCATCTTCTTTGGTTTAGCCCAAGTAATGACTTCCTTAAAA AGCTGAAGGAAAATCCAGAGTGACCAGATTATAAACTGTACTCTTGCATTTTCTCTCCCTCCTCTCACCCACAGCCTCTT GATGAACCGGAGGAAGTTTCTTTACCAATTCAAAAATGTCCGCTGGGCTAAGGGTCGGCGTGAGACCTACCTGTGCTACG TAGTGAAGAGGCGTGACAGTGCTACATCCTTTTCACTGGACTTTGGTTATCTTCGCAATAAGGTATCAATTAAAGTCGGC TTTGCAAGCAGTTTAATGGTCAACTGTGAGTGCTTTTAGAGCCACCTGCTGATGGTATTACTTCCATCCTTTTTTGGCAT TTGTGTCTCTATCACATTCCTCAAATCCTTTTTTTTATTTCTTTTTCCATGTCCATGCACCCATATTAGACATGGCCCAA AATATGTGATTTAATTCCTCCCCAGTAATGCTGGGCACCCTAATACCACTCCTTCCTTCAGTGCCAAGAACAACTGCTCC CAAACTGTTTACCAGCTTTCCTCAGCATCTGAATTGCCTTTGAGATTAATTAAGCTAAAAGCATTTTTATATGGGAGAAT ATTATCAGCTTGTCCAAGCAAAAATTTTAAATGTGAAAAACAAATTGTGTCTTAAGCATTTTTGAAAATTAAGGAAGAAG AATTTGGGAAAAAATTAACGGTGGCTCAATTCTGTCTTCCAAATGATTTCTTTTCCCTCCTACTCACATGGGTCGTAGGC CAGTGAATACATTCAACATGGTGATCCCCAGAAAACTCAGAGAAGCCTCGGCTGATGATTAATTAAATTGATCTTTCGGC TACCCGAGAGAATTACATTTCCAAGAGACTTCTTCACCAAAATCCAGATGGGTTTACATAAACTTCTGCCCACGGGTATC TCCTCTCTCCTAACACGCTGTGACGTCTGGGCTTGGTGGAATCTCAGGGAAGCATCCGTGGGGTGGAAGGTCATCGTCTG GCTCGTTGTTTGATGGTTATATTACCATGCAATTTTCTTTGCCTACATTTGTATTGAATACATCCCAATCTCCTTCCTAT TCGGTGACATGACACATTCTATTTCAGAAGGCTTTGATTTTATCAAGCACTTTCATTTACTTCTCATGGCAGTGCCTATT ACTTCTCTTACAATACCCATCTGTCTGCTTTACCAAAATCTATTTCCCCTTTTCAGATCCTCCCAAATGGTCCTCATAAA CTGTCCTGCCTCCACCTAGTGGTCCAGGTATATTTCCACAATGTTACATCAACAGGCACTTCTAGCCATTTTCCTTCTCA AAAGGTGCAAAAAGCAACTTCATAAACACAAATTAAATCTTCGGTGAGGTAGTGTGATGCTGCTTCCTCCCAACTCAGCG CACTTCGTCTTCCTCATTCCACAAAAACCCATAGCCTTCCTTCACTCTGCAGGACTAGTGCTGCCAAGGGTTCAGCTCTA CCTACTGGTGTGCTCTTTTGAGCAAGTTGCTTAGCCTCTCTGTAACACAAGGACAATAGCTGCAAGCATCCCCAAAGATC ATTGCAGGAGACAATGACTAAGGCTACCAGAGCCGCAATAAAAGTCAGTGAATTTTAGCGTGGTCCTCTCTGTCTCTCCA GAACGGCTGCCACGTGGAATTGCTCTTCCTCCGCTACATCTCGGACTGGGACCTAGACCCTGGCCGCTGCTACCGCGTCA CCTGGTTCACCTCCTGGAGCCCCTGCTACGACTGTGCCCGACATGTGGCCGACTTTCTGCGAGGGAACCCCAACCTCAGT CTGAGGATCTTCACCGCGCGCCTCTACTTCTGTGAGGACCGCAAGGCTGAGCCCGAGGGGCTGCGGCGGCTGCACCGCGC CGGGGTGCAAATAGCCATCATGACCTTCAAAGGTGCGAAAGGGCCTTCCGCGCAGGCGCAGTGCAGCAGCCCGCATTCGG GATTGCGATGCGGAATGAATGAGTTAGTGGGGAAGCTCGAGGGGAAGAAGTGGGCGGGGATTCTGGTTCACCTCTGGAGC CGAAATTAAAGATTAGAAGCAGAGAAAAGAGTGAATGGCTCAGAGACAAGGCCCCGAGGAAATGAGAAAATGGGGCCAGG GTTGCTTCTTTCCCCTCGATTTGGAACCTGAACTGTCTTCTACCCCCATATCCCCGCCTTTTTTTCCTTTTTTTTTTTTT GAAGATTATTTTTACTGCTGGAATACTTTTGTAGAAAACCACGAAAGAACTTTCAAAGCCTGGGAAGGGCTGCATGAAAA TTCAGTTCGTCTCTCCAGACAGCTTCGGCGCATCCTTTTGGTAAGGGGCTTCCTCGCTTTTTAAATTTTCTTTCTTTCTC TACAGTCTTTTTTGGAGTTTCGTATATTTCTTATATTTTCTTATTGTTCAATCACTCTCAGTTTTCATCTGATGAAAACT TTATTTCTCCTCCACATCAGCTTTTTCTTCTGCTGTTTCACCATTCAGAGCCCTCTGCTAAGGTTCCTTTTCCCTCCCTT TTCTTTCTTTTGTTGTTTCACATCTTTAAATTTCTGTCTCTCCCCAGGGTTGCGTTTCCTTCCTGGTCAGAATTCTTTTC TCCTTTTTTTTTTTTTTTTTTTTTTTTTTTAAACAAACAAACAAAAAACCCAAAAAAACTCTTTCCCAATTTACTTTCTT CCAACATGTTACAAAGCCATCCACTCAGTTTAGAAGACTCTCCGGCCCCACCGACCCCCAACCTCGTTTTGAAGCCATTC ACTCAATTTGCTTCTCTCTTTCTCTACAGCCCCTGTATGAGGTTGATGACTTACGAGACGCATTTCGTACTTTGGGACTT TGATAGCAACTTCCAGGAATGTCACACACGATGAAATATCTCTGCTGAAGACAGTGGATAAAAAACAGTCCTTCAAGTCT TCTCTGTTTTTATTCTTCAACTCTCACTTTCTTAGAGTTTACAGAAAAAATATTTATATACGACTCTTTAAAAAGATCTA TGTCTTGAAAATAGAGAAGGAACACAGGTCTGGCCAGGGACGTGCTGCAATTGGTGCAGTTTTGAATGCAACATTGTCCC CTACTGGGAATAACAGAACTGCAGGACCTGGGAGCATCCTAAAGTGTCAACGTTTTTCTATGACTTTTAGGTAGGATGAG AGCAGAAGGTAGATCCTAAAAGCATGGTGAGAGGATCAAATGTTTTTATATCAACATCCTTTATTATTTGATTCATTTG AGTTAACAGTGGTGTTAGTGATAGATTTTTCTATTCTTTTCCCTTGACGTTTACTTTCAAGTAACACAAACTCTTCCATC AGGCCATGATCTATAGGACCTCCTAATGAGAGTATCTGGGTGATTGTGACCCCAAACCATCTCTCCAAAGCATTAATATC CAATCATGGCCTGTATGTTTTAATCAGCAGAAGCATGTTTTTATGTTTGTACAAAAGAAGATTGTTATGGGTGGGGATGG AGGTATAGACCATGCATGGTCACCTTCAAGCTACTTTAATAAAGGATCTTAAAATGGGCAGGAGGACTGTGAACAAGACA CCCTAATAATGGGTTGATGTCTGAAGTAGCAAATCTTCTGGAAACGCAAACTCTTTTAAGGAAGTCCCTAATTTAGAAAC ACCCACAAACTTCACATATCATAATTAGCAAACAATTGGAAGGAAGTTGCTTGAATGTTGGGGAGAGGAAAATCTATTGG CTCTCGTGGGTCTCTTCATCTCAGAAATGCCAATCAGGTCAAGGTTTGCTACATTTTGTATGTGTGTGATGCTTCTCCCA AAGGTATATTAACTATATAAGAGAGTTGTGACAAAACAGAATGATAAAGCTGCGAACCGTGGCACACGCTCATAGTTCTA GCTGCTTGGGAGGTTGAGGAGGGAGGATGGCTTGAACACAGGTGTTCAAGGCCAGCCTGGGCAACATAACAAGATCCTGT CTCTCAAAAAAAAAAAAAAAAAAAAGAAAGAGAGAGGGCCGGGCGTGGTGGCTCACGCCTGTAATCCCAGCACTTTGGGA GGCCGAGCCGGGCGGATCACCTGTGGTCAGGAGTTTGAGACCAGCCTGGCCAACATGGCAAAACCCCGTCTGTACTCAAA ATGCAAAAATTAGCCAGGCGTGGTAGCAGGCACCTGTAATCCCAGCTACTTGGGAGGCTGAGGCAGGAGAATCGCTTGAA CCCAGGAGGTGGAGGTTGCAGTAAGCTGAGATCGTGCCGTTGCACTCCAGCCTGGGCGACAAGAGCAAGACTCTGTCTCA GAAAAAAAAAAAAAAAAGAGAGAGAGAGAGAAAGAGAACAATATTTGGGAGAGAAGGATGGGGAAGCATTGCAAGGAAAT TGTGCTTTATCCAACAAAATGTAAGGAGCCAATAAGGGATCCCTATTTGTCTCTTTTGGTGTCTATTTGTCCCTAACAAC TGTCTTTGACAGTGAGAAAAATATTCAGAATAACCATATCCCTGTGCCGTTATTACCTAGCAACCCTTGCAATGAAGATG A GCAGATCCACAGGAAAACTTGAATGCACAACTGTCTTATTTTAATCTTATTGTACATAAGTTTGTAAAAGAGTTAAAAAT TGTTACTTCATGTATTCATTTATATTTTATATTATTTTGCGTCTAATGATTTTTTATTAACATGATTTCCTTTTCTGATA TATTGAAATGGAGTCTCAAAGCTTCATAAATTTATAACTTTAGAAATGATTCTAATAACAACGTATGTAATTGTAACATT GCAGTAATGGTGCTACGAAGCCATTTCTCTTGATTTTTAGTAAACTTTTATGACAGCAAATTTGCTTCTGGCTCACTTTC AATCAGTTAAATAAATGATAAATAATTTTGGAAGCTGTGAAGATAAAATACCAAATAAAATAATAAAAGTGATTTATA TGAAGTTAAAATAAAAAATCAGTATGATGGAATAAACTTG

[0375] Apolipoprotein B mRNA editing enzyme, catalytic polypeptide-like (APOBEC) mRNA editing enzyme, catalytic polypeptide-like, The deaminase family. Members of this family are C to U editing enzymes. The N-terminal domain of APOBEC-like proteins is the catalytic domain, and the C-terminal domain is the pseudocatalytic domain. More specifically, the catalytic domain is a zinc-dependent cytidine deaminase domain. APOBEC family members are important for cytidine deamination. OBEC1, APOBEC2, APOBEC3A, APOBEC3B, APOBEC3C, APOBEC3D (now known as "APOBEC3E" (refers to APOBEC3F, APOBEC3G, APOBEC3H, APOBEC4, and activation-induced (cytidine) Deaminases include, but are not limited to, SaBE3, SaKKH-BE3, VQR-BE3, and EQR-B Numerous modifications including E3, VRER-BE3, VRER-BE3, YE1-BE3, EE-BE3, YE2-BE3, and YEE-BE3 Cytidine deaminases are commercially available and are available from Addgene (Pla Sumido 85169, 85170, 85171, 85172, 85173, 85174, 85175, 85176, 85177).

[0376] Other exemplary deaminases that can be fused to Cas9 according to embodiments of the present disclosure are provided below. In some embodiments, the active domain of each sequence, e.g., the localization signal, Domains without nulls (nuclear localization sequence, nuclear export signal, cytoplasmic localization signal) It should be understood that it can be used.

[0377] Human AID: JPEG2024174899000007.jpg33165 (Underline: nuclear localization sequence; Double underline: nuclear export signal)

[0378] Mouse AID: JPEG2024174899000008.jpg34165 (Underline: nuclear localization sequence; Double underline: nuclear export signal)

[0379] Canine AID: JPEG2024174899000009.jpg33165 (Underline: nuclear localization sequence; Double underline: nuclear export signal)

[0380] Bovine AID: JPEG2024174899000010.jpg34165 (Underline: nuclear localization sequence; Double underline: nuclear export signal)

[0381] Rat AID JPEG2024174899000011.jpg35165 (Underline: nuclear localization sequence; Double underline: nuclear export signal)

[0382] Mouse APOBEC-3 JPEG2024174899000012.jpg53164 (italics: nucleic acid editing domain)

[0383] Rat APOBEC-3: JPEG2024174899000013.jpg52166 (italics: nucleic acid editing domain)

[0384] Rhesus macaque APOBEC-3 G: MVEPMDPRTFVSNFNNRPILSGLNTVWLCCEVKTKDPSGPPLDAKIFQGKVYSKAKYHPEM RFLRWFHKWRQLHHDQEYK VTWYVSWSPCTRCANSVATFLAKDPKVTLTIFVARLYYFWKPDYQQALRILCQKRGGPHATMKIMNYNEFQDCWNKFVDG RGKPFKPRNNLPKHYTLLQATLGELLRHLMDPGTFTSNFNNKPWVSGQHETYLCYKVERLHNDTWVPLNQHRGFLRNQAP NIHGFPKGRHAELCFLDLIPFWKLDGQQYRVTCFTSWSPCFSCAQEMAKFISNNEHVSLCIFAARIYDDQGRYQEGLRAL HRDGAKIAMMNYSEFEYCWDTFVDRQGRPFQPWDGLDEHSQALSGRLRAI (Italics: nucleic acid editing domain; underline: cytoplasmic localization signal)

[0385] Chimpanzee APOBEC-3 G: JPEG2024174899000014.jpg44168

[0386] (Italics: nucleic acid editing domain; underline: cytoplasmic localization signal)

[0387] Green monkey APOBEC-3G: JPEG2024174899000015.jpg46168

[0388] (Italics: nucleic acid editing domain; underline: cytoplasmic localization signal)

[0389] Human APOBEC-3G: JPEG2024174899000016.jpg47168 (Italics: nucleic acid editing domain; Underline: cytoplasmic localization signal)

[0390] Human APOBEC-3F: JPEG2024174899000017.jpg44168 (italics: nucleic acid editing domain)

[0391] Human APOBEC-3B: JPEG2024174899000018.jpg45165 (italics: nucleic acid editing domain)

[0392] Rat APOBEC-3B: MQPQGLGPNAGMGPVCLGCSHRRPYSPIRNPLKKLYQQTFYFHFKNVRYAWGRKNNFLCYEVNGMDCALPVPLRQGVFRK QGHIHAELCFIYWFHDKVLRVLSPMEEFKVTWYMSWSPCSKCAEQVARFLAAHRNLSLAIFSSRLYYYLRNPNYQQKLCR LIQEGVHVAAMDLPEFKKCWNKFVDNDGQPFRPWMRLRINFSFYDCKLQEIFSRMNLLREDVFYLQFNNSHRVKPVQNRY YRRKSYLCYQLERANGQEPLKGYLLYKKGEQHVEILFLEKMRSMELSQVRITCYLTWSPCPNCARQLAAFKKDHPDLILR IYTSRLYFWRKKFQKGLCTLWRSGIHVDVMDLPQFADCWTNFVNPQRPFRPWNELEKNSWRIQRRLRRIKESWGL

[0393] Bovine APOBEC-3B: DGWEVAFRSGTVLKAGVLGVSMTEGWAGSGHPGQGACVWTPGTRNTMNLLREVLFKQQFGNQPRVPAPYYRRKTYLCYQL KQRNDLTLDRGCFRNKKQRHAERFIDKINSLDLNPSQSYKIICYITWSPCPNCANELVNFITRNNHLKLEIFASRLYFHW IKSFKMGLQDLQNAGISVAVMTHTEFEDCWEQFVDNQSRPFQPWDKLEQYSASIRRRLQRILTAPI

[0394] Chimpanzee APOBEC-3B: MNPQIRNPMEWMYQRTFYYNFENEPILYGRSYTWLCYEVKIRRGHSNLLWDTGVFRGQMYSQPEHHAEMCFLSWFCGNQL SAYKCFQITWFVSWTPCPDCVAKLAKFLAEHPNVTLTISAARLYYYWERDYRRALCRLSQAGARVKIMDDEEFAYCWENF VYNEGQPFMPWYKFDDNYAFLHRTLKEIIRHLMDPDTFTFNFNNDPLVLRRHQTYLCYEVERLDNGTWVLMDQHMGFLCN EAKNLLCGFYGRHAELRFLDLVPSLQLDPAQIYRVTWFISWSPCFSWGCAGQVRAFLQENTHVRLRIFAARIYDYDPLYK EALQMLRDAGAQVSIMTYDEFEYCWDTFVYRQGCPFQPWDGLEEHSQALSGRLRAILQVRASSLCMVPHRPPPPPQSPGP CLPLCSEPPLGSLLPTGRPAPSLPFLLTASFSFPPPASLPPLPSLSLSPGHLPVPSFHSLTSCSIQPPCSSRIRETEGWA SVSKEGRDLG

[0395] Human APOBEC-3C: JPEG2024174899000019.jpg29165 (italics: nucleic acid editing domain)

[0396] Gorilla APOBEC3C JPEG2024174899000020.jpg25165 (italics: nucleic acid editing domain)

[0397] Human APOBEC-3A: JPEG2024174899000021.jpg24165 (italics: nucleic acid editing domain)

[0398] Rhesus macaque APOBEC-3A: JPEG2024174899000022.jpg25165 (italics: nucleic acid editing domain)

[0399] Bovine APOBEC-3A: JPEG2024174899000023.jpg25168 (italics: nucleic acid editing domain)

[0400] Human APOBEC-3H: JPEG2024174899000024.jpg24168 (italics: nucleic acid editing domain)

[0401] Rhesus macaque APOBEC-3H: MALLTAKTFSLQFNNKRRVNKPYYPRKALLCYQLTPQNGSTPTRGHLKNKKKDHAEIRFINKIKSMGLDETQCYQVTCYL TWSPCPSCAGELVDFIKAHRHLNLRIFASRLYYHWRPNYQEGLLLLCGSQVPVEVMGLPEFTDCWENFVDHKEPPSFNPS EKLEELDKNSQAIKRRLERIKSRSVDVLENGLRSLQLGPVTPSSSIRNSR

[0402] Human APOBEC-3D: JPEG2024174899000025.jpeg44168

[0403] Human APOBEC-1: MTSEKGPSTGPDTLRRIEPWEFDVFYDPRELRKEACLLYEIKWGMSRKIWRSSGKNTTNHVEVNFIKKFTSERDFHPSM SCSITWFLSWSPCWECSQAIREFLSRHPGVTLVIYVARLFWHMDQQNRQGLRDLVNSGVTIQIMRASEYYHCWRNFVNYP PGDEAHWPQYPPLWMMLYALELHCIILSLPPCLKISRRWQNHLTFFRLHLQNCHYQTIPPHILLATGLIHPSVAWR

[0404] Mouse APOBEC-1: MSSETGPVAVDPTLRRRIEPHEFEVFFDPRELRKETCLLYEINWGGRHSVWRHTSQNTSNHVEVNFLEKFTTERYFRPNT RCSITWFLSWSPCGECSRAITEFLSRHPYVTLFIYIARLYHHTDQRNRQGLRDLISSGVTIQIMTEQEYCYCWRNFVNYP PSNEAYWPRYPHLWVKLYVLELYCIILGLPPCLKILRRKQPQLTFFTITLQTCHYQRIPPHLLWATGLK

[0405] Rat APOBEC-1: MSSETGPVAVDPTLRRRIEPHEFEVFFDPRELRKETCLLYEINWGGRHSIWRHTSQNTNKHVEVNFIEKFTTERYFCPNT RCSITWFLSWSPCGECSRAITEFLSRYPHVTLFIYIARLYHHADPRNRQGLRDLISSGVTIQIMTEQESGYCWRNFVNYS PSNEAHWPRYPHLWVRLYVLELYCIILGLPPCLNILRRKQPQLTFFTIALQSCHYQRLPPHILWATGLK

[0406] Human APOBEC-2: MAQKEEAAVATEAASQNGEDLENLDDPEKLKELIELPPFEIVTGERLPANFFKFQFRNVEYSSGRNKTFLCYVVEAQGKG GQVQASRGYLEDEHAAAHAEEAFFNTILPAFDPALRYNVTWYVSSSPCAACADRIIKTLSKTKNLRLLILVGRLFMWEEP EIQAALKKLKEAGCKLRIMKPQDFEYVWQNFVEQEEGESKAFQPWEDIQENFLYYEEKLADILK

[0407] Mouse APOBEC-2: MAQKEEAAEAAAPASQNGDDLENLEDPEKLKELIDLPPFEIVTGVRLPVNFFKFQFRNVEYSSGRNKTFLCYVVEVQSKG GQAQATQGYLEDEHAGAHAEEAFFNTILPAFDPALKYNVTWYVSSSPCAACADRILKTLSKTKNLRLLILVSRLFMWEEP EVQAALKKLKEAGCKLRIMKPQDFEYIWQNFVEQEEGESKAFEPWEDIQENFLYYEEKLADILK

[0408] Rat APOBEC-2: MAQKEEAAEAAAPASQNGDDLENLEDPEKLKELIDLPPFEIVTGVRLPVNFFKFQFRNVEYSSGRNKTFLCYVVEAQSKG GQVQATQGYLEDEHAGAHAEEAFFNTILPAFDPALKYNVTWYVSSSPCAACADRILKTLSKTKNLRLLILVSRLFMWEEP EVQAALKKLKEAGCKLRIMKPQDFEYLWQNFVEQEEGESKAFEPWEDIQENFLYYEEKLADILK

[0409] Bovine APOBEC-2: MAQKEEAAAAAEPASQNGEEVENLEDPEKLKELIELPPFEIVTGERLPAHYFKFQFRNVEYSSGRNKTFLCYVVEAQSKG GQVQASRGYLEDEHATNHAEEAFFNSIMPTFDPALRYMVTWYVSSSPCAACADRIVKTLNKTKNLRLLILVGRLFMWEEP EIQAALRKLKEAGCRLRIMKPQDFEYIWQNFVEQEEGESKAFEPWEDIQENFLYYEEKLADILK

[0410] Petromyzon marinus CDA1 (pmCDAl) MTDAEYVRIHEKLDIYTFKKQFFNNKKSVSHRCYVLFELKRRGERRACFWGYAVNKPQSGTERGIHAEIFSIRKVEEYLR DNPGQFTINWYSSWSPCADCAEKILEWYNQELRGNGHTLKIWACKLYYEKNARNQIGLWNLRDNGVGLNVMVSEHYQCCR KIFIQSSHNQLNENRWLEKTLKRAEKRRSELSFMIQVKILHTTKSPAV

[0411] Human APOBEC3G D316R D317R MKPHFRNTVERMYRDTFSYNFYNRPILSRRNTVWLCYEVKTKGPSRPPLDAKIFRGQVYSELKYHPEMRFFHWFSKWRKL HRDQEYEVTWYISWSPCTKCTRDMATFLAEDPKVTLTIFVARLYYFWDPDYQEALRSLCQKRDGPRATMKFNYDEFQHCW SKFVYSQRELFEPWNNLPKYYILLHFMLGEILRHSMDPPTFTFNFNNEPWVRGRHETYLCYEVERMHNDTWVLLNQRRGF LCNQAPHKHGFLEGRHAELCFLDVIPFWKLDLDQDYRVTC FTSWSPCFSCAQEMAKFISKKHVSLCIFTARIYRRQGRC QEGLRTLAEAGAKISFTYSEFKHCWDTFVDHQGCPFQPWDGLDEHSQDLSGRLRAILQNQEN

[0412] Human APOBEC3G chain A MDPPTFTFNFNNEPWWGRHETYLCYEVERMHNDTWVLLNQRRGFLCNQAPHKHGFLEGRHAELCFLDVIPFWKLDLDQDY RVTCFTSWSPCFSCAQEMAKFISKNKHVSLCIFTARIYDDQGRCQEGLRTLAEAGAKISF TYSEFKHCWDTFVDHQGCP FQPWDGLD EHSQDLSGRLRAILQ

[0413] Human APOBEC3G chain A D120R D121R MDPPTFTFNFNNEPWVRGRHETYLCYEVERMHNDTWVLLNQRRGFLCNQAPHKHGFLEGRHAELCFLDVIPFWKLDLDQD YRVTCFTSWSPCFSCAQEMAKFISKNKHVSLCIFTARIYRRQGRCQEGLRTLAEAGAKISFMTYSEFKHCWDTFVDHQGC PFQPWDGLDEHSQDLSGRLRAILQ

[0414] The term "deaminase" or "deaminase domain" refers to a protein that catalyzes a deamination reaction. In some embodiments, a deaminase or deaminase-binding protein is a protein or fragment thereof. The aminase domain is a variant of a naturally occurring deaminase derived from an organism. In some embodiments, the deaminase or deaminase domain is not naturally occurring. For example, in some embodiments, a deaminase or deaminase The domain has at least 50%, at least 55%, or at least at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least At least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% , at least 99%, or at least 99.5% identical. The aminase is a cytosine deaminase or an adenosine deaminase.

[0415] "Detect" refers to identifying the presence, absence, or amount of the analyte to be detected. vinegar.

[0416] A "detectable label" means a label that, when attached to a molecule of interest, is detectable by spectroscopic, photochemical, or biochemical means. By "detectable" is meant a composition that renders it detectable through biological, immunochemical, or chemical means. For example, useful labels include radioisotopes, magnetic beads, metal beads, colloidal particles, fluorescent Dyes, electron-dense reagents, enzymes (e.g., those commonly used in ELISA), biotin, dinucleotides, Goxigenin, or hapten.

[0417] "Disease" means any condition that damages or interferes with the normal function of a cell, tissue, or organ. In one embodiment, the disease refers to a neoplasia or cancer (e.g., multifocal cancer). myeloma).

[0418] As used herein, the term "effective amount" means an amount sufficient to elicit a desired biological response. In some embodiments, the term "biologically active agent" refers to an amount of a biologically active agent sufficient to provide a therapeutically effective dose. fusion proteins, e.g., a nCas9 domain and one or more deaminase domains. Cytidine deaminase, including enzymes (e.g., cytidine deaminase, adenosine deaminase) An effective amount of adenosine deaminase or adenosine deaminase nucleobase editor is specifically bound and edited by an adenosine deaminase or adenosine deaminase nucleobase editor It may refer to the amount of fusion protein that is sufficient to induce editing of the target site. As will be understood, an effective amount of an agent, e.g., a fusion protein, is determined by, for example, the amount of the agent that produces a desired biological response. The answer, e.g., the specific allele, genome, or target site to be edited, the targeted cells, or The relationship between CAR-T cells and the type of tumor can vary depending on various factors, such as the tumor tissue and the drug used. In this context, "effective amount" refers to the amount of cells required to be administered to a patient to achieve a therapeutic response. vinegar.

[0419] In some embodiments, the fusion proteins provided herein, e.g., nCas9 Fusion proteins containing the domain and cytidine deaminase or adenosine deaminase A quality effective amount is sufficient to effect editing of the target site specifically bound and edited by the fusion protein. As will be understood by those skilled in the art, the term "inducible" may refer to an amount of fusion protein that is sufficient to induce The agent, e.g., a fusion protein, a nuclease, a cytidine deaminase, or an adenosine deaminase, Aminase, hybrid protein, protein dimer, protein (or protein An effective amount of a complex of a polynucleotide (polydimer) and a polynucleotide, or a polynucleotide, is, for example, The desired biological response, e.g., the specific allele, genome, or target site to be edited, The time required for the treatment of ulcerative colitis may vary depending on various factors, such as the cells or tissue being treated and the agent being used.

[0420] As used herein, "epitope" means an antigenic determinant. of an antigen molecule, whose structure determines the specific antibody molecule that recognizes and binds it. It's a part of it.

[0421] By "fragment" is meant a portion of a polypeptide or nucleic acid molecule, which portion is identical to that of a reference nucleic acid At least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80% of the entire length of the molecule or polypeptide , or 90%. The fragments may be 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100, 200, 3 nucleotides or amino acids. do.

[0422] "Graft-versus-host disease" (GVHD) occurs when the transplanted cells of the donor become immune to the host's cells. Refers to a pathological condition that produces a response.

[0423] "Host-versus-graft disease" (HVGD) occurs when the host's immune system becomes immune to the transplanted cells of the donor. Refers to a pathological condition that produces an immune response.

[0424] "Hybridization" means hydrogen bonding between complementary nucleobases, as defined by Watson- Crick, Hoogsteen, or reversed Hoogsteen hydrogen bonds. Denine and thymine are complementary nucleobases that form hydrogen bonds to form pairs.

[0425] By "immune cell" is meant a cell of the immune system that is capable of producing an immune response.

[0426] "Immune effector cells" are cells that, once activated, initiate an immune response against target cells. T cells are exemplary immune effector cells.

[0427] "Immune response regulator gene" or "immune response regulator" refers to a gene involved in the regulation of immune responses. Immune response-regulating genes are those that regulate immune responses by multiple mechanisms, including For example, immune response-modulating genes may regulate immune cells, For example, they can inhibit or promote T cell activation. In some embodiments, the immune response modulating gene may increase or decrease the activation threshold. In some embodiments, the antibodies positively regulate immune cell signaling pathways. Nodal genes negatively regulate immune cell signaling pathways. In some embodiments, Immune response regulatory genes encode antigens, antibodies, cytokines, or neuroendocrine compounds. In some embodiments, the immune response modulating gene encodes a Cblb protein.

[0428] 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 some embodiments, the immunogenic gene is a cell surface antigen or cell surface marker. In some embodiments, the cell surface marker encodes a T cell marker or are B cell markers. In some embodiments, the immunogenic genes are CD2, CD3e, CD3 delta, CD3 gamma, TRAC, TRBC1, TRBC2, CD4, CD5, CD7, CD8, CD19, CD23, CD27, CD28, CD30, CD33, CD52, CD70, CD127, CD122, CD130, CD132, CD38, CD69, CD11a, CD5 8, CD99, CD103, CCR4, CCR5, CCR6, CCR9, CCR10, CXCR3, CXCR4, CLA, CD161, B2M, or a CIITA polypeptide.

[0429] The term "inhibitor of base repair" or "IBR" refers to an inhibitor of base repair by a nucleic acid repair enzyme, such as base excision repair. IBR refers to a protein that can inhibit the activity of an enzyme. It is an inhibitor of base excision repair. Examples of base repair inhibitors include APE1 and Endo III. , Endo IV, Endo V, Endo VIII, Fpg, hOGGl, hNEILl, T7 Endol, T4 PDG, UDG, hSMUGL and inhibitors of hAAG. In certain embodiments, the IBR is an inhibitor of Endo V or hAAG. In one embodiment, the IBR is a catalytically inactive EndoV or a catalytically inactive It is hAAG.

[0430] The terms "isolated," "purified," or "biologically pure" refer to a substance that is in its native state. Substances that have been removed to varying degrees from components normally associated with them when found in the natural state. "Isolated" refers to the degree of separation from the original source or surrounding environment. "Purified" refers to the degree of separation from the original source or surrounding environment. A "purified" or "biologically pure" protein is one that: Impurities may not materially affect the biological properties of the protein or cause other adverse consequences. other substances are sufficiently removed so as not to interfere with the synthesis of the nucleic acid or peptide of the present invention. A vaccine may contain cellular material, viral material, or culture medium if produced by recombinant DNA technology. Substantially free of chemical precursors or other chemicals if chemically synthesized A substance is purified if it is substantially free of other substances. Purity and homogeneity are typically measured by: Analytical chemistry techniques, such as polyacrylamide gel electrophoresis or high performance liquid chromatography The term "purified" refers to a nucleic acid or protein that has been purified by electrophoresis. This may mean that the protein produces essentially one band in the sample. For proteins that can undergo glycosylation, the different modifications are purified separately. This can result in different isolated proteins that can be isolated.

[0431] An "isolated polynucleotide" is a nucleic acid molecule that does not occur in the naturally occurring genome of the organism from which the nucleic acid molecule of the invention is derived. It means a nucleic acid (e.g., DNA) that does not include the genes adjacent to the gene. The term also applies to vectors, e.g., autonomously replicating plasmids and viruses. integrated into the genomic DNA of prokaryotes or eukaryotes; or other sequences A separate molecule (e.g., c generated by PCR or restriction endonuclease digestion) In addition, the term encompasses recombinant DNA present as either genomic DNA or genomic or cDNA fragments. The RNA molecule transcribed from the DNA molecule, as well as the RNA molecule encoding the additional polypeptide sequence, Contains recombinant DNA that is part of a hybrid gene.

[0432] An "isolated polypeptide" is a polypeptide of the invention separated from components that naturally accompany it. Typically, a polypeptide is a polypeptide that is a protein with which it is naturally associated. A substance is isolated if it is at least 60% by weight free from substances and naturally occurring organic molecules. Preferably, the preparation comprises at least 75% by weight, more preferably at least 90%, and most preferably Preferably, at least 99% of the isolated polypeptides of the present invention are polypeptides of the present invention. A peptide can be prepared by, for example, extraction from a natural source, recombinant nucleic acid encoding such a polypeptide, or the like. or by chemically synthesizing the protein. Any suitable method, e.g., column chromatography, polyacrylamide gel electrophoresis, or by HPLC analysis.

[0433] As used herein, the term "linker" refers to a molecule or moiety that connects two molecules or moieties, e.g., a fused Two domains of the fusion protein, e.g., a nuclease-inactive Cas9 domain and a nucleic acid editing domain, A bond (e.g., adenosine deaminase) that connects enzymes (e.g., cytidine deaminase, adenosine deaminase) a covalent bond), chemical group, or molecule, or in the context of a chimeric antigen receptor, It refers to the linker that connects the variable (VH) region to the heavy chain constant (CH) region. In this case, the linker separates two domains of the fusion protein, e.g., a nuclease-inactive Cas domain. 9 domain and nucleic acid editing domain (e.g., cytidine deaminase, adenosine deaminase) In one embodiment, the linker comprises a Cas9 nuclease domain. The gRNA-binding domain of the RNA-programmable nuclease and the catalytic domain of the nucleic acid editing protein In some embodiments, the linker connects the dCas9 and the nucleic acid editing protein. Typically, a linker is placed between two groups, molecules, or other moieties. or are adjacent to each other and linked to each other via covalent bonds, thus In some embodiments, the linker is an amino acid or a plurality of amino acids. (e.g., a peptide or protein). In some embodiments, the linker is an organic A linker is a molecule, group, polymer, or chemical moiety. In some embodiments, the linker is 5 to 100 amino acids, e.g., 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17 , 18, 19, 20, 25, 35, 45, 50, 55, 60, 60, 65, 70, 70, 75, 80, 85, 90, 90, 95, 10 0, 101, 102, 103, 104, 105, 110, 120, 130, 140, 150, 160, 175, 180, 190, or 2 00 amino acids. Longer or shorter linkers are also contemplated. In some embodiments, The linker comprises the amino acid sequence SGSETPGTSESATPES, which may also be referred to as an XTEN linker. In some embodiments, the linker comprises the amino acid sequence SGGS. GGS) n , (GGGS) n , (GGGGS) n , (G) n , (EAAAK) n , (GGS) n , SGSETPGTSESATPES, or (XP ) n motif, or any combination thereof, where n is independently an integer 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.

[0434] In some embodiments, the chimeric antigen receptor comprises at least one linker. At least one linker is a heavy chain variable (VH) sequence of the extracellular binding domain of the chimeric antigen receptor. The linker also connects or joins the heavy chain constant (CH) region to the extracellular binding domain. The light chain variable (VL) region of the amino acid may also be linked to a variable constant (VC) region.

[0435] In some embodiments, a cytidine deaminase or adenosine deaminase nucleus The acid-base editor domains are SGGSSGSETPGTSESATPESSGGS, SGGSSGGSSGSETPGTSESATPES SGGSSGGS, or GGSGGSPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSTE PSE The fusion occurs via a linker containing the amino acid sequence GSAPGTSTEPSEGSAPGTSESATPESGPGSEPATSGGSGGS. In some embodiments, cytidine deaminase or adenosine deaminase The aminoase nucleobase editor domain is an amino acid sequence that is sometimes called an XTEN linker. In some embodiments, the nucleotide sequence SGSETPGTSESATPES is fused to the nucleotide sequence SGSETPGTSESATPES. In some embodiments, the linker is 24 amino acids in length. In some embodiments, the linker comprises the amino acid sequence SGGSSGGSSGSETPGTSESATPES. In some embodiments, the linker has the amino acid sequence SGGSSGGSSGSE In some embodiments, the linker comprises a 64 amino acid sequence. In some embodiments, the linker has the amino acid sequence SGGSSGGSSGSETPGTSESATPESSGGS In some embodiments, the linker comprises SGGSSGGSSGGSSGSETPGTSESATPESSGGS SGGS. is 92 amino acids. In some embodiments, the linker has the amino acid sequence PGSPAGSPTSTEEGTSES Contains ATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAP GTSTEPSEGSAPGTSESATPESGGPGSEPATS nothing.

[0436] 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.

[0437] As used herein, the term "mutation" refers to a change in a sequence, e.g., a nucleic acid or amino acid sequence. The substitution of a residue in a sequence of amino acids by another residue, or the deletion of one or more residues in the sequence. Mutations, as used herein, are typically made by identifying the original residue and then and identifying the newly substituted residue, Various methods for making amino acid substitutions (mutations) are provided herein. are well known in the art and are described, for example, in Green and Sambrook, Molecular Cloning: A Laboratory Manual (4th ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (2012).

[0438] "Neoplasm" refers to cells or tissues that exhibit abnormal growth or proliferation. The term neoplasm encompasses cancer and solid tumors.

[0439] Nuclear factor of activated T cells 1 (NFATc1) polypeptide "Peptide" refers to a peptide having at least about 85% amino acid sequence identical to NCBI Accession No. NM_172390.2 or a fragment thereof. It refers to a protein with sequence identity to the ATP-binding protein 1 (ATP1A1), which is a component of the activated T cell DNA-binding transcription complex. Exemplary amino acid sequences are provided below.

[0440] >NP_765978.1 Nuclear factor of activated T cells, cytoplasmic 1 isoform A [Homo sapiens] MPSTSFPVPSKFPLGPAAAVFGRGETLGPAPRAGGTMKSAEEEHYGYASSNVSPALPLPTAHSTLPAPCHNLQTSTPGII PPADHPSGYGAALDGGPAGYFLSSGHTRPDGAPALESPRIEITSCLGLYHNNNQFFHDVEVEDVLPSSKRSPSTATLSLP SLEAYRDPSCLSPASSLSSRSCNSEASSYESNYSYPYASPQTSPWQSPCVSPKTTDPEEGFPRGLGACTLLGSPRHSPST SPRASVTEESWLGARSSRPASPCNKRKYSLNGRQPPYSPHHSPTPSPHGSPRVSVTDDSWLGNTTQYTSSAIVAAINALT TDSSLDLGDGVPVKSRKTTLEQPPSVALKVEPVGEDLGSPPPPADFAPEDYSSFQHIRKGGFCDQYLAVPQHPYQWAKPK PLSPTSYMSPTLPALDWQLPSHSGPYELRIEVQPKSHHRAHYETEGSRGAVKASAGGHPIVQLHGYLENEPLMLQLFIGT ADDRLLRPHAFYQVHRITGKTVSTTSHEAILSNTKVLEIPLLPENSMRAVIDCAGILKLRNSDIELRKGETDIGRKNTRV RLVFRVHVPQPSGRTLSLQVASNPIECSQRSAQELPLVEKQSTDSYPVVGGKKMVLSGHNFLQDSKVIFVEKAPDGHHVW EMEAKTDRDLCKPNSLVVEIPPFRNQRITSPVHVSFYVCNGKRKRSQYQRFTYLPANGNAIFLTVSREHERVGCFF

[0441] "Nuclear factor of activated T cells 1 (NFATc1) polynucleotide" refers to a polynucleotide encoding an NFATc1 polypeptide. The NFATc1 gene is a cytokine gene in T cells, particularly An exemplary nucleic acid sequence is Provided below.

[0442] >NM_172390.2 Homo sapiens nuclear factor of activated T cells 1 (NFATC1), transcript variant 1 , mRNA GGCGGGCGCTCGGCGACTCGTCCCCGGGGCCCCGCGCGGGCCCGGGCAGCAGGGGCGTGATGTCACGGCA GGGAGGGGG CGCGGGAGCCGCCGGGCCGGCGGGGAGGCGGGGGAGGTGTTTTCCAGCTTTAAAAAGGCAG GAGGCAGAGCGCGGCCCT GCGTCAGAGCGAGACTCAGAGGCTCCGAACTCGCCGGCGGAGTCGCCGCGCC AGATCCCAGCAGCAGGGCGCGGGCACC GGGGCGCGGGCAGGGCTCGGAGCCACCGCGCAGGTCCTAGGGC CGCGGCCGGGCCCCGCCACGCGCGCACACGCCCCTC GATGACTTTCCTCCGGGGCGCGCGGCGCTGAGCC CGGGGCGAGGGCTGTCTTCCCGGAGACCCGACCCCGGCAGCGCGG GGCGGCCGCTTCTCCTGTGCCTCCG CCCGCCGCTCCACTCCCCGCCGCCGCCGCGGATGCCAAGCACCAGCTTTCCA GTCCCTTCCAAGTTTC CACTTGGCCCTGCGGCTGCGGTCTTCGGGAGAGGAGAAACTTTGGGGCCCGCGCGCGCGCCG GCGGCAC CATGAAGTCAGCGGAAGGAAGAACACTATGGCTATGCATCCTCCAACGTCAGCCCCGCCCTGCCGCTCCCC A CGGCGCACTCCACCCTGCCGGCCCCGTGCCACAACCTTCAGACCTCCACACCGGGCATCATCCCGCCGG CGGATCACCC CTCGGGGTACGGAGCAGCTTTGGACGGTGGGCCCGCGGGCTACTTCCTCTCCTCCGGCCA CACCAGGCCTGATGGGGCC CCTGCCCTGGAGAGTCCTCGCATCGAGATAACCTCGTGCTTGGGCCTGTAC CACAACAATAACCAGTTTTTCCACGATG TGGAGGTGGAAGACGTCCTCCCTAGCTCCAAACGGTCCCCCT CCACGGCCACGCTGAGTCTGCCCAGCCTGGAGGCCTA CAGAGACCCCTCGTGCCTGAGCCCGGCCAGCAG CCTGTCCTCCCGGAGCTGCAACTCAGAGGCCTCCTCCTACGAGTCC AACTACTCGTACCCGTACGCGTCC CCCCAGACGTCGCCATGGCAGTCTCCCTGCGTGTCTCCCAAGACCACGGACCCCG AGGAGGGCTTTCCCC GCGGGCTGGGGGCCTGCACACTGCTGGGTTCCCCGCGGCACTCCCCCTCCACCTCGCCCCGCGC CAGCGT CACTGAGGAGAGCTGGCTGGGTGCCCGCTCCTCCAGACCCGCGTCCCCTTGCAACAAGAGGAAGTACAGC CT CAACGGCCGGCAGCCGCCCTACTCACCCCACCACTCGCCCACGCCGTCCCCGCACGGCTCCCCGCGGG TCAGCGTGACC GACGACTCGTGGTTGGGCAACACCACCCAGTACACCAGCTCGGCCATCGTGGCCGCCAT CAACGCGCTGACCACCGACA GCAGCCTGGACCTGGGAGATGGCGTCCCTGTCAAGTCCCGCAAGACCACC CTGGAGCAGCCGCCCTCAGTGGCGCTCAA GGTGGAGCCCGTCGGGGAGGACCTGGGCAGCCCCCCGCCCC CGGCCGACTTCGCGCCCGAAGACTACTCCTCTTTCCAG CACATCAGGAAGGGCGGCTTCTGCGACCAGTA CCTGGCGGTGCCGCAGCACCCCTACCAGTGGGCGAAGCCCAAGCCCC TGTCCCCTACGTCCTACATGAGC CCGACCCTGCCCGCCCTGGACTGGCAGCTGCCGTCCCACTCAGGCCCGTATGAGCT TCGGATTGAGGTGC AGCCCAAGTCCCACCACCGAGCCCACTACGAGACGGAGGGCAGCCGGGGGGCCGTGAAGGCGTCG GCCGG AGGACACCCCATCGTGCAGCTGCATGGCTACTTGGAGAATGAGCCGCTGATGCTGCAGCTTTTCATTGGG ACG GCGGACGACCGCCTGCTGCGCCCGCACGCCTTCTACCAGGTGCACCGCATCACAGGGAAGACCGTGT CCACCACCAGCC ACGAGGCCATCCTCTCCAAACACCAAAGTCCTGGAGATCCCACTCCTGCCGGAGAACAG CATGCGAGCCGTCATTGACTG TGCCGGAATCCTGAAACTCAGAAACTCCGACATTGAACTTCGGAAAGGA GAGACGGACATCGGGAGGAAGACACACGG GTACGGCTGGTGTTCCGCGTTCACGTCCCGCAACCCACGGCGCGCACGCTGTCCCTGCAGGTGGCTCCAACCCCATCG AATGCTCCCAGCGCTCAGCTCAGGAGCTGCC TCTGGTGGAGAAGCAGAGCACGGACAGCTATCCGGTCGTGGGCGGGAA GAAGATGGTCCTGTCTGGCCAC AACTTCCTGCAGGACTCCAAGGTCATTTTCGTGGAGAAAGCCCCAGATGGCCACCAT GTCTGGGAGATGG AAGCGAAAACTGACCGGGACCTGTGCAAGCCGAATTCTCTGGTGGTTGAGATCCCGCCATTTCGGA ATCA GAGGATAACCAGCCCCGTTCACGTCAGTTTCTACGTCTGCAACGGGAAGAGAAAGCGAAGCCAGTACCAG CGTT TCACCTACCTTCCCGCCAACGGTAACGCCATCTTCTAACCGTAAGCCGTGAACATGAGCGCGTGG GGTGCTTTTCTA AAGACGCAGAAACGACGTCGCCGTAAAGCAGCGTGGCGTGTTGCACATTTAACTGTG TGATGTCCCGTTAGTGAGACCG AGCCATCGATGCCCTGAAAGGAAAGGAAAGGGAAGCTTCGGATGCA TTTTCCTTGATCCCTGTTGGGGGTGGGGGGC GGGGGTTGCATACTCAGATAGTCACGGTTATTTTGCTTC TTGCGAATGTATAACAGCCAAGGGGAAAACATGGCTCTTC TGCTCCAAAAAACTGAGGGGGTCCTGGTGT GCATTTGCACCCTAAAGCTGCTTACGGTGAAAAGGCAAATAGGTATAGC TATTTTGCAGGCACCTTTAGG AATAAACTTTGCTTTTAAGCCTGTAAAAAAAAAAAAAAA

[0443] The term "nuclear localization sequence," "nuclear localization signal," or "NLS" refers to the cellular localization of a protein. refers to an amino acid sequence that promotes import into the nucleus. Nuclear localization sequences are known in the art. For example, patent application WO / 2001 / 038547 was filed on November 23, 2000 and published on May 31, 2001. The invention is described in the international PCT application PCT / EP2000 / 011690 by Plank et al., the contents of which are set forth in, e.g. The disclosure of exemplary nuclear localization sequences is incorporated herein by reference. In the present invention, NLS is, for example, Koblan et al., Nature Biotech. 2018 doi: 10.1038 / nbt.4172 Optimized NLSs useful in the methods of the present invention are described by The sequences are shown in Figures 8A-8E and 9. In some embodiments, the NLS has the amino acid sequence column PKKKRKVEGADKRTADGSEFES PKKKRKV, KRTADGSEFESPKKKRKV, KRPAATKKAGQAKKKK, KKTELQTTNA ENKTKKL, KRGINDRNFWRGENGRKTR, RKSGKIAAIVVKRPRK, PKKKRKV, or MDSLLMNRRKFLYQFKN Contains VRWAKGRRETYLC.

[0444] As used herein, the terms "nucleic acid" and "nucleic acid molecule" refer to a nucleic acid molecule comprising a nucleobase and Compounds containing an acidic moiety, such as nucleosides, nucleotides, or polynucleotides Typically, a polymeric nucleic acid, e.g., a nucleic acid molecule containing three or more nucleotides is a linear sequence in which adjacent nucleotides are linked to each other via phosphodiester bonds. In some embodiments, a "nucleic acid" refers to individual nucleic acid residues (e.g., nucleotides and In some embodiments, a "nucleic acid" refers to a group of three or more individual nucleotides. As used herein, the term "oligonucleotide" refers to an oligonucleotide chain containing nucleotide residues. "Dinucleotide," and "polynucleotide" refer to a polymer of nucleotides (e.g., a small molecule). These may be used interchangeably to refer to a chain of at least three nucleotides. In this context, "nucleic acid" encompasses RNA and single- and / or double-stranded DNA. For example, genome, transcript, mRNA, tRNA, rRNA, siRNA, snRNA, plasmid, cosmid, staining may occur naturally in the context of a nucleic acid molecule, a chromatid, or other naturally occurring nucleic acid molecule On the other hand, nucleic acid molecules can be, for example, non-naturally occurring molecules, recombinant DNA or RNA, artificial chromosomes, etc. , engineered genomes or fragments thereof, or synthetic DNA, RNA, DNA / RNA hybrids; or non-naturally occurring molecules, including non-naturally occurring nucleotides or nucleosides. Additionally, the terms "nucleic acid," "DNA," "RNA," and / or similar terms may be used interchangeably. Nucleic acids include nucleic acid analogs, e.g., analogs having other than a phosphodiester backbone. purified from natural sources; produced using recombinant expression systems and optionally purified; chemically In the case of chemically synthesized molecules, the nucleic acid may, where appropriate, Nucleotides, such as analogs with chemically modified bases or sugars, and backbone modifications, are also included. Nucleic acid sequences are shown in the 5' to 3' direction unless otherwise indicated. In some embodiments, nucleic acids contain natural nucleosides (e.g., adenosine, thymidine, guanine, adenosine, cytidine, uridine, deoxyadenosine, deoxythymidine, deoxyguanosine cytidine, and deoxycytidine; nucleoside analogs (e.g., 2-aminoadenosine, 2- Thiothymidine, inosine, pyrrolo-pyrimidine, 3-methyladenosine, 5-methylcytidine , 2-aminoadenosine, C5-bromouridine, C5-fluorouridine, C5-iodouridine , C5-propynyl-uridine, C5-propynyl-cytidine, C5-methylcytidine, 2-amino adenosine, 7-deazaadenosine, 7-deazaguanosine, 8-oxoadenosine, 8-oxoglucan guanine, O(6)-methylguanine, and 2-thiocytidine); chemically modified bases; biologically modified Modified bases (e.g., methylated bases); inserted bases; modified sugars (e.g., 2'-fluororibose , ribose, 2'-deoxyribose, arabinose, and hexose); and / or Modified phosphate groups (e.g., phosphorothioate and 5'-N-phosphoramidite linkages) or containing them.

[0445] The term "nucleic acid programmable DNA binding protein" or "napDNAbp" refers to the Associating with a nucleic acid (e.g., DNA or RNA), such as a guide nucleic acid, that directs the Abp to a specific nucleic acid sequence For example, the Cas9 protein refers to a protein that binds the Cas9 protein to a guide RNA. It may bind to a guide RNA that directs it to a complementary specific DNA sequence. , napDNAbp is a Cas9 domain, e.g., nuclease-active Cas9, Cas9 nickase ( nCas9), or nuclease-inactivated Cas9 (dCas9). Examples of binding proteins include Cas9 (e.g., dCas9 and nCas9), CasX, CasY, Cpfl, Ca Other nucleic acid promoters include, but are not limited to, s12b / C2c1, and Cas12c / C2c3. Ramming-capable DNA binding proteins are also disclosed herein, even if not specifically listed in the disclosure. is within the range.

[0446] As used herein, "obtaining," as in "obtaining a drug," means This includes synthesizing, purchasing, or otherwise obtaining the drug.

[0447] "Programmed cell death 1 (PDCD1 or PD-1) polypeptide" refers to NCBI accession number AJS1 0360.1 or a fragment thereof. The PD-1 protein is thought to be involved in regulating T cell function and tolerance during immune responses. Exemplary B2M polypeptide sequences are provided below.

[0448] >AJS10360.1 Programmed cell death 1 protein [Homo sapiens] MQIPQAPWPVVWAVLQLGWRPGWFLDSPDRPWNPPTFSPALLVVTEGDNATFTCSFSNTSESFVLNWYRMSPSNQTDKLA AFPEDRSQPGQDCRFRVTQLPNGRDFHMSVVRARRNDSGTYLCGAISLAPKAQIKESLRAELRVTERRAEVPTAHPSSPSP RPAGQFQTLVVGVVGGLLGSLVLLVWVLAVICSRAARGTIGARRTGQPLKEDPSAVPVFSVDYGELDFQWREKTPEPPVP CVPEQTEYATIVFPSGMGTSSPARRGSADGPRSAQPLRPEDGHCSWPL

[0449] "Programmed cell death 1 (PDCD1 or PD-1) polynucleotide" refers to a PD-1 polypeptide. The PDCD1 gene is a nucleic acid molecule that encodes a T cell effector function specific to an antigen. Exemplary PDCD1 nucleic acid sequences are provided below. It is served.

[0450] AY238517.1 Homo sapiens programmed cell death 1 (PDCD1) mRNA, complete cds ATGCAGATCCCACAGGCGCCCTGGCCAGTCGTCTGGGCGGTGCTACAACTGGGCTGGCGGCCAGGATGGTTCTTAGACTC CCCAGACAGGCCCTGGAACCCCCCACCTTCTCCCCAGCCCTGCTCGTGGTGACCGAAGGGGACAACGCCACCTTCACCT GCAGCTTCTCCAACACATCGGAGAGCTTCGTGCTAAACTGGTACCGCATGAGCCCCAGCAACCAGACGGACAAGCTGGCC GCCTTCCCCGAGGACCGCAGCCAGCCCGGCCAGGACTGCCGCTTCCGTGTCACACAACTGCCCAACGGGCGTGACTTCCA CATGAGCGTGGTCAGGGCCCGGCGCAATGACAGCGGCACCTACCTCTGTGGGGCCATCTCCCTGGCCCCCAAGGCGCAGA TCAAAGAGAGCCTGCGGGCAGAGCTCAGGGTGACAGAGAGAAGGGCAGAAGTGCCCACAGCCCACCCCAGCCCCTCACCC AGGCCAGCCGGCCAGTTCCAAACCCTGGTGGTTGGTGTCGTGGGCGGCCTGCTGGGCAGCCTGGTGCTGCTAGTCTGGGT CCTGGCCGTCATCTGCTCCCGGGCCGCACGAGGGACAATAGGAGCCAGGCGCACCGGCCAGCCCCTGAAGGAGGACCCCT CAGCCGTGCCTGTGTTCTCTGTGGACTATGGGGAGCTGGATTTCCAGTGGCGAGAGAAGACCCCGGAGCCCCCCGTGCCC TGTGTCCCTGAGCAGACGGAGTATGCCACCATTGTCTTTCCTAGCGGAATGGGCACCTCATCCCCCGCCCGCAGGGGCTC AGCTGACGGCCCTCGGAGTGCCCAGCCACTGAGGCCTGAGGATGGACACTGCTCTTGGCCCCTCTGA

[0451] As used herein in the context of a protein or nucleic acid, the term "recombinant" means Refers to a protein or nucleic acid that does not occur in nature and is the product of human manipulation. For example, In some embodiments, the recombinant protein or nucleic acid molecule may be any naturally occurring sequence. Compared to at least one, at least two, at least three, at least four, at least Amino acid or nucleotide sequences containing five, at least six, or at least seven mutations Contains columns.

[0452] "Reduce" or "increase" means to reduce by at least 10%, 25%, 50%, 75%, or 100% means a negative or positive change, respectively.

[0453] "Reference" means a standard or control condition.

[0454] A "reference sequence" is a defined sequence used as a basis for sequence comparison. The sequence may be a subset or the entirety of a specified sequence; for example, a full-length cDNA or A segment of a gene sequence, or the complete cDNA or gene sequence. For polypeptides, see The length of the reference polypeptide sequence is generally at least about 16 amino acids, at least about 20 amino acids. more preferably at least about 25 amino acids, even more preferably about 35 amino acids, For nucleic acids, the length of a reference nucleic acid sequence is generally , at least about 50 nucleotides, at least about 60 nucleotides, at least about 75 nucleotides tide, and about 100 nucleotides or about 300 nucleotides, or their surroundings or is any integer between them.

[0455] The terms "RNA programmable nuclease" and "RNA-guided nuclease" refer to It is used in conjunction with one or more non-target RNAs (e.g., those that bind to or associate with it). In some embodiments, the RNA-programmable nuclease, when complexed with RNA, Typically, the bound RNA is a guide RNA (gRNA) and gRNAs can exist as a complex of two or more RNAs, or as a single RNA molecule. A gRNA that exists as a single RNA molecule is called a single guide RNA (sgRNA). Although sometimes referred to as "gRNA," "gRNA" can be used as a single molecule or as a complex of two or more molecules. It is used interchangeably to refer to guide RNAs that exist as a single RNA species. A gRNA exists as a target nucleic acid that has (1) a domain that shares homology with the target nucleic acid (e.g., a C domain to the target) (2) a domain that binds to the Cas9 protein; In one embodiment, domain (2) is a sequence encoding the tracrRNA. For example, in some embodiments, the domain (2 ) is disclosed in Jinek et al., Science 337: 816-821 (2012), the entire contents of which are incorporated herein by reference. The gRNA (e.g., the driver RNA) is identical to or homologous to the tracrRNA provided in the Other examples (including those containing main 2) include "Switchable Cas9 Nucleases and Uses Thereof" and No. 61 / 874,682, filed September 6, 2013, entitled "Delivery System" and U.S. Provisional Patent Application No. 61 / 8 filed September 6, 2013, entitled "m For Functional Nucleases" 74,746, the entire contents of each of which are incorporated herein by reference. In some embodiments, the gRNA comprises two or more of domains (1) and (2) and is referred to as an "extension" domain. By way of example, an extended gRNA may be referred to as a "longered gRNA" as described herein. , e.g., by binding two or more Cas9 proteins to target nucleic acids in two or more different regions. The gRNA contains a nucleotide sequence complementary to the target site, which binds to the target site. Mediates binding of nuclease / RNA complexes to the target site and determines the sequence specificity of the nuclease:RNA complex. In some embodiments, the RNA programmable nuclease is a CRISPR-associated system. Cas9 endonuclease, e.g., Cas9 (Csnl) from Streptococcus pyogenes. (e.g., "Complete genome sequence of an M1 strain of Streptococcus pyogenes." " Ferretti JJ, McShan WM, Ajdic DJ, Savic DJ, Savic G., Lyon K., Primeau x C, Sezate S., Suvorov AN, Kenton S., Lai HS, Lin SP, Qian Y., Jia HG, Najar FZ, Ren Q., Zhu H., Song L., White J., Yuan X., Clifton SW, Roe BA, McLaughlin RE, Proc. Natl. Acad. Sci. USA 98: 4658-4663(2001); "CRISPR RNA maturation by trans-encoded small RNA and host factor RNase III." Deltcheva E., Chylinski K., Sharma CM., Gonzales K., Chao Y., Pirzada ZA, Eckert MR, Vogel J., Charpentier E., Nature 471: 602-607 (2011)).

[0456] "Specifically binds" means to bind to a nucleic acid molecule, polypeptide, or complex thereof (e.g., a nucleic acid molecule, polypeptide, or complex thereof). (antigen-programmable DNA-binding proteins, guide nucleic acids, and chimeric antigen receptors) However, it is possible for the antibody to recognize and bind to other molecules in a sample, e.g., a biological sample. For example, chimeric antigen receptors specifically bind to specific markers expressed on the surface of cells. bind specifically to other polypeptides, carbohydrates, lipids, or any other compounds on the surface of the cell. It doesn't bond with things.

[0457] Nucleic acid molecules useful in the methods of the present invention may encode a polypeptide of the present invention or a fragment thereof. Such nucleic acid molecules include any nucleic acid molecule that is 100% identical to the endogenous nucleic acid sequence. Typically, but not necessarily, substantial identity is shown. A polynucleotide having a nucleotide sequence typically hybridizes with at least one strand of a double-stranded nucleic acid molecule. Nucleic acid molecules useful in the methods of the present invention can be modified to The present invention also includes any nucleic acid molecule encoding an endogenous nucleotide sequence or a fragment thereof. It need not be 100% identical to the nucleic acid sequence, but typically will show substantial identity. A polynucleotide having "substantial identity" to a double-stranded nucleic acid molecule "Hybridize" means to hybridize with at least one strand of , complementary polynucleotide sequences (e.g., those described herein) under various stringency conditions. This means that the DNA fragments (or portions thereof) of the nucleotide sequence of the nucleotide sequence described in the present specification form a double-stranded molecule. (See, e.g., Wahl, GM and SL Berger (1987) Methods Enzymol. 152: 399; Kimme l, AR (1987) Methods Enzymol. 152: 507).

[0458] For example, a stringent salt concentration is typically less than about 750 mM NaCl and 75 mM citrate. trisodium, preferably less than about 500 mM NaCl and 50 mM trisodium citrate, more preferably Preferably, the concentration is less than about 250 mM NaCl and 25 mM trisodium citrate. See hybridization can be obtained in the absence of organic solvents, e.g., formamide. whereas high stringency hybridization requires at least about 35% formaldehyde. It can be obtained in the presence of at least about 50% formamide. Stringent temperature conditions are typically at least about 30°C, more preferably at least about 37°C. 0 C, and most preferably at least about 42°C. The concentration of detergent (e.g., sodium dodecyl sulfate (SDS)) and the content of carrier DNA Various additional parameters, such as inclusion or exclusion, are well known to those skilled in the art. By combining these various conditions, various levels of stringency can be achieved. In one embodiment, hybridization is achieved at 30° C. in 750 mM NaCl, 75 In another embodiment, hybridization occurs in 10 mM trisodium citrate and 1% SDS. The solution was incubated at 37°C in 500 mM NaCl, 50 mM trisodium citrate, 1% SDS, 35% formamide. In another embodiment, the hybridization occurs in 100 μg / ml denatured salmon sperm DNA (ssDNA). Hybridization was performed at 42°C in 250 mM NaCl, 25 mM trisodium citrate, 1% The reaction takes place in SDS, 50% formamide, and 200 μg / ml ssDNA. The implementation will be apparent to those skilled in the art.

[0459] For most applications, the washing steps that follow hybridization are also stringent. Wash stringency conditions are defined by salt concentration and temperature. As mentioned above, wash stringency can be increased by decreasing the salt concentration or increasing the temperature. This can be increased by increasing the thickness of the strip for the cleaning process. The appropriate salt concentration is preferably less than about 30 mM NaCl and 3 mM trisodium citrate. and most preferably less than about 15 mM NaCl and 1.5 mM trisodium citrate. Stringent temperature conditions for the process are typically at least about 25°C, more preferably In one embodiment, the temperature is at least about 42°C, and even more preferably at least about 68°C. Washing steps were performed at 25°C in 30 mM NaCl, 3 mM trisodium citrate, and 0.1% SDS. In a more preferred embodiment, the wash steps are carried out in 15 mM NaCl, 1.5 mM NaCl, at 42°C. 10 mM trisodium citrate, and 0.1% SDS. Washing steps were performed at 68°C in 15 mM NaCl, 1.5 mM trisodium citrate, and 0.1% SDS. Further variations of these conditions will be apparent to those skilled in the art. Hybridization techniques are well known to those skilled in the art and are described, for example, in Benton and Dav is (Science 196: 180, 1977); Grunstein and Hogness (Proc. Natl. Acad. Sci., USA 72: 3961, 1975); Ausubel et al. (Current Protocols in Molecular Biology, Wiley I terscience, New York, 2001); Berger and Kimmel (Guide to Molecular Cloning Tech Niques, 1987, Academic Press, New York); and Sambrook et al., Molecular Cloning g: A Laboratory Manual, Cold Spring Harbor Laboratory Press, New York are.

[0460] "Subject" means a mammal, including a human or non-human mammal, e.g., a rat. The subject may be a livestock animal, including, but not limited to, a dog, horse, dog, sheep, or cat. , domestic animals kept to produce labor and to supply goods such as food including, but not limited to, cattle, goats, chickens, horses, pigs, rabbits, and sheep. Not limited.

[0461] "Substantially identical" means that the amino acid sequence of a reference amino acid sequence (e.g., an amino acid sequence described herein) is substantially identical to the amino acid sequence of a reference amino acid sequence (e.g., an amino acid sequence described herein). any one of the sequences) or nucleic acid sequences (e.g., any one of the nucleic acid sequences described herein ) means a polypeptide or nucleic acid molecule that exhibits at least 50% identity to In embodiments, such sequences are identical to the sequences used for comparison at the amino acid level or have at least 60%, 80%, or even 85%, 90%, 95%, or even 99% nucleic acid identity. do.

[0462] Sequence identity is typically determined using sequence analysis software (e.g., Genetics Computer Group p, University of Wisconsin Biotechnology Center, 1710 University Avenue, Madison , Wis. 53705 Sequence Analysis Software Package, BLAST, BESTFIT, GAP, or PIL Such software is used in various places. By assigning degrees of homology to substitutions, deletions, and / or other modifications, it is possible to identify identical or Conservative substitutions typically include substitutions within the following groups: Lysine, Alanine; Valine, Isoleucine, Leucine; Aspartic Acid, Glutamic Acid, Asparagine, Glutamine; Serine, Threonine; Lysine, Arginine; Phenylanine In an exemplary approach to determining the degree of identity, the BLAST protocol grams can be used, e -3 and e -100 Probability scores between indicate closely related sequences .

[0463] RNA-programmable nucleases (e.g., Cas9) use RNA to target DNA cleavage sites. A: Because DNA hybridization is used, these proteins are, in principle, Any sequence specified by the RNA can be targeted. Methods that use RNA-programmable nucleases such as Cas9 for this purpose (e.g., genome modification) Methods for modifying the chromatin (for example, to change the chromatin) are known in the art (see, e.g., Cong, L. et al., Multi plex genome engineering using CRISPR / Cas systems. Science 339, 819-823 (2013); M ali, P. et al., RNA-guided human genome engineering via Cas9. Science 339, 823-8 26 (2013); Hwang, WY et al., Efficient genome editing in zebrafish using a CRI SPR-Cas system. Nature biotechnology 31, 227-229 (2013); Jinek, M. et al., RNA-p rogrammed genome editing in human cells. eLife 2, e00471 (2013); Dicarlo, JE e t al., Genome engineering in Saccharomyces cerevisiae using CRISPR-Cas systems. Nucleic acids research (2013); Jiang, W. et al., RNA-guided editing of bacteria genomes using CRISPR-Cas systems. Nature biotechnology 31, 233-239 (2013) (The entire contents of each of which are incorporated herein by reference).

[0464] "Tet methylcytosine dioxygenase 2 (TET2) ) polypeptide" refers to a polypeptide that is at least about 85% identical to NCBI Accession No. FM992369.1 or a fragment thereof It has amino acid sequence identity and converts methylcytosine to 5-hydroxymethylcytosine. Defects in the gene for this protein cause myeloproliferative disorders. and its ability to methylate cytosines contributes to transcriptional regulation. An exemplary TET2 amino acid sequence is provided below. >CAX30492.1 tet oncogene family member 2 [Homo sapiens] MEQDRTNHVEGNRLSPFLIPSPPICQTEPLATKLQNGSPLPERAHPEVNGDTKWHSFKSYYGIPCMKGSQNSRVSPDFTQ ESRGYSKCLQNGGIKRTVSEPSLSGLLQIKKLKQDQKANGERRNFGVSQERNPGESSQPNVSDLSDKKESVSSVAQENAV KDFTSFSTHNCSGPENPELQILNEQEGKSANYHDKNIVLLKNKAVLMPNGATVSASSVEHTHGELLEKTLSQYYPDCVSI AVQKTTSHINAINSQATNELSCEITHPSHTSGQINSAQTSNSELPPKPAAVVSEACDADDADNASKLAAMLNTCSFQKPE QLQQQKSVFEICPSPAENNIQGTTKLASGEEFCSGSSSNLQAPGGSSERYLKQNEMNGAYFKQSSVFTKDSFSATTTPPP PSQLLLSPPPPLPQVPQLPSEGKSTLNGGVLEEHHHYPNQSNTTLLREVKIEGKPEAPPSQSPNPSTHVCSPSPMLSERP QNNCVNRNDIQTAGTMTVPLCSEKTRPMSEHLKHNPPIFGSGELQDNCQQLMRNKEQEILKGRDKEQTRDLVPPTQHYL KPGWIELKAPRFHQAESHLKRNEASLPSILQYQPNLSNQMTSKQYTGNSNMPGGLPRQAYTQKTTQLEHKSQMYQVEMNQ GQSQGTVDQHLQFQKPSHQVHFSKTDHLPKAHVQSLCGTRFHFQQRADSQTEKLMSPVLKQHLNQQASETEPFSNSHLLQ HKPHKQAAQTQPSQSSHLPQNQQQQQKLQIKNKEEILQTFPHPQSNNDQQREGSFFGQTKVEECFHGENQYSKSSEFETH NVQMGLEEVQNINRRNSPYSQTMKSSACKIQVSCSNNTHLVSENKEQTTHPELFAGNKTQNLHHMQYFPNNVIPKQDLLH RCFQEQEQKSQQASVLQGYKNRNQDMSGQQAAQLAQQRYLIHNHANVFPVPDQGGSHTQTPPQKDTQKHAALRWHLLQKQ EQQQTQQPQTESCHSQMHRPIKVEPGCKPHACMHTAPPENKTWKKVTKQENPPASCDNVQQKSIIETMEQHLKQFHAKSL FDHKALTLKSQKQVKVEMSGPVTVLTRQTTAAELDSHTPALEQQTTSSEKTPTKRTAASVLNNFIESPSKLLDTPIKNLL DTPVKTQYDFPSCRCVEQIIEKDEGPFYTHLGAGPNVAAIREIMEERFGQKGKAIRIERVIYTGKEGKSSQGCPIAKWVV RRSSSEEKLLCLVRERAGHTCEAAVIVILILVWEGIPLSLADKLYSELTETLRKYGTLTNRRCALNEERTCACQGLDPET CGASFSFGCSWSMYYNGCKFARSKIPRKFKLLGDDPKEEEKLESHLQNLSTLMAPTYKKLAPDAYNNQIEYEHRAPECRL GLKEGRPFSGVTACLDFCAHAHRDLHNMQNGSTLVCTLTREDNREFGGKPEDEQLHVLPLYKVSDVDEFGSVEAQEEKKR SGAIQVLSSFRRKVRMLAEPVKTCRQRKLEAKKAAAEKLSSLENSSNKNEKEKSAPSRTKQTENASQAKQLAELLRLSGP VMQQSQQPQPLQKQPPQPQQQQRPQQQQPHHPQTESVNSYSASGSTNPYMRRPNPVSPYPNSSHTSDIYGSTSPMNFYST SSQAAGSYLNSSNPMNPYPGLLNQNTQYPSYQCNGNLSVDNCSPYLGSYSPQSQPMDLYRYPSQDPLSKLSLPPIHTLYQ PRFGNSQSFTSKYLGYGNQNMQGDGFSSCTIRPNVHHVGKLPPYPTHEMDGHFMGATSRLPPNLSNPNMDYKNGEHHSPS HIIHNYSAAPGMFNSSLHALHLQNKENDMLSHTANGLSKMLPALNHDRTACVQGGLHKLSDANGQEKQPLALVQGVASGA EDNDEVWSDSEQSFLDPDIGGVAVAPTHGSILIECAKRELHATTPLKNPNRNHPTRISLVFYQHKSMNEPKHGLALWEAK MAEKAREKEEECEKYGPDYVPQKSHGKKVKREPAEPHETSEPTYLRFIKSLAERTMSVTTDSTVTTSPYAFTRVTGPYNR YI

[0465] "Tet methylcytosine dioxygenase 2 (TET2) polynucleotide" means a TET2 polypeptide. TET polypeptide refers to a nucleic acid molecule encoding a methylcytosine dioxygenase (TET) polypeptide. Exemplary TET2 nucleic acids are provided below. >FM992369.1 Homo sapiens mRNA for tet oncogene family member 2 (TET2 gene) CCGTGCCATCCCAACCTCCCACCTCGCCCCAACCTTCGCGCTTGCTCTGCTTCTTCTCCCAGGGGTGGAGACCCGCCGA GGTCCCCGGGGTTCCCGAGGGCTGCACCCTTCCCCGCGCTCGCCAGCCCTGGCCCCTACTCCGCGCTGGTCCGGGCGCAC CACTCCCCCCGCGCCACTGCACGGCGTGAGGGCAGCCCAGGTCTCCACTGCGCGCCCCGCTGTACGGCCCCAGGTGCCGC CGGCCTTTGTGCTGGACGCCCGGTGCGGGGGGCTAATTCCCTGGGAGCCGGGGCTGAGGGCCCCAGGGCGGCGGCGCAGG CCGGGGCGGAGCGGGAGGAGGCCGGGGCGGAGCAGGAGGAGGCCCGGGCGGAGGAGGAGAGCCGGCGGTAGCGGCAGTGG CAGCGGCGAGAGCTTGGGCGGCCGCCGCCGCCTCCTCGCGAGCGCCGCGCGCCCGGGTCCCGCTCGCATGCAAGTCACGT CCGCCCCCTCGGCGCGGCCGCCCCGAGACGCCGGCCCCGCTGAGTGATGAGAACAGACGTCAAACTGCCTTATGAATATT GATGCGGAGGCTAGGCTGCTTTCGTAGAGAAGCAGAAGGAAGCAAGATGGCTGCCCTTTAGGATTTGTTAGAAAGGAGAC CCGACTGCAACTGCTGGATTGCTGCAAGGCTGAGGGACGAGAACGAGGCTGGCAAACATTCAGCAGCACACCCTCTCAAG ATTGTTTACTTGCCTTTGCTCCTGTTGAGTTACAACGCTTGGAAGCAGGAGATGGGCTCAGCAGCAGCCAATAGGACATG ATCCAGGAAGAGCAAATTCAACTAGAGGGCAGCCTTGTGGATGGCCCCGAAGCAAGCCTGATGGAACAGGATAGAACCAA CCATGTTGAGGGCAACAGACTAAGTCCATTCCTGATACCATCACCTCCCATTTGCCAGACAGAACCTCTGGCTACAAAGC TCCAGAATGGAAGCCCACTGCCTGAGAGAGCTCATCCAGAAGTAAATGGAGACACCAAGTGGCACTCTTTCAAAAGTTAT TATGGAATACCCTGTATGAAGGGAAGCCAGAATAGTCGTGTGAGTCCTGACTTTACACAAGAAAGTAGAGGGTATTCCAA GTGTTTGCAAAATGGAGGAATAAAACGCACAGTTAGTGAACCTTCTCTCTCTGGGCTCCTTCAGATCAAGAAATTGAAAC AAGACCAAAAGGCTAATGGAGAAAGACGTAACTTCGGGGTAAGCCAAGAAAGAAATCCAGGTGAAAGCAGTCAACCAAAT GTCTCCGATTTGAGTGATAAGAAAGAATCTGTGAGTTCTGTAGCCCAAGAAAATGCAGTTAAAGATTTCACCAGTTTTTC AACACATAACTGCAGTGGGCCTGAAAATCCAGAGCTTCAGATTCTGAATGAGCAGGAGGGGAAAAGTGCTAATTACCATG ACAAGAACATTGTATTACTTAAAAACAAGGCAGTGCTAATGCCTAATGGTGCTACAGTTTCTGCCTCTTCCGTGGAACAC ACACATGGTGAACTCCTGGAAAAAACACTGTCTCAATATTATCCAGATTGTGTTTCCATTGCGGTGCAGAAAACCACATC TCACATAAATGCCATTAACAGTCAGGCTACTAATGAGTTGTCCTGTGAGATCACTCACCCATCGCATACCTCAGGGCAGA TCAATTCCGCACAGACCTCTAACTCTGAGCTGCCTCCAAAGCCAGCTGCAGTGGTGAGTGAGGCCTGTGATGCTGATGAT GCTGATAATGCCAGTAAACTAGCTGCAATGCTAAATACCTGTTCCTTTCAGAAACCAGAACAACTACAACAACAAAAATC AGTTTTTGAGATATGCCCATCTCCTGCAGAAAATAACATCCAGGGAACCACAAAGCTAGCGTCTGGTGAAGAATTCTGTT CAGGTTCCAGCAGCAATTTGCAAGCTCCTGGTGGCAGCTCTGAACGGTATTTAAAACAAAATGAAATGAATGGTGCTTAC TTCAAGCAAAGCTCAGTGTTCACTAAGGATTCCTTTTCTGCCACTACCACACCACCACCACCATCACAATTGCTTCTTTC TCCCCCTCCTCCTCTTCCACAGGTTCCTCAGCTTCCTTCAGAAGGAAAAAGCACTCTGAATGGTGGAGTTTTAGAAGAAC ACCACCACTACCCCAACCAAAGTAACACAACACTTTTAAGGGAAGTGAAAATAGAGGGTAAACCTGAGGCACCACCTTCC CAGAGTCCTAATCCATCTACACATGTATGCAGCCCTTCTCCGATGCTTTCTGAAAGGCCTCAGAATAATTGTGTGAACAG GAATGACATACAGACTGCAGGGACAATGACTGTTCCATTGTGTTCTGAGAAAACAAGACCAATGTCAGAACACCTCAAGC ATAACCCACCAATTTTTGGTAGCAGTGGAGAGCTACAGGACAACTGCCAGCAGTTGATGAGAAACAAAGAGCAAGAGATT CTGAAGGGTCGAGACAAGGAGCAAACACGAGATCTTGTGCCCCCAACACAGCACTATCTGAAACCAGGATGGATTGAATT GAAGGCCCCTCGTTTTCACCAAGCGGAATCCCATCTAAAACGTAATGAGGCATCACTGCCATCAATTCTTCAGTATCAAC CCAATCTCTCCAATCAAATGACCTCCAAACAATACACTGGAAATTCCAACATGCCTGGGGGGCTCCCAAGGCAAGCTTAC ACCCAGAAAACAACACAGCTGGAGCACAAGTCACAAATGTACCAAGTTGAAATGAATCAAGGGCAGTCCCAAGGTACAGT GGACCAACATCTCCAGTTCCAAAAACCCTCACACCAGGTGCACTTCTCCAAAACAGACCATTTACCAAAAGCTCATGTGC AGTCACTGTGTGGCACTAGATTTCATTTTCAACAAAGAGCAGATTCCCAAACTGAAAAACTTATGTCCCCAGTGTTGAAA CAGCACTTGAATCAACAGGCTTCAGAGACTGAGCCATTTTCAAACTCACACCTTTTGCAACATAAGCCTCATAAACAGGC AGCACAAACACAACCATCCCAGAGTTCACATCTCCCTCAAAACCAGCAACAGCAGCAAAAATTACAAATAAAGAATAAAG AGGAAATACTCCAGACTTTTCCTCACCCCCAAAGCAACAATGATCAGCAAAGAGAAGGATCATTCTTTGGCCAGACTAAA GTGGAAGAATGTTTTCATGGTGAAAATCAGTATTCAAAATCAAGCGAGTTCGAGACTCATAATGTCCAAATGGGACTGGA GGAAGTACAGAATATAAATCGTAGAAATTCCCCTTATAGTCAGACCATGAAATCAAGTGCATGCAAAATACAGGTTTCTT GTTCAAACAATACACACCTAGTTTCAGAGAATAAAGAACAGACTACACATCCTGAACTTTTTGCAGGAAACAAGACCCAA AACTTGCATCACATGCAATATTTTCCAAATAATGTGATCCCAAAGCAAGATCTTCTTCACAGGTGCTTTCAAGAACAGGA GCAGAAGTCACAACAAGCTTCAGTTCTACAGGGATATAAAAATAGAAACCAAGATATGTCTGGTCAACAAGCTGCGCAAC TTGCTCAGCAAAGGTACTTGATACATAACCATGCAAATGTTTTTCCTGTGCCTGACCAGGGAGGAAGTCACACTCAGACC CCTCCCCAGAAGGACACTCAAAAGCATGCTGCTCTAAGGTGGCATCTCTTACAGAAGCAAGAACAGCAGCAAACACAGCA ACCCCAAACTGAGTCTTGCCATAGTCAGATGCACAGGCCAATTAAGGTGGAACCTGGATGCAAGCCACATGCCTGTATGC ACACAGCACCACCAGAAAACAAAACATGGAAAAAGGTAACTAAGCAAGAGAATCCACCTGCAAGCTGTGATAATGTGCAG CAAAAGAGCATCATTGAGACCATGGAGCAGCATCTGAAGCAGTTTCACGCCAAGTCGTTATTTGACCATAAGGCTCTTAC TCTCAAATCACAGAAGCAAGTAAAAGTTGAAATGTCAGGGCCAGTCACAGTTTTGACTAGACAAACCACTGCTGCAGAAC TTGATAGCCACACCCCAGCTTTAGAGCAGCAAACAACTTCTTCAGAAAAGACACCAACCAAAAAGACAGCTGCTTCTGTT CTCAATAATTTATAGAGTCACCTTCCAAATTACTAGATACTCCTATAAAAAATTATTGGATACACCTGTCAAGACTCA ATATGATTTCCCATCTTGCAGATGTGTAGAGCAAATTATTGAAAAAGATGAAGGTCCTTTTTATACCCATCTAGGAGCAG GTCCTAATGTGGCAGCTATTAGAGAAATCATGGAAGAAAGGTTTGGACAGAAGGGTAAAGCTATTAGGATTGAAAGAGTC ATCTATACTGGTAAAGAAGGCAAAGTTCTCAGGGATGTCCTATTGCTAAGTGGGTGGTTCCGCAGAAGCAGCAGTGAAGA GAAGCTACTGTGTTTGGTGCGGGAGCGAGCTGGCCACACCTGTGAGGCTGCAGTGATTGTGATTCTCATCCTGGTGTGGG AAGGAATCCCGCTGTCTCTGGCTGACAAACTCTACTCGGAGCTTACCGAGACGTGAGGAAATACGGCACGCTCACCAAT CGCCGGTGTGCCTTGAATGAAGAGAGAACTTGCGCCTGTCAGGGGCTGGATCCAGAAACCTGTGGTGCCTCCTTCTCTTT TGGTTGTTCATGGAGCATGTACTACAATGGATGTAAGTTTGCCAGAAGCAAGATCCCAAGGAAGTTTAAGCTGCTTGGGG ATGACCCAAAAAGGAAGAGAAACTGGAGTCTCATTTGCAAAACCTGTCCACTCTTATGGCACCAACATATAAGAAACTT GCACCTGATGCATATAATAATCAGATTGAATATGAACACAGAGCACCAGAGTGCCGTCTGGGTCTGAAGGAAGGCCGTCC ATTCTCAGGGGTCACTGCATGTTTGGACTTCTGTGCTCATGCCCACAGAGACTTGCACAACATGCAGAATGGCAGCACAT TGGTATGCACTCTCACTAGAGAAGACAATCGAGAATTTGGAGGAAAACCTGAGGATGAGCAGCTTCACGTTCTGCCTTTA TACAAAGTCTCTGACGTGGATGAGTTTGGGAGTGTGGAAGCTCAGGAGGAGAAAAAACGGAGTGGTGCCATTCAGGTACT GAGTTCTTTTCGGCGAAAAGTCAGGATGTTAGCAGAGCCAGTCAAGACTTGCCGACAAAGGAAACTAGAAGCCAAGAAAG CTGCAGCTGAAAAGCTTTCCTCCCTGGAGAACAGCTCAAATAAAAATGAAAAGGAAAAGTCAGCCCCATCACGTACAAAA CAAACTGAAAACGCAAGCCAGGCTAAACAGTTGGCAGAACTTTTGCGACTTTCAGGACCAGTCATGCAGCAGTCCCAGCA GCCCCAGCCTCTACAGAAGCAGCCACCACAGCCCCAGCAGCAGCAGAGACCCCAGCAGCAGCAGCCACATCACCCTCAGA CAGAGTCTGTCAACTCTTATTCTGCTTCTGGATCCACCAATCCATACATGAGACGGCCCAATCCAGTTAGTCCTTATCCA AACTCTTCACACACTTCAGATATCTATGGAAGCACCAGCCCTATGAACTTCTATTCCACCTCATCTCAAGCTGCAGGTTC ATATTTGAATTCTTCTAATCCCATGAACCCTTACCCTGGGCTTTTGAATCAGAATACCCAATATCCATCATATCAATGCA ATGGAAACCTATCAGTGGACAACTGCTCCCCATATCTGGGTTCCTATTCTCCCCAGTCTCAGCCGATGGATCTGTATAGG TATCCAAGCCAAGACCCTCTGTCTAAGCTCAGTCTACCACCCATCCATACACTTTACCAGCCAAGGTTTGGAAATAGCCA GAGTTTTACATCTAAATACTTAGGTTATGGAAACCAAAATATGCAGGGAGATGGTTTCAGCAGTTGTACCATTAGACCAA ATGTACATCATGTAGGGAAATTGCCTCCTTATCCCACTCATGAGATGGATGGCCACTTCATGGGAGCCACCTCTAGATTA CCACCCAATCTGAGCAATCCAAACATGGACTATAAAAATGGTGAACATCATTCACCTTCTCACATAATCCATAACTACAG TGCAGCTCCGGGCATGTTCAACAGCTCTCTTCATGCCCTGCATCTCCAAAACAAGGAGAATGACATGCTTTCCCACACAG CTAATGGGTTATCAAAGATGCTTCCAGCTCTTAACCATGATAGAACTGCTTGTGTCCAAGGAGGCTTACACAAATTAAGT GATGCTAATGGTCAGGAAAAGCAGCCATTGGCACTAGTCCAGGGTGTGGCTTCTGGTGCAGAGGACAACGATGAGGTCTG GTCAGACAGCGAGCAGAGCTTTCTGGATCCTGACATTGGGGGAGTGGCCGTGGCTCCAACTCATGGGTCAATTCTCATTG AGTGTGCAAAGCGTGAGCTGCATGCCACAACCCCTTTAAAGAATCCCAATAGGAATCACCCCACCAGGATCTCCCTCGTC TTTTACCAGCATAAGAGCATGAATGAGCCAAAACATGGCTTGGCTCTTTGGGAAGCCAAAATGGCTGAAAAAGCCCGTGA GAAAGAGGAAGAGTGTGAAAAGTATGGCCCAGACTATGTGCCTCAGAAATCCCATGGCAAAAAAGTGAAACGGGAGCCTG CTGAGCCACATGAAACTTCAGAGCCCACTTACCTGCGTTTCATCAAGTCTCTTGCCGAAAGGACCATGTCCGTGACCACA GACTCCACAGTAACTACATCTCCATATGCCTTCACTCGGGTCACAGGGCCTTACAACAGATATATATGAAGATATATATG ATATCACCCCCTTTTGTTGGTTACCTCACTTGAAAAGACCACAACCAACCTGTCAGTAGTATAGTTCTCATGACGTGGGC AGTGGGGAAAGGTCACAGTATTCATGACAAATGTGGTGGGAAAAACCTCAGCTCACCAGCAACAAAAGAGGTTATCTTAC CATAGCACTTAATTTTCACTGGCTCCCAAGTGGTCACAGATGGCATCTAGGAAAAGACCAAAGCATTCTATGCAAAAAGA AGGTGGGGAAGAAAGTGTTCCGCAATTTACATTTTTAAACACTGGTTCTATTATTGGACGAGATGATATGTAAATGTGAT CCCCCCCCCCCGCTTACAACTCTACACATCTGTGACCACTTTTAATAATATCAAGTTTGCATAGTCATGGAACACAAATC AAACAAGTACTGTAGTATTACAGTGACAGGAATCTTAAAATACCATCTGGTGCTGAATATATGATGTACTGAAATACTGG AATTATGGCTTTTTGAAATGCAGTTTTTACTGTAATCTTAACTTTTATTTATCAAAATAGCTACAGGAAACATGAATAGC AGGAAAACACTGAATTTGTTTGGATGTTCTAAGAAATGGTGCTAAGAAAATGGTGTCTTTAATAGCTAAAAATTTAATGC CTTTATATCATCAAGATGCTATCAGTGTACTCCAGTGCCCTTGAATAATAGGGGTACCTTTTCATTCAAGTTTTTATCAT AATTACCTATTCTTACACAAGCTTAGTTTTTAAAATGTGGACATTTTAAAGGCCTCTGGATTTTGCTCATCCAGTGAAGT CCTTGTAGGACAATAAACGTATATATGTACATATATACACAAACATGTATATGTGCACACACATGTATATGTATAAATAT TTTAAATGGTGTTTTAGAAGCACTTTGTCTACCTAAGCTTTGACAACTTGAACAATGCTAAGGTACTGAGATGTTTAAAA AACAAGTTTACTTTCATTTTAGAATGCAAAGTTGATTTTTTTAAGGAAACAAAGAAAGCTTTTAAAATATTTTTGCTTTT AGCCATGCATCTGCTGATGAGCAATTGTGTCCATTTTTAACACAGCCAGTTAAATCCACCATGGGGCTTACTGGATTCAA GGGAATACGTTAGTCCACAAAACATGTTTTCTGGTGCTCATCTCACATGCTATACTGTAACACAGTTTTATACAAAATTG TATGACAAGTTCATTGCTCAAAAATGTACAGTTTTAAGAATTTTCTATTAACTGCAGGTAATAATTAGCTGCATGCTGCA GACTCAACAAAGCTAGTTCACTGAAGCCTATGCTATTTTATGGATCATAGGCTCTTCAGAGAACTGAATGGCAGTCTGCC TTTGTGTTGATAATTATGTACATTGTGACGTTGTCATTTCTTAGCTTAAGTGTCCTCTTTAACAAGAGGATTGAGCAGAC TGATGCCTGCATAAGATGAATAAACAGGGTTAGTTCCATGTGAATCTGTCAGTTAAAAAGAAACAACAAGGCAGCTGG TTTGCTGTGGTGGTTTTAAATCATTAATTTGTATAAAGAAGTGAAAGAGTTGTATAGTAAATTAAATTGTAAACAAAACT TTTTTAATGCAATGCTTTAGTATTTTAGTACTGTAAAAAAATTAAATATATACATATATATATATATATATATATATATATA TATATGAGTTTGAAGCAGAATTCACATCATGATGGTGCTACTCAGCCTGCTACAAATATATCATAATGTGAGCTAAGAAT TCATTAAATGTTTGAGTGATGTTCCTACTTGTCATATACCTCAACACTAGTTTGGCAATAGGATATTGAACTGAGAGTGA AAGCATTGTGTACCATCATTTTTTTCCAAGTCCTTTTTTTTATTGTTAAAAAAGCATACCTTTTTTCAATACTTGA TTTCTTAGCAAGTATAACTTGAACTTCAACCTTTTTGTTCTAAAAATTCAGGGATATTTCAGCTCATGCTCTCCCTATGC CAACATGTCACCTGTGTTTATGTAAAATTGTTGTAGGTTAATAAAATATATTCTTTGTCAGGGATTTAACCCTTTTATTTT GAATCCCTTCTATTTTACTTGTACATGTGCTGATGTAACTAAAACTAATTTTTGTAAATCTGTTGGCTCTTTTTATTGTAA AGAAAAGCATTTTAAAAGTTTGAGGAATCTTTTGACTGTTTCAAGCAGGAAAAAAAAAATTACATGAAAATAGAAATGCACT GAGTTGATAAAGGGAAAAAATTGTAAGGCAGGAGTTTGGCAAGTGGCTGTTGGCCAGAGACTTACTTGTAACTCTCTAAAT GAAGTTTTTTGATCCTGTAATCACTGAAGGTACATACTCCATGTGGACTTCCCTTAAACAGGCAAACACCTACAGGTAT GGTGTGCAACAGATTGTACAAATTACATTTTGGCCTAAATACATTTTTGCTTACTAGTATTTAAAATAAATTCTTAATCAG AGGAGGCCTTTGGGTTTTATTGGTCAAATCTTTGTAAGCTGGCTTTTGTCTTTTAAAAAATTTCTTGAATTTGTGGTTG TGTCCAATTTGCAAACATTTCCAAAAATGTTTGCTTTGCTTACAAACCACATGATTTTAATGTTTTTTGTATACCATAAT ATCTAGCCCCAAACATTTGATTACTACATGTGCATTGGTGATTTTGATCATCCATTCTTAATATTTGATTTCTGTGTCAC CTACTGTTCATTGTTAAACTGCTGGCCAACAAGAACAGGAAGTATAGTTTGGGGGGTTGGGGAGAGTTTACATAAGGAAG AGAAGAAATTGAGTGGCATATTGTAAATATCAGATCTATAATTGAAAATATAAAACCTGCCTCAGTTAGAATGAATGGAA AGCAGATCTACAATTTGCTAATATAGGAATATCAGGTTGACTATATAGCCATACTTGAAAATGCTTCTGAGTGGTGTCAA CTTTACTTGAATGAATTTTTCATCTTGATTGACGCACAGTGATGTACAGTTCACTTCTGAAGCTAGTGGTTAACTTGTGT AGGAAACTTTTGCAGTTTGACACTAAGATAACTTCTGTGTGCATTTTTCTATGCTTTTTTAAAAACTAGTTTCATTTCAT TTTCATGAGATGTTTGGTTTATAAGATCTGAGGATGGTTATAAATACTGTAAGTATTGTAATGTTATGAATGCAGGTTAT TTGAAAGCTGTTTATTATTATATCATTCCTGATAATGCTATGTGAGTGTTTTTAATAAAATTTATATTTATTTAATGCAC TCTAAGTGTTGTCTTCCT

[0466] "Transforming growth factor receptor 2 (TGFBRII) polypeptide" refers to the NCBI Accession No. having at least about 85% sequence identity with sequence number ABG65632.1 or a fragment thereof, and having immunosuppressive activity Exemplary amino acid sequences are provided below. >ABG65632.1 Transforming growth factor beta receptor II [Homo sapiens] MGRGLLRGLWPLHIVLWTRIASTIPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKP QEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSSDECNDNIIFSEEYNTSNPDL LLVIFQVTGISLLPPLGVAISVIIIFYCYRVNRQQKLSSTWETGKTRKLMEFSEHCAIILEDDRSDISSTCANNINHNTE LLPIELDTLVGKGRFAEVYKAKLKQNTSEQFETVAVKIFPYEEYASWKTEKDIFSDINLKHENILQFLTAEERKTELGKQ YWLITAFHAKGNLQEYLTRHVISWEDLRKLGSSLARGIAHLHSDHTPCGRPKMPIVHRDLKSSNILVKNDLTCCLCDFGL SLRLDPTLSVDDLANSGQVGTARYMAPEVLESRMNLENVESFKQTDVYSMALVLWEMTSRCNAVGEVKDYEPPFGSKVRE HPCVESMKDNVLRDRGRPEIPSFWLNHQGIQMVCETLTECWDHDPEARLTAQCVAERFSELEHLDRLSGRSCSEEKIPED GSLNTTK

[0467] "Transforming growth factor receptor 2 (TGFBRII) polynucleotide" means TGFBRI The TGFBRII gene refers to a nucleic acid encoding a serine / threonine kinase I polypeptide. Exemplary TGFBRII nucleic acids are provided below. do. >M85079.1 Human TGF-beta type II receptor mRNA, complete cds GTTGGCGAGGAGTTTCCTGTTTCCCCCGCAGCGCTGAGTTGAAGTTGAGTGAGTCACTCGCGCGCACGGAGCGACGACAC CCCCGCGCGTGCACCCGCTCGGGACAGGAGCCGGACTCCTGTGCAGCTTCCCTCGGCCGCCGGGGGCCTCCCCGCGCCTC GCCGGCCTCCAGGCCCCTCCTGGCTGGCGAGCGGGCGCCACATCTGGCCCGCACATCTGCGCTGCCGGCCCGGCGCGGGG TCCGGAGAGGGCGCGGCGCGGAGCGCAGCCAGGGGTCCGGGAAGGCGCCGTCCGTGCGCTGGGGGCTCGGTCTATGACGA GCAGCGGGGTCTGCCATGGGTCGGGGGCTGCTCAGGGGCCTGTGGCCGCTGCACATCGTCCTGTGGACGCGTATCGCCAG CACGATCCCACCGCACGTTCAGAAGTCGGTTAATAACGACATGATAGTCACTGACAACAACGGTGCAGTCAAGTTTCCAC AACTGTGTAAATTTTGTGATGTGAGATTTTCCACCTGTGACAACCAGAAATCCTGCATGAGCAACTGCAGCATCACCTCC ATCTGTGAGAAGCCACAGGAAGTCTGTGTGGCTGTATGGAGAAAGAATGACGAGAACATAACACTAGAGACAGTTTGCCA TGACCCCAAGCTCCCCTACCATGACTTTATTCTGGAAGATGCTGCTTCTCCAAAGTGCATTATGAAGGAAAAAAAAAAGC CTGGTGAGACTTTCTTCATGTGTTCCTGTAGCTCTGATGAGTGCAATGACAACATCATCTTCTCAGAAGAATATAACACC AGCAATCCTGACTTGTTGCTAGTCATATTTCAAGTGACAGGCATCAGCCTCCTGCCACCACTGGGAGTTGCCATATCTGT CATCATCATCTTCTACTGCTACCGCGTTAACCGGCAGCAGAAGCTGAGTTCAACCTGGGAAACCGGCAAGACGCGGAAGC TCATGGAGTTCAGCGAGCACTGTGCCATCATCCTGGAAGATGACCGCTCTGACATCAGCTCCACGTGTGCCAACAACATC AACCACAACACAGAGCTGCTGCCCATTGAGCTGGACACCCTGGTGGGGAAAGGTCGCTTTGCTGAGGTCTATAAGGCCAA GCTGAAGCAGAACACTTCAGAGCAGTTTGAGACAGTGGCAGTCAAGATCTTTCCCTATGAGGAGTATGCCTCTTGGAAGA CAGAGAAGGACATCTTCTCAGACATCAATCTGAAGCATGAGAACATACTCCAGTTCCTGACGGCTGAGGAGCGGAAGACG GAGTTGGGGAAACAATACTGGCTGATCACCGCCTTCCACGCCAAGGGCAACCTACAGGAGTACCTGACGCGGCATGTCAT CAGCTGGGAGGACCTGCGCAAGCTGGGCAGCTCCCTCGCCCGGGGGATTGCTCACCTCCACAGTGATCACACTCCATGTG GGAGGCCCAAGATGCCCATCGTGCACAGGGACCTCAAGAGCTCCAATATCCTCGTGAAGAACGACCTAACCTGCTGCCTG TGTGACTTTGGGCTTTCCCTGCGTCTGGACCCTACTCTGTCTGTGGATGACCTGGCTAACAGTGGGCAGGTGGGAACTGC AAGATACATGGCTCCAGAAGTCCTAGAATCCAGGATGAATTTGGAGAATGCTGAGTCCTTCAAGCAGACCGATGTCTACT CCATGGCTCTGGTGCTCTGGGAAATGACATCTCGCTGTAATGCAGTGGGAGAAGTAAAAGATTATGAGCCTCCATTTGGT TCCAAGGTGCGGGAGCACCCCTGTGTCGAAAGCATGAAGGACAACGTGTTGAGAGATCGAGGGCGACCAGAAATTCCAG CTTCTGGCTCAACCACCAGGGCATCCAGATGGTGTGTGAGACGTTGACTGAGTGCTGGGACCACGACCCAGAGGCCCGTC TCACAGCCCAGTGTGTGGCAGAACGCTTCAGTGAGCTGGAGCATCTGGACAGGCTCTCGGGGAGGAGCTGCTCGGAGGAG AAGATTCCTGAAGACGGCTCCCTAAACACTACCAAATAGCTCTTATGGGGCAGGCTGGGCATGTCCAAAGAGGCTGCCCC TCTCACCAAA

[0468] "T Cell Immunoreceptor with Ig and ITIM Domains" "TIGIT polypeptide" refers to the polypeptide identified in NCBI accession number ACD74757.1 or its equivalent. The present invention relates to a protein having at least about 85% sequence identity with a fragment of the present invention and having immunomodulatory activity. Exemplary TIGIT amino acid sequences are provided below. >ACD74757.1 T cell immunoreceptor with Ig and ITIM domains [Homo sapiens] MRWCLLLIWAQGLRQAPLASGMMTGTIETTGNISAEKGGSIILQCHLSSTTAQVTQVNWEQQDQLLAICNADLGWHISPS FKDRVAPGPGLGLTLQSLTVNDTGEYFCIYHTYPDGTYTGRIFLEVLESSVAEHGARFQIPLLGAMAATLVVICTAVIVV VALTRKKKALRIHSVEGDLRRKSAGQEEWSPSAPSPPGSCVQAEAAPAGLCGEQRGEDCAELHDYFNVLSYRSLGNCSFF TETG

[0469] "T cell immunoreceptor (TIGIT) polynucleotide having Ig and ITIM domains" means The TIGIT gene is a nucleic acid encoding a TIGIT polypeptide. Exemplary nucleic acid sequences are provided below. . >EU675310.1 Homo sapiens Ig and T cell immunoreceptor with ITIM domains (TIGIT)m RNA, complete CDS CGTCCTATCTGCAGTCGGCTACTTTCAGTGGCAGAAGAGGCCACATCTGCTTCCTGTAGGCCCTCTGGGCAGAAGCATGC GCTGGTGTCTCCTCCTGATCTGGGCCCAGGGGCTGAGGCAGGCTCCCCTCGCCTCAGGAATGATGACAGGCACAATAGAA ACAACGGGGAACATTTCTGCAGAGAAAGGTGGCTCTATCATCTTACAATGTCACCTCTCCTCCACCACGGCACAAGTGAC CCAGGTCAACTGGGAGCAGCAGGACCAGCTTCTGGCCATTTGTAATGCTGACTTGGGTGGCACATCTCCCCATCCTTCA AGGATCGAGTGGCCCCAGGTCCCGGCCTGGGCCTCACCCTCCAGTCGCTGACCGTGAACGATACAGGGGAGTACTTCTGC ATCTATCACACCTACCCTGATGGGACGTACACTGGGAGAATCTTCCTGGAGGTCCTAGAAAGCTCAGTGGCTGAGCACGG TGCCAGGTTCCAGATTCCATTGCTTGGAGCCATGGCCGCGACGCTGGTGGTCATCTGCACAGCAGTCATCGTGGTGGTCG CGTTGACTAGAAAGAAGAAAGCCCTCAGAATCCATTCTGTGGAAGGTGACCTCAGGAGAAAATCAGCTGGACAGGAGGAA TGGAGCCCCAGTGCTCCCTCACCCCAGGAAGCTGTTGTCCAGGCAGAAGCTGCACCTGCTGGGCTCTGTGGAGAGCAGCG GGGAGAGGACTGTGCCGAGCTGCATGACTACTTCAATGTCCTGAGTTACAGAAGCCTGGGTAACTGCAGCTTCTTCACAG AGACTGGTTAGCAACCAGAGGCATCTTCTGG

[0470] T Cell Receptor Alpha Constant (TRAC) Polypeptide "Tid" refers to a polypeptide having at least about 85% amino acid sequence identical to NCBI Accession No. P01848.2 or a fragment thereof. and have immunomodulatory activity. is provided below. >sp|P01848.2|TRAC_HUMAN RecName: Full=T cell receptor alpha constant region IQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKTVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSI IPEDTFFSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRILLLKVAGFNLLMTLRLWSS

[0471] "T cell receptor alpha constant region (TRAC) polynucleotide" refers to a polynucleotide that encodes a TRAC polypeptide. An exemplary TRAC nucleic acid sequence is provided below. UCSC Human Genome Database, gene ENSG00000277734.8 human T cell receptor alpha chain ( TCR-alpha) catgctaatcctccggcaaacctctgtttcctcctcaaaaggcaggaggtcggaaagaataaacaatgagagtcacatta aaaacacaaaatcctacggaaatactgaagaatgagtctcagcactaaggaaaagcctccagcagctcctgctttctgag ggtgaaggatagacgctgtggctctgcatgactcactagcactctatcacggccatattctggcagggtcagtggctcca actaacatttgtttggtactttacagtttattaaatagatgtttatatggagaagctctcatttctttctcagaagagcc tggctaggaaggtggatgaggcaccatattcattttgcaggtgaaattcctgagatgtaaggagctgctgtgacttgctc aaggccttatatcgagtaaacggtagtgctggggcttagacgcaggtgttctgatttatagttcaaaacctctatcaatg agagagcaatctcctggtaatgtgatagatttcccaacttaatgccaacataccataaacctcccattctgctaatgccc agcctaagttggggagaccactccagattccaagatgtacagtttgctttgctgggcctttttcccatgcctgcctttac tctgccagagttatattgctggggttttgaagaagatcctattaaataaaagaataagcagtattattaagtagccctgc atttcaggtttccttgagtggcaggccaggcctggccgtgaacgttcactgaaatcatggcctcttggccaagattgata gcttgtgcctgtccctgagtcccagtccatcacgagcagctggtttctaagatgctatttcccgtataaagcatgagacc gtgacttgccagccccacagagccccgcccttgtccatcactggcatctggactccagcctgggttggggcaaagaggga aatgagatcatgtcctaaccctgatcctcttgtcccacagATATCCAGAACCCTGACCCTGCCGTGTACCAGCTGAGAGA CTCTAAATCCAGTGACAAGTCTGTCTGCCTATTCACCGATTTTGATTCTCAAACAAATGTGTCACAAAGTAAGGATTCTG ATGTGTATATCACAGACAAAACTGTGCTAGACATGAGGTCTATGGACTTCAAGAGCAACAGTGCTGTGGCCTGGAGCAAC AAATCTGACTTTGCATGTGCAAACGCCTTCAACAACAGCATTATTCCAGAAGACACCTTCTTCCCCAGCCCAGgtaaggg cagctttggtgccttcgcaggctgtttccttgcttcaggaatggccaggttctgcccagagctctggtcaatgatgtcta aaactcctctgattggtggtctcggccttatccattgccaccaaaaccctctttttactaagaaacagtgagccttgttc tggcagtccagagaatgacacgggaaaaaagcagatgaagagaaggtggcaggagagggcacgtggcccagcctcagtct ctccaactgagttcctgcctgcctgcctttgctcagactgtttgccccttactgctcttctaggcctcattctaagcccc ttctccaagttgcctctccttatttctccctgtctgccaaaaaatctttcccagctcactaagtcagtctcacgcagtca ctcattaacccaccaatcactgattgtgccggcacatgaatgcaccaggtgttgaagtggaggaattaaaaagtcagatg aggggtgtgcccagaggaagcaccattctagttgggggagcccatctgtcagctgggaaaagtccaaataacttcagatt ggaatgtgttttaactcagggttgagaaaacagctaccttcaggacaaaagtcagggaagggctctctgaagaaatgcta cttgaagataccagccctaccaagggcagggagaggaccctatagaggcctgggacaggagctcaatgagaaaggagaag agcagcaggcatgagttgaatgaaggaggcagggccgggtcacagggccttctaggccatgagagggtagacagtattct aaggacgccagaaagctgttgatcggcttcaagcaggggagggacacctaatttgcttttcttttttttttttttttttt tttttttttttgagatggagttttgctcttgttgcccaggctggagtgcaatggtgcatcttggctcactgcaacctccg cctcccaggttcaagtgattctcctgcctcagcctcccgagtagctgagattacaggcacccgccaccatgcctggctaa ttttttgtatttttagtagagacagggtttcactatgttggccaggctggtctcgaactcctgacctcaggtgatccacc cgcttcagcctcccaaagtgctgggattacaggcgtgagccaccacacccggcctgcttttcttaaagatcaatctgagt gctgtacggagagtgggttgtaagccaagagtagaagcagaaagggagcagttgcagcagagagatgatggaggcctggg cagggtggtggcagggaggtaaccaacaccattcaggtttcaaaggtagaaccatgcagggatgagaaagcaaagagggg atcaaggaaggcagctggattttggcctgagcagctgagtcaatgatagtgccgtttactaagaagaaaccaaggaaaaa atttggggtgcagggatcaaaactttttggaacatatgaaagtacgtgtttatactctttatggcccttgtcactatgta tgcctcgctgcctccattggactctagaatgaagccaggcaagagcagggtctatgtgtgatggcacatgtggccagggt catgcaacatgtactttgtacaaacagtgtatattgagtaaatagaaatggtgtccaggagccgaggtatcggtcctgcc agggccaggggctctccctagcaggtgctcatatgctgtaagttccctccagatctctccacaaggaggcatggaaaggc tgtagttgttcacctgcccaagaactaggaggtctggggtgggagagtcagcctgctctggatgctgaaagaatgtctgt ttttccttttagAAAGTTCCTGTGATGTCAAGCTGGTCGAGAAAAGCTTTGAAACAGgtaagacaggggtctagcctggg tttgcacaggattgcggaagtgatgaacccgcaataaccctgcctggatgagggagtgggaagaaattagtagatgtggg aatgaatgatgaggaatggaaacagcggttcaagacctgcccagagctgggtggggtctctcctgaatccctctcaccat ctctgactttccattctaagcactttgaggatgagtttctagcttcaatagaccaaggactctctcctaggcctctgtat tcctttcaacagctccactgtcaagagagccagagagagcttctgggtggcccagctgtgaaatttctgagtcccttagg gatagccctaaacgaaccagatcatcctgaggacagccaagaggttttgccttctttcaagacaagcaacagtactcaca taggctgtgggcaatggtcctgtctctcaagaatcccctgccactcctcacacccaccctgggcccatattcatttccat ttgagttgttcttattgagtcatccttcctgtggtagcggaactcactaaggggcccatctggacccgaggtattgtgat gataaattctgagcacctaccccatccccagaagggctcagaaataaaataagagccaagtctagtcggtgtttcctgtc ttgaaacacaatactgttggccctggaagaatgcacagaatctgtttgtaaggggatatgcacagaagctgcaagggaca ggaggtgcaggagctgcaggcctcccccacccagcctgctctgccttggggaaaaccgtgggtgtgtcctgcaggccatg caggcctgggacatgcaagcccataaccgctgtggcctcttggttttacagATACGAACCTAAACTTTCAAAACCTGTCA GTGATTGGGTTCCGAATCCTCCTCCTGAAAGTGGCCGGGTTTAATCTGCTCATGACGCTGCGGCTGTGGTCCAGCTGAGg tgaggggccttgaagctgggagtggggtttagggacgcgggtctctgggtgcatcctaagctctgagagcaaacctccct gcagggtcttgcttttaagtccaaagcctgagcccaccaaactctcctacttcttcctgttacaaattcctcttgtgcaa taataatggcctgaaacgctgtaaaatatcctcatttcagccgcctcagttgcacttctcccctatgaggtaggaagaac agttgtttagaaacgaagaaactgaggccccacagctaatgagtggaggaagagagacacttgtgtacaccacatgcctt gtgttgtacttctctcaccgtgtaacctcctcatgtcctctctccccagtacggctctcttagctcagtagaaagaagac attacactcatattacaccccaatcctggctagagtctccgcaccctcctcccccagggtccccagtcgtcttgctgaca actgcatcctgttccatcaccatcaaaaaaaaactccaggctgggtgcgggggctcacacctgtaatcccagcactttgg gaggcagaggcaggaggagcacaggagctggagaccagcctgggcaacacagggagaccccgcctctacaaaaagtgaaa aaattaaccaggtgtggtgctgcacacctgtagtcccagctacttaagaggctgagatgggaggatcgcttgagccctgg aatgttgaggctacaatgagctgtgattgcgtcactgcactccagcctggaagacaaagcaagatcctgtctcaaataat aaaaaaaataagaactccagggtacatttgctcctagaactctaccacatagccccaaacagagccatcaccatcacatc cctaacagtcctgggtcttcctcagtgtccagcctgacttctgttcttcctcattccagATCTGCAAGATTGTAAGACAG CCTGTGCTCCCTCGCTCCTTCCTCTGCATTGCCCCTCTTCTCCCTCTCCAAACAGAGGGAACTCTCCTACCCCCAAGGAG GTGAAAGCTGCTACCACCTCTGTGCCCCCCCGGCAATGCCACCAACTGGATCCTACCCGAATTTATGATTAAGATTGCTG AAGAGCTGCCAAACACTGCTGCCACCCCCTCTGTTCCCTTATTGCTGCTTGTCACTGCCTGACATTCACGGCAGAGGCAA GGCTGCTGCAGCCTCCCCTGGCTGTGCACATTCCCTCCTGCTCCCCAGAGACTGCCTCCGCCATCCCACAGATGATGGAT CTTCAGTGGGTTCTCTTGGGCTCTAGGTCCTGCAGAATGTTGTGAGGGGTTTATTTTTTTTTAATAGTGTTCATAAAGAA ATACATAGTATTCTTCTTCTCAAGACGTGGGGGGAAATTATCTCATTATCGAGGCCCTGCTATGCTGTGTATCTGGGCGT GTTGTATGTCCTGCTGCCGATGCCTTCATTAAAATGATTTGGAAGAGCAGA

[0472] Nucleotides in lower case above are untranslated regions or introns, The nucleotides in are exons.

[0473] >X02592.1 Human mRNA for T cell receptor alpha chain (TCR-alpha) TTTTGAAACCCTTCAAAGGCAGAGACTTGTCCAGCCTAACCTGCCTGCTGCTCCTAGCTCCTGAGGCTCAGGGCCCTTGG CTTCTGTCCGCTCTGCTCAGGGCCCTCCAGCGTGGCCACTGCTCAGCCATGCTCCTGCTGCTCGTCCCAGTGCTCGAGGT GATTTTTACCCTGGGAGGAACCAGAGCCCAGTCGGTGACCCAGCTTGGCAGCCACGTCTCTGTCTCTGAAGGAGCCCTGG TTCTGCTGAGGTGCAACTACTCATCGTCTGTTCCACCATATCTCTTCTGGTATGTGCAATACCCCAACCAAGGACTCCAG CTTCTCCTGAAGTACACATCAGCGGCCACCCTGGTTAAAGGCATCAACGGTTTTGAGGCTGAATTTAAGAAGAGTGAAAC CTCCTTCCACCTGACGAAACCCTCAGCCCATATGAGCGACGCGGCTGAGTACTTCTGTGCTGTGAGTGATCTCGAACCGA ACAGCAGTGCTTCCAAGATAATCTTTGGATCAGGGACCAGACTCAGCATCCGGCCAAATATCCAGAACCCTGACCCTGCC GTGTACCAGCTGAGAGACTCTAAATCCAGTGACAAGTCTGTCTGCCTATTCACCGATTTTGATTCTCAAACAAATGTGTC ACAAAGTAAGGATTCTGATGTGTATATCACAGACAAAACTGTGCTAGACATGAGGTCTATGGACTTCAAGAGCAACAGTG CTGTGGCCTGGAGCAACAAATCTGACTTTGCATGTGCAAACGCCTTCAACAACAGCATTATTCCAGAAGACACCTTCTTC CCCAGCCCAGAAAGTTCCTGTGATGTCAAGCTGGTCGAGAAAAGCTTTGAAACAGATACGAACCTAAACTTTCAAAACCT GTCAGTGATTGGGTTCCGAATCCTCCTCCTGAAAGTGGCCGGGTTTAATCTGCTCATGACGCTGCGGCTGTGGTCCAGCT GAGATCTGCAAGATTGTAAGACAGCCTGTGCTCCCTCGCTCCTTCCTCTGCATTGCCCCTCTTCTCCCTCTCCAAACAGA GGGAACTCTCCTACCCCCAAGGAGGTGAAAGCTGCTACCACCTCTGTGCCCCCCCGGTAATGCCACCAACTGGATCCTAC CCGAATTTATGATTAAGATTGCTGAAGAGCTGCCAAACACTGCTGCCACCCCCTCTGTTCCCTTATTGCTGCTTGTCACT GCCTGACATTCACGGCAGAGGCAAGGCTGCTGCAGCCTCCCCTGGCTGTGCACATTCCCTCCTGCTCCCCAGAGACTGCC TCCGCCATCCCACAGATGATGGATCTTCAGTGGGTTCTCTTGGGCTCTAGGTCCTGGAGAATGTTGTGAGGGGTTTATTT TTTTTTAATAGTGTTCATAAAGAAATACATAGTATTCTTCTTCTCAAGACGTGGGGGGAAATTATCTCATTATCGAGGCC CTGCTATGCTGTGTGTCTGGGCGTGTTGTATGTCCTGCTGCCGATGCCTTCATTAAAATGATTTGGAA

[0474] T cell receptor beta constant 1 (TRBC1) polypeptide "Tide" refers to a polypeptide having at least about 85% amino acid sequence identity with NCBI Accession No. P01850 or a fragment thereof. An exemplary amino acid sequence is: Provided below.

[0475] >sp|P01850|TRBC1_HUMAN T cell receptor beta constant region 1 OS=Homo sapiens OX=9606 GN=TRBC1 PE=1 SV=4DLNKVFPPEVAVFEPSEAEISHTQKATLVCLATGFFPDHVELSWWVNGKEVHSGVSTDPQPLKEQPALNDSRYCLS SRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSVSYQQGVLSATILYEILLGKAT LYAVLVSALVLMAMVKRKDF

[0476] "T cell receptor beta constant region 1 (TRBC1) polynucleotide" refers to a polynucleotide that encodes a TRBC1 polypeptide. By "TRBC1" is meant a nucleic acid that encodes the TRBC1. Exemplary TRBC1 nucleic acid sequences are provided below. >X00437.1 CTGGTCTAGAATATTCCACATCTGCTCTCACTCTGCCATGGACTCCTGGACCTTCTGCTGTGTGTCCCTTTGCATCCTGG TAGCGAAGCATACAGATGCTGGAGTTATCCAGTCACCCCGCCATGAGGTGACAGAGATGGGACAAGAAGTGACTCTGAGA TGTAAACCAATTTCAGGCCACAACTCCCTTTTCTGGTACAGACAGACCATGATGCGGGGACTGGAGTTGCTCATTTACTT TAACAACAACGTTCCGATAGATGATTCAGGGATGCCCGAGGATCGATTCTCAGCTAAGATGCCTAATGCATCATTCTCCA CTCTGAAGATCCAGCCCTCAGAACCCAGGGACTCAGCTGTGTACTTCTGTGCCAGCAGTTTCTCGACCTGTTCGGCTAAC TATGGCTACACCTTCGGTTCGGGGACCAGGTTAACCGTTGTAGAGGACCTGAACAAGGTGTTCCCACCCGAGGTCGCTGT GTTTGAGCCATCAGAAGCAGAGATCTCCCACACCCAAAAGGCCACACTGGTGTGCCTGGCCACAGGCTTCTTCCCCGACC ACGTGGAGCTGAGCTGGTGGGTGAATGGGAAGGAGGTGCACAGTGGGGTCAGCACAGACCCGCAGCCCCTCAAGGAGCAG CCCGCCCTCAATGACTCCAGATACTGCCTGAGCAGCCGCCTGAGGGTCTCGGCCACCTTCTGGCAGAACCCCCGCAACCA CTTCCGCTGTCAAGTCCAGTTCTACGGGCTCTCGGAGAATGACGAGTGGACCCAGGATAGGGCCAAACCCGTCACCCAGA TCGTCAGCGCCGAGGCCTGGGGTAGAGCAGACTGTGGCTTTACCTCGGTGTCCTACCAGCAAGGGGTCCTGTCTGCCACC ATCCTCTATGAGATCCTGCTAGGGAAGGCCACCCTGTATGCTGTGCTGGTCAGCGCCCTTGTGTTGATGGCCATGGTCAA GAGAAAGGATTTCTGAAGGCAGCCCTGGAAGTGGAGTTAGGAGCTTCTAACCCGTCATGGTTCAATACACATTCTTCTTT TGCCAGCGCTTCTGAAGAGCTGCTCTCACCTCTCTGCATCCCAATAGATATCCCCCTATGTGCATGCACACCTGCACACT CACGGCTGAAATCTCCCTAACCCAGGGGGAC

[0477] T cell receptor beta constant 2 (TRBC2) polypeptide "Tide" refers to a polypeptide having at least about 85% amino acid sequence identity with NCBI Accession No. A0A5B9 or a fragment thereof. An exemplary amino acid sequence is: Provided below. >sp|A0A5B9|TRBC2_HUMAN T cell receptor beta constant region 2 OS=Homo sapiens OX=9606 GN =TRBC2 PE=1 SV=2DLKNVFPPKVAVFEPSEAEISHTQKATLVCLATGFYPDHVELSWWVNGKEVHSGVSTDPQPLKEQPALNDSRYCLS SRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSESYQQGVLSATILYEILLGKAT LYAVLVSALVLMAMVKRKDSRG

[0478] "T cell receptor beta constant region 2 (TRBC2) polynucleotide" refers to a polynucleotide that encodes a TRAC polypeptide. By "TRBC2" is meant a nucleic acid that encodes the TRBC2. Exemplary TRBC2 nucleic acid sequences are provided below.

[0479] >NG_001333.2: T cell receptor beta locus (TRB) on chromosome 7 of Homo sapiens, positions 655095 - 656583 locus (TRB) AGGACCTGAAAAACGTGTTCCCACCCGAGGTCGCTGTGTTTGAGCCATCAGAAGCAGAGATCTCCCACACCCAAAAGGCC ACACTGGTATGCCTGGCCACAGGCTTCTACCCCGACCACGTGGAGCTGAGCTGGTGGGTGAATGGGAAGGAGGTGCACAG TGGGGTCAGCACAGACCCGCAGCCCCTCAAGGAGCAGCCCGCCCTCAATGACTCCAGATACTGCCTGAGCAGCCGCCTGA GGGTCTCGGCCACCTTCTGGCAGAACCCCCGCAACCACTTCCGCTGTCAAGTCCAGTTCTACGGGCTCTCGGAGAATGAC GAGTGGACCCAGGATAGGGCCAAACCCGTCACCCAGATCGTCAGCGCCGAGGCCTGGGGTAGAGCAGGTGAGTGGGGCCT<00044​​​​​​​​ATAGTGACCACTTCGCTGGCACTGGAGCAGCATGAGGGAGACAGAACCAGGGCTATCAAAGGAGGCTGACTTTGTACTAT CTGATATGCATGTGTTTGTGGCCTGTGAGTCTGTGATGTAAGGCTCAATGTCCTTACAAAGCAGCATTCTCTCATCCATT TTTCTTCCCCTGTTTTCTTTCAGACTGTGGCTTCACCTCCGGTAAGTGAGTCTCTCCTTTTTCTCTCTATCTTTCGCCGT CTCTGCTCTCGAACCAGGGCATGGAGAATCCACGGACACAGGGGCGTGAGGGAGGCCAGAGCCACCTGTGCACAGGTGCC TACATGCTCTGTTCTTGTCAACAGAGTCTTACCAGCAAGGGGTCCTGTCTGCCACCATCCTCTATGAGATCTTGCTAGGG AAGGCCACCTTGTATGCCGTGCTGGTCAGTGCCCTCGTGCTGATGGCCATGGTAAGGAGGAGGGTGGGATAGGGCAGATG ATGGGGGCAGGGGATGGAACATCACACATGGGCATAAAGGAATCTCAGAGCCAGAGCACAGCCTAATATATCCTATCACC TCAATGAAACCATAATGAAGCCAGACTGGGGAGAAAATGCAGGGAATATCACAGAATGCATCATGGGAGGATGGAGACAA CCAGCGAGCCCTACTCAAATTAGGCCTCAGAGCCCGCCTCCCCTGCCCTACTCCTGCTGTGCCATAGCCCCTGAAACCCT GAAAATGTTCTCTCTTCCACAGGTCAAGAGAAAGGATTCCAGAGGCTAG

[0480] As used herein, "transduction" refers to the transfer of genes or genetic material into cells by a virus. This means transferring via a vector.

[0481] As used herein, "transformation" refers to the process by which exogenous nucleic acid is introduced into a cell. This refers to the process of introducing genetic changes that occur.

[0482] "Transfection" is the chemical or physical introduction of genes or genetic material into cells. Refers to transfer through stages.

[0483] "Translocation" refers to the rearrangement of nucleic acid segments between non-homologous chromosomes.

[0484] As used herein, the terms "treat," "treating," "Treatment" and the like means any treatment that alleviates or improves a disorder and / or its associated symptoms. Treating a disorder or condition means alleviating the associated disorder, condition or symptoms. It will be understood that removal is not required (and removal is not excluded). ).

[0485] The term "uracil glycosylase inhibitor" or "UGI" as used herein refers to a In this case, a protein capable of inhibiting uracil-DNA glycosylase base excision repair enzyme is In some embodiments, the polypeptide comprises one or more (e.g., one , 2, 3, 4, 5) uracil glycosylase inhibitors. In some embodiments, the UGI domain comprises wild-type UGI or a modified version thereof. In this regard, the UGI proteins provided herein include fragments of UGI and proteins that are compatible with UGI or UGI fragments. For example, in some embodiments, the UGI domain comprises a protein similar to In some embodiments, the UGI fragment comprises a fragment of the amino acid sequence provided in At least 60%, at least 65%, at least 70%, at least a few of the exemplary UGI sequences provided herein. at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least an amino acid sequence containing 96%, at least 97%, at least 98%, at least 99%, or 100% of the amino acid sequence; In some embodiments, the UGI comprises the amino acid sequence shown herein below. or a fragment of an amino acid sequence shown herein below. In some embodiments, UGI or a fragment of UGI, or Proteins containing homologs of UGI or fragments of UGI are called "UGI variants." GI variants share homology with UGI or fragments thereof. For example, UGI variants At least 70% identical, at least 75% identical to a native UGI or UGI as set forth herein; At least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, at least In some embodiments, the UGI variant is 99.5% identical, or at least 99.9% identical. The variants are at least 70% identical to wild-type UGI or the corresponding fragments of UGI as shown below. Identical, at least 80% identical, at least 90% identical, at least 95% identical, at least 96% identical , at least 97% identical, at least 98% identical, at least 99% identical, at least 99.5% identical, or a fragment of UGI that is at least 99.9% identical. UGI contains the following amino acid sequence:

[0486] >splP14739IUNGI_BPPB2 Uracil-DNA glycosylase inhibitor

[0487] MTNLSDIIEKETGKQLVIQESILMLPEEVEEVIGNKPESDILVHTAYDESTDENVMLLT SD APE YKPW ALVIQDS NGENKIKML

[0488] The term "vector" refers to a vector that transfers a nucleic acid sequence into a cell, resulting in a transformed cell. Vectors include plasmids, transposons, phages, viruses, and ribosomal An "expression vector" includes a vector that is expressed in a recipient cell. An expression vector is a nucleic acid sequence containing a nucleotide sequence to be expressed. and / or drive expression of the introduced sequences, such as sequences, promoters, and secretory sequences. It may contain additional nucleic acid sequences to facilitate transcription.

[0489] T cell receptor zeta chain-associated protein kinase 70 (zeta chain of T cell receptor a "ZAP70 (associated protein kinase 70) polypeptide" is NCBI accession number AAH538 A protein having at least about 85% amino acid sequence identity with 78.1 and having kinase activity. Exemplary amino acid sequences are provided below.

[0490] >AAH53878.1 Zeta chain (TCR)-associated protein kinase 70kDa [Homo sapiens] MPDPAAHLPFFYGSISRAEAEEHLKLAGMADGLFLLRQCLRSLGGYVLSLVHDVRFHHFPIERQLNGTYAIAGGKAHCGP AELCEFYSRDPDGLPCNLRKPCNRPSGLEPQPGVFDCLRDAMVRDYVRQTWKLEGEALEQAIISQAPQVEKLIATTAHER MPWYHSSLTREEAERKLYSGAQTDGKFLLRPRKEQGTYALSLIYGKTVYHYLISQDKAGKYCIPEGTKFDTLWQLVEYLK LKADGLIYCLKEACPNSSASNASGAAAPTLPAHPSTLTHPQRRIDTLNSDGYTPEPARITSPDKPRPMPMDTSVYESPYS DPEELKDKKLFLKRDNLLIADIELGCGNFGSVRQGVYRMRKKQIDVAIKVLKQGTEKADTEEMMREAQIMHQLDNPYIVR LIGVCQAEALMLVMEMAGGGPLHKFLVGKREEIPVSNVAELLHQVSMGMKYLEEKNFVHRDLAARNVLLVNRHYAKISDF GLSKALGADDSYYTARSAGKWPLKWYAPECINFRKFSSRSDVWSYGVTMWEALSYGQKPYKKMKGPEVMAFIEQGKRMEC PPECPPELYALMSDCWIYKWEDRPDFLTVEQRMRACYYSLASKVEGPPGSTQKAEAACA

[0491] "T cell receptor zeta chain-associated protein kinase 70 (ZAP70) polynucleotide" means The ZAP70 gene is a nucleic acid encoding a ZAP70 polypeptide. It encodes a tyrosine kinase involved in lymphocyte activation. Absence of functional ZAP10 inhibits CD8+ T cell activation. This can result in severe combined immunodeficiency characterized by a lack of ZAP70. The sequences are provided below.

[0492] >BC053878.1 Homo sapiens zeta chain (TCR)-associated protein kinase 70 kDa, mRNA (c DNA clone MGC: 61743 IMAGE: 5757161), complete cds GCTTGCCGGAGCTCAGCAGACACCAGGCCTTCCGGGCAGGCCTGGCCCACCGTGGGCCTCAGAGCTGCTGCTGGGGCATT CAGAACCGGCTCTCCATTGGCATTGGGACCAGAGACCCCGCAAGTGGCCTGTTTGCCTGGACATCCACCTGTACGTCCCC AGGTTTCGGGAGGCCCAGGGGCGATGCCAGACCCCGCGGCGCACCTGCCCTTCTTCTACGGCAGCATCTCGCGTGCCGAG GCCGAGGAGCACCTGAAGCTGGCGGGCATGGCGGACGGGCTCTTCCTGCTGCGCCAGTGCCTGCGCTCGCTGGGCGGCTA TGTGCTGTCGCTCGTGCACGATGTGCGCTTCCACCACTTTCCCATCGAGCGCCAGCTCAACGGCACCTACGCCATTGCCG GCGGCAAAGCGCACTGTGGACCGGCAGAGCTCTGCGAGTTCTACTCGCGCGACCCCGACGGGCTGCCCTGCAACCTGCGC AAGCCGTGCAACCGGCCGTCGGGCCTCGAGCCGCAGCCGGGGGTCTTCGACTGCCTGCGAGACGCCATGGTGCGTGACTA CGTGCGCCAGACGTGGAAGCTGGAGGGCGAGGCCCTGGAGCAGGCCATCATCAGCCAGGCCCCGCAGGTGGAGAAGCTCA TTGCTACGACGGCCCACGAGCGGATGCCCTGGTACCACAGCAGCCTGACGCGTGAGGAGGCCGAGCGCAAACTTTACTCT GGGGCGCAGACCGACGGCAAGTTCCTGCTGAGGCCGCGGAAGGAGCAGGGCACATACGCCCTGTCCCTCATCTATGGGAA GACGGTGTACCACTACCTCATCAGCCAAGACAAGGCGGGCAAGTACTGCATTCCCGAGGGCACCAAGTTTGACACGCTCT GGCAGCTGGTGGAGTATCTGAAGCTGAAGGCGGACGGGCTCATCTACTGCCTGAAGGAGGCCTGCCCCAACAGCAGTGCC AGCAACGCCTCAGGGGCTGCTGCTCCCACACTCCCAGCCCACCCATCCACGTTGACTCATCCTCAGAGACGAATCGACAC CCTCAACTCAGATGGATACACCCCTGAGCCAGCACGCATAACGTCCCCAGACAAACCGCGGCCGATGCCCATGGACACGA GCGTGTATGAGAGCCCCTACAGCGACCCAGAGGAGCTCAAGGACAAGAAGCTCTTCCTGAAGCGCGATAACCTCCTCATA GCTGACATTGAACTTGGCTGCGGCAACTTTGGCTCAGTGCGCCAGGGCGTGTACCGCATGCGCAAGAAGCAGATCGACGT GGCCATCAAGGTGCTGAAGCAGGGCACGGAGAAGGCAGACACGGAAGAGATGATGCGCGAGGCGCAGATCATGCACCAGC TGGACAACCCCTACATCGTGCGGCTCATTGGCGTCTGCCAGGCCGAGGCCCTCATGCTGGTCATGGAGATGGCTGGGGGC GGGCCGCTGCACAAGTTCCTGGTCGGCAAGAGGGAGGAGATCCCTGTGAGCAATGTGGCCGAGCTGCTGCACCAGGTGTC CATGGGGATGAAGTACCTGGAGGAGAAGAACTTTGTGCACCGTGACCTGGCGGCCCGCAACGTCCTGCTGGTTAACCGGC ACTACGCCAAGATCAGCGACTTTGGCCTCTCCAAAGCACTGGGTGCCGACGACAGCTACTACACTGCCCGCTCAGCAGGG AAGTGGCCGCTCAAGTGGTACGCACCCGAATGCATCAACTTCCGCAAGTTCTCCAGCCGCAGCGATGTCTGGAGCTATGG GGTCACCATGTGGGAGGCCTTGTCCTACGGCCAGAAGCCCTACAAGAAGATGAAAGGGCCGGAGGTCATGGCCTTCATCG AGCAGGGCAAGCGGATGGAATGCCCACCAGAGTGTCCACCCGAACTGTACGCACTCATGAGTGACTGCTGGATCTACAAG TGGGAGGATCGCCCCGACTTCCTGACCGTGGAGCAGCGCATGCGAGCCTGTTACTACAGCCTGGCCAGCAAGGTGGAAGG GCCCCCAGGCAGCACACAGAAGGCTGAGGCTGCCTGTGCCTGAGCTCCCGCTGCCCAGGGGAGCCCTCCACACCGGCTCT TCCCCACCCTCAGCCCCACCCCAGGTCCTGCAGTCTGGCTGAGCCCTGCTTGGTTGTCTCCACACACAGCTGGGCTGTGG TAGGGGGTGTCTCAGGCCACACCGGCCTTGCATTGCCTGCCTGGCCCCCTGTCCTCTCTGGCTGGGGAGCAGGGAGGTCC GGGAGGGTGCGGCTGTGCAGCCTGTCCTGGGCTGGTGGCTCCCGGAGGGCCCTGAGCTGAGGGCATTGCTTACACGGATG CCTTCCCCTGGGCCCTGACATTGGAGCCTGGGCATCCTCAGGTGGTCAGGCGTAGATCACCAGAATAAACCCAGCTTCCC TCTTGAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA

[0493] Unless otherwise stated or clear from the context, as used herein, " The term "or" is understood to be inclusive. Unless otherwise clear, as used herein, the terms "a," "an," and "the" refer to It is understood to be singular or plural.

[0494] As used herein, unless otherwise stated or clear from the context, The term "about" means within normal tolerance in the art, e.g., within 2 standard deviations of the mean. Approximately means 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5% of the stated value. %, 0.1%, 0.05%, or 0.01%. Unless otherwise clear from the context, All numerical values ​​provided herein are modified by the term about.

[0495] Ranges provided herein are understood to be shorthand for all values ​​within that range. For example, the range 1 to 50 is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 , 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36 , 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 It is understood that the terms "a," "b," "c," "d," "e," "f," "g," "h" and "i" are intended to include any number, combination of numbers, or subrange.

[0496] The recitation of a list of chemical groups in a variable definition herein is intended to encompass any single This includes the definition of the variables as a group or combination of the listed groups. The description of an embodiment with respect to a variable or aspect does not necessarily mean that it should be taken as any single embodiment or as a single or multiple embodiment. or any other embodiment or portion thereof.

[0497] The compositions or methods provided herein may be used in combination with any other compositions and methods provided herein. The method may be combined with one or more of the following methods: [Brief explanation of the drawings]

[0498] [Figure 1] Figures 1A-1B are diagrams of three proteins that influence T cell function. Figure 1A is a diagram of the TRAC protein, a key component of graft-versus-host disease. Figure 1B is a diagram of the B2M protein, a component of the MHC class 1 antigen-presenting complex present on nucleated cells that can be recognized by host CD8+ T cells. Figure 1C is a diagram of T cell signaling that leads to expression of the PDCD1 gene, and the resulting PD-1 protein, which acts to inhibit T cell signaling.

[0499] [Figure 2] Figure 1 shows the percentage of cells in which target gene expression was knocked down after base editing. "EP" stands for electroporation.

[0500] [Figure 3] Graph of the percentage of types of gene modifications observed in untransduced cells or cells transduced with the BE4 base editing system or Cas9 nuclease.

[0501] [Figure 4]Graph showing the percentage of targeted nucleotide modifications, as measured by the percentage of cells that are negative for target protein expression or generate a stop (STOP) codon, as determined by flow cytometry (FC) in cells transduced with BE4 and sgRNAs directing BE4 to the splice site acceptor (SA) or donor (SD). Control cells were mock electroporated (EP).

[0502] [Figure 5] FIG. 1 is a diagram of the BE4 system, which disrupts the splice site acceptor (SA), splice donor (SD), or generates a stop (STOP) codon.

[0503] [Figure 6] 1 is a chart summarizing the off-target binding sites of sgRNAs used to disrupt target genes.

[0504] [Figure 7] Figure 11 is a graph summarizing flow cytometry (FC) data for the percentage of BE4- or Cas9-edited cells showing reduced protein expression. Cells were gated on either B2M or CD3, the latter being a proxy for TRAC expression.

[0505] [Figure 8] Figure 8A is a scatter plot of FACS data for non-edited control cells, and Figure 8B is a scatter plot of FACS data for cells edited at the B2M, TRAC, and PD1 loci.

[0506] [Figure 9] 1A-1C are graphs showing the effectiveness of base editing techniques described herein to modify specific genes that may adversely affect CAR-T immunotherapy.

[0507] [Figure 10]FIG. 1 shows a droplet digital PCR (ddPCR) protocol for detecting and quantifying genetic alterations and translocations.

[0508] [Figure 11] Two graphs are presented showing data generated from next-generation sequencing (NGS) analysis or ddPCR of cells edited using either the BE4 system or the Cas9 system.

[0509] [Figure 12] FIG. 1 is a schematic diagram showing the role of Cbl-b in suppressing T cell activation.

[0510] [Figure 13] Graph showing the efficiency of Cbl-b knockdown by splice site disruption. SA = splice acceptor; SD = splice donor; STOP - generated stop codon; 2° Only = secondary antibody only; C373 refers to the loss-of-function variant (C373R); RL1-A: : APC-A = laser; ICS = intracellular staining.

[0511] [Figure 14] Figure 1 shows the rate of Cas12b-mediated indels in the GRIN2B and DNMT1 genes in T cells. EP indicates electroporation.

[0512] [Figure 15] Graph summarizing fluorescence-assisted cell sorting (FACS) data for cells transduced via electroporation (EP) with bvCas12b and guide RNAs specific for TRAC, GRIN2B, and DNMT1, and gated on CD3.

[0513] [Figure 16] Scatter plot of fluorescence-assisted cell sorting data of cells transduced with CAR-P2A-mCherry lentivirus demonstrating CAR expression.

[0514] [Figure 17] FIG. 1 is a scatter plot of fluorescence-assisted cell sorting data demonstrating CAR expression in cells transduced with poly(1,8-octanediol citrate) (POC) lentiviral vectors.

[0515] [Figure 18] 1 is a graph showing that BE4 produced efficient and durable gene knockouts with high product purity.

[0516] [Figure 19] Figure 19A is a representative FACS analysis showing loss of protein surface expression due to gene knockout with BE4 or spCas9. Figure 19B is a graph showing that gene knockout with BE4 or spCas9 results in loss of B2M surface expression.

[0517] [Figure 20] Schematic diagram showing the locations of B2M, TRAC, and PD-1 target sites. Translocations can be detected when B2M, TRAC, and PD-1 sequences recombine.

[0518] [Figure 21] 10A-10C are graphs showing that multiplexed base editing does not significantly impair cell expansion.

[0519] [Figure 22] 10A-B are graphs showing that BE4 generated triple-edited T cells with on-target editing efficiency and cell phenotype similar to spCas9.

[0520] [Figure 23] Flow cytometry analysis showing the generation of triple-edited CD3-, B2M-, PD1- T cells.

[0521] [Figure 24]Flow cytometry analysis showing CAR expression in BE4 and Cas9 edited cells.

[0522] [Figure 25] Graph showing CAR-T cell killing or antigen-positive cells.

[0523] [Figure 26] 1 is a graph showing that Cas12b and BE4 can be paired for efficient multiplex editing in T cells.

[0524] [Figure 27]

[0023] Figure 10 is a graph showing that Cas12b can direct the insertion of a chimeric antigen receptor (CAR) into a locus by introducing into cells a double-stranded DNA template encoding the CAR in the presence of Cas12 nuclease and an sgRNA targeting the locus.

[0525] [Figure 28] Figures 28A and 28B are graphs showing protein knockdown (% negative) using base editing targeted to the genes shown in the figures, as determined by flow cytometry, gated relative to unedited controls. The figures represent the results of replicate experiments. The bars for each set of conditions are displayed in the order (left to right) listed in the key (top to bottom). The identifiers for each bar in the grouping of eight bars correspond, from left to right, to CD3, CD7, CD52, PD1, B2M CD2, HLADR (CIITA surrogate), and CD5. DETAILED DESCRIPTION OF THE INVENTION ...

Claims

1. Modified immune cells with reduced immunogenicity and / or increased anti-neoplastic activity A method for producing by multiplex editing at least four genes in an immune cell, The sequence or its regulatory elements are modified at a single target nucleic acid base in each thereof, Thus, the modified immune system has reduced immunogenicity and / or increased anti-neoplastic activity. generating an immune cell.

2. Modified immune cells with reduced immunogenicity and / or increased anti-neoplastic activity The present invention relates to a method for producing a population of immune cells by multiplex editing, the method comprising: Modify four gene sequences or their regulatory elements with a single target nucleobase in each of them. thereby reducing said immunogenicity and / or increasing said antineoplastic activity. A method comprising generating a population of modified immune cells.

3. At least one of the at least four gene sequences is a checkpoint inhibitor gene.

3. The nucleic acid sequence according to claim 1 or 2, which is a nucleic acid sequence, an immune response regulating gene sequence, or an immunogenic gene sequence. Method of posting.

4. The modifying reduces expression of at least one of the at least four gene sequences. The method according to any one of claims 1 to 3,

5. The expression of at least one of the at least four genes is compared to a control cell not having the modification. The method of claim 1, wherein the concentration of 100% or more of the ...

6. The expression of each of the at least four genes is increased, compared to a control cell not having the modification. The method of claim 5, wherein the concentration is reduced by at least 80%.

7. Expression of at least one of the at least four genes is at least one of the population of immune cells. The method of claim 2, wherein the concentration of β-aminobutyric acid in the blood is also reduced by 50%.

8. Expression of each of the at least four genes is reduced in at least 50% of the population of immune cells. The method of claim 7, wherein the

9. Any of the preceding claims, wherein the at least four gene sequences include TCR complex gene sequences.

3. The method according to claim 1 .

10. The method of claim 9, wherein the at least four gene sequences include a TRAC gene sequence.

11. The at least four gene sequences include a checkpoint inhibitor gene sequence. The method according to any one of claims 1 to 8.

12. The method of claim 10, wherein the at least four gene sequences include a PDCD1 gene sequence.

13. The method according to any one of claims 1 to 8, wherein the at least four gene sequences include a T cell marker gene sequence. The method according to any one of claims 1 to 5.

14. The method of claim 13, wherein the at least four gene sequences include a CD52 gene sequence.

15. The method of claim 13, wherein the at least four gene sequences include a CD7 gene sequence.

16. The at least four gene sequences are a TRAC gene sequence, a PDCD1 gene sequence, a CD52 gene sequence, 16. The method of claim 1, comprising a sequence of a CD7 gene, or a CD7 gene sequence.

17. The at least four sequences are a TCR complex gene sequence, a CD7 gene sequence, a CD52 gene sequence, , as well as CD2 gene sequence, CD4 gene sequence, CD5 gene sequence, CD7 gene sequence, CD30 gene sequence sequence, CD33 gene sequence, CD52 gene sequence, CD70 gene sequence, B2M gene sequence, and CIITA The method according to any one of claims 1 to 16, comprising a gene sequence selected from the group consisting of gene sequences The method described.

18. The at least four gene sequences are a CD2 gene sequence, a TRAC gene sequence, a CD3 epsilon gene sequence, CD3 gamma gene sequence, CD3 delta gene sequence, TRBC1 gene sequence, TRBC2 gene sequence CD4 gene sequence, CD5 gene sequence, CD7 gene sequence, CD30 gene sequence, CD33 gene sequence The sequence consists of the CD52 gene sequence, the CD70 gene sequence, the B2M gene sequence, and the CIITA gene sequence. The method of any one of claims 1 to 17, comprising a gene sequence selected from the group consisting of:

19. The five gene sequences or their regulatory elements in the immune cells are each The method according to any one of claims 1, 5 and 6, further comprising modifying at a target nucleobase of Law.

20. The six gene sequences or their regulatory elements in the immune cells are The method according to any one of claims 1, 5 and 6, further comprising modifying at a target nucleobase of Law.

21. The seven gene sequences or their regulatory elements in the immune cells are The method according to any one of claims 1, 5 and 6, further comprising modifying at a target nucleobase of Law.

22. The eight gene sequences or their regulatory elements in the immune cells are The method according to any one of claims 1, 5 and 6, further comprising modifying at a target nucleobase of Law.

23. Five gene sequences or regulatory elements thereof in the population of immune cells are The method according to any one of claims 2, 7 and 8, further comprising modifying at a single target nucleobase. Method of posting.

24. Six gene sequences or regulatory elements thereof in the population of immune cells are The method according to any one of claims 2, 7 and 8, further comprising modifying at a single target nucleobase. Method of posting.

25. Seven gene sequences or their regulatory elements in the population of immune cells are The method according to any one of claims 2, 7 and 8, further comprising modifying at a single target nucleobase. Method of posting.

26. The eight gene sequences or their regulatory elements in the population of immune cells are The method according to any one of claims 2, 7 and 8, further comprising modifying at a single target nucleobase. Method of posting.

27. The five, six, seven, or eight gene sequences or regulatory elements thereof are sequence, TRAC gene sequence, CD3 epsilon gene sequence, CD3 gamma gene sequence, CD3 delta gene sequence gene sequence, TRBC1 gene sequence, TRBC2 gene sequence, CD4 gene sequence, CD5 gene sequence, CD7 gene sequence gene sequence, CD30 gene sequence, CD33 gene sequence, CD52 gene sequence, CD70 gene sequence, B2M gene Any of claims 19 to 26, selected from the group consisting of a gene sequence, a CIITA gene sequence, and a CIITA gene sequence.

13. The method according to claim 1.

28. The five, six, seven, or eight gene sequences or regulatory elements thereof are sequence, CD7 gene sequence, CD2 gene sequence, CD5 gene sequence, and CD52 gene sequence, 28. The method according to any one of claims 19 to 27.

29. The modifying comprises deaminating the single target nucleobase. The method according to any one of the items.

30. The deaminating step is performed by a polypeptide comprising a deaminase.

30. The method according to item 29.

31. The deaminase associates with a nucleic acid programmable DNA binding protein (napDNAbp) to form a salt 31. The method of claim 30, further comprising forming a group editor.

32. The deaminase is fused to a nucleic acid programmable DNA binding protein (napDNAbp). The method of claim 31 .

33. 33. The method of Claim 32, wherein said napDNAbp comprises a Cas9 polypeptide or a portion thereof.

34. 34. The method of claim 33, wherein the napDNAbp comprises Cas9 nickase or a nuclease-inactive Cas9. How to.

35. 35. The method according to any one of claims 30 to 34, wherein the deaminase is a cytidine deaminase. How to.

36. the single target nucleobase is a cytosine (C) and the modification is to convert the C to a thymine (T).

36. The method of claim 35, comprising converting

37. 37. The method of claim 36, wherein the base editor further comprises a uracil glycosylase inhibitor. Method of posting.

38. The method according to any one of claims 30 to 34, wherein the deaminase is adenosine deaminase. Method of posting.

39. the single target nucleobase is an adenosine (A) and the modification is a guanine (G 40. The method of claim 38, comprising converting

40. 4. The method of claim 3, wherein the modifying comprises contacting the immune cell with a guide nucleic acid sequence.

40. The method of any one of claims 30 to 39.

41. The modifying step comprises contacting the immune cell with at least four guide nucleic acid sequences. and each guide nucleic acid sequence is adapted to link said napDNAbp to said at least four gene sequences or The method of claim 40, which targets one of the regulatory elements.

42. The guide nucleic acid sequence is selected from the guide RNA sequences of Table 8A, Table 8B, or Table 8C.

41. The method of claim 40, comprising:

43. The guide nucleic acid sequence is CCAUAACC, CACCUACCUAAGAACCAUCC, ACUCACGCUGGAUAGCCUCC, ACUCACCCAGCAUCCCCAGC, CACU CACCUUAGCCUGAGCA, and CACGCACCUGGACAGCUGAC, The method of claim 40.

44. The modifying step comprises translating the target primer using a reverse transcriptase and an extended guide nucleic acid sequence. and replacing said single target nucleobase with a different nucleobase by reverse transcription. The method of any one of claims 1 to 28.

45. The extended guide nucleic acid sequence comprises a reverse transcription template sequence, a reverse transcription primer binding site, 45. The method of claim 44, comprising:

46. The method of any one of claims 1 to 45, wherein the single target nucleobase is in an exon. Law.

47. 47. The method of claim 46, wherein said modifying generates a premature stop codon in said exon. method.

48. The single target nucleobase is located in exon 1, exon 2, or exon 3 of the TRAC gene sequence. The method of claim 46 or 47, wherein the method is in Son 3.

49. The single target nucleobase is located in exon 1, exon 2, or exon 3 of the PCDC1 gene sequence.

48. The method of claim 46 or 47, wherein the nucleic acid is in Chthon 5.

50. The single target nucleobase is within exon 1 or exon 2 of the CD52 gene sequence. The method according to claim 46 or 47.

51. The single target nucleobase is located in exon 1, exon 2, or exon 3 of the CD7 gene sequence. The method of claim 46 or 47, wherein the method is in Son 3.

52. The single target nucleobase is within exon 1 or exon 2 of the B2M gene sequence. The method according to claim 46 or 47.

53. The single target nucleobase is selected from exon 2, exon 3, exon 4, The method of claim 46 or 47, wherein the nucleic acid sequence is within exon 5, exon 6, exon 7, or exon 8. How to.

54. The single target nucleobase is selected from exon 2, exon 3, exon 4, or within exon 5.

55. The single target nucleic acid base is selected from exon 1, exon 2, exon 4 of the CIITA gene sequence. , exon 7, exon 8, exon 9, exon 10, exon 11, exon 12, exon 1 48. The method according to claim 46 or 47, wherein the nucleic acid sequence is within exon 4, exon 15, exon 18, or exon 19. Law.

56. The single target nucleobase is a splice donor site or a splice acceptor site. The method according to any one of claims 1 to 45,

57. The single target nucleobase is an exon 1 splice acceptor of the TRAC gene sequence. site, exon 1 splice donor site, or exon 3 splice acceptor site The method of claim 50.

58. The single target nucleobase is an exon 1 splice acceptor of the PDCD1 gene sequence. site, exon 1 splice donor site, exon 2 splice acceptor site, exon exon 3 splice donor site, exon 4 splice acceptor site, exon 4 splice 51. The method of claim 50, wherein the nucleotide sequence is located at the exon 5 donor site, or at the exon 5 splice acceptor site. method.

59. the single target nucleobase is an exon 1 splice donor site of the CD52 gene sequence; or in the exon 2 splice acceptor site.

60. The single target nucleobase is located at the exon 1 splice donor site, exon 2 splice donor site, exon 3 splice donor site, exon 4 splice donor site, exon 5 splice donor site, exon 6 splice donor site, exon 7 splice donor site, exon 8 splice donor site, exon 9 splice donor site, exon 1 splice donor site, exon 1 sp exon 2 splice donor site, exon 2 splice acceptor site, or exon 51. The method of claim 50, wherein the nucleotide sequence is located at the 3 splice acceptor site.

61. The single target nucleobase is a splice donor site of exon 1 of the B2M gene sequence. exon 2 splice donor site, exon 2 splice acceptor site, or exon 51. The method of claim 50, wherein the nucleotide sequence is located at the 3 splice acceptor site.

62. The single target nucleobase is located in the exon 3 splice donor site of the CD2 gene sequence. The method of claim 50,

63. The single target nucleobase is located at the exon 1 splice donor site, exon 2 splice donor site, exon 3 splice donor site, exon 4 splice donor site, exon 5 splice donor site, exon 6 splice donor site, exon 7 splice donor site, exon 8 splice donor site, exon 9 splice donor site, exon 1 splice donor site, exon 1 sp exon 1 splice acceptor site, exon 3 splice acceptor site, exon 3 splice donor site, exon 4 splice acceptor site, exon 5 splice donor site, exon 6 splice acceptor site, exon 9 splice donor site, or in the exon 10 splice acceptor site.

64. the single target nucleobase is an exon 1 splice donor site of the CIITA gene sequence; Exon 7 splice donor site, exon 8 splice acceptor site, exon 9 splice Splice donor site, exon 10 splice acceptor site, exon 11 splice acceptor site acceptor site, exon 14 splice acceptor site, exon 14 splice donor site, exon 15 splice donor site, exon 16 splice acceptor site, exon Exon 16 splice donor site, exon 17 splice acceptor site, exon 17 splice 51. The method of claim 50, wherein the nucleotide sequence is located at the Rice donor site or at the exon 19 splice acceptor site. Method of posting.

65. The method of any one of claims 4 to 64, wherein the immune cells are human cells.

66. The immune cells include cytotoxic T cells, regulatory T cells, helper T cells, dendritic cells, B cells, or a NK cell.

67. 67. The method of any one of claims 7 to 66, wherein the population of immune cells is human cells.

68. The population of immune cells may include cytotoxic T cells, regulatory T cells, helper T cells, dendritic cells, B 68. The method of claim 67, wherein the cell is a human NK cell.

69. The method of any one of claims 1 to 68, wherein the modifying is ex vivo.

70. Claims 1 to 6, wherein the immune cells or the population of immune cells are derived from a single human donor.

10. The method of any one of claims 9 to 9.

71. The immune cell or population of immune cells is coupled to an exogenous functional chimeric antigen receptor (CAR) or or a functional fragment thereof. Item 71. The method according to any one of items 1 to 70.

72. The immune cell or population of immune cells is treated with the polynucleotide encoding the CAR.

72. The method of claim 71, comprising contacting the vector with a lentivirus comprising the vector.

73. The immune cell or population of immune cells is treated with napDNAbp and the polypeptide encoding the CAR.

72. The method of claim 71, comprising contacting said nucleic acid sequence with a donor DNA sequence comprising a nucleotide sequence selected from the group consisting of nucleotides 1 and 2. 。

74. 74. The method of Claim 73, wherein said napDNAbp is Cas12b.

75. Any of claims 71 to 74, wherein the CAR specifically binds to a marker associated with a neoplasm.

13. The method according to claim 1.

76. The neoplasm is a T-cell cancer, a B-cell cancer, a lymphoma, a leukemia, or multiple myeloma.

76. The method of claim 75.

77. 77. The method of claim 76, wherein the CAR specifically binds to CD7.

78. The method of claim 76, wherein the CAR specifically binds to BCMA.

79. Claims 2-78, wherein said immune cell or said population of immune cells does not contain a detectable translocation.

2. The method according to claim 1 .

80. 80. The method of Claim 79, wherein at least 50% of the population of immune cells express the CAR.

81. 80. The method of claim 79, wherein at least 50% of the population of immune cells are viable.

82. at least 50% of said population of immune cells are a population of control cells of the same type that do not have said modification; 80. The method of claim 79, wherein the cell expands at a rate of at least 80% of the rate of expansion.

83. Claims 4 to 7, wherein the modifying generates less than 1% indels in the immune cells.

10. The method of any one of claims 9 to 9.

84. 4. The method of claim 3, wherein the modifying produces less than 5% non-targeted editing in the immune cell.

80. The method according to any one of claims 1 to 79.

85. The modifying produces less than 5% off-target editing in the immune cell.

80. The method of any one of claims 4 to 79.

86. 86. A modified immune cell produced according to the method of any one of claims 4 to 85.

87. A modified immune cell produced according to the method of any one of claims 7 to 85. Group.

88. 1. A modified immune cell having reduced immunogenicity or increased anti-neoplastic activity, A single target nucleobase modification in each of at least four gene sequences or their regulatory elements. Modified immune cells, including those with

89. Each of the at least four gene sequences is a checkpoint inhibitor gene sequence, an immunoreactive gene sequence, The modified polypeptide of claim 88 is an immune response regulating gene sequence or an immunogenic gene sequence. immune cells.

90. 89. The method of claim 88, wherein the at least four gene sequences include TCR complex gene sequences. The modified immune cells described herein.

91. 91. The modified polypeptide of claim 90, wherein the at least four gene sequences include a TRAC gene sequence. Immune cells.

92. The at least four gene sequences include a checkpoint inhibitor gene sequence.

90. The modified immune cell of claim 88 or 89.

93. 93. The modified polypeptide of claim 92, wherein the at least four gene sequences include a PDCD1 gene sequence. Immune cells.

94. 88. The method of claim 87, wherein the at least four gene sequences comprise T cell marker gene sequences.

89. A modified immune cell according to claim 89.

95. The modified polypeptide of claim 94, wherein the at least four gene sequences include a CD52 gene sequence. Immune cells.

96. The modified polypeptide of claim 94, wherein the at least four gene sequences include a CD7 gene sequence. Immune cells.

97. The expression of one of the at least four genes is reduced compared to a control cell not having the modification.

97. The modified immune cell of any one of claims 88 to 96, wherein the expression level of the immune cell is reduced by at least 80%.

98. The expression of each of the at least four genes is increased, compared to a control cell not having the modification.

98. The modified immune cell of claim 97, which is reduced by at least 90%.

99. Alterations to a single targeted nucleobase in each of the five gene sequences or their regulatory elements Each of the five gene sequences comprises a checkpoint inhibitor gene sequence, an immune response gene sequence, The nucleic acid sequence according to any one of claims 88 to 98, which is an antigen-regulating gene sequence or an immunogenic gene sequence. Modified immune cells.

100. Modifications to a single targeted nucleobase in each of six gene sequences or their regulatory elements Each of the six gene sequences comprises a checkpoint inhibitor gene sequence, an immune response gene sequence, The nucleic acid sequence according to any one of claims 88 to 98, which is an antigen-regulating gene sequence or an immunogenic gene sequence. Modified immune cells.

101. Alterations to a single targeted nucleobase in each of the seven gene sequences or their regulatory elements Each of the seven gene sequences comprises a checkpoint inhibitor gene sequence, an immune response gene sequence, The nucleic acid sequence according to any one of claims 88 to 98, which is an antigen-regulating gene sequence or an immunogenic gene sequence. Modified immune cells.

102. Alterations to a single targeted nucleobase in each of the eight gene sequences or their regulatory elements Each of the eight gene sequences comprises a checkpoint inhibitor gene sequence, an immune response gene sequence, The nucleic acid sequence according to any one of claims 88 to 98, which is an antigen-regulating gene sequence or an immunogenic gene sequence. Modified immune cells.

103. The expression of at least one of the five, six, seven or eight genes does not have the alteration. The modified embodiment of any one of claims 99 to 102, which is at least 90% reduced compared to control cells. Altered immune cells.

104. The expression of each of the five, six, seven, or eight genes is greater than or equal to that of a control cell not having the modification. The modified composition according to any one of claims 99 to 102, immune cells.

105. The five, six, seven, or eight gene sequences or regulatory elements thereof are sequence, TRAC gene sequence, CD3 epsilon gene sequence, CD3 gamma gene sequence, CD3 delta gene sequence gene sequence, TRBC1 gene sequence, TRBC2 gene sequence, CD4 gene sequence, CD5 gene sequence, CD7 gene sequence gene sequence, CD30 gene sequence, CD33 gene sequence, CD52 gene sequence, CD70 gene sequence, B2M gene Claims 99-104, comprising a sequence selected from the group consisting of a gene sequence, a CIITA gene sequence, and a CIITA gene sequence. The modified immune cell of any one of claims 1 to 4.

106. A single gene sequence was identified for each of the CD3 gene sequence, the CD5 gene sequence, the CD52 gene sequence, and the CD7 gene sequence. a modified immune cell of the same type that does not have said modification, comprising a modification of the target nucleobase of Modified immune cells that exhibit reduced immunogenicity or increased anti-neoplastic activity compared to control cells. Cell.

107. A single targeted nucleobase modification in the CD2 gene sequence, CIITA or their respective regulatory elements The modified immune cell of claim 106, further comprising:

108. TRAC gene sequence, CD3 epsilon gene sequence, CD3 gamma gene sequence, CD3 delta gene sequence a single targeted nucleic acid base modification in the sequence, the TRBC1 gene sequence, or the TRBC2 gene sequence; 4 gene sequence, CD30 gene sequence, CD33 gene sequence, CD70 gene sequence, B2M gene sequence, and and CIITA gene sequences or their respective regulatory elements.

107. The modified immune cell of Claim 106, further comprising a single targeted nucleobase modification at said site.

109. TRAC gene sequence, PDCD1 gene sequence, CD52 gene sequence, CD7 gene sequence, CD2 gene sequence , CD5 gene sequence, CIITA gene sequence, and B2M gene sequence each have a single nucleic acid base modification. The modified immune cell of claim 107, comprising a mutation.

110. The modified immune cell of any one of claims 88 to 109, which does not contain a detectable translocation. Cell.

111. The modified immune cell of any one of claims 88 to 110, comprising less than 1% indels. 。

112. The modified immune cell of any one of claims 88 to 110, comprising less than 5% non-targeted editing. Cell.

113. The modified polypeptide of any one of claims 88 to 110, comprising less than 5% off-target editing. Immune cells.

114. 111. The modified immune cell of any one of claims 88 to 110, which is a mammalian cell.

115. 111. The modified immune cell of any one of claims 88 to 110, which is a human cell.

116. Cytotoxic T cells, regulatory T cells, helper T cells, dendritic cells, B cells, or NK cells. The modified immune cell of any one of claims 88 to 115.

117. 117. The modified immune cell of any one of Claims 88-116, in ex vivo culture.

118. The modified immune system according to any one of claims 88 to 117, derived from a single human donor. cell.

119. A polynucleotide encoding an exogenous functional chimeric antigen receptor (CAR) or a functional fragment thereof. The modified immune cell of any one of claims 88 to 118, further comprising an octide.

120. The polynucleotide encoding the CAR is integrated into the genome of the immune cell. The modified immune cell of claim 119.

121. 121. The method of claim 119 or 120, wherein the CAR specifically binds to a marker associated with a neoplasm. Modified immune cells.

122. The neoplasm is a T-cell cancer, a B-cell cancer, a lymphoma, a leukemia, or multiple myeloma.

121. The modified immune cell of claim 119 or 120.

123. The modified CAR of any one of claims 119 to 122, wherein the CAR specifically binds to CD7. Immune cells.

124. The modified CAR of any one of claims 119 to 122, wherein the CAR specifically binds to BCMA. Immune cells.

125. The nucleic acid according to any one of claims 88 to 124, wherein the single target nucleobase is in an exon. Engineered immune cells.

126. The single target nucleobase is located in exon 1, exon 2, or exon 3 of the TRAC gene sequence. The modified immune cell of claim 125, wherein the cell is in Son 3.

127. The single target nucleobase is located in exon 1, exon 2, or exon 3 of the PCDC1 gene sequence. The modified immune cell of claim 125, wherein the cell is in Chthon 5.

128. The single target nucleobase is within exon 1 or exon 2 of the CD52 gene sequence.

126. The modified immune cell of claim 125.

129. The single target nucleobase is located in exon 1, exon 2, or exon 3 of the CD7 gene sequence.

126. The modified immune cell of Claim 125,

130. The single target nucleobase is a splice donor site or a splice acceptor site. The modified immune cell of any one of claims 88 to 124.

131. The single target nucleobase is an exon 1 splice acceptor of the TRAC gene sequence. site, exon 1 splice donor site, or exon 3 splice acceptor site The modified immune cell of claim 130.

132. The single target nucleobase is an exon 1 splice acceptor of the PDCD1 gene sequence. site, exon 1 splice donor site, exon 2 splice acceptor site, exon exon 3 splice donor site, exon 4 splice acceptor site, exon 4 splice 131. The method of claim 130, wherein the nucleotide sequence is located at the exon 5 donor site or at the exon 5 splice acceptor site. Engineered immune cells.

133. the single target nucleobase is an exon 1 splice donor site of the CD52 gene sequence; or at the exon 2 splice acceptor site. cell.

134. The single target nucleobase is located at the exon 1 splice donor site, exon 2 splice donor site, exon 3 splice donor site, exon 4 splice donor site, exon 5 splice donor site, exon 6 splice donor site, exon 7 splice donor site, exon 8 splice donor site, exon 9 splice donor site, exon 1 splice donor site, exon 1 sp exon 2 splice donor site, exon 2 splice acceptor site, or exon 131. The modified immune cell of claim 130, wherein the nucleotide sequence is at the 3 splice acceptor site.

135. A population of modified immune cells, wherein a plurality of said populations of cells comprise at least four genetic variants. Each of the child sequences or their regulatory elements contains a single targeted nucleobase modification, said plurality of populations of cells having the modification compared to a plurality of control cells of the same type not having the modification. A population of modified immune cells exhibiting reduced immunogenicity or increased anti-neoplastic activity.

136. 136. The modified cell of claim 135, wherein said plurality of cells constitutes at least 50% of said population. A population of immune cells.

137. Each of the at least four gene sequences is a checkpoint inhibitor gene sequence, an immunoreactive gene sequence, 137. The method of claim 135 or 136, which is an immune response regulating gene sequence or an immunogenic gene sequence. Modified immune cell populations.

138. The at least four gene sequences include a TCR component gene sequence, a checkpoint inhibitor gene sequence, 138. The modified immune cell of claim 137, comprising a gene sequence, or a T cell marker gene sequence. A cluster of cells.

139. The modified polypeptide of claim 137, wherein the at least four gene sequences include a TRAC gene sequence. A population of isolated immune cells.

140. The modification of claim 137, wherein said at least four gene sequences include a PDCD1 gene sequence. A population of isolated immune cells.

141. The modified polypeptide of claim 137, wherein the at least four gene sequences include a CD52 gene sequence. A population of isolated immune cells.

142. The modified polypeptide of claim 137, wherein the at least four gene sequences include a CD7 gene sequence. A population of isolated immune cells.

143. The expression of at least one of the at least four genes is compared to a control cell not having the modification.

135. The method of claim 134, wherein the expression level of said plurality of cells having said modification is at least 80% reduced in said plurality of cells having said modification compared to said plurality of cells having said modification. A population of modified immune cells according to any one of claims 1 to 142.

144. The expression of each of the at least four genes is increased, compared to a control cell not having the modification. The method of claim 143, wherein the alteration is reduced by at least 80% in the plurality of cells having the alteration. Modified immune cell populations.

145. The plurality of said populations each contain a single gene sequence or a regulatory element thereof. Each of the five gene sequences comprises an alteration in one target nucleobase, and each of the five gene sequences inhibits checkpoint inhibition.

13. The gene sequence of claim 13, which is a factor gene sequence, an immune response modulating gene sequence, or an immunogenic gene sequence. A population of modified immune cells according to any one of claims 5 to 144.

146. The plurality of said populations are each a single gene sequence or a regulatory element thereof. each of the six sequences comprises an alteration at one target nucleobase, Claims 135-144 are gene sequences, immune response regulating gene sequences, or immunogenic gene sequences.

2. The population of modified immune cells of any one of claims 1 to 11.

147. The plurality of said populations are each a single gene sequence or a regulatory element thereof. Each of the seven gene sequences comprises an alteration in one target nucleobase, and each of the seven gene sequences inhibits checkpoint inhibition.

13. The gene sequence of claim 13, which is a factor gene sequence, an immune response modulating gene sequence, or an immunogenic gene sequence. A population of modified immune cells according to any one of claims 5 to 144.

148. The plurality of said populations are each a single gene sequence or a regulatory element thereof. Each of the eight gene sequences comprises an alteration in one target nucleobase, and each of the eight gene sequences inhibits checkpoint inhibition.

13. The gene sequence of claim 13, which is a factor gene sequence, an immune response modulating gene sequence, or an immunogenic gene sequence. A population of modified immune cells according to any one of claims 5 to 144.

149. The expression of at least one of the five, six, seven, or eight genes is not affected by the modification. is reduced by at least 90% in said plurality of cells having said modification compared to control cells 149. The population of modified immune cells of any one of claims 145 to 148.

150. The expression of each of the five, six, seven, or eight genes is greater than or equal to that of a control cell not having the modification. 1, wherein the alteration is reduced by at least 90% in the plurality of cells having the alteration compared to A population of modified immune cells according to any one of claims 45 to 148.

151. The five, six, seven, or eight gene sequences or regulatory elements thereof are sequence, TRAC gene sequence, CD3 epsilon gene sequence, CD3 gamma gene sequence, CD3 delta gene sequence gene sequence, TRBC1 gene sequence, TRBC2 gene sequence, CD4 gene sequence, CD5 gene sequence, CD7 gene sequence gene sequence, CD30 gene sequence, CD33 gene sequence, CD52 gene sequence, CD70 gene sequence, B2M gene Any of claims 145 to 148, wherein the sequence of the gene is selected from the group consisting of a CIITA gene sequence and a CIITA gene sequence. A population of modified immune cells according to any one of claims 1 to 4.

152. A population of modified immune cells, a plurality of said populations comprising a TRAC gene sequence, a PDCD1 gene sequence, The nucleic acid sequence of the nucleotide ... and wherein said plurality of said populations having said alteration are compared with a plurality of control cells of the same type not having said alteration. and a modified collection of immune cells that exhibit reduced immunogenicity or increased antineoplastic activity compared to human cells. Group.

153. The plurality of populations include a CD2 gene sequence, a CD5 gene sequence, a CIITA gene sequence, a B2M gene sequence, The sequence, or each of its regulatory elements, further comprises a single targeted nucleobase modification.

153. The population of modified immune cells of paragraph 152.

154. The plurality of said populations comprises a CD2 gene sequence, a TRAC gene sequence, a CD3 epsilon gene sequence, CD3 gamma gene sequence, CD3 delta gene sequence, TRBC1 gene sequence, TRBC2 gene sequence, CD4 gene sequence, CD5 gene sequence, CD7 gene sequence, CD30 gene sequence, CD33 gene sequence, CD52 gene sequence a gene sequence selected from the group consisting of a CD70 gene sequence, a B2M gene sequence, and a CIITA gene sequence. A single target nucleic acid base modification in the gene sequence of the gene to be treated or in each of its regulatory elements.

153. The population of modified immune cells of Claim 152, further comprising:

155. The plurality of populations include a TRAC gene sequence, a PDCD1 gene sequence, a CD52 gene sequence, a CD7 gene sequence, Each of the CD2 gene sequence, the CD5 gene sequence, the CIITA gene sequence, and the B2M gene sequence 154. The population of modified immune cells of Claim 153, comprising a single nucleobase modification in

156. 156. The method of any one of claims 135 to 155, wherein said plurality of said populations does not contain a detectable translocation.

2. A population of engineered immune cells.

157. The modified vector according to any one of claims 135 to 156, at least 60% of which are viable. A population of immune cells.

158. at least 60% faster than the expansion rate of a population of control cells of the same type that do not have the modification.

157. The population of modified immune cells of any one of claims 135-156, wherein said population of modified immune cells is expanded by 80%.

159. 159. The population of modified immune cells of any one of claims 135-158, which are human cells.

160. Cytotoxic T cells, regulatory T cells, helper T cells, dendritic cells, B cells, or NK cells.

160. The population of modified immune cells of any one of claims 135 to 159.

161. The modified immune system according to any one of claims 135 to 160, derived from a single human donor. A population of cells.

162. The plurality of cells having the modification comprises an exogenous functional chimeric antigen receptor (CAR) or Any one of claims 135 to 161 further comprising a polynucleotide encoding a functional fragment of A population of modified immune cells as described in paragraph 1.

163. The population of modified immune cells of Claim 162, wherein at least 50% express said CAR.

164. The method of claim 162 or 163, wherein the CAR specifically binds to a marker associated with a neoplasm. A population of engineered immune cells.

165. The neoplasm is a T-cell cancer, a B-cell cancer, a lymphoma, a leukemia, or multiple myeloma.

165. The population of modified immune cells of claim 164.

166. The population of modified immune cells of Claim 165, wherein the CAR specifically binds to CD7.

167. The population of modified immune cells of Claim 165, wherein the CAR specifically binds to BCMA.

168. The nucleic acid according to any one of claims 135 to 167, wherein the single target nucleobase is in an exon. Modified immune cell populations.

169. The single target nucleobase is located in exon 1, exon 2, or exon 3 of the TRAC gene sequence. The population of modified immune cells of claim 168, wherein the population is within son 3.

170. The single target nucleobase is located in exon 1, exon 2, or exon 3 of the PCDC1 gene sequence. The population of modified immune cells of claim 168, wherein the population is within Chthon 5.

171. The single target nucleobase is within exon 1 or exon 2 of the CD52 gene sequence.

169. The population of modified immune cells of claim 168.

172. The single target nucleobase is located in exon 1, exon 2, or exon 3 of the CD7 gene sequence.

169. The population of modified immune cells of Claim 168, within

173. The single target nucleobase is a splice donor site or a splice acceptor site.

168. The population of modified immune cells of any one of claims 135 to 167.

174. The single target nucleobase is an exon 1 splice acceptor of the TRAC gene sequence. site, exon 1 splice donor site, or exon 3 splice acceptor site 174. The population of modified immune cells of Claim 173.

175. The single target nucleobase is an exon 1 splice acceptor of the PDCD1 gene sequence. site, exon 1 splice donor site, exon 2 splice acceptor site, exon exon 3 splice donor site, exon 4 splice acceptor site, exon 4 splice 174. The method of claim 173, wherein the nucleotide sequence is located at the exon 5 donor site or at the exon 5 splice acceptor site. Modified immune cell populations.

176. the single target nucleobase is an exon 1 splice donor site of the CD52 gene sequence; or at the exon 2 splice acceptor site. A population of cells.

177. The single target nucleobase is located at the exon 1 splice donor site, exon 2 splice donor site, exon 3 splice donor site, exon 4 splice donor site, exon 5 splice donor site, exon 6 splice donor site, exon 7 splice donor site, exon 8 splice donor site, exon 9 splice donor site, exon 1 splice donor site, exon 1 sp exon 2 splice donor site, exon 2 splice acceptor site, or exon 174. The population of modified immune cells of Claim 173, wherein said target polypeptide is located at a 3 splice acceptor site.

178. A composition comprising a deaminase and a nucleic acid sequence, wherein the guide nucleic acid sequence is AAACCAAG, CCUACCUGUCACCAGGACCA, CUCUUACCUGUACCAUAACC, CACCUACCUAAGAACCAUCC, ACUC ACGCUGGAUAGCCUCC, ACUCACCCAGCAUCCCCAGC, CACUCACCUUAGCCUGAGCA, and CACGCACCUGGA A composition comprising a sequence selected from the group consisting of: CAGCUGAC.

179. The deaminase associates with a nucleic acid programmable DNA binding protein (napDNAbp) to form a salt The composition of claim 178, which forms a group editor.

180. The napDNAbp comprises a Cas9 nickase or a nuclease-inactive Cas9, and the deaminase The composition of claim 179, wherein the enzyme is a cytidine deaminase.

181. 181. The method of claim 180, wherein the base editor further comprises a uracil glycosylase inhibitor. The composition described.

182. The napDNAbp comprises a Cas9 nickase or a nuclease-inactive Cas9, and the deaminase The composition of claim 166, wherein the enzyme is adenosine deaminase.

183. A composition comprising a polymerase and a guide nucleic acid sequence, wherein the guide nucleic acid sequence is CGUAUCUGUAAAACCAAG, CCUACCUGUCACCAGGACCA, CUCUUACCUGUACCAUAACC, CACCUACCUAAGAACC AUCC, ACUCACGCUGGAUAGCCUCC, ACUCACCCAGCAUCCCCAGC, CACUCACCUUAGCCUGAGCA, and CA A composition comprising a sequence selected from the group consisting of: CGCACCUGGACAGCUGAC.

184. The polymerase is a reverse transcriptase and the guide nucleic acid sequence is a reverse transcription template sequence. a sequence, a reverse transcription primer binding site, or a combination thereof, The composition of claim 170,

185. Modified immune cells with reduced immunogenicity and / or increased anti-neoplastic activity A method for producing a) a single target nucleic acid salt in a first gene sequence or a regulatory element thereof in an immune cell; modifying the group, and b) using a Cas12 polypeptide to transduce a second gene sequence or a regulatory element thereof in said immune cell. The step of modifying a portion of the gene sequence, wherein the Cas12 polypeptide comprises a site in the second gene sequence. Producing specific cleavage Including, Each of the first gene and the second gene is an immunogenic gene, a checkpoint gene, an inhibitor gene or an immune response regulator gene, Modified IL-18 / ... A method for generating immune cells.

186. expressing an exogenous functional chimeric antigen receptor (CAR) or a functional fragment thereof in said immune cells.

186. The method of claim 185, further comprising:

187. The polynucleotide encoding the CAR or a functional fragment thereof is linked to a Cas12 polypeptide. The method of claim 186, wherein the site-specific cleavage is inserted into the site-specific cleavage site generated thereby.

188. The method of Claim 187, wherein the Cas12 polypeptide is a Cas12b polypeptide.

189. Modified immune cells with reduced immunogenicity and / or increased anti-neoplastic activity A method for producing a) a single target nucleic acid salt in a first gene sequence or a regulatory element thereof in an immune cell; modifying the group, and b) The second gene comprises an exogenous functional chimeric antigen receptor (CAR) or a functional fragment thereof, or The present invention provides a method for the production of said immune cells by inserting an exogenous functional T cell receptor or a functional fragment thereof. modifying said second gene sequence or its regulatory element in the cell. Including, Each of the first gene and the second gene is an immunogenic gene, a checkpoint gene, an inhibitor gene or an immune response regulator gene, Modified IL-18 / ... A method for generating immune cells.

190. Step b) comprises ligating the second gene using a nucleic acid programmable DNA binding protein (napDNAbp).

190. The method of claim 189, further comprising generating a site-specific cleavage in the gene sequence.

191. 191. The method of Claim 190, wherein said napDNAbp is Cas12b.

192. compared to a control cell of the same type not having the modification, the expression of the first gene is at least or the expression of the second gene is reduced by at least 60%.

191. The method of any one of claims 191.

193. The first gene is selected from the group consisting of CD3 epsilon, CD3 gamma, CD3 delta, CD4, TRAC, TRBC1, TRBC 2, from the group consisting of PDCD1, CD30, CD33, CD7, CD52, B2M, CD70, CIITA, CD2, and CD5 The method of any one of claims 138 to 192, selected.

194. The first gene or the second gene is selected from the group consisting of TRAC, CIITA, CD2, CD5, CD7, and CD52.

194. The method of claim 193, selected from the group consisting of:

195. The method of any one of claims 185 to 194, wherein the second gene is TRAC.

196. Step a) comprises detecting a single target nucleic acid salt in two other gene sequences or in their regulatory elements.

196. The method of any one of claims 185 to 195, further comprising modifying the group.

197. Step a) comprises detecting a single target nucleic acid salt in three other gene sequences or regulatory elements thereof.

196. The method of any one of claims 185 to 195, further comprising modifying the group.

198. Step a) comprises detecting a single target nucleic acid salt in four other gene sequences or regulatory elements thereof.

196. The method of any one of claims 185 to 195, further comprising modifying the group.

199. Step a) comprises detecting a single target nucleic acid salt in five other gene sequences or regulatory elements thereof.

196. The method of any one of claims 185 to 195, further comprising modifying the group.

200. Step a) comprises detecting a single target nucleic acid salt in six other gene sequences or regulatory elements thereof.

196. The method of any one of claims 185 to 195, further comprising modifying the group.

201. Step a) comprises detecting a single target nucleic acid salt in seven other gene sequences or regulatory elements thereof.

196. The method of any one of claims 185 to 195, further comprising modifying the group.

202. The modifying in step a) comprises deaminase and nucleic acid programmable DNA binding Deamination of a single targeted nucleobase using a base editor containing a protein (napDNAbp) The method of any one of claims 185 to 201, comprising:

203. The napDNAbp of claim 202, wherein the napDNAbp comprises a Cas9 nickase or a nuclease-inactive Cas9. Method of posting.

204. the deaminase is a cytidine deaminase, and the modification is a cytidine (C) to thymine The method of claim 203, comprising converting the aryl group to a cyclic amine (T).

205. The deaminase is adenosine deaminase, and the modification is adenine (A) 204. The method of claim 203, comprising conversion to anine (G).

206. The modifying in a) comprises contacting the immune cell with a guide nucleic acid sequence. The method of any one of claims 185 to 205, comprising:

207. The guide nucleic acid sequence is CCAUAACC, CACCUACCUAAGAACCAUCC, ACUCACGCUGGAUAGCCUCC, ACUCACCCAGCAUCCCCAGC, CACU CACCUUAGCCUGAGCA, and CACGCACCUGGACAGCUGAC, The method of claim 206.

208. The modifying step in b) comprises contacting the immune cell with a guide nucleic acid sequence. The method of any one of claims 185 to 207, comprising:

209. The method of Claim 208, wherein the guide nucleic acid sequence comprises a sequence selected from the sequences in Table 1. 。

210. The modifying step in a) can be carried out using a reverse transcriptase and an extended guide nucleic acid sequence. The replacement of a single target nucleobase with a different nucleobase by target-primed reverse transcription wherein the extended guide nucleic acid sequence is a reverse transcription template sequence, a reverse transcription primer sequence, The method of any one of claims 185 to 209, comprising a binding site, a binding moiety, or a combination thereof.

211. The modifying in a) and b) reduces the percentage of indels in the immune cells to less than 1%. The method of any one of claims 185 to 210, wherein

212. The modifying in a) and b) reduces the off-target activity of less than 5% in the immune cells. The method of any one of claims 185 to 211, wherein the method produces a mitotic modification.

213. The modifying in a) and b) results in less than 5% non-target modification in the immune cells. The method of any one of claims 185 to 211, wherein

214. The method of any one of claims 185 to 213, wherein the immune cells are human cells.

215. The immune cells include cytotoxic T cells, regulatory T cells, helper T cells, dendritic cells, B cells, or a NK cell.

216. Any of claims 186 to 215, wherein the CAR specifically binds to a marker associated with a neoplasm.

3. The method according to claim 1 .

217. The method of claim 216, wherein the CAR specifically binds to CD7.

218. Modified immune cells with reduced immunogenicity and / or increased anti-neoplastic activity There was, a) modifying a single targeted nucleic acid base in a first gene sequence or a regulatory element thereof; and Call b) a modification in the second gene sequence or in its regulatory element, Modifications that are site-specific cleavage generated by tides Including, Each of the first gene and the second gene is an immunogenic gene, a checkpoint gene, The modified immune cells are inhibitory genes or immune response modulating genes.

219. Claim 218, further comprising an exogenous functional chimeric antigen receptor (CAR) or a functional fragment thereof. The modified immune cell according to claim 1.

220. The polynucleotide encoding the CAR or a functional fragment thereof is linked to a Cas12 polypeptide. The modified immune system of claim 219, wherein the modified immune system is inserted into the site-specific cleavage site generated by the modified immune system. cell.

221. Modified immune cells with reduced immunogenicity and / or increased anti-neoplastic activity There was, a) a single target nucleic acid salt in a first gene sequence or a regulatory element thereof in an immune cell; Modification of groups, and b) modifications in the second gene sequence or in its regulatory elements, A native T cell receptor (CAR) or a functional fragment thereof or an exogenous T cell receptor or a functional fragment thereof An alteration that is the insertion of a piece Including, Each of the first gene and the second gene is an immunogenic gene, a checkpoint gene, The modified immune cells are inhibitory genes or immune response modulating genes.

222. The modification in b) is generated by site-specific cleavage using Cas12b.

22. The modified immune cell according to 21.

223. compared to a control cell of the same type not having the modification, the expression of the first gene is at least or the expression of the second gene is reduced by at least 60%.

223. The modified immune cell of any one of claims 222.

224. The first gene or the second gene is selected from the group consisting of CD3 epsilon, CD3 gamma, CD3 delta, C D4, TRAC, TRBC1, TRBC2, PDCD1, CD30, CD33, CD7, CD52, B2M, CD70, CIITA, CD2, The modified antibody according to any one of claims 218 to 223, selected from the group consisting of CD5 and CD6. immune cells.

225. The first gene or the second gene comprises TRAC, CD2, CD5, CD7, and CD52. The method of claim 193, wherein the compound is selected from the group consisting of:

226. The modified immune cell of Claim 225, wherein said second gene is TRAC.

227. Alterations to a single target nucleic acid base in two other gene sequences or their regulatory elements 227. The modified immune cell of any one of claims 218-226, further comprising:

228. Alterations to a single target nucleic acid base in three other gene sequences or their regulatory elements 227. The modified immune cell of any one of claims 218-226, further comprising:

229. Alterations to single target nucleic acid bases in four other gene sequences or their regulatory elements 227. The modified immune cell of any one of claims 218-226, further comprising:

230. Alterations to single target nucleic acid bases in five other gene sequences or their regulatory elements 227. The modified immune cell of any one of claims 218-226, further comprising:

231. Alterations to single target nucleobases in six other gene sequences or their regulatory elements 227. The modified immune cell of any one of claims 218-226, further comprising:

232. Alterations to single target nucleobases in seven other gene sequences or their regulatory elements 227. The modified immune cell of any one of claims 218-226, further comprising:

233. a) the modification of the deaminase and nucleic acid programmable DNA binding protein ( napDNAbp) The modified immune cells described herein.

234. the deaminase is a cytidine deaminase, and the modification is a cytidine (C) to thymine The modified immune cell of Claim 233, comprising conversion to a tyrosine (T).

235. The deaminase is adenosine deaminase, and the modification is adenine (A) The modified immune cell of Claim 233, comprising a conversion to anine (G).

236. The modification according to any one of claims 218 to 235, comprising less than 1% indels in the genome Immune cells.

237. 237. The modified immune cell of any one of claims 218-236, which is a human cell.

238. Cytotoxic T cells, regulatory T cells, helper T cells, dendritic cells, B cells, or NK cells. The modified immune cell of any one of claims 218 to 237.

239. Any of claims 218 to 238, wherein the CAR specifically binds to a marker associated with a neoplasm. The modified immune cell of any one of claims 1 to 4.

240. The modified immune cell of Claim 239, wherein the CAR specifically binds to CD7.

241. b) is an insertion in exon 1 in the TRAC gene sequence. The modified immune cell of any one of paragraphs 218 to 240.

242. A population of modified immune cells, comprising a plurality of said population of immune cells comprising: a) a single target nucleic acid salt in a first gene sequence or a regulatory element thereof in an immune cell; Modification of groups, and b) a modification in the second gene sequence or in its regulatory element, Modifications that are site-specific cleavage generated by tides Including, Each of the first gene and the second gene is an immunogenic gene, a checkpoint gene, an inhibitor gene or an immune response regulator gene, The plurality of said populations comprises an exogenous chimeric antigen receptor (CAR) or a functional fragment thereof. 、 Modified immune cell populations.

243. The polynucleotide encoding the CAR or a functional fragment thereof is linked to a Cas12 polypeptide. The modified immune system of claim 242, wherein the modified immune system is inserted into the site-specific cleavage site generated by the modified immune system. A population of cells.

244. A population of modified immune cells, comprising a plurality of said population of immune cells comprising: a) modifying a single targeted nucleic acid base in a first gene sequence or a regulatory element thereof; and Call b) a modification in the second gene sequence or its regulatory sequence, which is capable of regulating the acceptance of the exogenous chimeric antigen; Insertion of a CAR or a functional fragment thereof or an exogenous T cell receptor or a functional fragment thereof Changes that are Including, Each of the first gene and the second gene is an immunogenic gene, a checkpoint gene, an inhibitor gene or an immune response regulator gene, having an alteration in a) or b) The cells are modified immune cells that exhibit reduced immunogenicity and / or increased anti-neoplastic activity. A group of infectious cells.

245. The modification in b) is generated by site-specific cleavage using Cas12b.

44. A population of modified immune cells according to claim 44.

246. The modification in a) or b) is compared to a plurality of control cells of the same type that do not have the modification. the expression of the first gene is reduced by at least 60% in the plurality of cells having the mutation; or any one of claims 242 to 245, wherein expression of the second gene is reduced by at least 60%. A population of modified immune cells as described in paragraph 1.

247. The first gene or the second gene is selected from the group consisting of CD3 epsilon, CD3 gamma, CD3 delta, C D4, TRAC, TRBC1, TRBC2, PDCD1, CD30, CD33, CD7, CD52, B2M, CD70, CIITA, CD2, The modified antibody according to any one of claims 242 to 246, selected from the group consisting of CD5 and CD6. A population of immune cells.

248. The first gene or the second gene is selected from the group consisting of TRAC, CIITA, CD2, CD5, CD7, and CD52.

248. The population of modified immune cells of Claim 247, selected from the group consisting of:

249. The modified immune system of claim 248, wherein said first gene is TRAC, CD7, or CD52. A group of infectious cells.

250. The population of modified immune cells of Claim 248, wherein said second gene is TRAC.

251. The plurality of cells having the alteration in a) or b) are Claims 242-250 further comprise an alteration at a single targeted nucleobase in said regulatory element.

2. The population of modified immune cells of any one of claims 1 to 11.

252. The plurality of cells having the modification in a) or b) is 3, 4, 5, or 6 Further comprising a single target nucleic acid base in another gene sequence or a regulatory element thereof.

252. The population of modified immune cells of claim 251.

253. The modification in a) is carried out by reacting a deaminase and a nucleic acid protease to form a base editor. Generated by a base editor containing a programmable DNA-binding protein (napDNAbp) 253. The population of modified immune cells of any one of claims 242-252.

254. the deaminase is a cytidine deaminase, and the modification is a cytidine (C) to thymine 254. The modified immune cell population of Claim 253, comprising conversion to a T cell.

255. The deaminase is adenosine deaminase, and the modification is adenine (A) 254. The modified immune cell population of Claim 253, comprising an anine (G) conversion.

256. 255. The method of claim 254, wherein the base editor further comprises a uracil glycosylase inhibitor. A population of modified immune cells as described herein.

257. The modified vector according to any one of claims 242 to 256, at least 60% of which are viable. A population of immune cells.

258. at least 60% faster than the expansion rate of a population of control cells of the same type that do not have the modification. The population of modified immune cells of any one of claims 242-256, wherein said population of modified immune cells is expanded by 80%.

259. The modified immune cell of any one of claims 242 to 258, wherein the immune cell is a human cell. A group of infectious cells.

260. The immune cells include cytotoxic T cells, regulatory T cells, helper T cells, dendritic cells, B cells, or NK cells. 。

261. Any of claims 242 to 260, wherein the CAR specifically binds to a marker associated with a neoplasm. A population of modified immune cells according to any one of claims 1 to 4.

262. The population of modified immune cells of Claim 261, wherein said CAR specifically binds to CD7.

263. b) is an insertion in exon 1 in the TRAC gene sequence. The population of modified immune cells of any one of paragraphs 242-262.

264. A method for producing modified immune cells with increased antineoplastic activity, comprising: A single gene sequence or its regulatory element in the Cbl proto-oncogene B (CBLB) gene in immune cells wherein the modification is compared to an immune cell lacking the modification. The modified immunocytes have a lower activation threshold, thereby increasing the anti-neoplastic activity. A method for generating engineered immune cells.

265. A composition comprising modified immune cells having increased anti-neoplastic activity, the modified immune cells comprising Immune cells express a single gene in the Cbl proto-oncogene B (CBLB) gene sequence or its regulatory elements. and the modified immune cells are of the same type but without the modification. A composition that exhibits a reduced activation threshold compared to control immune cells.

266. A population of immune cells, wherein a plurality of said population of immune cells are characterized by a CBLB gene sequence or a sequence thereof. The plurality of target nucleic acid bases comprising a modification at a single target nucleic acid base at a node element, The population of immune cells is activated relative to a control population of the same type of immune cells that does not have the modification. A population of immune cells exhibiting a reduced activation threshold.

267. 1. A method for producing a population of modified immune cells with increased anti-neoplastic activity, comprising: In a population of immune cells, the Cbl proto-oncogene B (CBLB) gene sequence or its regulatory elements wherein at least 50% of a population of immune cells are modified by targeting a single nucleobase in the population of immune cells. % are modified to include said single targeted nucleobase modification.

268. A composition comprising at least four different guide nucleic acid sequences for base editing.

269. The method further comprises the step of: The editor polypeptide is a nucleic acid programmable DNA binding protein (napDNAbp) and a The composition of claim 268, comprising an aminase.

270. 269. Claim 269, wherein the polynucleotide encoding the base editor is an mRNA sequence. The composition described in

271. The method of claim 1, wherein the deaminase is a cytidine deaminase or an adenosine deaminase. Item 269 or 270. The composition according to item 269 or 270.

272. and further comprising a base editor polypeptide, wherein the base editor polypeptide is The method of claim 268, comprising a programmable DNA binding protein (napDNAbp) and a deaminase. The composition described.

273. The method of claim 1, wherein the deaminase is a cytidine deaminase or an adenosine deaminase. Item 273. The composition according to item 272.

274. 274. The composition of claim 272 or 273, further comprising lipid nanoparticles.

275. Each of the at least four guide nucleic acid sequences is selected from the group consisting of CD2, CD3 epsilon, CD3 gamma, CD4 epsilon, CD4 ... 3delta, CD4, CD5, CD7, CD30, CD33, CD52, CD70, and CIITA. In some embodiments, the hybridization sequence is at least one, two, or three. , 3, 4, 5, 6, 7, or 8 or more genes or their regulatory elements are , CD3 epsilon, CD3 gamma, CD3 delta, CD4, CD5, CD7, CD30, CD33, CD52, CD70, and and CIITA, and in some embodiments, at least one, two, three, four , 5, 6, 7, or 8 or more genes or their regulatory elements are involved in the CD2, CD3 ipsilateral CD3 gamma, CD3 delta, CD4, CD5, CD7, CD30, CD33, CD52, CD70, and CIITA. In some embodiments, the gene comprises at least one selected from the group consisting of One, two, three, four, five, six, seven, eight or more genes or their regulatory elements But ACAT1, ACLY, ADORA2A, AXL, B2M, BATF, BCL2L11, BTLA, CAMK2D, cAMP, CASP8, Cb lb, CCR5, CD2, CD3D, CD3E, CD3G, CD4, CD5, CD7, CD8A, CD33, CD38, CD52, CD70, CD 82, CD86, CD96, CD123, CD160, CD244, CD276, CDK8, CDKN1B, Chi3l1, CIITA, CISH, C SF2CSK, CTLA-4, CUL3, Cyp11a1, DCK, DGKA, DGKZ, DHX37, ELOB(TCEB2), ENTPD1(CD39) , FADD, FAS, GATA3, IL6, IL6R, IL10, IL10RA, IRF4, IRF8, JUNB, Lag3, LAIR-1(CD30 5), LDHA, LIF, LYN, MAP4K4, MAPK14, MCJ, MEF2D, MGAT5, NR4A1, NR4A2, NR4A3, NT5E (CD73), ODC1, OTULINL(FAM105A), PAG1, PDCD1, PDIA3, PHD1(EGLN2), PHD2(EGLN1), PH D3(EGLN3), PIK3CD, PIKFYVE, PPARa, PPARd, PRDMI1, PRKACA, PTEN, PTPN2, PTPN6, PT PN11, PVRIG(CD112R), RASA2, RFXANK, SELPG / PSGL1, SIGLEC15, SLA, SLAMF7, SOCS1, S pry1, Spry2, STK4, SUV39, H1TET2, TGFbRII, TIGIT, Tim-3, TMEM222, TNFAIP3, TNFRS F8(CD30), TNFRSF10B, TOX, TOX2, TRAC, TRBC1, TRBC2, UBASH3A, VHL, VISTA, XBP1, Y The composition of any one of claims 267-274, wherein the composition is selected from: AP1, and ZC3H12A.

276. Each of the at least four guide nucleic acid sequences is selected from the group consisting of CD3 epsilon, CD3 delta, CD3 cancer, The group consisting of TRAC, TRBC1, TRBC2, CD2, CD5, CD7, CD52, CD70, and CIITA The method according to any one of claims 267 to 274, which hybridizes with a gene sequence selected from composition.

277. The at least four guide nucleic acid sequences are A, CUCUUACCUGUACCAUAACC, CACCUACCUAAGAACCAUCC, ACUCACGCUGGAUAGCCUCC, ACUCACCCAGC AUCCCCAGC, CACUCACCUUAGCCUGAGCA, and CACGCACCUGGACAGCUGAC. The composition of any one of claims 267 to 274, comprising a sequence

278. Claims 267-2, wherein the composition is introduced into immune cells using electroporation.

78. An immune cell comprising the composition of any one of claims 77.

279. The composition may be used for electroporation, nucleofection, viral transduction, or are introduced into immune cells using a combination thereof according to any one of claims 267 to 277. An immune cell comprising the composition.

280. Any of claims 86 and 88-134, which have increased growth or viability compared to a reference cell. The modified immune cell of any one of claims 1 to 4.

281. The method of claim 280, wherein the reference cell is an immune cell modified with Cas9 nuclease. The modified immune cells described herein.

282. 87. The method of claim 87, wherein the yield of the modified immune cells is increased as compared to a reference population of cells. and 135-179, a population of modified immune cells.

283. The reference population is a population of immune cells modified with Cas9 nuclease.

282. The population of modified immune cells according to 282.

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