Gene-regulating composition and method for improved immunotherapy

Abstract

The present disclosure provides methods and compositions related to the modification of immune effector cells to increase therapeutic efficacy. In some embodiments, immune effector cells are provided that are modified to reduce the expression of one or more endogenous target genes, or to reduce the function of one or more endogenous proteins to enhance the effector function of immune cells. In some embodiments, immune effector cells are provided that are further modified by the introduction of a transgene conferring antigen specificity, e.g., an exogenous T cell receptor (TCR) or a chimeric antigen receptor (CAR). A method for treating a cell proliferative disorder, e.g., cancer, using the modified immune effector cells described herein is provided.

Description

Technology Field

[0001] 관련 출원의 상호 참조

[0002] The present application is U.S. Provisional Application No. 62 / 643,578 (filed March 15, 2018), U.S. Provisional Application No. 62 / 692,010 (filed June 29, 2018), U.S. Provisional Application No. 62 / 768,428 (filed November 16, 2018), U.S. Provisional Application No. 62 / 643,584 (filed March 15, 2018), U.S. Provisional Application No. 62 / 692,016 (filed June 29, 2018), U.S. Provisional Application No. 62 / 768,441 (filed November 16, 2018), U.S. Provisional Application No. 62 / 790,179 (filed January 2019 Claiming priority to U.S. Provisional Application No. 62 / 804,261 (filing date: February 12, 2019), U.S. Provisional Application No. 62 / 736,185 (filing date: September 25, 2018) and U.S. Provisional Application No. 62 / 790,192 (filing date: January 9, 2019), the full text of which is incorporated herein by reference.

[0003] 전자 제출된 텍스트 파일의 설명

[0004] The contents of the attached electronically submitted text file, in their entirety, are incorporated herein by reference: a computer-readable format copy of the sequence list (filename: KSQT_006_05WO_SeqList_ST25.txt; date of entry: March 14, 2019; file size 266 kilobytes).

[0005] 기술분야

[0006] The present disclosure relates to methods, compositions, and components for editing a target nucleic acid sequence or controlling the expression of a target nucleic acid sequence, and to the application thereof in immunotherapy, including use with receptor-engineered immune effector cells in the treatment of proliferative diseases, inflammatory diseases, and / or infectious diseases. Background Technology

[0007] Adoptive cell transfer using genetically modified T cells, particularly CAR-T cells, has entered clinical trials as a therapeutic agent for solid and hematological malignancies. Results to date are mixed. In hematological malignancies (particularly lymphoma, CLL, and ALL), in several Phase 1 and Phase 2 trials, most patients showed at least a partial response, with some showing a complete response (Kochenderfer et al. , 2012 Blood 1 19, 2709-2720). In 2017, the FDA approved two CAR-T therapies for the treatment of blood cancer, Kymriah (상표명) and Yescarta (상표명) It was approved. However, in most tumor types (including melanoma, renal cell carcinoma, and colorectal cancer), a lower response was observed (Johnson et al. , 2009 Blood 1 14, 535-546; lamers et al. , 2013 Mol. Ther. 21, 904-912; Warren et al. , 1998 Cancer Gene Ther. 5, S1-S2). As such, since the success of CAR-T cell approaches targeting B-cell lineage hematological malignancies has been significantly limited, there is room for significant improvement in adoptive T cell therapy.

[0008] There is a need to improve the efficacy of adoptive delivery of modified immune cells in cancer treatment and, in particular, to increase the efficacy of adoptive cell therapy for solid malignancies, because reduced response is observed in these tumor types (melanoma, renal cell carcinoma, and colorectal cancer; Yong, 2017, Imm Cell Biol., 95:356-363). Furthermore, even in hematological malignancies where the benefits of adoptive delivery have been observed, not all patients respond due to reduced function of adoptive T cells, and recurrence occurs at a higher frequency than intended.

[0009] Factors limiting the efficacy of genetically modified immune cells as cancer treatments include (1) cell proliferation, e.g., limited proliferation of T cells after adoptive delivery; (2) cell survival, e.g., induction of T cell apoptosis by factors in the tumor environment; and (3) cell function, e.g., depletion of immune cells after the manufacturing process and / or delivery, and inhibition of cytotoxic T cell function by inhibitory factors secreted by host immune cells and cancer cells.

[0010] Specific features thought to enhance the anti-tumor effect of immune cells include: 1) the ability of the cell to proliferate in the host after adoptive delivery; 2) the ability of the cell to infiltrate the tumor; 3) the ability of the cell to persist in the host and / or exhibit resistance to immune cell attrition; and 4) the ability of the cell to function in a manner capable of killing tumor cells. The present disclosure provides immune cells comprising reduced expression and / or function of one or more endogenous target genes, wherein the modified immune cells exhibit enhancement of one or more effector functions, including increased proliferation, increased infiltration into the tumor, persistence of the immune cell in the subject, and / or increased resistance to immune cell attrition. The present disclosure also provides methods and compositions for modifying immune effector cells to derive enhanced immune cell activity against tumor cells, as well as methods and compositions suitable for use in the context of adoptive immune cell delivery therapy.

[0011] In some embodiments, the present disclosure is (a) BCL2L11 , FLI1 , CALM2 , DHODH , UMPS , RBM39 , SEMA7A , CHIC2 , PCBP1 , PBRM1 , WDR6 , E2F8 , SERPINA3 and GNAS A group consisting of; or (b) SOCS1 and ANKRD11 The present invention provides a modified immune effector cell comprising a gene-regulatory system capable of reducing the reduced expression and / or function of one or more endogenous target genes selected from the group consisting of, wherein the reduced expression and / or function of one or more endogenous genes enhances the effector function of the immune effector cell. In some embodiments, the gene-regulatory system comprises (a) BCL2L11 , FLI1 , CALM2 , DHODH , UMPS , RBM39 , SEMA7A , CHIC2 , PCBP1 , PBRM1 , WDR6 , E2F8 , SERPINA3 and GNAS A group consisting of; (b) SOCS1 and ANKRD11 It can reduce the expression and / or function of two or more endogenous target genes selected from a group consisting of

[0012] In some embodiments, the present disclosure is IKZF1, IKZF3, GATA3, BCL3, TNIP1, TNFAIP3, NFKBIA, SMAD2, TGFBR1, TGFBR2, TANK, FOXP3 , RC3H1 , TRAF6 , IKZF2, CBLB, PPP2R2D, NRP1, HAVCR2, LAG3, TIGIT, CTLA4, PTPN6, PDCD1 and BCOR A gene-regulatory system capable of reducing the expression and / or function of one or more endogenous target genes selected from the group consisting of, wherein the reduced expression and / or function of the one or more endogenous genes enhances the effector function of the immune effector cells. In some embodiments, the gene-regulatory system IKZF1, IKZF3, GATA3, BCL3, TNIP1, TNFAIP3, NFKBIA, SMAD2, TGFBR1, TGFBR2, TANK, FOXP3 , RC3H1 , TRAF6 , IKZF2, CBLB, PPP2R2D, NRP1, HAVCR2, LAG3, TIGIT, CTLA4, PTPN6, PDCD1 and BCOR The expression and / or function of two or more endogenous target genes selected from the group consisting of can be reduced. In some embodiments, at least one of the endogenous target genes is IKZF1, IKZF3, GATA3, BCL3, TNIP1, TNFAIP3, NFKBIA, SMAD2, TGFBR1, TGFBR2, TANK, FOXP3 , RC3H1 , TRAF6 and IKZF2 Selected from a group consisting of, and at least one of the endogenous target genes CBLB, PPP2R2D, NRP1, HAVCR2, LAG3, TIGIT, CTLA4, PTPN6, PDCD1 and BCOR It is selected from a group consisting of.

[0013] In some embodiments, the present disclosure is (a) BCL2L11 , FLI1 , CALM2 , DHODH , UMPS , RBM39 , SEMA7A , CHIC2 , PCBP1 , PBRM1 , WDR6 , E2F8 , SERPINA3 and GNAS A group consisting of; or (b) SOCS1 and ANKRD11 , and IKZF1, IKZF3, GATA3, BCL3, TNIP1, TNFAIP3, NFKBIA, SMAD2, TGFBR1, TGFBR2, TANK, FOXP3 , RC3H1 , TRAF6 , IKZF2, CBLB, PPP2R2D, NRP1, HAVCR2, LAG3, TIGIT, CTLA4, PTPN6, PDCD1 and BCOR The present invention provides a modified immune effector cell comprising a gene-regulatory system capable of reducing the expression and / or function of one or more endogenous target genes selected from a group consisting of one or more endogenous target genes selected from. In some embodiments, the gene-regulatory system BCL2L11 , FLI1 , CALM2 , DHODH , UMPS , RBM39 , SEMA7A , CHIC2 , PCBP1 , PBRM1 , WDR6 , E2F8 , SERPINA3 and GNAS At least one endogenous target gene selected from the group consisting of and IKZF1, IKZF3, GATA3, BCL3, TNIP1, TNFAIP3, NFKBIA, SMAD2, TGFBR1, TGFBR2, TANK, FOXP3 , RC3H1 , TRAF6 , IKZF2, CBLB, PPP2R2D, NRP1, HAVCR2, LAG3, TIGIT, CTLA4, PTPN6, PDCD1 and BCORIt can reduce the expression and / or function of at least one endogenous target gene selected from a group consisting of . In some embodiments, the gene-regulatory system herein SOCS1 and ANKRD11 At least one endogenous target gene selected from the group consisting of and IKZF1, IKZF3, GATA3, BCL3, TNIP1, TNFAIP3, NFKBIA, SMAD2, TGFBR1, TGFBR2, TANK, FOXP3 , RC3H1 , TRAF6 , IKZF2, CBLB, PPP2R2D, NRP1, HAVCR2, LAG3, TIGIT, CTLA4, PTPN6, PDCD1 and BCOR It can reduce the expression and / or function of at least one endogenous target gene selected from a group consisting of .

[0014] In some embodiments, the gene-regulatory system SOCS1 and CBLB It may reduce the expression and / or function of . In some embodiments, the gene-regulatory system SOCS1 and BCOR It may reduce the expression and / or function of . In some embodiments, the gene-regulatory system SOCS1 and TNFAIP3 It may reduce the expression and / or function of . In some embodiments, the gene-regulatory system ANKRD11 and CBLB It may reduce the expression and / or function of . In some embodiments, the gene-regulatory system ANKRD11 and BCOR It may reduce the expression and / or function of . In some embodiments, the gene-regulatory system ANKRD11 and TNFAIP3 It may reduce the expression and / or function of . In some embodiments, the gene-regulatory system ANKRD11 and SOCS1 It may reduce the expression and / or function of .

[0015] In some embodiments, the gene-regulatory system comprises (i) one or more nucleic acid molecules; (ii) one or more enzyme proteins; or (iii) one or more guide nucleic acid molecules and enzyme proteins. In some embodiments, one or more nucleic acid molecules are selected from siRNA, shRNA, microRNA (miR), antagomiR, or antisense RNA. In some embodiments, the gene-regulatory system comprises siRNA or shRNA nucleic acid molecules.

[0016] In some embodiments, one or more endogenous target genes are IKZF1, IKZF3, GATA3, BCL3, TNIP1, TNFAIP3, NFKBIA, SMAD2, TGFBR1, TGFBR2, TANK, FOXP3 , RC3H1 , TRAF6 , IKZF2, CBLB, PPP2R2D, NRP1, HAVCR2, LAG3, TIGIT, CTLA4, PTPN6, PDCD1 and BCOR Selected from the group consisting of, said siRNA or shRNA molecule comprises about 19 to 30 nucleotides that bind to an RNA sequence encoded by a DNA sequence defined by the set of genome coordinates presented in Tables 5A and 5B. In some embodiments, the siRNA or shRNA comprises about 19 to 30 nucleotides that bind to an RNA sequence encoded by a DNA sequence selected from the group consisting of SEQ ID NOs 154 to 813.

[0017] In some embodiments, one or more endogenous target genes are BCL2L11 , FLI1 , CALM2 , DHODH , UMPS , RBM39 , SEMA7A , CHIC2 , PCBP1 , PBRM1 , WDR6 , E2F8 , SERPINA3 and GNASSelected from the group consisting of, said siRNA or shRNA molecule comprises about 19 to 30 nucleotides that bind to an RNA sequence encoded by a DNA sequence defined by the set of genome coordinates presented in Tables 6A and 6B. In some embodiments, the siRNA or shRNA comprises about 19 to 30 nucleotides that bind to an RNA sequence encoded by a DNA sequence selected from the group consisting of SEQ ID NOs 814 to 1064.

[0018] In some embodiments, one or more endogenous target genes are SOCS1 And, the siRNA or shRNA molecule comprises about 19 to 30 nucleotides that bind to an RNA sequence encoded by a DNA sequence defined by the set of genome coordinates presented in Tables 6C and 6D. In some embodiments, the siRNA or shRNA comprises about 19 to 30 nucleotides that bind to an RNA sequence encoded by a DNA sequence selected from the group consisting of SEQ ID NOs 1088 to 1232. In some embodiments, one or more endogenous target genes are ANKRD11 And, the siRNA or shRNA molecule comprises about 19 to 30 nucleotides that bind to an RNA sequence encoded by a DNA sequence defined by the set of genome coordinates presented in Tables 6E and 6F. In some embodiments, the siRNA or shRNA comprises about 19 to 30 nucleotides that bind to an RNA sequence encoded by a DNA sequence selected from the group consisting of SEQ ID NOs 1065 to 1087.

[0019] In some embodiments, the gene-regulatory system comprises a plurality of siRNA or shRNA molecules and can reduce the expression and / or function of two or more endogenous target genes.

[0020] In some embodiments, at least one of the endogenous target genes is BCL2L11 , FLI1 , CALM2 , DHODH , UMPS , RBM39 , SEMA7A , CHIC2 , PCBP1 , PBRM1 , WDR6 , E2F8 , SERPINA3 and GNAS Selected from a group consisting of, and at least one of the endogenous target genes IKZF1, IKZF3, GATA3, BCL3, TNIP1, TNFAIP3, NFKBIA, SMAD2, TGFBR1, TGFBR2, TANK, FOXP3 , RC3H1 , TRAF6 , IKZF2, CBLB, PPP2R2D, NRP1, HAVCR2, LAG3, TIGIT, CTLA4, PTPN6, PDCD1 and BCOR Selected from the group consisting of. In some embodiments, at least one of the plurality of siRNA or shRNA molecules comprises about 19 to 30 nucleotides that bind to an RNA sequence encoded by a DNA sequence defined by the set of genome coordinates presented in Tables 6A and 6B, and at least one of the plurality of siRNA or shRNA molecules comprises about 19 to 30 nucleotides that bind to an RNA sequence encoded by a DNA sequence defined by the set of genome coordinates presented in Tables 5A and 5B. In some embodiments, at least one of the plurality of siRNA or shRNA molecules comprises about 19 to 30 nucleotides that bind to an RNA sequence encoded by a DNA sequence selected from the group consisting of SEQ ID NOs 814 to 1064, and at least one of the plurality of siRNA or shRNA molecules comprises about 19 to 30 nucleotides that bind to an RNA sequence encoded by a DNA sequence selected from the group consisting of SEQ ID NOs 154 to 813.

[0021] In some embodiments, at least one of the endogenous target genes is BCL2L11 , FLI1 , CALM2 , DHODH , UMPS , RBM39 , SEMA7A , CHIC2 , PCBP1 , PBRM1 , WDR6 , E2F8 , SERPINA3 and GNAS Selected from a group consisting of, and at least one of the endogenous target genes CBLB is. In some embodiments, at least one of a plurality of siRNA or shRNA molecules comprises about 19 to 30 nucleotides that bind to an RNA sequence encoded by a DNA sequence selected from the group consisting of SEQ ID NOs 814 to 1064, and at least one of a plurality of siRNA or shRNA molecules comprises about 19 to 30 nucleotides that bind to an RNA sequence encoded by a DNA sequence selected from the group consisting of SEQ ID NOs 499 to 524. In some embodiments, at least one of the endogenous target genes is BCL2L11 , FLI1 , CALM2 , DHODH , UMPS , RBM39 , SEMA7A , CHIC2 , PCBP1 , PBRM1 , WDR6 , E2F8 , SERPINA3 and GNAS Selected from a group consisting of, and at least one of the endogenous target genes CBLB, TNFAIP3, or BCOR am.

[0022] In some embodiments, at least one of the endogenous target genes is SOCS1 and at least one of the endogenous target genes is IKZF1, IKZF3, GATA3, BCL3, TNIP1, TNFAIP3, NFKBIA, SMAD2, TGFBR1, TGFBR2, TANK, FOXP3, RC3H1, TRAF6, IKZF2, CBLB, PPP2R2D, NRP1, HAVCR2, LAG3, TIGIT, CTLA4, PTPN6, PDCD1 and BCORSelected from the group consisting of. In some embodiments, at least one of the plurality of siRNA or shRNA molecules comprises about 19 to 30 nucleotides that bind to an RNA sequence encoded by a DNA sequence defined by the set of genome coordinates presented in Tables 6C and 6D, and at least one of the plurality of siRNA or shRNA molecules comprises about 19 to 30 nucleotides that bind to an RNA sequence encoded by a DNA sequence defined by the set of genome coordinates presented in Tables 5A and 5B. In some embodiments, at least one of the plurality of siRNA or shRNA molecules comprises about 19 to 30 nucleotides that bind to an RNA sequence encoded by a DNA sequence selected from the group consisting of SEQ ID NOs 1088 to 1232, and at least one of the plurality of siRNA or shRNA molecules comprises about 19 to 30 nucleotides that bind to an RNA sequence encoded by a DNA sequence selected from the group consisting of SEQ ID NOs 154 to 813.

[0023] In some embodiments, at least one of the endogenous target genes is SOCS1 and at least one of the endogenous target genes is CBLB am . In some embodiments, at least one of a plurality of siRNA or shRNA molecules comprises about 19 to 30 nucleotides that bind to an RNA sequence encoded by a DNA sequence selected from the group consisting of SEQ ID NOs 1088 to 1232, and at least one of a plurality of siRNA or shRNA molecules comprises about 19 to 30 nucleotides that bind to an RNA sequence encoded by a DNA sequence selected from the group consisting of SEQ ID NOs 499 to 524. In some embodiments, at least one of the endogenous target genes is SOCS1 and at least one of the endogenous target genes is CBLB, TNFAIP3 or BCOR am.

[0024] In some embodiments, at least one of the endogenous target genes is ANKRD11 and at least one of the endogenous target genes is IKZF1, IKZF3, GATA3, BCL3, TNIP1, TNFAIP3, NFKBIA, SMAD2, TGFBR1, TGFBR2, TANK, FOXP3, RC3H1, TRAF6, IKZF2, CBLB, PPP2R2D, NRP1, HAVCR2, LAG3, TIGIT, CTLA4, PTPN6, PDCD1 and BCOR Selected from the group consisting of. In some embodiments, at least one of the plurality of siRNA or shRNA molecules comprises about 19 to 30 nucleotides that bind to an RNA sequence encoded by a DNA sequence defined by the set of genome coordinates presented in Tables 6E and 6F, and at least one of the plurality of siRNA or shRNA molecules comprises about 19 to 30 nucleotides that bind to an RNA sequence encoded by a DNA sequence defined by the set of genome coordinates presented in Tables 5A and 5B. In some embodiments, at least one of the plurality of siRNA or shRNA molecules comprises about 19 to 30 nucleotides that bind to an RNA sequence encoded by a DNA sequence selected from the group consisting of SEQ ID NOs 1065 to 1087, and at least one of the plurality of siRNA or shRNA molecules comprises about 19 to 30 nucleotides that bind to an RNA sequence encoded by a DNA sequence selected from the group consisting of SEQ ID NOs 154 to 813.

[0025] In some embodiments, at least one of the endogenous target genes is ANKRD11 and at least one of the endogenous target genes is CBLB am .In some embodiments, at least one of a plurality of siRNA or shRNA molecules comprises about 19 to 30 nucleotides that bind to an RNA sequence encoded by a DNA sequence selected from the group consisting of SEQ ID NOs 1065 to 1087, and at least one of a plurality of siRNA or shRNA molecules comprises about 19 to 30 nucleotides that bind to an RNA sequence encoded by a DNA sequence selected from the group consisting of SEQ ID NOs 499 to 524. In some embodiments, at least one of the endogenous target genes is ANKRD11 and at least one of the endogenous target genes is CBLB, TNFAIP3 or BCOR am.

[0026] In some embodiments, at least one of the endogenous target genes is SOCS1 and at least one of the endogenous target genes is ANKRD11 am . In some embodiments, at least one of the plurality of siRNA or shRNA molecules comprises about 19 to 30 nucleotides that bind to an RNA sequence encoded by a DNA sequence defined by the set of genome coordinates presented in Tables 6C and 6D, and at least one of the plurality of siRNA or shRNA molecules comprises about 19 to 30 nucleotides that bind to an RNA sequence encoded by a DNA sequence defined by the set of genome coordinates presented in Tables 6E and 6F. In some embodiments, at least one of the plurality of siRNA or shRNA molecules comprises about 19 to 30 nucleotides that bind to an RNA sequence encoded by a DNA sequence selected from the group consisting of SEQ ID NOs 1065 to 1087, and at least one of the plurality of siRNA or shRNA molecules comprises about 19 to 30 nucleotides that bind to an RNA sequence encoded by a DNA sequence selected from the group consisting of SEQ ID NOs 1088 to 1232.

[0027] In some embodiments, the gene-regulatory system comprises an enzyme protein, said enzyme protein is engineered to specifically bind to a target sequence within one or more of the endogenous genes. In some embodiments, the protein is a transcription activator-like effector nuclease (TALEN), a zinc-finger nuclease, or a meganuclease.

[0028] In some embodiments, the gene-regulatory system comprises a guide nucleic acid molecule and an enzyme protein, said nucleic acid molecule is a guide RNA (gRNA) molecule, and said enzyme protein is a Cas protein or a Cas ortholog.

[0029] In some embodiments, one or more endogenous target genes are IKZF1, IKZF3, GATA3, BCL3, TNIP1, TNFAIP3, NFKBIA, SMAD2, TGFBR1, TGFBR2, TANK, FOXP3 , RC3H1 , TRAF6 , IKZF2, CBLB, PPP2R2D, NRP1, HAVCR2, LAG3, TIGIT, CTLA4, PTPN6, PDCD1 and BCOR Selected from the group consisting of, the gRNA molecule comprises a targeting domain sequence that binds to a nucleic acid sequence defined by the set of genome coordinates presented in Tables 5A and 5B. In some embodiments, the gRNA molecule comprises a targeting domain sequence that binds to a target DNA sequence selected from the group consisting of SEQ ID NOs 154 to 813. In some embodiments, the gRNA molecule comprises a targeting domain sequence encoded by a nucleic acid sequence selected from the group consisting of SEQ ID NOs 154 to 813.

[0030] In some embodiments, one or more endogenous target genes are BCL2L11 , FLI1 , CALM2 , DHODH , UMPS , RBM39 , SEMA7A , CHIC2 , PCBP1 , PBRM1 , WDR6 , E2F8, SERPINA3 and GNAS Selected from the group consisting of, wherein the gRNA molecule comprises a targeting domain sequence that binds to a nucleic acid sequence defined by the set of genome coordinates presented in Tables 6A and 6B. In some embodiments, the gRNA molecule comprises a targeting domain sequence that binds to a target DNA sequence selected from the group consisting of SEQ ID NOs 814 to 1064. In some embodiments, the gRNA molecule comprises a targeting domain sequence encoded by a nucleic acid sequence selected from the group consisting of SEQ ID NOs 814 to 1064.

[0031] In some embodiments, one or more endogenous target genes are SOCS1 And, the gRNA molecule comprises a targeting domain sequence that binds to a nucleic acid sequence defined by the set of genome coordinates presented in Tables 6C and 6D. In some embodiments, the gRNA molecule comprises a targeting domain sequence that binds to a target DNA sequence selected from the group consisting of SEQ ID NOs 1088 to 1232. In some embodiments, the gRNA molecule comprises a targeting domain sequence encoded by a nucleic acid sequence selected from the group consisting of SEQ ID NOs 1088 to 1232.

[0032] In some embodiments, one or more endogenous target genes are ANKRD11 And, the gRNA molecule comprises a targeting domain sequence that binds to a nucleic acid sequence defined by the set of genome coordinates presented in Tables 6E and 6F. In some embodiments, the gRNA molecule comprises a targeting domain sequence that binds to a target DNA sequence selected from the group consisting of SEQ ID NOs 1065 to 1087. In some embodiments, the gRNA molecule comprises a targeting domain sequence encoded by a nucleic acid sequence selected from the group consisting of SEQ ID NOs 1065 to 1087.

[0033] In some embodiments, the gene-regulatory system comprises a plurality of gRNA molecules and can reduce the expression and / or function of two or more endogenous target genes.

[0034] In some embodiments, at least one of the endogenous target genes is BCL2L11 , FLI1 , CALM2 , DHODH , UMPS , RBM39 , SEMA7A , CHIC2 , PCBP1 , PBRM1 , WDR6 , E2F8 , SERPINA3 and GNAS Selected from a group consisting of, and at least one of the endogenous target genes IKZF1, IKZF3, GATA3, BCL3, TNIP1, TNFAIP3, NFKBIA, SMAD2, TGFBR1, TGFBR2, TANK, FOXP3 , RC3H1 , TRAF6 , IKZF2, CBLB, PPP2R2D, NRP1, HAVCR2, LAG3, TIGIT, CTLA4, PTPN6, PDCD1 and BCORSelected from the group consisting of. In some embodiments, at least one of the plurality of gRNA molecules comprises a targeting domain sequence that binds to a nucleic acid sequence defined by the set of genome coordinates presented in Tables 6A and 6B, and at least one of the plurality of gRNA molecules comprises a targeting domain sequence that binds to a nucleic acid sequence defined by the set of genome coordinates presented in Tables 5A and 5B. In some embodiments, at least one of the plurality of gRNA molecules comprises a targeting domain sequence that binds to a target DNA sequence selected from the group consisting of SEQ ID NOs 814 to 1064, and at least one of the plurality of gRNA molecules comprises a targeting domain sequence that binds to a target DNA sequence selected from the group consisting of SEQ ID NOs 154 to 813. In some embodiments, at least one of the plurality of gRNA molecules comprises a targeting domain sequence encoded by a nucleic acid sequence selected from the group consisting of SEQ ID NOs 814 to 1064, and at least one of the plurality of gRNA molecules comprises a targeting domain sequence encoded by a nucleic acid sequence selected from the group consisting of SEQ ID NOs 154 to 813.

[0035] In some embodiments, at least one of the endogenous target genes is BCL2L11 , FLI1 , CALM2 , DHODH , UMPS , RBM39 , SEMA7A , CHIC2 , PCBP1 , PBRM1 , WDR6 , E2F8 , SERPINA3 and GNAS Selected from a group consisting of, and at least one of the endogenous target genes CBLBis. In some embodiments, at least one of the plurality of gRNA molecules comprises a targeting domain sequence that binds to a target DNA sequence selected from the group consisting of SEQ ID NOs 814 to 1064, and at least one of the plurality of gRNA molecules comprises a targeting domain sequence that binds to a target DNA sequence selected from the group consisting of SEQ ID NOs 499 to 524. In some embodiments, at least one of the plurality of gRNA molecules comprises a targeting domain sequence encoded by a nucleic acid sequence selected from the group consisting of SEQ ID NOs 814 to 1064, and at least one of the plurality of gRNA molecules comprises a targeting domain sequence encoded by a nucleic acid sequence selected from the group consisting of SEQ ID NOs 499 to 524. In some embodiments, at least one of the endogenous target gene is BCL2L11 , FLI1 , CALM2 , DHODH , UMPS , RBM39 , SEMA7A , CHIC2 , PCBP1 , PBRM1 , WDR6 , E2F8 , SERPINA3 and GNAS Selected from a group consisting of, and at least one of the endogenous target genes CBLB, TNFAIP3 or BCOR am.

[0036] In some embodiments, at least one of the endogenous target genes is SOCS1 and at least one of the endogenous target genes is IKZF1, IKZF3, GATA3, BCL3, TNIP1, TNFAIP3, NFKBIA, SMAD2, TGFBR1, TGFBR2, TANK, FOXP3 , RC3H1 , TRAF6 , IKZF2, CBLB, PPP2R2D, NRP1, HAVCR2, LAG3, TIGIT, CTLA4, PTPN6, PDCD1 and BCORSelected from the group consisting of. In some embodiments, at least one of the plurality of gRNA molecules comprises a targeting domain sequence that binds to a nucleic acid sequence defined by the set of genome coordinates presented in Tables 6C and 6D, and at least one of the plurality of gRNA molecules comprises a targeting domain sequence that binds to a nucleic acid sequence defined by the set of genome coordinates presented in Tables 5A and 5B. In some embodiments, at least one of the plurality of gRNA molecules comprises a targeting domain sequence that binds to a target DNA sequence selected from the group consisting of SEQ ID NOs 1088 to 1232, and at least one of the plurality of gRNA molecules comprises a targeting domain sequence that binds to a target DNA sequence selected from the group consisting of SEQ ID NOs 154 to 813. In some embodiments, at least one of the plurality of gRNA molecules comprises a targeting domain sequence encoded by a nucleic acid sequence selected from the group consisting of SEQ ID NOs 1088 to 1232, and at least one of the plurality of gRNA molecules comprises a targeting domain sequence encoded by a nucleic acid sequence selected from the group consisting of SEQ ID NOs 154 to 813.

[0037] In some embodiments, at least one of the endogenous target genes is SOCS1 and at least one of the endogenous target genes is CBLB am .In some embodiments, at least one of the plurality of gRNA molecules comprises a targeting domain sequence that binds to a target DNA sequence selected from the group consisting of SEQ ID NOs 1088 to 1232, and at least one of the plurality of gRNA molecules comprises a targeting domain sequence. In some embodiments, at least one of the plurality of gRNA molecules comprises a targeting domain sequence encoded by a nucleic acid sequence selected from the group consisting of SEQ ID NOs 1088 to 1232, and at least one of the plurality of gRNA molecules comprises a targeting domain sequence encoded by a nucleic acid sequence selected from the group consisting of SEQ ID NOs 499 to 524. In some embodiments, at least one of the endogenous target gene is SOCS1 and at least one of the endogenous target genes is CBLB, TNFAIP3 or BCOR am .

[0038] In some embodiments, at least one of the endogenous target genes is ANKRD11 and at least one of the endogenous target genes is IKZF1, IKZF3, GATA3, BCL3, TNIP1, TNFAIP3, NFKBIA, SMAD2, TGFBR1, TGFBR2, TANK, FOXP3 , RC3H1 , TRAF6 , IKZF2, CBLB, PPP2R2D, NRP1, HAVCR2, LAG3, TIGIT, CTLA4, PTPN6, PDCD1 and BCORSelected from the group consisting of. In some embodiments, at least one of the plurality of gRNA molecules comprises a targeting domain sequence that binds to a nucleic acid sequence defined by the set of genome coordinates presented in Tables 6E and 6F, and at least one of the plurality of gRNA molecules comprises a targeting domain sequence that binds to a nucleic acid sequence defined by the set of genome coordinates presented in Tables 5A and 5B. In some embodiments, at least one of the plurality of gRNA molecules comprises a targeting domain sequence that binds to a target DNA sequence selected from the group consisting of SEQ ID NOs 1065 to 1087, and at least one of the plurality of gRNA molecules comprises a targeting domain sequence that binds to a target DNA sequence selected from the group consisting of SEQ ID NOs 154 to 813. In some embodiments, at least one of the plurality of gRNA molecules comprises a targeting domain sequence encoded by a nucleic acid sequence selected from the group consisting of SEQ ID NOs 1065 to 1087, and at least one of the plurality of gRNA molecules comprises a targeting domain sequence that binds to a target DNA sequence selected from the group consisting of SEQ ID NOs 154 to 813.

[0039] In some embodiments, at least one of the endogenous target genes is ANKRD11 and at least one of the endogenous target genes is CBLB am .In some embodiments, at least one of the plurality of gRNA molecules comprises a targeting domain sequence that binds to a target DNA sequence selected from the group consisting of SEQ ID NOs 1065 to 1087, and at least one of the plurality of gRNA molecules comprises a targeting domain sequence that binds to a target DNA sequence selected from the group consisting of SEQ ID NOs 499 to 524. In some embodiments, at least one of the plurality of gRNA molecules comprises a targeting domain sequence encoded by a nucleic acid sequence selected from the group consisting of SEQ ID NOs 1065 to 1087, and at least one of the plurality of gRNA molecules comprises a targeting domain sequence encoded by a nucleic acid sequence selected from the group consisting of SEQ ID NOs 499 to 524. In some embodiments, at least one of the endogenous target gene is ANKRD11 and at least one of the endogenous target genes is CBLB, TNFAIP3 or BCOR am .

[0040] In some embodiments, at least one of the endogenous target genes is SOCS1 and at least one of the endogenous target genes is ANKRD11 am .In some embodiments, at least one of the plurality of gRNA molecules comprises a targeting domain sequence that binds to a nucleic acid sequence defined by the set of genome coordinates presented in Tables 6E and 6F, and at least one of the plurality of gRNA molecules comprises a targeting domain sequence that binds to a nucleic acid sequence defined by the set of genome coordinates presented in Tables 6C and 6D. In some embodiments, at least one of the plurality of gRNA molecules comprises a targeting domain sequence that binds to a target DNA sequence selected from the group consisting of SEQ ID NOs 1065 to 1087, and at least one of the plurality of gRNA molecules comprises a targeting domain sequence that binds to a target DNA sequence selected from the group consisting of SEQ ID NOs 1088 to 1232. In some embodiments, at least one of the plurality of gRNA molecules comprises a targeting domain sequence encoded by DNA selected from the group consisting of SEQ ID NOs 1065 to 1087, and at least one of the plurality of gRNA molecules comprises a targeting domain sequence encoded by a DNA sequence selected from the group consisting of SEQ ID NOs 1088 to 1232.

[0041] In some embodiments, the modified immune effector cell comprises a Cas protein, wherein the Cas protein is a wild-type Cas protein comprising two enzyme-active domains and capable of inducing a double-stranded DNA break; the Cas protein is a Cas nickase mutant comprising one enzyme-active domain and capable of inducing a single-stranded DNA break; or the Cas protein is a deactivated Cas protein (dCas) and is associated with a heterologous protein capable of regulating the expression of one or more endogenous target genes. In some embodiments, the Cas protein is a Cas9 protein. In some embodiments, the heterologous protein is selected from the group consisting of MAX-interacting protein 1 (MXI1), Krueppel-associated box (KRAB) domain, methyl-CpG binding protein 2 (MECP2) and four concatenated mSin3 domains (SID4X).

[0042] In some embodiments, the gene-regulatory system introduces an inactivation mutation into one or more endogenous target genes. In some embodiments, the inactivation mutation comprises a deletion, substitution, or insertion of one or more nucleotides within the genomic sequences of two or more endogenous genes. In some embodiments, the deletion is a partial or complete deletion of two or more endogenous target genes. In some embodiments, the inactivation mutation is a frame shift mutation. In some embodiments, the inactivation mutation reduces the expression and / or function of two or more endogenous target genes.

[0043] In some embodiments, the gene-regulatory system is introduced into an immune effector cell by transfusion, transduction, electroporation, or physical disruption of the cell membrane by a microfluidics device. In some embodiments, the gene-regulatory system is introduced as a polynucleotide encoding one or more components of the system, a protein or a ribonucleoprotein (RNP) complex.

[0044] In some embodiments, the present disclosure is IKZF1, IKZF3, GATA3, BCL3, TNIP1, TNFAIP3, NFKBIA, SMAD2, TGFBR1, TGFBR2, TANK, FOXP3 , RC3H1 , TRAF6 , IKZF2, CBLB, PPP2R2D, NRP1, HAVCR2, LAG3, TIGIT, CTLA4, PTPN6, PDCD1 and BCOR Provides modified immune effector cells comprising reduced expression and / or function of one or more endogenous genes selected from a group consisting of, wherein the reduced expression and / or function of one or more endogenous genes enhances the effector function of the immune effector cells.

[0045] In some embodiments, the present disclosure is (a) IKZF1, IKZF3, GATA3, BCL3, TNIP1, TNFAIP3, NFKBIA, SMAD2, TGFBR1, TGFBR2, TANK, FOXP3 , RC3H1 , TRAF6 and IKZF2 A group consisting of; or (b) CBLB, PPP2R2D, NRP1, HAVCR2, LAG3, TIGIT, CTLA4, PTPN6, PDCD1 and BCOR Provides modified immune effector cells comprising reduced expression and / or function of one or more endogenous genes selected from a group consisting of, wherein the reduced expression and / or function of one or more endogenous genes enhances the effector function of the immune effector cells.

[0046] In some embodiments, the present disclosure is (a) BCL2L11 , FLI1 , CALM2 , DHODH , UMPS , RBM39 , SEMA7A , CHIC2 , PCBP1 , PBRM1 , WDR6 , E2F8 , SERPINA3 and GNASA group consisting of; (b) SOCS1 ; or (c) ANKRD11 Provides a modified immune effector cell comprising reduced expression and / or function of one or more endogenous genes selected from, wherein the reduced expression and / or function of one or more endogenous genes enhances the effector function of the modified immune effector cell. In some embodiments, the modified immune effector cell is SOCS and ANKRD11 Reduced expression and / or function of Includes.

[0047] In some embodiments, the present disclosure is IKZF1, IKZF3, GATA3, BCL3, TNIP1, TNFAIP3, NFKBIA, SMAD2, TGFBR1, TGFBR2, TANK, FOXP3 , RC3H1 , TRAF6 , IKZF2, CBLB, PPP2R2D, NRP1, HAVCR2, LAG3, TIGIT, CTLA4, PTPN6, PDCD1 and BCOR Provides a modified immune effector cell comprising reduced expression and / or function of two or more target genes selected from, wherein the reduced expression and / or function of two or more endogenous genes enhances the effector function of the modified immune effector cell. In some embodiments, the modified immune effector cell is CBLB and BCOR Reduced expression and / or function of Includes.

[0048] In some embodiments, the present disclosure provides a modified immune effector cell comprising reduced expression and / or function of two or more target genes, wherein at least one target gene is BCL2L11 , FLI1 , CALM2 , DHODH , UMPS , RBM39 , SEMA7A , CHIC2 , PCBP1 , PBRM1 , WDR6 , E2F8 , SERPINA3 and GNAS Selected from a group consisting of, At least one type of target gene is IKZF1, IKZF3, GATA3, BCL3, TNIP1, TNFAIP3, NFKBIA, SMAD2, TGFBR1, TGFBR2, TANK, FOXP3 , RC3H1 , TRAF6 , IKZF2 , CBLB, PPP2R2D, NRP1, HAVCR2, LAG3, TIGIT, CTLA4, PTPN6, PDCD1 and BCOR Selected from a group consisting of, reduced expression and / or function of two or more endogenous genes enhances the effector function of modified immune effector cells.

[0049] In some embodiments, the present disclosure provides a modified immune effector cell comprising reduced expression and / or function of two or more target genes, wherein at least one target gene is BCL2L11 , FLI1 , CALM2 , DHODH , UMPS , RBM39 , SEMA7A , CHIC2 , PCBP1 , PBRM1 , WDR6 , E2F8 , SERPINA3 and GNAS Selected from a group consisting of, At least one type of target gene is CBLB am.

[0050] In some embodiments, the present disclosure provides a modified immune effector cell comprising reduced expression and / or function of two or more target genes, wherein at least one target gene is SOCS1 Selected from a group consisting of, At least one type of target gene is IKZF1, IKZF3, GATA3, BCL3, TNIP1, TNFAIP3, NFKBIA, SMAD2, TGFBR1, TGFBR2, TANK, FOXP3 , RC3H1 , TRAF6 , IKZF2 , CBLB, PPP2R2D, NRP1, HAVCR2, LAG3, TIGIT, CTLA4, PTPN6, PDCD1 and BCOR Selected from the group consisting of, and reduced expression and / or function of two or more endogenous genes enhances the effector function of modified immune effector cells. In some embodiments, the modified immune effector cells are SOCS1 and CBLB Reduced expression and / or function of Includes. In some embodiments, modified immune effector cells SOCS1 and TNFAIP3Reduced expression and / or function of Includes. In some embodiments, modified immune effector cells SOCS1 and BCOR Reduced expression and / or function of Includes.

[0051] In some embodiments, the present disclosure provides a modified immune effector cell comprising reduced expression and / or function of two or more target genes, wherein at least one target gene is ANKRD11 Selected from a group consisting of, At least one type of target gene is IKZF1, IKZF3, GATA3, BCL3, TNIP1, TNFAIP3, NFKBIA, SMAD2, TGFBR1, TGFBR2, TANK, FOXP3 , RC3H1 , TRAF6 , IKZF2 , CBLB, PPP2R2D, NRP1, HAVCR2, LAG3, TIGIT, CTLA4, PTPN6, PDCD1 and BCOR Selected from the group consisting of, and reduced expression and / or function of two or more endogenous genes enhances the effector function of modified immune effector cells. In some embodiments, the modified immune effector cells are ANKRD11 and CBLB Reduced expression and / or function of Includes. In some embodiments, modified immune effector cells ANKRD11 and TNFAIP3 Reduced expression and / or function of Includes. In some embodiments, modified immune effector cells ANKRD11 and BCOR Reduced expression and / or function of Includes.

[0052] In some embodiments, the present disclosure is IKZF1, IKZF3, GATA3, BCL3, TNIP1, TNFAIP3, NFKBIA, SMAD2, TGFBR1, TGFBR2, TANK, FOXP3 , RC3H1 , TRAF6 , IKZF2, CBLB, PPP2R2D, NRP1, HAVCR2, LAG3, TIGIT, CTLA4, PTPN6, PDCD1 and BCOR Provides modified immune effector cells containing inactivated mutations within one or more endogenous genes selected from a group consisting of

[0053] In some embodiments, the present disclosure is (a) IKZF1, IKZF3, GATA3, BCL3, TNIP1, TNFAIP3, NFKBIA, SMAD2, TGFBR1, TGFBR2, TANK, FOXP3 , RC3H1, TRAF6 , and IKZF2 A group composed of ; or (b) CBLB, PPP2R2D, NRP1, HAVCR2, LAG3, TIGIT, CTLA4, PTPN6, PDCD1 and BCOR Provides modified immune effector cells containing inactivated mutations within one or more endogenous genes selected from a group consisting of

[0054] In some embodiments, the present disclosure is (a) BCL2L11 , FLI1 , CALM2 , DHODH , UMPS , RBM39 , SEMA7A , CHIC2 , PCBP1 , PBRM1 , WDR6 , E2F8 , SERPINA3 and GNAS A group consisting of; (b) SOCS1 ; or (c) ANKRD11 Provides a modified immune effector cell comprising an inactivated mutation within one or more endogenous genes selected from. In some embodiments, the modified immune effector cell is SOCS1 and ANKRD11 It contains inactivated mutations.

[0055] A modified immune effector cell comprising inactivated mutations within two or more target genes selected from IKZF1, IKZF3, GATA3, BCL3, TNIP1, TNFAIP3, NFKBIA, SMAD2, TGFBR1, TGFBR2, TANK, FOXP3, RC3H1, TRAF6, IKZF2, CBLB, PPP2R2D, NRP1, HAVCR2, LAG3, TIGIT, CTLA4, PTPN6, PDCD1, and BCOR. In some embodiments, the modified immune effector cell is CBLB and BCOR It contains inactivated mutations within the gene.

[0056] In some embodiments, the present disclosure provides a modified immune effector cell comprising an inactivated mutation in two or more target genes, wherein at least one target gene is BCL2L11 , FLI1 , CALM2 , DHODH , UMPS , RBM39 , SEMA7A , CHIC2 , PCBP1 , PBRM1 , WDR6 , E2F8 , SERPINA3 and GNAS Selected from a group consisting of, At least one type of target gene is IKZF1, IKZF3, GATA3, BCL3, TNIP1, TNFAIP3, NFKBIA, SMAD2, TGFBR1, TGFBR2, TANK, FOXP3 , RC3H1 , TRAF6 , IKZF2 , CBLB, PPP2R2D, NRP1, HAVCR2, LAG3, TIGIT, CTLA4, PTPN6, PDCD1 and BCOR It is selected from a group consisting of.

[0057] In some embodiments, the present disclosure provides a modified immune effector cell comprising an inactivated mutation in two or more target genes, wherein at least one target gene is BCL2L11 , FLI1 , CALM2 , DHODH , UMPS , RBM39 , SEMA7A , CHIC2 , PCBP1 , PBRM1 , WDR6 , E2F8 , SERPINA3 and GNAS Selected from a group consisting of, At least one type of target gene is CBLB am.

[0058] In some embodiments, the present disclosure provides a modified immune effector cell comprising an inactivated mutation in two or more target genes, wherein at least one target gene is SOCS1 And, At least one type of target gene is IKZF1, IKZF3, GATA3, BCL3, TNIP1, TNFAIP3, NFKBIA, SMAD2, TGFBR1, TGFBR2, TANK, FOXP3 , RC3H1 , TRAF6 , IKZF2 , CBLB, PPP2R2D, NRP1, HAVCR2, LAG3, TIGIT, CTLA4, PTPN6, PDCD1 and BCOR It is selected from the group consisting of. In some embodiments, the modified immune effector cells are SOCS1 and TNFAIP3 It contains inactivated mutations within the gene. In some embodiments, the modified immune effector cells are SOCS1 and BCOR It contains inactivated mutations within the gene. In some embodiments, the modified immune effector cells are SOCS1 and CBLB It contains inactivated mutations within the gene.

[0059] In some embodiments, the present disclosure provides a modified immune effector cell comprising an inactivated mutation in two or more target genes, wherein at least one target gene is ANKRD11 And, At least one type of target gene is IKZF1, IKZF3, GATA3, BCL3, TNIP1, TNFAIP3, NFKBIA, SMAD2, TGFBR1, TGFBR2, TANK, FOXP3 , RC3H1 , TRAF6 , IKZF2 , CBLB, PPP2R2D, NRP1, HAVCR2, LAG3, TIGIT, CTLA4, PTPN6, PDCD1 and BCOR It is selected from the group consisting of. In some embodiments, the modified immune effector cells are ANKRD11 and TNFAIP3 It contains inactivated mutations within the gene. In some embodiments, the modified immune effector cells are ANKRD11 and BCOR It contains inactivated mutations within the gene. In some embodiments, the modified immune effector cells are ANKRD11 and CBLB It contains inactivated mutations within the gene.

[0060] In some embodiments, the inactivation mutation comprises the deletion, substitution, or insertion of one or more nucleotides within the genomic sequences of two or more endogenous genes. In some embodiments, the deletion is a partial or complete deletion of two or more endogenous target genes. In some embodiments, the inactivation mutation is a frameshift mutation. In some embodiments, the inactivation mutation reduces the expression and / or function of two or more endogenous target genes. In some embodiments, the expression of one or more endogenous target genes is reduced by at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% compared to unmodified or control immune effector cells. In some embodiments, the function of one or more endogenous target genes is reduced by at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% compared to unmodified or control immune effector cells.

[0061] In some embodiments, the modified immune effector cell further comprises a modified immune receptor presented on the cell surface. In some embodiments, the modified immune receptor is a CAR comprising an antigen-binding domain, a transmembrane domain, and an intracellular signaling domain. In some embodiments, the modified immune receptor is a modified TCR. In some embodiments, the modified immune receptor specifically binds to an antigen expressed on a target cell, wherein the antigen is a tumor-associated antigen.

[0062] In some embodiments, the modified immune effector cell further comprises an exogenous transgene expressing an immune-activating molecule. In some embodiments, the immune-activating molecule is selected from the group consisting of cytokines, chemokines, co-stimulating molecules, activating peptides, antibodies, or antigen-binding fragments thereof. In some embodiments, the antibody or its binding fragment is NRP1, HAVCR2, LAG3, TIGIT, CTLA4 or PDCD1It specifically binds to proteins encoded by and inhibits their function.

[0063] In some embodiments, the immune effector cell is, and in some embodiments, the lymphocyte is a tumor infiltrating lymphocyte (TIL).

[0064] In some embodiments, the effector function is selected from cell proliferation, cell survival, tumor infiltration, cytotoxicity, anti-tumor immune response, and / or resistance to consumption.

[0065] In some embodiments, the present disclosure provides a composition comprising the modified immune effector cells described herein. In some embodiments, the composition further comprises a pharmaceutically acceptable carrier or diluent. In some embodiments, the composition comprises at least 1 × 10 4 , 1×10 5 or 1×10 6 It comprises modified immune effector cells. In some embodiments, the composition is suitable for administration to a subject in need of it. In some embodiments, the composition comprises autologous immune effector cells derived from a subject in need of it. In some embodiments, the composition comprises allogeneic immune effector cells derived from a donor subject.

[0066] In some embodiments, the present disclosure is (a) IKZF1, IKZF3, GATA3, BCL3, TNIP1, TNFAIP3, NFKBIA, SMAD2, TGFBR1, TGFBR2, TANK, FOXP3 , RC3H1 , TRAF6 and IKZF2 A group consisting of; or (b) CBLB, PPP2R2D, NRP1, HAVCR2, LAG3, TIGIT, CTLA4, PTPN6, PDCD1 and BCOR A gene-regulatory system capable of reducing the expression and / or function of one or more endogenous target genes in cells selected from a group consisting of, wherein the system comprises (i) a nucleic acid molecule; (ii) an enzyme; or (iii) a guide nucleic acid molecule and an enzyme protein.

[0067] In some embodiments, the present disclosure is (a) BCL2L11 , FLI1 , CALM2 , DHODH , UMPS , RBM39 , SEMA7A , CHIC2 , PCBP1 , PBRM1 , WDR6 , E2F8 , SERPINA3 and GNAS A group consisting of; (b) SOCS1 ; or (b) ANKRD11 A gene-regulatory system capable of reducing the expression of one or more endogenous target genes in cells selected from, wherein the system comprises (i) a nucleic acid molecule; (ii) an enzyme; or (iii) a guide nucleic acid molecule and an enzyme protein.

[0068] In some embodiments, the system includes a guide RNA (gRNA) nucleic acid molecule and a Cas endonuclease.

[0069] In some embodiments, one or more endogenous target genes are IKZF1, IKZF3, GATA3, BCL3, TNIP1, TNFAIP3, NFKBIA, SMAD2, TGFBR1, TGFBR2, TANK, FOXP3 , RC3H1 , TRAF6 and IKZF2 Selected from, CBLB, PPP2R2D, NRP1, HAVCR2, LAG3, TIGIT, CTLA4, PTPN6, PDCD1 and BCOR Selected from, wherein the gRNA molecule comprises a targeting domain sequence complementary to the target DNA sequence defined by the set of genomic coordinates presented in Tables 5A and 5B. In some embodiments, one or more endogenous target genes are IKZF1, IKZF3, GATA3, BCL3, TNIP1, TNFAIP3, NFKBIA, SMAD2, TGFBR1, TGFBR2, TANK, FOXP3 , RC3H1 , TRAF6 and IKZF2 Selected from, the gRNA molecule comprises a targeting domain sequence that binds to a target DNA sequence selected from the group consisting of SEQ ID NOs 154 to 498. In some embodiments, the gRNA molecule comprises a targeting domain sequence encoded by a nucleic acid sequence selected from the group consisting of SEQ ID NOs 154 to 498.

[0070] In some embodiments, one or more endogenous target genes are CBLB, PPP2R2D, NRP1, HAVCR2, LAG3, TIGIT, CTLA4, PTPN6, PDCD1 and BCOR Selected from, wherein the gRNA molecule comprises a targeting domain sequence that binds to a target DNA sequence selected from the group consisting of SEQ ID NOs 499 to 813. In some embodiments, the gRNA molecule comprises a targeting domain sequence encoded by a nucleic acid sequence selected from the group consisting of SEQ ID NOs 499 to 813.

[0071] In some embodiments, one or more endogenous target genes are BCL2L11 , FLI1 , CALM2 , DHODH , UMPS , RBM39 , SEMA7A , CHIC2 , PCBP1 , PBRM1 , WDR6 , E2F8 , SERPINA3 and GNAS Selected from, wherein the gRNA molecule comprises a targeting domain sequence that binds to a target DNA sequence defined by the set of genome coordinates presented in Tables 6A and 6B. In some embodiments, the gRNA molecule comprises a targeting domain sequence that binds to a target DNA sequence selected from the group consisting of SEQ ID NOs 814 to 1064. In some embodiments, the gRNA molecule comprises a targeting domain sequence encoded by a nucleic acid sequence selected from the group consisting of SEQ ID NOs 814 to 1064.

[0072] In some embodiments, one or more endogenous target genes are SOCS1It comprises, wherein the gRNA molecule comprises a targeting domain sequence that binds to a target DNA sequence defined by the set of genome coordinates presented in Tables 6C and 6D. In some embodiments, the gRNA molecule comprises a targeting domain sequence that binds to a target DNA sequence selected from the group consisting of SEQ ID NOs 1088 to 1232. In some embodiments, the gRNA molecule comprises a targeting domain sequence encoded by a nucleic acid sequence selected from the group consisting of SEQ ID NOs 1088 to 1232.

[0073] In some embodiments, one or more endogenous target genes are ANKRD11 It comprises, wherein the gRNA molecule comprises a targeting domain sequence that binds to a target DNA sequence defined by the set of genome coordinates presented in Tables 6E and 6F. In some embodiments, the gRNA molecule comprises a targeting domain sequence that binds to a target DNA sequence selected from the group consisting of SEQ ID NOs 1065 to 1087. In some embodiments, the gRNA molecule comprises a targeting domain sequence encoded by a nucleic acid sequence selected from the group consisting of SEQ ID NOs 1065 to 1087.

[0074] In some embodiments, the gene-regulatory system comprises a siRNA or shRNA nucleic acid molecule. In some embodiments, one or more endogenous target genes are IKZF1, IKZF3, GATA3, BCL3, TNIP1, TNFAIP3, NFKBIA, SMAD2, TGFBR1, TGFBR2, TANK, FOXP3 , RC3H1 , TRAF6 and IKZF2 Selected from, CBLB, PPP2R2D, NRP1, HAVCR2, LAG3, TIGIT, CTLA4, PTPN6, PDCD1 and BCOR Selected from, said siRNA or shRNA molecule comprises about 19 to 30 nucleotides that bind to an RNA sequence encoded by a DNA sequence defined by the set of genome coordinates presented in Tables 5A and 5B. In some embodiments, one or more endogenous target genes are IKZF1, IKZF3, GATA3, BCL3, TNIP1, TNFAIP3, NFKBIA, SMAD2, TGFBR1, TGFBR2, TANK, FOXP3 , RC3H1 , TRAF6 , and IKZF2 Selected from, wherein the siRNA or shRNA molecule comprises about 19 to 30 nucleotides that bind to an RNA sequence encoded by a DNA sequence selected from SEQ ID NOs 154 to 498. In some embodiments, one or more endogenous target genes are CBLB, PPP2R2D, NRP1, HAVCR2, LAG3, TIGIT, CTLA4, PTPN6, PDCD1 및 BCOR Selected from, wherein the siRNA or shRNA molecule comprises about 19 to 30 nucleotides that bind to an RNA sequence encoded by a DNA sequence selected from SEQ ID NOs 499 to 813.

[0075] In some embodiments, one or more endogenous target genes are BCL2L11 , FLI1 , CALM2 , DHODH , UMPS , RBM39 , SEMA7A , CHIC2 , PCBP1 , PBRM1 , WDR6 , E2F8 , SERPINA3 and GNAS Selected from, the siRNA or shRNA molecule comprises about 19 to 30 nucleotides that bind to an RNA sequence encoded by a DNA sequence defined by the set of genome coordinates presented in Tables 6A and 6B. In some embodiments, the siRNA or shRNA molecule comprises about 19 to 30 nucleotides that bind to an RNA sequence encoded by a DNA sequence selected from SEQ ID NOs 814 to 1064.

[0076] In some embodiments, one or more endogenous target genes are SOCS1It comprises, wherein the siRNA or shRNA molecule comprises about 19 to 30 nucleotides that bind to an RNA sequence encoded by a DNA sequence defined by the set of genome coordinates presented in Tables 6C and 6D. In some embodiments, the siRNA or shRNA molecule comprises about 19 to 30 nucleotides that bind to an RNA sequence encoded by a DNA sequence selected from SEQ ID NOs 1088 to 1232. In some embodiments, one or more endogenous target genes are ANKRD11 It comprises, wherein the siRNA or shRNA molecule comprises about 19 to 30 nucleotides that bind to an RNA sequence encoded by a DNA sequence defined by the set of genome coordinates presented in Tables 6E and 6F. In some embodiments, the siRNA or shRNA molecule comprises about 19 to 30 nucleotides that bind to an RNA sequence encoded by a DNA sequence selected from SEQ ID NOs 1065 to 1087.

[0077] In some embodiments, the present disclosure provides a gene-regulatory system capable of reducing the expression and / or function of two or more endogenous target genes in a cell, wherein at least one of the endogenous target genes is (a) BCL2L11 , FLI1 , CALM2 , DHODH , UMPS , RBM39 , SEMA7A , CHIC2 , PCBP1 , PBRM1 , WDR6 , E2F8 , SERPINA3 and GNAS A group consisting of; (b) SOCS1 ; or (b) ANKRD11 Selected from, and at least one of the endogenous target genes is (a) IKZF1, IKZF3, GATA3, BCL3, TNIP1, TNFAIP3, NFKBIA, SMAD2, TGFBR1, TGFBR2, TANK, FOXP3 , RC3H1 , TRAF6 and IKZF2 A group consisting of; or (b) CBLB, PPP2R2D, NRP1, HAVCR2, LAG3, TIGIT, CTLA4, PTPN6, PDCD1 and BCOR Selected from the group consisting of, wherein the system comprises (i) a nucleic acid molecule; (ii) an enzyme; or (iii) a guide nucleic acid molecule and an enzyme protein.

[0078] In some embodiments, the system comprises a plurality of guide RNA (gRNA) nucleic acid molecules and Cas endonucleases.

[0079] In some embodiments, at least one of the endogenous target genes is BCL2L11 , FLI1 , CALM2 , DHODH , UMPS , RBM39 , SEMA7A , CHIC2 , PCBP1 , PBRM1 , WDR6 , E2F8 , SERPINA3 and GNAS Selected from a group consisting of, and at least one of the endogenous target genes IKZF1, IKZF3, GATA3, BCL3, TNIP1, TNFAIP3, NFKBIA, SMAD2, TGFBR1, TGFBR2, TANK, FOXP3 , RC3H1 , TRAF6 , IKZF2, CBLB, PPP2R2D, NRP1, HAVCR2, LAG3, TIGIT, CTLA4, PTPN6, PDCD1 and BCORSelected from the group consisting of. In some embodiments, at least one of the plurality of gRNAs binds to a target DNA sequence defined by the set of genome coordinates presented in Tables 6A and 6B, wherein at least one of the plurality of gRNAs binds to a target DNA sequence defined by the set of genome coordinates presented in Tables 5A and 5B. In some embodiments, at least one of the plurality of gRNA molecules comprises a targeting domain sequence that binds to a target DNA sequence selected from the group consisting of SEQ ID NOs 814 to 1064, wherein at least one of the plurality of gRNA molecules comprises a targeting domain sequence that binds to a target DNA sequence selected from the group consisting of SEQ ID NOs 154 to 498 or SEQ ID NOs 499 to 813. In some embodiments, at least one of the plurality of gRNA molecules comprises a targeting domain sequence encoded by a nucleic acid sequence selected from the group consisting of SEQ ID NOs 814 to 1064, wherein at least one of the plurality of gRNA molecules comprises a targeting domain sequence encoded by a nucleic acid sequence selected from SEQ ID NOs 154 to 498 or SEQ ID NOs 499 to 813.

[0080] In some embodiments, at least one of the endogenous target genes is BCL2L11 , FLI1 , CALM2 , DHODH , UMPS , RBM39 , SEMA7A , CHIC2 , PCBP1 , PBRM1 , WDR6 , E2F8 , SERPINA3 and GNAS Selected from a group consisting of, and at least one of the endogenous target genes CBLBIn some embodiments, at least one of the plurality of gRNA molecules comprises a targeting domain sequence that binds to a target DNA sequence selected from the group consisting of SEQ ID NOs 814 to 1064, wherein at least one of the plurality of gRNA molecules comprises a targeting domain sequence that binds to a target DNA sequence selected from the group consisting of SEQ ID NOs 499 to 524. In some embodiments, at least one of the plurality of gRNA molecules comprises a targeting domain sequence encoded by a nucleic acid sequence selected from the group consisting of SEQ ID NOs 814 to 1064, wherein at least one of the plurality of gRNA molecules comprises a targeting domain sequence encoded by a nucleic acid sequence selected from SEQ ID NOs 499 to 524.

[0081] In some embodiments, at least one of the endogenous target genes is SOCS1 and at least one of the endogenous target genes is IKZF1, IKZF3, GATA3, BCL3, TNIP1, TNFAIP3, NFKBIA, SMAD2, TGFBR1, TGFBR2, TANK, FOXP3 , RC3H1 , TRAF6 , IKZF2, CBLB, PPP2R2D, NRP1, HAVCR2, LAG3, TIGIT, CTLA4, PTPN6, PDCD1 and BCORSelected from the group consisting of. In some embodiments, at least one of the plurality of gRNAs binds to a target DNA sequence defined by the set of genome coordinates presented in Tables 6C and 6D, wherein at least one of the plurality of gRNAs binds to a target DNA sequence defined by the set of genome coordinates presented in Tables 5A and 5B. In some embodiments, at least one of the plurality of gRNA molecules comprises a targeting domain sequence that binds to a target DNA sequence selected from the group consisting of SEQ ID NOs 1088 to 1232, wherein at least one of the plurality of gRNA molecules comprises a targeting domain sequence that binds to a target DNA sequence selected from the group consisting of SEQ ID NOs 154 to 498 or SEQ ID NOs 499 to 813. In some embodiments, at least one of the plurality of gRNA molecules comprises a targeting domain sequence encoded by DNA selected from the group consisting of SEQ ID NOs 1088 to 1232, and at least one of the plurality of gRNA molecules comprises a targeting domain sequence encoded by a DNA sequence selected from the group consisting of SEQ ID NOs 154 to 498 or SEQ ID NOs 499 to 813. In some embodiments, at least one of the endogenous target gene is SOCS1 and at least one of the endogenous target genes is CBLB am . In some embodiments, at least one of the endogenous target genes is SOCS1 and at least one of the endogenous target genes is TNFAIP3 am . In some embodiments, at least one of the endogenous target genes is SOCS1 and at least one of the endogenous target genes is BCOR am .In some embodiments, at least one of the plurality of gRNA molecules comprises a targeting domain sequence that binds to a target nucleic acid sequence selected from the group consisting of SEQ ID NOs 1088 to 1232, wherein at least one of the plurality of gRNA molecules comprises a targeting domain sequence that binds to a target nucleic acid sequence selected from the group consisting of SEQ ID NOs 499 to 524. In some embodiments, at least one of the plurality of gRNA molecules comprises a targeting domain sequence encoded by a nucleic acid sequence selected from the group consisting of SEQ ID NOs 1088 to 1232, wherein at least one of the plurality of gRNA molecules comprises a targeting domain sequence encoded by a nucleic acid sequence selected from SEQ ID NOs 499 to 524.

[0082] In some embodiments, at least one of the endogenous target genes is ANKRD11 and at least one of the endogenous target genes is IKZF1, IKZF3, GATA3, BCL3, TNIP1, TNFAIP3, NFKBIA, SMAD2, TGFBR1, TGFBR2, TANK, FOXP3 , RC3H1 , TRAF6 , IKZF2, CBLB, PPP2R2D, NRP1, HAVCR2, LAG3, TIGIT, CTLA4, PTPN6, PDCD1 and BCORSelected from the group consisting of. In some embodiments, at least one of the plurality of gRNAs binds to a target DNA sequence defined by the set of genome coordinates presented in Tables 6E and 6F, wherein at least one of the plurality of gRNAs binds to a target DNA sequence defined by the set of genome coordinates presented in Tables 5A and 5B. In some embodiments, at least one of the plurality of gRNA molecules comprises a targeting domain sequence that binds to a target DNA sequence selected from the group consisting of SEQ ID NOs 1065 to 1087, wherein at least one of the plurality of gRNA molecules comprises a targeting domain sequence that binds to a target DNA sequence selected from the group consisting of SEQ ID NOs 154 to 498 or SEQ ID NOs 499 to 813. In some embodiments, at least one of the plurality of gRNA molecules comprises a targeting domain sequence encoded by DNA selected from the group consisting of SEQ ID NOs 1065 to 1087, and at least one of the plurality of gRNA molecules comprises a targeting domain sequence encoded by a DNA sequence selected from the group consisting of SEQ ID NOs 154 to 498 or SEQ ID NOs 499 to 813. In some embodiments, at least one of the endogenous target genes is ANKRD11 and at least one of the endogenous target genes is CBLB am . In some embodiments, at least one of the endogenous target genes is ANKRD11 and at least one of the endogenous target genes is TNFAIP3 am . In some embodiments, at least one of the endogenous target genes is ANKRD11 and at least one of the endogenous target genes is BCOR am .In some embodiments, at least one of the plurality of gRNA molecules comprises a targeting domain sequence that binds to a target nucleic acid sequence selected from the group consisting of SEQ ID NOs 1065 to 1087, wherein at least one of the plurality of gRNA molecules comprises a targeting domain sequence that binds to a target nucleic acid sequence selected from the group consisting of SEQ ID NOs 499 to 524. In some embodiments, at least one of the plurality of gRNA molecules comprises a targeting domain sequence encoded by a nucleic acid sequence selected from the group consisting of SEQ ID NOs 1065 to 1087, wherein at least one of the plurality of gRNA molecules comprises a targeting domain sequence encoded by a nucleic acid sequence selected from SEQ ID NOs 499 to 524.

[0083] In some embodiments, at least one of the endogenous target genes is ANKRD11 and at least one of the endogenous target genes is SOCS1 am .In some embodiments, at least one of the plurality of gRNAs binds to a target DNA sequence defined by a set of genome coordinates presented in Tables 6E and 6F, wherein at least one of the plurality of gRNAs binds to a target DNA sequence defined by a set of genome coordinates presented in Tables 6C and 6D. In some embodiments, at least one of the plurality of gRNA molecules comprises a targeting domain sequence that binds to a target nucleic acid sequence selected from the group consisting of SEQ ID NOs 1065 to 1087, wherein at least one of the plurality of gRNA molecules comprises a targeting domain sequence that binds to a target nucleic acid sequence selected from the group consisting of SEQ ID NOs 1088 to 1232. In some embodiments, at least one of the plurality of gRNA molecules comprises a targeting domain sequence encoded by DNA selected from the group consisting of SEQ ID NOs 1065 to 1087, wherein at least one of the plurality of gRNA molecules comprises a targeting domain sequence encoded by a DNA sequence selected from SEQ ID NOs 1088 to 1232.

[0084] In some embodiments, the Cas protein is a wild-type Cas protein comprising two enzyme-active domains capable of inducing a double-stranded DNA break; a Cas nikcase mutant comprising one enzyme-active domain capable of inducing a single-stranded DNA break; and an inactivated Cas protein (dCas) which is associated with a heterologous protein capable of regulating the expression of one or more endogenous target genes. In some embodiments, the heterologous protein is selected from the group consisting of MAX-interacting protein 1 (MXI1), a Krupel-associated box (KRAB) domain, and four consecutive mSin3 domains (SID4X). In some embodiments, the Cas protein is a Cas9 protein.

[0085] In some embodiments, the system comprises a nucleic acid molecule, said nucleic acid molecule is siRNA, shRNA, microRNA (miR), antagomiR, or antisense RNA. In some embodiments, the system comprises a plurality of shRNA or siRNA molecules.

[0086] In some embodiments, at least one of the endogenous target genes is BCL2L11 , FLI1 , CALM2 , DHODH , UMPS , RBM39 , SEMA7A , CHIC2 , PCBP1 , PBRM1 , WDR6 , E2F8 , SERPINA3 and GNAS Selected from a group consisting of, and at least one of the endogenous target genes IKZF1, IKZF3, GATA3, BCL3, TNIP1, TNFAIP3, NFKBIA, SMAD2, TGFBR1, TGFBR2, TANK, FOXP3 , RC3H1 , TRAF6 , IKZF2, CBLB, PPP2R2D, NRP1, HAVCR2, LAG3, TIGIT, CTLA4, PTPN6, PDCD1 and BCORIt is selected from the group consisting of. In some embodiments, at least one of the plurality of siRNA or shRNA molecules comprises about 19 to 30 nucleotides that bind to an RNA sequence encoded by a DNA sequence defined by the set of genome coordinates presented in Tables 6A and 6B, and at least one of the plurality of siRNA or shRNA molecules comprises about 19 to 30 nucleotides that bind to an RNA sequence encoded by a DNA sequence defined by the set of genome coordinates presented in Tables 5A and 5B. In some embodiments, at least one of a plurality of siRNA or shRNA molecules comprises about 19 to 30 nucleotides that bind to an RNA sequence encoded by a DNA sequence selected from the group consisting of SEQ ID NOs 814 to 1064, wherein at least one of a plurality of siRNA or shRNA molecules comprises about 19 to 30 nucleotides that bind to an RNA sequence encoded by a DNA sequence selected from the group consisting of SEQ ID NOs 154 to 498 or SEQ ID NOs 499 to 813.

[0087] In some embodiments, at least one of the endogenous target genes is BCL2L11 , FLI1 , CALM2 , DHODH , UMPS , RBM39 , SEMA7A , CHIC2 , PCBP1 , PBRM1 , WDR6 , E2F8 , SERPINA3 and GNAS Selected from a group consisting of, and at least one of the endogenous target genes CBLBIn some embodiments, at least one of the plurality of siRNA or shRNA molecules comprises about 19 to 30 nucleotides that bind to an RNA sequence encoded by a DNA sequence selected from the group consisting of SEQ ID NOs 814 to 1064, and at least one of the plurality of siRNA or shRNA molecules comprises about 19 to 30 nucleotides that bind to an RNA sequence encoded by a DNA sequence selected from the group consisting of SEQ ID NOs 499 to 524.

[0088] In some embodiments, at least one of the endogenous target genes is SOCS1 and at least one of the endogenous target genes is IKZF1, IKZF3, GATA3, BCL3, TNIP1, TNFAIP3, NFKBIA, SMAD2, TGFBR1, TGFBR2, TANK, FOXP3 , RC3H1 , TRAF6 , IKZF2, CBLB, PPP2R2D, NRP1, HAVCR2, LAG3, TIGIT, CTLA4, PTPN6, PDCD1 and BCORIt is selected from the group consisting of. In some embodiments, at least one of the plurality of siRNA or shRNA molecules comprises about 19 to 30 nucleotides that bind to an RNA sequence encoded by a DNA sequence defined by the set of genome coordinates presented in Tables 6C and 6D, and at least one of the plurality of siRNA or shRNA molecules comprises about 19 to 30 nucleotides that bind to an RNA sequence encoded by a DNA sequence defined by the set of genome coordinates presented in Tables 5A and 5B. In some embodiments, at least one of a plurality of siRNA or shRNA molecules comprises about 19 to 30 nucleotides that bind to an RNA sequence encoded by a DNA sequence selected from the group consisting of SEQ ID NOs 1088 to 1232, wherein at least one of a plurality of siRNA or shRNA molecules comprises about 19 to 30 nucleotides that bind to an RNA sequence encoded by a DNA sequence selected from the group consisting of SEQ ID NOs 154 to 498 or SEQ ID NOs 499 to 813. In some embodiments, at least one of the endogenous target genes is SOCS1 and at least one of the endogenous target genes is CBLB am . In some embodiments, at least one of a plurality of siRNA or shRNA molecules comprises about 19 to 30 nucleotides that bind to an RNA sequence encoded by a DNA sequence selected from the group consisting of SEQ ID NOs 1088 to 1232, and at least one of a plurality of siRNA or shRNA molecules comprises about 19 to 30 nucleotides that bind to an RNA sequence encoded by a DNA sequence selected from the group consisting of SEQ ID NOs 499 to 524.

[0089] In some embodiments, at least one of the endogenous target genes is ANKRD11and at least one of the endogenous target genes is IKZF1, IKZF3, GATA3, BCL3, TNIP1, TNFAIP3, NFKBIA, SMAD2, TGFBR1, TGFBR2, TANK, FOXP3 , RC3H1 , TRAF6 , IKZF2, CBLB, PPP2R2D, NRP1, HAVCR2, LAG3, TIGIT, CTLA4, PTPN6, PDCD1 and BCOR It is selected from the group consisting of. In some embodiments, at least one of the plurality of siRNA or shRNA molecules comprises about 19 to 30 nucleotides that bind to an RNA sequence encoded by a DNA sequence defined by the set of genome coordinates presented in Tables 6E and 6F, and at least one of the plurality of siRNA or shRNA molecules comprises about 19 to 30 nucleotides that bind to an RNA sequence encoded by a DNA sequence defined by the set of genome coordinates presented in Tables 5A and 5B. In some embodiments, at least one of a plurality of siRNA or shRNA molecules comprises about 19 to 30 nucleotides that bind to an RNA sequence encoded by a DNA sequence selected from the group consisting of SEQ ID NOs 1065 to 1087, wherein at least one of a plurality of siRNA or shRNA molecules comprises about 19 to 30 nucleotides that bind to an RNA sequence encoded by a DNA sequence selected from the group consisting of SEQ ID NOs 154 to 498 or SEQ ID NOs 499 to 813.

[0090] In some embodiments, at least one of the endogenous target genes is ANKRD11 and at least one of the endogenous target genes is CBLB am .In some embodiments, at least one of a plurality of siRNA or shRNA molecules comprises about 19 to 30 nucleotides that bind to an RNA sequence encoded by a DNA sequence selected from the group consisting of SEQ ID NOs 1065 to 1087, and at least one of a plurality of siRNA or shRNA molecules comprises about 19 to 30 nucleotides that bind to an RNA sequence encoded by a DNA sequence selected from the group consisting of SEQ ID NOs 499 to 524.

[0091] In some embodiments, at least one of the endogenous target genes is ANKRD11 and at least one of the endogenous target genes is SOCS1 am . In some embodiments, at least one of the plurality of siRNA or shRNA molecules comprises about 19 to 30 nucleotides that bind to an RNA sequence encoded by a DNA sequence defined by the set of genome coordinates presented in Tables 6C and 6D, and at least one of the plurality of siRNA or shRNA molecules comprises about 19 to 30 nucleotides that bind to an RNA sequence encoded by a DNA sequence defined by the set of genome coordinates presented in Tables 6E and 6F. In some embodiments, at least one of the plurality of siRNA or shRNA molecules comprises about 19 to 30 nucleotides that bind to an RNA sequence encoded by a DNA sequence selected from the group consisting of SEQ ID NOs 1088 to 1232, and at least one of the plurality of siRNA or shRNA molecules comprises about 19 to 30 nucleotides that bind to an RNA sequence encoded by a DNA sequence selected from the group consisting of SEQ ID NOs 1065 to 1087.

[0092] In some embodiments, the system comprises a protein having a DNA binding domain and an enzyme domain, and is selected from zinc finger nucleases and transcription-activator-like effector nucleases (TALEN).

[0093] In some embodiments, the present disclosure provides a gene-regulatory system comprising a vector encoding one or more gRNAs and a vector encoding a Cas endonuclease protein, wherein one or more gRNAs comprise a targeting domain sequence encoded by a nucleic acid sequence selected from SEQ ID NOs 814 to 1064, SEQ ID NOs 1065 to 1087, SEQ ID NOs 1088 to 1232, SEQ ID NOs 154 to 498 or SEQ ID NOs 499 to 813.

[0094] In some embodiments, the present disclosure provides a gene-regulatory system comprising a vector encoding a plurality of gRNAs and a vector encoding a Cas endonuclease protein, wherein at least one of the plurality of gRNAs comprises a targeting domain sequence encoded by a nucleic acid sequence selected from SEQ ID NOs 814 to 1064, SEQ ID NOs 1065 to 1087 and SEQ ID NOs 1088 to 1232, and at least one of the plurality of gRNAs comprises a targeting domain sequence encoded by a nucleic acid sequence selected from SEQ ID NOs 154 to 498 or SEQ ID NOs 499 to 813.

[0095] In some embodiments, the present disclosure provides a gene-regulatory system comprising a vector encoding one or more gRNAs and an mRNA molecule encoding a Cas endonuclease protein, wherein one or more gRNAs comprise a targeting domain sequence encoded by a nucleic acid sequence selected from SEQ ID NOs 814 to 1064, SEQ ID NOs 1065 to 1087, SEQ ID NOs 1088 to 1232, SEQ ID NOs 154 to 498 or SEQ ID NOs 499 to 813.

[0096] In some embodiments, the present disclosure provides a gene-regulatory system comprising a vector encoding a plurality of gRNAs and an mRNA molecule encoding a Cas endonuclease protein, wherein at least one of the plurality of gRNAs comprises a targeting domain sequence encoded by a nucleic acid sequence selected from SEQ ID NOs 814 to 1064, SEQ ID NOs 1065 to 1087 and SEQ ID NOs 1088 to 1232, and at least one of the plurality of gRNAs comprises a targeting domain sequence encoded by a nucleic acid sequence selected from SEQ ID NOs 154 to 498 or SEQ ID NOs 499 to 813.

[0097] In some embodiments, the present disclosure provides a gene-regulatory system comprising one or more gRNAs and a Cas endonuclease protein, wherein one or more gRNAs comprise a targeting domain sequence encoded by a nucleic acid sequence selected from SEQ ID NOs 814 to 1064, SEQ ID NOs 1065 to 1087, SEQ ID NOs 1088 to 1232, SEQ ID NOs 154 to 498 or SEQ ID NOs 499 to 813, wherein one or more gRNAs and a Cas endonuclease protein form a complex to form a ribonucleoprotein (RNP) complex.

[0098] In some embodiments, the present disclosure provides a gene-regulatory system comprising a plurality of gRNAs and a Cas endonuclease protein, wherein at least one of the plurality of gRNAs comprises a targeting domain sequence encoded by a nucleic acid sequence selected from SEQ ID NOs 814 to 1064, SEQ ID NOs 1065 to 1087 and SEQ ID NOs 1088 to 1232, wherein at least one of the plurality of gRNAs comprises a targeting domain sequence encoded by a nucleic acid sequence selected from SEQ ID NOs 154 to 498 or SEQ ID NOs 499 to 813, and one or more of the gRNAs and Cas endonuclease proteins form a complex to form a ribonucleoprotein (RNP) complex.

[0099] In some embodiments, the present disclosure provides a kit comprising a gene-regulatory system as described herein.

[0100] In some embodiments, the present disclosure is (a) BCL2L11 , FLI1 , CALM2 , DHODH , UMPS , RBM39 , SEMA7A , CHIC2 , PCBP1 , PBRM1 , WDR6 , E2F8 , SERPINA3 and GNAS A group consisting of; (b) SOCS1; (c) ANKRD11 ; (d) IKZF1, IKZF3, GATA3, BCL3, TNIP1, TNFAIP3, NFKBIA, SMAD2, TGFBR1, TGFBR2, TANK, FOXP3 , RC3H1 , TRAF6 and IKZF2 A group consisting of; or (e) CBLB, PPP2R2D, NRP1, HAVCR2, LAG3, TIGIT, CTLA4, PTPN6, PDCD1 and BCOR Provides a gRNA nucleic acid molecule comprising a targeting domain nucleic acid sequence that is complementary to a target sequence within an endogenous target gene selected from a group consisting of

[0101] In some embodiments, the endogenous gene is BCL2L11 , FLI1 , CALM2 , DHODH , UMPS , RBM39 , SEMA7A , CHIC2 , PCBP1 , PBRM1 , WDR6 , E2F8 , SERPINA3 and GNAS Selected from the group consisting of, and said gRNA comprises a targeting domain sequence complementary to a target DNA sequence located at genomic coordinates selected from those presented in Tables 6A and 6B; said endogenous gene SOCS1 and, the gRNA comprises a targeting domain sequence complementary to a target DNA sequence located at genomic coordinates selected from those presented in Tables 6C and 6D; and the endogenous gene ANKRD11 and, the gRNA comprises a targeting domain sequence complementary to a target DNA sequence located at genomic coordinates selected from those presented in Tables 6E and 6F; and the endogenous gene IKZF1, IKZF3, GATA3, BCL3, TNIP1, TNFAIP3, NFKBIA, SMAD2, TGFBR1, TGFBR2, TANK, FOXP3 , RC3H1 , TRAF6 and IKZF2 Selected from the group consisting of, and the gRNA comprises a targeting domain sequence complementary to a target DNA sequence located at genomic coordinates selected from those presented in Tables 5A and 5B; or the endogenous gene is CBLB, PPP2R2D, NRP1, HAVCR2, LAG3, TIGIT, CTLA4, PTPN6, PDCD1 and BCOR Selected from a group consisting of, said gRNA, the gRNA includes a targeting domain sequence that is complementary to a target DNA sequence located at a genomic coordinate selected from those presented in Tables 5A and 5B.

[0102] In some embodiments, the gRNA comprises a targeting domain sequence that binds to a target DNA sequence selected from SEQ ID NOs 814 to 1064, SEQ ID NOs 1088 to 1232, SEQ ID NOs 1065 to 1087, SEQ ID NOs 154 to 498, or SEQ ID NOs 499 to 813. In some embodiments, the gRNA comprises a targeting domain sequence encoded by a sequence selected from SEQ ID NOs 814 to 1064, SEQ ID NOs 1088 to 1232, SEQ ID NOs 1065 to 1087, SEQ ID NOs 154 to 498, or SEQ ID NOs 499 to 813. In some embodiments, the target sequence comprises a PAM sequence.

[0103] In some embodiments, the gRNA is a modular gRNA molecule. In some embodiments, the gRNA is a double gRNA molecule. In some embodiments, the targeting domain is 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, or greater than the nucleotide length. In some embodiments, the gRNA includes a modification at or near its 5' end (e.g., within 1 to 10, 1 to 5, or 1 to 2 nucleotides of its 5' end) and / or a modification at or near its 3' end (e.g., within 1 to 10, 1 to 5, or 1 to 2 nucleotides of its 3' end). In some embodiments, the modified gRNA exhibits increased stability against nucleases when introduced into T cells. In some embodiments, modified gRNA exhibits a reduced innate immune response when introduced into T cells.

[0104] In some embodiments, the present disclosure provides a polynucleotide molecule encoding a gRNA molecule described herein. In some embodiments, the present disclosure provides a composition comprising one or more gRNA molecules described herein or a polynucleotide encoding the same. In some embodiments, the present disclosure provides a kit comprising a gRNA molecule described herein or a polynucleotide encoding the same.

[0105] In some embodiments, the present disclosure is a method for producing modified immune effector cells, the method comprising: obtaining immune effector cells from a subject; introducing a gene-regulatory system of any one of claims 157 to 244 into the immune effector cells; and culturing the immune effector cells such that the expression and / or function of one or more endogenous target genes is reduced compared to the immune effector cells that are not modified.

[0106] In some embodiments, the present disclosure is a method for producing modified immune effector cells, the method comprising the step of introducing a gene-regulatory system of any one of claims 157 to 244 into said immune effector cells. In some embodiments, the method further comprises the step of introducing a polynucleotide sequence encoding a engineered immune receptor selected from CARs and TCRs. In some embodiments, the polynucleotide encoding the gene-regulatory system and / or the engineered immune receptor is introduced into the immune effector cells by transfusion, transduction, electroporation, or physical disruption of the cell membrane by a microfluidic device. In some embodiments, the gene-regulatory system is introduced as a polynucleotide sequence encoding one or more components of the system, as a protein or as a ribonucleoprotein (RNP) complex.

[0107] In some embodiments, the present disclosure provides a method for producing modified immune effector cells, the method comprising the steps of: expanding a population of immune effector cells in a culture; and introducing a gene-regulatory system described herein into the population of immune effector cells. In some embodiments, the method further comprises the step of obtaining a population of immune effector cells from a subject. In some embodiments, the gene-regulatory system is introduced into the population of immune effector cells before, during, or after expansion. In some embodiments, the expansion of the population of immune effector cells includes a first round of expansion and a second round of expansion. In some embodiments, the gene-regulatory system is introduced into the population of immune effector cells before, during, or after the first round of expansion. In some embodiments, the gene-regulatory system is introduced into the population of immune effector cells before, during, or after the second round of expansion. In some embodiments, the gene-regulatory system is introduced into the population of immune effector cells before the first round and the second round of expansion. In some embodiments, the gene-regulatory system is introduced into the population of immune effector cells after the first round and the second round of expansion. In some embodiments, a gene-regulatory system is introduced into a population of immune effector cells after the first round of expansion and before the second round of expansion.

[0108] In some embodiments, the present disclosure provides a method for treating a disease or disorder in a subject requiring treatment, the method comprising the step of administering an effective amount of the modified immune effector cells or a composition thereof described herein. In some embodiments, the disease or disorder is a proliferative disorder, an inflammatory disorder, or an infectious disease. In some embodiments, the disease or disorder is cancer or a viral infection. In some embodiments, the cancer is selected from leukemia, lymphoma, or a solid tumor. In some embodiments, the solid tumor is melanoma, pancreatic tumor, bladder tumor, lung tumor or metastasis, colorectal cancer, or head and neck cancer. In some embodiments, the cancer is PD1-resistant or non-sensitive cancer. In some embodiments, the subject has been previously treated with a PD1 inhibitor or a PDL1 inhibitor. In some embodiments, the method to the subject NRP1, HAVCR2, LAG3, TIGIT, CTLA4 or PDCD1 The method further includes the step of administering an antibody or a binding fragment thereof that specifically binds to a protein encoded by and inhibits its function.

[0109] In some embodiments, the modified immune effector cells are autologous to the subject. In some embodiments, the modified immune effector cells are homologous to the subject.

[0110] In some embodiments, the present disclosure provides a method for killing cancer cells, the method comprising the step of exposing cancer cells to modified immune effector cells or compositions thereof as described herein. In some embodiments, exposure occurs in vitro, in vivo, or in vitro.

[0111] In some embodiments, the present disclosure provides a method for enhancing one or more effector functions of an immune effector cell, the method comprising the step of introducing a gene-regulatory system described herein into the immune effector cell. In some embodiments, the present disclosure provides a method for enhancing one or more effector functions of an immune effector cell, the method comprising the step of introducing a gene-regulatory system described herein into the immune effector cell, wherein the modified immune effector cell exhibits one or more enhanced effector functions compared to an unmodified immune effector cell. In some embodiments, one or more effector functions are selected from cell proliferation, cell viability, cytotoxicity, tumor invasion, increased cytokine production, anti-tumor immune response, and / or resistance to consumption. Brief explanation of the drawing

[0112] FIGS. 1a and FIGS. 1b are drawings illustrating combinations of endogenous target genes that can be modified by the method described herein. FIGS. 2a and FIGS. 2b illustrate combinations of endogenous target genes that can be modified by the method described herein. FIGS. 3a and FIGS. 3b illustrate combinations of endogenous target genes that can be modified by the method described herein. FIGS. 4A and FIGS. 4D are drawings illustrating the editing of the TRAC gene and the B2M gene using the method described herein. Figures 5A and 5B show primary human T cells CBLB A drawing illustrating TIDE analysis data for editing. Figure 6 shows the comparison with the unedited control group (D6551-WT). CBLB A diagram showing the Western blot of the CBLB protein in primary human T cells (D6551-CBLB) edited with sgRNA. Figures 7A and 7B illustrate tumor growth over time in a murine B16 / Ova syngeneic tumor model. Figure 7A is compared with control-edited OT1 T cells. Cblb - A diagram illustrating tumor growth in mice treated with edited OT1 T cells. Fig. 7B shows the control group and Pd1- Compared to edited OT1 T cells Socs1 - Diagram illustrating tumor growth in mice treated with edited OT1 T cells. Figures 8A and 8B illustrate tumor growth over time in a murine MC38 / gp100 synonym tumor model. Figure 8A is compared with control-edited T cells. Socs1 - A diagram illustrating tumor growth in mice treated with edited PMEL T cells. Fig. 8B is compared to the control group and edited T cells. Ankrd11 - Diagram illustrating tumor growth in mice treated with edited PMEL T cells. Figure 9 shows the comparison with control-edited T cells. CBLB - Diagram illustrating tumor growth over time in a murine A375 xenograft model for mice treated with edited T cells. Fig. 10 is BCOR -Edited, CBLB -Edited or BCOR / CBLB A diagram illustrating tumor growth over time in mice treated with double-edited anti-CD19 CAR T cells. Tumor growth is compared to mice not treated with CAR T cells or non-edited anti-CD19 CAR T cells. Figure 11 is a diagram illustrating the accumulation of BCOR-edited or BCOR / CBLB-edited CD19 CAR T cells in an in vitro culture system. Figure 12 illustrates IL-2 production by BCOR-edited or BCOR / CBLB-edited CD19 CAR T cells in an in vitro culture system. Figure 13 illustrates IFNγ production by BCOR-edited or BCOR / CBLB-edited CD19 CAR T cells in an in vitro culture system. Figure 14 shows a murine B16 / Ova synonymous tumor model. Cblb / Socs1 Diagram illustrating tumor growth over time in mice treated with double-edited OT1 T cells. Figure 15 is a diagram illustrating the anti-tumor efficacy of PD1 / Lag3 double-edited transgenic T cells in a B16-Ova murine tumor model. Figure 16 illustrates the increase in pSTAT5 levels in primary human CD8 T cells in response to IL-2 signaling after SOCS1 deletion. Fig. 17 is a diagram illustrating gRNA enrichment from a SOCS1 tiling screen. Figure 18 is a diagram illustrating the accumulation of CD4+ and C8+ human T cells with SOCS1-targeting gRNA in vitro. FIG. 19 is a diagram illustrating the accumulation of CD4+ and C8+ human T cells with SOCS1-targeting gRNA or CBLB-targeting gRNA compared to a guide, PP2R2D, CBL, and SOCS3 targeting known regulators of T cell function in vitro. Figure 20 is a diagram illustrating the accumulation of murine CD8+ T cells with SOCS1-targeting gRNA in vitro. Figure 21 is a diagram showing the accumulation of murine CD8+ T cells with SOCS1-targeting gRNA compared to guide, PDCD1, and target-A targeting other regulators of T cell function in vitro. FIG. 22 is a diagram illustrating the accumulation of human CAR-T cells having a guide that targets SOCS1 and CBLB in response to antigen-specific stimulation in vitro. Figures 23A and 23B illustrate the surface expression of PD1 and CD25 (Figure 23A) and 4-1BB (Figure 23B) on SOCS1-edited and control-edited tumor-infiltrating lymphocytes. Specific details for implementing the invention

[0113] The present disclosure provides methods and compositions related to modifying immune effector cells to increase therapeutic efficacy in the context of immunotherapy. In some embodiments, immune effector cells are modified by the methods of the present invention to reduce the expression of one or more endogenous target genes or to reduce the function of one or more endogenous proteins so as to enhance one or more effector functions of the immune cells. In some embodiments, immune effector cells are further modified by the introduction of transgenes conferring antigen specificity, e.g., T cell receptor (TCR) or chimeric antigen receptor (CAR) expression constructs. In some embodiments, the present disclosure provides compositions and methods for modifying immune effector cells, e.g., compositions of gene-regulatory systems. In some embodiments, the present disclosure provides a method for treating a cell proliferative disorder, e.g., cancer, and the method comprises the step of administering the modified immune effector cells described herein to a subject requiring treatment.

[0114] I.정의

[0115] As used in this specification and the appended claims, the singular form includes plural objects unless the context otherwise clearly indicates.

[0116] As used in this specification, the term "and / or" is used in this specification to mean "and" or "or" unless otherwise indicated.

[0117] Throughout this specification, unless the context otherwise requires, the word “comprising,” or variations such as “comprising” or “comprising,” means the inclusion of the mentioned element or integer or group of elements or integers, but does not exclude any other element or integer or group of elements or integers.

[0118] As used in this application, the terms “about” and “approximately” are used as synonyms. Any numerical value used in this application with or without “about” and “approximately” is meant to include any general variation recognized by those skilled in the art. In certain embodiments, the terms “approximately” or “about” refer to a range of values ​​included in 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1% or less (greater or less) of both directions of the mentioned reference value, unless otherwise indicated or not evident from the context (except where such a number exceeds 100% of the possible values).

[0119] "Decrease" or "reduce" refers to a decrease or reduction of at least 5%, e.g., 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% from a specific value when compared to a reference value. A decrease or reduction of a specific value may also be expressed as a change in value by a multiple compared to a reference value, for example, as a decrease of at least 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 500, 1000 times or more compared to a reference value.

[0120] "Increases" refers to an increase of at least 5% from a specific value when compared to a reference value, for example, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, 100%, 200%, 300%, 400%, 500% or more. An increase in a specific value may also be expressed as a change in value by a multiple compared to a reference value, for example, as an increase of at least 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 500, 1000 times or more compared to a reference value.

[0121] The terms “peptide,” “polypeptide,” and “protein” are used interchangeably herein and refer to polymeric forms of amino acids of any length, which may include encoded and non-coded amino acids, chemically or biochemically modified or derivatized amino acids, and polypeptides having a modified peptide backbone.

[0122] The terms “polynucleotide” and “nucleic acid,” used interchangeably herein, refer to polymeric forms of nucleotides, ribonucleotides, or deoxyribonucleotides of any length. Accordingly, the term includes, but is not limited to, polymers comprising single-, double-, or multi-stranded DNA or RNA, genomic DNA, cDNA, DNA-RNA hybrids, or purine and pyrimidine bases or other natural, chemically or biochemically modified, non-natural, or derivatized nucleotide bases. “Oligonucleotide” generally refers to a polynucleotide of about 5 to about 100 nucleotides of single- or double-stranded DNA. However, for the purposes of this disclosure, there is no upper limit on the length of the oligonucleotide. Oligonucleotides are also known as “oligomers” or “oligos” and may be chemically synthesized from genes or by methods known in the art. The terms “polynucleotide” and “nucleic acid” should be understood to include single-stranded (e.g., sense or antisense) and double-stranded polynucleotides where applicable to the embodiments to be described.

[0123] "Fragment" refers to a portion of a polypeptide or polynucleotide molecule containing a portion smaller than the entire polypeptide or polynucleotide sequence. In some embodiments, the fragment of the polypeptide or polynucleotide comprises at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% of the total length of the reference polypeptide or polynucleotide. In some embodiments, the polypeptide or polynucleotide fragment may contain 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000 or more nucleotides or amino acids.

[0124] The term "sequence identity" refers to the percentage of bases or amino acids between two polynucleotide or polypeptide sequences at the same and same relative positions. Thus, one polynucleotide or polypeptide sequence has a specific percentage of sequence identity compared to another polynucleotide or polypeptide sequence. For sequence comparison, typically one sequence functions as a reference sequence against which the test sequence is compared. The term "reference sequence" refers to the molecule against which the test sequence is compared.

[0125] "Complementary" refers to the ability of two sequences to couple, including natural or non-natural bases or analogs thereof, through base stacking and specific hydrogen bonding. For example, if a base at one position of a nucleic acid can hydrogen bond with a base at a corresponding position of a target, those bases are considered complementary to each other at that position. Nucleic acids may include universal bases that provide neither a positive nor a negative contribution to hydrogen bonding, or inert abasic spacers. Base couplings may include regular Watson-Crick base couplings and non-Watson-Crick base couplings (e.g., Wobble base couplings and Hoogsteen base couplings). In the case of complementary base coupling, it is understood that an adenosine-type base (A) is complementary to a thymidine-type base (T) or a uracil-type base (U), and a cytosine-type base (C) is complementary to a guanosine-type base (G); and a universal base, e.g., 3-nitropyrrole or 5-nitroindole, can hybridize to any A, C, U, or T and is considered to be complementary (Nichols et al. , Nature, 1994;369:492-493 and Loakes et al. , Nucleic Acids Res., 1994;22:4039-4043). Inosine (I) has also been considered a universal base in the art and is regarded as complementary to any A, C, U, or T (see [Watkins and SantaLucia, Nucl. Acids Research, 2005; 33 (19): 6258-6267]).

[0126] As referred to in this specification, a “complementary nucleic acid sequence” is a nucleic acid sequence comprising a sequence of nucleotides that enables non-covalent binding to another nucleic acid in a sequence-specific antiparallel manner under appropriate in vitro and / or in vivo temperature conditions and solution ionic strength (i.e., the nucleic acid specifically binds to the complementary nucleic acid).

[0127] Sequence alignment methods for comparing and determining the percentages of sequence identity and complementarity are widely known in the art. Optimal sequence alignment for comparison is determined, for example, by homology alignment algorithms from the literature [Needleman and Wunsch, (1970) J. Mol. Biol. 48:443], by searching for similarity methods from the literature [Pearson and Lipman, (1988) Proc. Nat'l. Acad. Sci. USA 85:2444], by computer implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA (Wisconsin Genetics Software Package, Genetics Computer Group (575 Science Drive, Madison, Wisconsin))), by manual alignment and visual inspection (e.g., the literature [Brent et al. , (2003) Current Protocols in Molecular Biology]) by, respectively, literature [Altschul et al. , (1977) Nuc. Acids Res. 25:3389-3402; and Altschul et al. This can be performed by using algorithms known in the art, including the BLAST and BLAST 2.0 algorithms described in [J. Mol. Biol. 215:403-410]. Software for performing BLAST analysis is publicly available through the National Center for Biotechnology Information.

[0128] In this specification, the term “hybridize” refers to forming a double-stranded nucleic acid molecule by pairing complementary nucleotide bases (e.g., adenine (A) forms a base pair with thymine (T) in DNA molecules and with uracil (U) in RNA molecules, and guanine (G) forms a base pair with cytosine (C) in both DNA and RNA molecules) (e.g., see literature [Wahl and Berger (1987) Methods Enzymol. 152:399; Kimmel, (1987) Methods Enzymol. 152:507]). Additionally, for hybridization between two RNA molecules (e.g., dsRNA), it is known in the art that the guanine (G) base is paired with uracil (U). For example, G / U base pairing is partly the cause of the degeneracy (i.e., redundancy) of the genetic code in terms of codon and tRNA anti-codon base pairing within mRNA. In the context of this disclosure, guanine (G) of the protein-binding segment (dsRNA duplex) of a guide RNA molecule is considered complementary to uracil (U), and vice versa. Thus, if a G / U base pair can construct the protein-binding segment (dsRNA duplex) of a guide RNA molecule at a given nucleotide position, that position is not considered non-complementary, but instead is considered complementary. It is understood that the sequence of a polynucleotide does not need to be 100% complementary to the sequence of a target nucleic acid capable of specific hybridization. Additionally, the polynucleotide may hybridize onto one or more segments so that intervening or adjacent segments are not involved in the hybridization event (e.g., loop structure or hairpin structure). The polynucleotide may have at least 70%, at least 80%, at least 90%, at least 95%, at least 99%, or 100% sequence complementarity with the target region within the target nucleic acid sequence being targeted.

[0129] The term "modified" refers to a substance or compound that is altered or changed compared to a corresponding unmodified substance or compound (e.g., cells, polynucleotide sequences, and / or polypeptide sequences).

[0130] As applied to nucleic acids, polypeptides, cells, or organisms, the term “naturally occurring” as used herein refers to nucleic acids, polypeptides, cells, or organisms found in nature. For example, polypeptide or polynucleotide sequences present in organisms (including viruses) that can be isolated from natural sources and have not been intentionally modified by humans in a laboratory are naturally occurring.

[0131] "Isolated" refers to a substance that is not present or is present to varying degrees from the accompanying components when found in its native state.

[0132] "Expression cassette" or "expression construct" refers to a DNA polynucleotide sequence operably linked to a promoter. "Operably linked" refers to the existence of a relationship that allows the component described hereto to function in an intended manner. For example, a promoter is operably linked to a polynucleotide sequence when the promoter influences the transcription or expression of the polynucleotide sequence.

[0133] The term "recombinant vector" refers to a polynucleotide molecule capable of delivering or transporting another polynucleotide inserted into the vector. The inserted polynucleotide may be an expression cassette. In some embodiments, the recombinant vector may be a viral vector or a non-viral vector (e.g., a plasmid).

[0134] The term “sample” refers to a biological composition (e.g., a part of a cell or tissue) to be applied for analysis and / or genetic modification. In some embodiments, the sample is a “primary sample” in that it is obtained directly from the subject; in some embodiments, the “sample” is the result of processing the primary sample to, for example, remove a specific component and / or isolate or purify a specific component of interest.

[0135] The term “subject” includes animals, e.g., mammals. In some embodiments, the mammal is a primate. In some embodiments, the mammal is a human. In some embodiments, the subject is livestock, e.g., cattle, sheep, goats, cattle, pigs, etc.; or domesticated animals such as dogs and cats. In some embodiments (e.g., in a research context), the subject is a rodent (e.g., mouse, rat, hamster), rabbit, primate, or pig, e.g., inbred pigs, etc. The terms “subject” and “patient” are used interchangeably herein.

[0136] "Administration" in this specification refers to the introduction of an agent or composition into a subject.

[0137] As used herein, "treating" refers to delivering an agent or composition to a subject to influence physiological results.

[0138] As used herein, the term “effective amount” refers to the minimum amount of agent or composition required to produce a specific physiological effect. The effective amount of a specific agent may be expressed in terms of the nature of the agent, e.g., mass / volume, number of cells / volume, particle / volume, (mass of agent)(mass of target), number of cells / (mass of target), or particle / (mass of target). The effective amount of a specific agent may also be expressed as the half-maximal effective concentration (EC50), which refers to the concentration of the agent that results in a magnitude of a specific physiological response that is half between the reference level and the maximum response level.

[0139] The "population" of cells exceeds one, but preferably at least 1×10⁻⁶ 3 Dog cells, at least 1×10⁶ 4 Dog cells, at least 1×10⁶ 5 Dog cells, at least 1×10⁶ 6 Dog cells, at least 1×10⁶ 7 Dog cells, at least 1×10⁶ 8 Dog cells, at least 1×10⁶ 9 Dog cells, at least 1×10⁶ 10 It refers to any number of cells, or more than one cell. A cell population may refer to an in vitro population (e.g., a population of cells in a culture) or an in vivo population (e.g., a population of cells present in a specific tissue).

[0140] Common methods in molecular and cellular biochemistry are found in the literature [Molecular Cloning: A Laboratory Manual, 3rd Ed. (Sambrook et al. , HaRBor Laboratory Press 2001); Short Protocols in Molecular Biology, 4th Ed. (Ausubel et al. eds., John Wiley & Sons 1999); Protein Methods (Bollag et al. , John Wiley & Sons 1996); Nonviral Vectors for Gene Therapy (Wagner et al. It can be found in standard textbooks such as [I. Lefkovits eds., Academic Press 1999); Viral Vectors (Kaplift & Loewy eds., Academic Press 1995); Immunology Methods Manual (I. Lefkovits ed., Academic Press 1997); and Cell and Tissue Culture: Laboratory Procedures in Biotechnology (Doyle & Griffiths, John Wiley & Sons 1998)], the contents of which are incorporated herein by reference.

[0141] II. 변형된 면역 효과기 세포

[0142] In some embodiments, the present disclosure provides modified immune effector cells. As specified herein, the term “modified immune effector cell” includes immune effector cells comprising one or more modifications resulting in reduced expression and / or function of one or more endogenous target genes, as well as immune effector cells comprising gene-regulatory systems capable of reducing the expression and / or function of one or more endogenous target genes. As specified herein, “unmodified immune effector cell” or “control immune effector cell” refers to a cell or group of cells whose genome is not modified and which does not contain a gene-regulatory system or contains a control gene-regulatory system (e.g., empty vector control, non-targeted gRNA, scrambled siRNA, etc.).

[0143] The term “immune effector cell” refers to a cell involved in the establishment of innate immune and adaptive immune responses, including but not limited to lymphocytes (e.g., T-cells (thymocytes) and B-cells), natural killer (NK) cells, NKT cells, macrophages, monocytes, eosinophils, basophils, neutrophils, dendritic cells, and mast cells. In some embodiments, the modified immune effector cell is a T cell, e.g., CD4+ T cell, CD8+ T cell (also referred to as cytotoxic T cell or CTL), regulatory T cell (Treg), Th1 cell, Th2 cell, or Th17 cell.

[0144] In some embodiments, the immune effector cells are T cells isolated from a tumor sample (referred herein to as “tumor-infiltrating lymphocytes” or “TILs”). Without intending to be confined to theory, TILs increase specificity for tumor antigens (Radvanyi et al ., 2012 Clin Canc Res 18:6758-6770), therefore, by mediating tumor antigen-specific immune responses (e.g., activation, proliferation, and cytotoxic activity against cancer cells) without introducing exogenous engineered receptors, cancer cell destruction (Brudno et al It is thought that this may lead to ., 2018 Nat Rev Clin Onc 15:31-46). Accordingly, in some embodiments, the TIL is isolated from the tumor in the subject, expanded in vitro, and reinjected into the subject. In some embodiments, the TIL is modified to express one or more exogenous receptors specific to autologous tumor antigens, expanded in vitro, and reinjected into the subject. These embodiments can be modeled using an in vivo mouse model, wherein a cancer cell line expressing a cancer antigen (e.g., CD19) is transplanted into the mouse and treated with modified T cells expressing exogenous receptors specific to the cancer antigen (e.g., see Examples 10 and 11).

[0145] In some embodiments, the immune effector cells are animal cells or are derived from animal cells, including invertebrates and vertebrates (e.g., fish, amphibians, reptiles, birds, or mammals). In some embodiments, the immune effector cells are mammalian cells or are derived from mammalian cells (e.g., pigs, cattle, goats, sheep, rodents, non-human primates, humans, etc.). In some embodiments, the immune effector cells are rodent cells or are derived from rodent cells (e.g., rats or mice). In some embodiments, the immune effector cells are human cells or are derived from human cells.

[0146] In some embodiments, modified immune effector cells include one or more modifications (e.g., insertions, deletions, or mutations of one or more nucleic acids) within the genomic DNA sequence of an endogenous target gene, resulting in reduced expression and / or function of the endogenous gene. Such modifications are referred to herein as “inactivating mutations,” and the endogenous gene containing the inactivating mutations is referred to as “modified endogenous target gene.” In some embodiments, the inactivating mutations reduce or inhibit mRNA transcription, thereby reducing the expression levels of the encoded mRNA transcripts and proteins. In some embodiments, the inactivating mutations reduce or inhibit mRNA translation, thereby reducing the expression levels of the encoded proteins. In some embodiments, the inactivating mutations encode a modified endogenous protein having reduced or altered function compared to an unmodified (i.e., wild-type) version of the endogenous protein (e.g., the dominant-negative mutant described below).

[0147] In some embodiments, the modified immune effector cells include one or more genomic modifications at genomic locations other than the endogenous target gene that result in reduced expression and / or function of the endogenous target gene or in the expression of a modified version of the endogenous protein. For example, in some embodiments, a polynucleotide sequence encoding a gene-regulatory system is inserted at one or more locations within the genome, which reduces the expression and / or function of the endogenous target gene upon expression of the gene-regulatory system. In some embodiments, a polynucleotide sequence encoding a modified version of the endogenous protein is inserted at one or more locations within the genome, wherein the function of the modified version of the protein is reduced compared to the unmodified or wild-type version of the protein (e.g., the negative-dominant mutant described below).

[0148] In some embodiments, the modified immune effector cells described herein comprise one or more modified endogenous target genes, wherein one or more modifications result in reduced expression and / or function of the gene product (i.e., mRNA transcript or protein) encoded by the endogenous target gene compared to the unmodified immune effector cells. For example, in some embodiments, the modified immune effector cells exhibit reduced expression of the mRNA transcript and / or reduced expression of the protein. In some embodiments, the expression of the gene product in the modified immune effector cells is reduced by at least 5% compared to the expression of the gene product in the unmodified immune effector cells. In some embodiments, the expression of the gene product in the modified immune effector cells is reduced by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or more compared to the expression of the gene product in the unmodified immune effector cells. In some embodiments, the modified immune effector cells described herein exhibit reduced expression and / or function of gene products encoded by a plurality (e.g., two or more) of endogenous target genes compared to the expression of gene products in non-modified immune effector cells. For example, in some embodiments, the modified immune effector cells exhibit reduced expression and / or function of gene products from 2, 3, 4, 5, 6, 7, 8, 9, 10 or more endogenous target genes compared to the expression of gene products in non-modified immune effector cells.

[0149] In some embodiments, the present disclosure provides a modified immune effector cell, wherein one or more endogenous target genes, or parts thereof, are deleted (i.e., “knocked out”) so that the modified immune effector cell does not express an mRNA transcript or protein. In some embodiments, the modified immune effector cell comprises deletions of a plurality of endogenous target genes or parts thereof. In some embodiments, the modified immune effector cell comprises deletions of two, three, four, five, six, seven, eight, nine, ten or more endogenous target genes.

[0150] In some embodiments, the modified immune effector cell described herein comprises one or more modified endogenous target genes, wherein one or more modifications within the target DNA sequence result in the expression of a protein (e.g., "modified endogenous protein") having a reduced or altered function compared to the function of the corresponding protein (e.g., "unmodified endogenous protein") expressed in the unmodified immune effector cell. In some embodiments, the modified immune effector cell described herein comprises two, three, four, five, six, seven, eight, nine, ten, or more modified endogenous target genes encoding two, three, four, five, six, seven, eight, nine, ten, or more modified endogenous proteins. In some embodiments, the modified endogenous protein exhibits a reduced or altered binding affinity for another protein expressed by the modified immune effector cell or by another cell; It exhibits reduced or altered signaling ability; reduced or altered enzyme activity; reduced or altered DNA-binding activity; or reduced or altered ability to function as a scaffold protein.

[0151] In some embodiments, the modified endogenous target gene comprises one or more dominant negative mutations. As used herein, “dominant negative mutation” refers to one or more substitutions, deletions, or insertions of nucleotides in the target gene that cause the encoded protein to act antagonistically to the protein encoded by the unmodified target gene. The mutation is dominant because the negative phenotype is genetically dominant over the positive phenotype of the corresponding unmodified gene. A gene comprising one or more dominant negative mutations and the protein encoded by it are referred to as “dominant negative mutants,” e.g., the dominant negative gene and the dominant negative protein. In some embodiments, the dominant negative mutant protein is encoded by an exogenous transgene inserted at one or more locations within the genome of an immune effector cell.

[0152] Various mechanisms for negative dominance are known. Typically, the gene product of a negative-dominant mutant retains some functions of the unmodified gene product but lacks one or more other important functions of the unmodified gene product. This causes the negative-dominant mutant to antagonize the unmodified gene product. For example, as an exemplary embodiment, a negative-dominant mutant of a transcription factor may lack a functional activation domain but retain a functional DNA binding domain. In this example, the negative-dominant transcription factor cannot activate DNA transcription as an unmodified transcription factor, but the negative-dominant transcription factor can indirectly inhibit gene expression by preventing the unmodified transcription factor from binding to the transcription factor binding site. As another exemplary embodiment, a negative-dominant mutant of a protein that functions as a dimer is known. A negative-dominant mutant of such a dimer protein may retain the ability to dimerize with the unmodified protein but cannot otherwise function. The negative-dominant monomer prevents the formation of a functional homomer of the unmodified monomer by dimerizing with the unmodified monomer to form a heteromer.

[0153] In some embodiments, the modified immune effector cell comprises a gene-regulatory system capable of reducing the expression or function of one or more endogenous target genes. The gene-regulatory system may reduce the expression and / or function of the modified endogenous target gene by various mechanisms by modifying the genomic DNA sequence of the endogenous target gene (e.g., by insertion, deletion, or mutation of one or more nucleic acids within the genomic DNA sequence); by regulating the transcription of the endogenous target gene (e.g., inhibition or repression of mRNA transcription); and / or by regulating the translation of the endogenous target gene (e.g., by mRNA degradation).

[0154] In some embodiments, the modified immune effector cell described herein comprises a gene-regulatory system (e.g., a nucleic acid-based gene-regulatory system, a protein-based gene-regulatory system, or a combination protein / nucleic acid-based gene-regulatory system). In these embodiments, the gene-regulatory system included in the modified immune effector cell may modify one or more endogenous target genes. In some embodiments, the modified immune effector cell described herein comprises a gene-regulatory system comprising the following:

[0155] (a) one or more nucleic acid molecules capable of reducing the expression or modifying the function of a gene product encoded by one or more endogenous target genes;

[0156] (b) one or more nucleotides encoding a nucleic acid molecule capable of reducing the expression or modifying the function of a gene product encoded by one or more endogenous target genes;

[0157] (c) one or more proteins capable of reducing the expression or modifying the function of a gene product encoded by one or more endogenous target genes;

[0158] (d) one or more nucleotides encoding a protein capable of reducing the expression or modifying the function of a gene product encoded by one or more endogenous target genes;

[0159] (e) one or more guide RNAs (gRNAs) capable of binding to a target DNA sequence within an endogenous gene;

[0160] (f) one or more polynucleotides encoding one or more gRNAs capable of binding to a target DNA sequence within an endogenous gene;

[0161] (g) One or more site-directed modified polypeptides capable of interacting with gRNA to modify target DNA sequences within endogenous genes;

[0162] (h) One or more polypeptides encoding a site-directed modification polypeptide capable of modifying a polypeptide that interacts with gRNA to modify a target DNA sequence within an endogenous gene;

[0163] (i) one or more guide DNAs (gDNA) capable of binding to a target DNA sequence within an endogenous gene;

[0164] (j) One or more polynucleotides encoding one or more gDNAs capable of binding to a target DNA sequence within an endogenous gene;

[0165] (k) One or more site-directed modified polypeptides capable of interacting with gDNA to modify target DNA sequences within endogenous genes;

[0166] (l) One or more polypeptides encoding a site-directed modification polypeptide capable of modifying a polypeptide that interacts with gDNA to modify a target DNA sequence within an endogenous gene;

[0167] (m) One or more gRNAs capable of binding to a target mRNA sequence encoded by an endogenous gene;

[0168] (n) One or more polynucleotides encoding one or more gRNAs capable of binding to a target mRNA sequence encoded by an endogenous gene;

[0169] (o) One or more site-directed modified polypeptides capable of interacting with gRNA to modify a target mRNA sequence encoded by an endogenous gene;

[0170] (p) One or more polypeptides encoding a site-directed modification polypeptide capable of interacting with gRNA to modify a target mRNA sequence encoded by an endogenous gene;

[0171] (q) Any combination of these.

[0172] In some embodiments, one or more polynucleotides encoding a gene-regulatory system are inserted into the genome of an immune effector cell. In some embodiments, one or more polynucleotides encoding a gene-regulatory system are expressed episomatically and are not inserted into the genome of an immune effector cell.

[0173] In some embodiments, the modified immune effector cells described herein comprise reduced expression and / or function of one or more endogenous target genes and further comprise one or more exogenous transgenes inserted into one or more genomic loci (e.g., gene “knock-in”). In some embodiments, one or more exogenous transgenes encode a detectable label, a stability-switching system, a chimeric switch receptor, and / or a engineered antigen-specific receptor.

[0174] In some embodiments, the modified immune effector cells described herein further comprise an exogenous transgene encoding a detectable tag. The detectable tag includes, but is not limited to, FLAG tags, poly-histidine tags (e.g., 6xHis), SNAP tags, Halo tags, cMyc tags, glutathione-S-transferase tags, avidin, enzymes, fluorescent proteins, luminescent proteins, chemiluminescent proteins, bioluminescent proteins, and phosphorescent proteins. In some embodiments, the fluorescent protein is a blue / UV protein (e.g., BFP, TagBFP, mTagBFP2, Azurite, EBFP2, mKalama1, Sirius, Sapphire, and T-Sapphire); a cyanide protein (e.g., CFP, eCFP, Cerulean, SCFP3A, mTurquoise, mTurquoise2, monomer Midoriishi-Cyan, TagCFP, and mTFP1); Green proteins (e.g., GFP, eGFP, meGFP (A208K mutant), Emerald, Superfolder GFP, Monomeric Azami Green, TagGFP2, mUKG, mWasabi, Clover, and mNeonGreen); yellow proteins (e.g., YFP, eYFP, Citrine, Venus, SYFP2, and TagYFP); orange proteins (e.g., Monomeric Kusabira-Orange, mKOκ, mKO2, mOrange, and mOrange2); red proteins (e.g., RFP, mRaspberry, mCherry, mStrawberry, mTangerine, tdTomato, TagRFP, TagRFP-T, mApple, mRuby, and mRuby2); far-red proteins (e.g., mPlum, HcRed-Tandem, mKate2, mNeptune, and NirFP); Near-infrared proteins (e.g., TagRFP657, IFP1).It is selected from the group consisting of: 4 and iRFP); long stroke shift proteins (e.g., mKeima Red, LSS-mKate1, LSS-mKate2 and mBeRFP); photoactivatible proteins (e.g., PA-GFP, PAmCherry1 and PATagRFP); photoconvertible proteins (e.g., Kaede (green), Kaede (red), KikGR1 (green), KikGR1 (red), PS-CFP2, PS-CFP2, mEos2 (green), mEos2 (red), mEos3.2 (green), mEos3.2 (red), PSmOrange and PSmOrange); and photoswitchable proteins (e.g., Dronpa). In some embodiments, the detectable tag may be selected from AmCyan, AsRed, DsRed2, DsRed Express, E2-Crimson, HcRed, ZsGreen, ZsYellow, mCherry, mStrawberry, mOrange, mBanana, mPlum, mRasberry, tdTomato, DsRed Monomer and / or AcGFP, all of which are available from Clontech.

[0175] In some embodiments, the modified immune effector cells described herein further comprise an exogenous transgene encoding a safety-switch system. The safety-switch system (referred to in the art as a suicide gene system) comprises an exogenous transgene encoding one or more proteins that enable the removal of the modified immune effector cells after the cells are administered to a subject. Examples of safety-switch systems are known in the art. For example, a safety-switch system may be a protein that converts a non-toxic prodrug into a toxic compound, such as herpes simplex thymidine kinase (Hsv- tk) and the gene encoding the gancyclovir (GCV) system (Hsv- tk Includes / GCV). Hsv- tk It converts non-toxic GCV into cytotoxic compounds, which induce cell apoptosis. In this way, GCV, Hsv- tk Administering to subjects treated with modified immune effector cells containing protein-coding transgenes can selectively eliminate modified immune effector cells while sparing endogenous immune effector cells (e.g., literature [Bonini et al ., Science, 1997, 276(5319):1719-1724; Ciceri et al ., Blood, 2007, 109(11):1828-1836; Bondanza et al [See ., Blood 2006, 107(5):1828-1836]).

[0176] An additional safety-switching system comprises a gene encoding a cell-surface marker that enables the removal of modified immune effector cells by administration of a monoclonal antibody specific to the cell-surface marker via ADCC. In some embodiments, the cell-surface marker is CD20, and the modified immune effector cells can be removed by administration of an anti-CD20 monoclonal antibody, e.g., rituximab (e.g., the literature [Introna et al ., Hum Gene Ther, 2000, 11(4):611-620; Serafini et al ., Hum Gene Ther, 2004, 14, 63-76; van Meerten et al[Refer to Gene Ther, 2006, 13, 789-797]. A similar system using EGF-R and cetuximab or fenitumumab is described in PCT Publication No. WO 2018006880. An additional safety-switch system comprises a transgene encoding a pro-apoptotic molecule comprising one or more binding sites to a chemical inducer of dimerization (CID), which enables the elimination of immune effector cells modified by the administration of a CID that induces the oligomerization of the pro-apoptotic molecule and the activation of the apoptosis pathway. In some embodiments, the pro-apoptotic molecule is Fas (also known as CD95) (Thomis et al ., Blood, 2001, 97(5), 1249-1257). In some embodiments, the pro-apoptotic molecule is caspase-9 (Straathof et al ., Blood, 2005, 105(11), 4247-4254).

[0177] In some embodiments, the modified immune effector cells described herein further comprise an exogenous transgene encoding a chimeric switch receptor. The chimeric switch receptor is a engineered cell-surface receptor comprising an extracellular domain from an endogenous cell-surface receptor and a heterologous intracellular signaling domain, such that ligand recognition by the extracellular domain results in a signaling cascade activation different from that activated by the wild-type form of the cell-surface receptor. In some embodiments, the chimeric switch receptor comprises an extracellular domain of an inhibitory cell-surface receptor fused to an intracellular domain that leads to the transmission of an activating signal rather than the inhibitory signal typically transmitted by the inhibitory cell-surface receptor. In certain embodiments, an extracellular domain derived from a cell-surface receptor known to inhibit immune effector cell activation may be fused to an activating intracellular domain. Subsequently, engagement with the corresponding ligand will activate a signaling cascade that increases, rather than inhibits, the activation of the immune effector cell. For example, in some embodiments, the modified immune effector cells described herein comprise a transgene encoding a PD1-CD28 switch receptor, wherein the extracellular domain of PD1 is fused to the intracellular signaling domain of CD28 (e.g., literature [Liu et al ., Cancer Res 76:6 (2016), 1578-1590 and Moon. et al [Refer to ., Molecular Therapy 22 (2014), S201]). In some embodiments, the modified immune effector cells described herein comprise a transgene encoding the extracellular domain of CD200R and the intracellular signaling domain of CD28 (Reference [Oda et al [Refer to ., Blood 130:22 (2017), 2410-2419]).

[0178] In some embodiments, the modified immune effector cells described herein further comprise a modified antigen-specific receptor that recognizes a protein target expressed by a target cell, e.g., a tumor cell or an antigen-presenting cell (APC), referred herein as a “modified receptor-modified cell” or a “modified RE-cell.” The term “modified antigen receptor” refers to a non-naturally occurring antigen-specific receptor, e.g., a chimeric antigen receptor (CAR) or a recombinant T cell receptor (TCR). In some embodiments, the modified antigen receptor is a CAR comprising an extracellular antigen-binding domain fused to a cytoplasmic domain containing a signaling domain through hinge and transmembrane domains. In some embodiments, the CAR extracellular domain binds to an antigen expressed by the target cell in an MHC-independent manner, which leads to the activation and proliferation of the RE cell. In some embodiments, the extracellular domain of the CAR recognizes a tag fused to an antibody or its antigen-binding fragment. In these embodiments, the antigen-specificity of the CAR depends on the antigen-specificity of the labeled antibody, so that a single CAR construct can be used to target multiple different antigens by replacing another antibody with one antibody (see, for example, U.S. Patents No. 9,233,125 and 9,624,279; U.S. Patent Application Publications No. 20150238631 and 20180104354). In some embodiments, the extracellular domain of the CAR may comprise an antigen-binding fragment derived from an antibody. Antigen-binding domains useful in this disclosure include, for example, scFv; antibody; antigen-binding region of antibody; variable region of heavy / light chain; and single-chain antibody.

[0179] In some embodiments, the intracellular signaling domain of the CAR may be derived from the TCR complex zeta chain (e.g., CD3ξ signaling domain), FcγRIII, FcεRI, or T-lymphocyte activation domain. In some embodiments, the intracellular signaling domain of the CAR further comprises a co-stimulating domain, e.g., a 4-1BB, CD28, CD40, MyD88, or CD70 domain. In some embodiments, the intracellular signaling domain of the CAR further comprises any two of two co-stimulating domains, e.g., a 4-1BB, CD28, CD40, MyD88, or CD70 domain. Exemplary CAR structures and intracellular signaling domains are known in the art (see, for example, WO 2009 / 091826; US 20130287748; WO 2015 / 142675; WO 2014 / 055657; and WO 2015 / 090229, incorporated herein by reference).

[0180] CARs specific to various tumor antigens are known in the art, for example, CD171-specific CARs (Park et al ., Mol Ther (2007) 15(4):825-833), EGFRvIII-specific CAR(Morgan et al ., Hum Gene Ther (2012) 23(10):1043-1053), EGF-R-specific CAR(Kobold et al ., J Natl Cancer Inst (2014) 107(1):364), carbonic anhydrase K-specific CAR(Lamers et al ., Biochem Soc Trans (2016) 44(3):951-959), FR-α-specific CAR(Kershaw et al., Clin Cancer Res (2006) 12(20):6106-6015), HER2-specific CAR(Ahmed et al ., J Clin Oncol (2015) 33(15)1688-1696;Nakazawa et al., Mol Ther (2011) 19(12):2133-2143; Ahmed et al ., Mol Ther (2009) 17(10):1779-1787; Luo et al ., Cell Res (2016) 26(7):850-853; Morgan et al ., Mol Ther (2010) 18(4):843-851; Grada et al ., Mol Ther Nucleic Acids (2013) 9(2):32), CEA-specific CAR (Katz et al ., Clin Cancer Res (2015) 21(14):3149-3159), IL13Rα2-specific CAR(Brown et al ., Clin Cacner Res (2015) 21(18):4062-4072), GD2-specific CAR(Louis et al ., Blood (2011) 118(23):6050-6056; Caruana et al ., Nat Med (2015) 21(5):524-529), ErbB2-specific CAR(Wilkie et al ., J Clin Immunol (2012) 32(5):1059-1070), VEGF-R-specific CAR(Chinnasamy et al ., Cancer Res (2016) 22(2):436-447), FAP-specific CAR(Wang et al ., Cancer Immunol Res (2014) 2(2):154-166), MSLN-specific CAR(Moon et al , Clin Cancer Res (2011) 17(14):4719-30), NKG2D-specific CAR(VanSeggelen et al ., Mol Ther (2015) 23(10):1600-1610), CD19-specific CAR(Axicabtagene ciloleucel(Yescarta (등록상표) ) and Tisagenlecleucel (Kymriah (등록상표)) is. For a review of clinical trials of tumor-specific CARs, please refer to the literature [Li et al., J Hematol and Oncol (2018) 11(22)].

[0181] In some embodiments, the engineered antigen receptor is an engineered TCR. The engineered TCR comprises TCRα and / or TCRβ chains isolated and cloned from a T cell population that recognizes a specific target antigen. For example, TCRα and / or TCRβ genes (i.e., TRAC and TRBC The engineered TCR can be cloned from humanized mice immunized with specific tumor antigens or tumor cells, or from T cell populations isolated from individuals with specific malignancies or T cell populations. The engineered TCR recognizes antigens through the same mechanism as its endogenous counterpart (e.g., by recognition of cognate antigens presented in the context of major histocompatibility complex (MHC) proteins expressed on the surface of target cells). This antigen coupling stimulates endogenous signaling pathways, leading to the activation and proliferation of TCR-engineered cells.

[0182] Engineered TCRs specific to tumor antigens, e.g., WT1-specific TCR (JTCR016, Juno Therapeutics; WT1-TCRc4, described in U.S. Patent Application Publication No. 20160083449), MART-1 specific TCR (Liberty [Morgan et al Including the DMF4T clone described in [., Science 314 (2006) 126-129]); literature [Johnson et al DMF5T clones described in ., Blood 114 (2009) 535-546); and literature [van den Berg et al ID3T described in ., Mol. Ther. 23 (2015) 1541-1550), gp100-specific TCR (Johnson et al., Blood 114 (2009) 535-546), CEA-specific TCR(Parkhurst et al ., Mol Ther. 19 (2011) 620-626), NY-ESO and LAGE-1 specific TCR(Literature [Robbins et al ., J Clin Oncol 26 (2011) 917-924; Robbins et al ., Clin Cancer Res 21 (2015) 1019-1027; and Rapoport et al 1G4T clone described in [Nature Medicine 21 (2015) 914-921], and MAGE-A3-specific TCR (Morgan et al ., J Immunother 36 (2013) 133-151) and Linette et al ., Blood 122 (2013) 227-242) is known in the art (also the literature [Debets et al [Refer to ., Seminars in Immunology 23 (2016) 10-21]).

[0183] In some embodiments, the engineered antigen receptor is a cluster of differentiated molecules, e.g., CD3, CD4, CD8, CD16, CD24, CD25, CD33, CD34, CD45, CD64, CD71, CD78, CD80 (also known as B7-1), CD86 (also known as B7-2), CD96, CD116, CD117, CD123, CD133 and CD138, CD371 (also known as CLL1); Tumor-associated surface antigens, e.g., 5T4, BCMA (also known as CD269 and TNFRSF17, Uniprot No. Q02223), Carcinoembryonic antigen (CEA), carbonic anhydrase 9 (CAIX or MN / CAIX), CD19, CD20, CD22, CD30, CD40, disialogangliosides, e.g., GD2, ELF2M, ductal-epithelial mucin, ephrine B2, epithelial cell adhesion molecule (EpCAM), ErbB2 (HER2 / neu), FCRL5 (Uniprot No. Q68SN8), FKBP11 (Uniprot No. Q9NYL4), gliomar-associated antigen, gligosphingolipid, gp36, GPRC5D (Uniprot No. Q9NZD1), mut hsp70-2, intestinal carboxylate (intestinal carboxyl esterase), IGF-I receptor, ITGA8 (Uniprot No. P53708), KAMP3, LAGE-1a, MAGE, mesothelin, neutrophil elastase, NKG2D, Nkp30, NY-ESO-1, PAP, prostase, prostate-carcinoma tumor antigen-1 (PCTA-1), prostate-specific antigen (PSA), PSMA, prosteine, RAGE-1, ROR1, RU1 (SFMBT1), RU2 (DCDC2), SLAMF7 (Uniprot No. Q9NQ25), survivin, TAG-72, and telomerase; major histocompatibility complex (MHC) molecule-presented tumor-specific peptide epitopes;Tumor stromal antigens, e.g., the extra domain A (EDA) and extra domain B (EDB) of fibronectin; the A1 domain of tenacin-C (TnC A1) and fibroblast-associated protein (FAP); cytokine receptors, e.g., epidermal growth factor receptor (EGFR), EGFR variant III (EGFRvIII), TFGβ-R or components thereof, e.g., endoglin; major histocompatibility complex (MHC) molecules; virus-specific surface antigens, e.g., HIV-specific antigen (e.g., HIV gp120); EBV-specific antigen, CMV-specific antigen, HPV-specific antigen, Lassa virus-specific antigen, influenza virus-specific antigen, as well as any derivatives or variants of these surface antigens, are directed toward a target antigen selected from.

[0184] A. 효과기 기능

[0185] In some embodiments, the modified immune effector cells described herein exhibit an increase in one or more immune cell effector functions. In this specification, the term “effector function” refers to the function of an immune cell associated with the generation, maintenance, and / or enhancement of an immune response toward a target cell or a target antigen. In some embodiments, the modified immune effector cells described herein exhibit one or more of the following characteristics compared to non-modified immune effector cells: increased infiltration or migration into a tumor; increased proliferation; increased or prolonged cell survival; increased resistance to inhibitory factors in the surrounding microenvironment that cause the cell’s active state to be prolonged or increased; increased production of pro-inflammatory immune factors (e.g., pro-inflammatory cytokines, chemokines, and / or enzymes); increased cytotoxicity; and / or increased resistance to vesicles.

[0186] In some embodiments, the modified immune effector cells described herein exhibit increased infiltration into the tumor compared to non-modified immune effector cells. In some embodiments, increased tumor infiltration by modified immune effector cells refers to the number of modified immune effector cells infiltrating the tumor during a given time period compared to the number of non-modified immune effector cells infiltrating the tumor during the same time period. In some embodiments, modified immune effector cells exhibit a multiple increase of 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.5, 3, 3.5, 4, 4.5, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100 or more in tumor infiltration compared to non-modified immune cells. Tumor infiltration may be measured by isolating one or more tumors from a subject and evaluating the number of modified immune cells in the sample by flow cytometry, immunohistochemistry, and / or immunofluorescence.

[0187] In some embodiments, the modified immune effector cells described herein exhibit increased cell proliferation compared to unmodified immune effector cells. In these embodiments, this result increases the number of modified immune effector cells present compared to unmodified immune effector cells after a given time period. For example, in some embodiments, the modified immune effector cells exhibit an increased proliferation rate compared to unmodified immune effector cells, wherein the modified immune effector cells differentiate at a faster rate than the unmodified immune effector cells. In some embodiments, the modified immune effector cells exhibit a proliferation rate of 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.5, 3, 3.5, 4, 4.5, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100 or more multiples of the above compared to the non-modified immune cells. In some embodiments, the modified immune effector cells exhibit an extended proliferation period compared to the non-modified immune effector cells, wherein the modified immune effector cells and the non-modified immune effector cells differentiate at similar rates, but the modified immune effector cells maintain a proliferative state for a longer period of time. In some embodiments, the modified immune effector cells maintain a proliferative state for a longer period of time than the non-modified immune cells, such as 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.5, 3, 3.5, 4, 4.5, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100 or more multiples thereof.

[0188] In some embodiments, the modified immune effector cells described herein exhibit increased or prolonged cell survival compared to unmodified immune effector cells. In these embodiments, this result increases the number of modified immune effector cells present compared to unmodified immune effector cells after a given time period. For example, in some embodiments, the modified immune effector cells described herein maintain survival and persist for a longer period of time than non-modified immune cells, by 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.5, 3, 3.5, 4, 4.5, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100 or more multiples thereof.

[0189] In some embodiments, the modified immune effector cells described herein exhibit increased resistance to inhibitory factors compared to unmodified immune effector cells. Exemplary inhibitory factors include signaling by immune checkpoint molecules (e.g., PD1, PDL1, CTLA4, LAG3, IDO) and / or inhibitory cytokines (e.g., IL-10, TGFβ).

[0190] In some embodiments, the modified T cells described herein exhibit resistance to T cell depletion compared to non-modified T cells. T cell depletion is a state of antigen-specific T cell dysfunction characterized by reduced effector function, which leads to the subsequent deletion of antigen-specific T cells. In some embodiments, depleted T cells lack the ability to proliferate in response to an antigen and / or exhibit reduced cytokine production or / or exhibit reduced cytotoxicity toward target cells, e.g., tumor cells. In some embodiments, depleted T cells are identified by altered expression of cell surface markers and transcription factors, e.g., reduced cell surface expression of CD122 and CD127; increased expression of inhibitory cell surface markers, e.g., PD1, LAG3, CD244, CD160, TIM3, and / or CTLA4; and / or increased expression of transcription factors, e.g., Blimp1, NFAT, and / or BATF. In some embodiments, depleted T cells exhibit altered sensitivity to cytokine signaling, e.g., increased sensitivity to TGFβ signaling and / or decreased sensitivity to IL-7 and IL-15 signaling. T cell depletion can be determined, for example, by co-culturing T cells with a population of target cells and measuring T cell proliferation, cytokine production, and / or lysis of target cells. In some embodiments, the modified immune effector cells described herein are co-cultured with a population of target cells (e.g., autologous tumor cells or cell lines engineered to express a target tumor antigen) and effector cell proliferation, cytokine production, and / or lysis of target cells are measured. These results are then compared with results obtained from co-culturing the target cells with a population of control immune cells (e.g., unmodified immune effector cells or immune effector cells having a control modification).

[0191] In some embodiments, resistance to T cell consumption is demonstrated by increased production of one or more cytokines (e.g., IFNγ, TNFα, or IL-2) from modified immune effector cells compared to cytokine production observed from a control immune cell population. In some embodiments, an increase of 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5, 6, 7, 8, 9, 10, 15, 20, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100 or more multiples thereof in cytokine production from modified immune effector cells compared to cytokine production from a control immune cell population indicates increased resistance to T cell consumption. In some embodiments, resistance to T cell consumption is demonstrated by increased proliferation of modified immune effector cells compared to proliferation observed from a control immune cell population. In some embodiments, compared to the proliferation of a control immune cell population, an increase of 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5, 6, 7, 8, 9, 10, 15, 20, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100 or more multiples in the proliferation of modified immune effector cells indicates increased resistance to T cell consumption. In some embodiments, resistance to T cell consumption is demonstrated by increased lysis of target cells by modified immune effector cells compared to the lysis of target cells observed by the control immune cell population. In some embodiments, compared to target cell lysis by a control immune cell population, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.5, 3.0, 3.5, 4.0, 4. in target cell lysis by modified immune effector cells.An increase of 5, 5, 6, 7, 8, 9, 10, 15, 20, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100 or more multiples indicates increased resistance to T cell consumption.

[0192] In some embodiments, the consumption of modified immune effector cells compared to a control immune cell population is measured during the in vitro or in vitro manufacturing process. For example, in some embodiments, TILs isolated from a tumor fragment are isolated according to the method described herein and subsequently expanded by one or more rounds of expansion to produce a population of modified TILs. In these embodiments, the consumption of modified TILs may be determined immediately after collection and before the first round of expansion, after the first round of expansion before the second round of expansion, and / or after the first and second rounds of expansion. In some embodiments, the consumption of modified immune effector cells compared to a control immune cell population is measured at one or more time points after the modified immune effector cells are delivered to a subject. For example, in some embodiments, the modified cells are produced according to the method described herein and administered to a subject. The consumption of modified immune effector cells in vivo over time may then be determined by collecting samples from the subject at various time points after delivery.

[0193] In some embodiments, the modified immune effector cells described herein exhibit increased expression or production of pro-inflammatory immune factors compared to non-modified immune effector cells. Examples of pro-inflammatory immune factors include cytolytic factors, e.g., granzyme B, perforin, and granulisin; and pro-inflammatory cytokines, e.g., interferons (IFNα, IFNβ, IFNγ), TNFα, IL-1β, IL-12, IL-2, IL-17, CXCL8, and / or IL-6.

[0194] In some embodiments, the modified immune effector cells described herein exhibit increased cytotoxicity toward target cells compared to unmodified immune effector cells. In some embodiments, the modified immune effector cells exhibit a multiple increase of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100 or more in cytotoxicity toward target cells compared to unmodified immune cells.

[0195] Assays for measuring immune effector function are known in the art. For example, tumor invasion can be measured by isolating a tumor from a subject and determining the total number and / or phenotype of lymphocytes present in the tumor by flow cytometry, immunohistochemistry, and / or immunofluorescence. Cell-surface receptor expression can be determined by flow cytometry, immunohistochemistry, immunofluorescence, Western blot, and / or qPCR. Cytokine and chemokine expression and production can be measured by flow cytometry, immunohistochemistry, immunofluorescence, Western blot, ELISA, and / or qPCR. Response or sensitivity to extracellular stimuli (e.g., cytokines, inhibitory ligands, or antigens) can be measured by assessing cell proliferation and / or activation of downstream signaling pathways (e.g., phosphorylation of downstream signaling intermediates) in response to the stimulation. Cytotoxicity can be measured by in vitro or ex vivo co-culture of modified immune effector cells and target cells and by target-cell lysis assays known in the art, such as those described in all examples.

[0196] B.

[0197] In some embodiments, the modified immune effector cells described herein comprise a gene-regulatory system (described below) capable of exhibiting reduced expression or function of one or more endogenous target genes or reducing the expression and / or function of one or more endogenous target genes. In some embodiments, one or more endogenous target genes are present in pathways involved in the activation and regulation of the effector cell response. In these embodiments, the reduced expression or function of one or more endogenous target genes enhances one or more effector functions of the immune cell.

[0198] Table 1 presents exemplary pathways suitable for control by the method described herein. In some embodiments, the expression of endogenous target genes in a specific pathway is reduced in modified immune effector cells. In some embodiments, the expression of multiple (e.g., two or more) endogenous target genes in a specific pathway is reduced in modified immune effector cells. For example, the expression of 2, 3, 4, 5, 6, 7, 8, 9, 10 or more endogenous target genes in a specific pathway may be reduced. In some embodiments, the expression of endogenous target genes in one pathway and the expression of endogenous target genes in another pathway are reduced in modified immune effector cells. In some embodiments, the expression of multiple endogenous target genes in one pathway and the expression of multiple endogenous target genes in another pathway are reduced in modified immune effector cells. For example, the expression of 2, 3, 4, 5, 6, 7, 8, 9, 10 or more endogenous target genes in one pathway may be reduced, and the expression of 2, 3, 4, 5, 6, 7, 8, 9, 10 or more endogenous target genes in another specific pathway may be reduced.

[0199] In some embodiments, the expression of multiple endogenous target genes in multiple pathways is reduced. For example, one endogenous gene from each of multiple pathways (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10 or more pathways) may be reduced. In additional aspects, multiple endogenous genes (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10 or more pathways) from each of multiple pathways (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10 or more pathways) may be reduced.

[0200]

[0201] Exemplary endogenous target genes are presented in Tables 2 and 3 below.

[0202] In some embodiments, the modified effector cell is IKZF1, IKZF3, GATA3, BCL3, TNIP1, TNFAIP3, NFKBIA, SMAD2, TGFBR1, TGFBR2, TANK, FOXP3, RC3H1 , TRAF6 , IKZF2, CBLB, PPP2R2D, NRP1, HAVCR2, LAG3, TIGIT, CTLA4, PTPN6, PDCD1 , or BCOR It includes reduced expression and / or function of one or more of (e.g., one or more endogenous target genes selected from Table 2). In some embodiments, the modified effector cell is TNFAIP3, CBLB or BCOR middle Includes one or more reduced expressions and / or functions.

[0203] In some embodiments, modified immune effector cells are IKZF1, IKZF3, GATA3, BCL3, TNIP1, TNFAIP3, NFKBIA, SMAD2, TGFBR1, TGFBR2, TANK, FOXP3, RC3H1 , TRAF6 , IKZF2, CBLB, PPP2R2D, NRP1, HAVCR2, LAG3, TIGIT, CTLA4, PTPN6, PDCD1 , and BCOR It includes reduced expression and / or function of at least two genes selected from (e.g., at least two genes selected from Table 2). For example, in some embodiments, the modified immune effector cell includes reduced expression and / or function of at least two genes selected from combination numbers 1 to 600 as presented in FIGS. 1a and 1b. In some embodiments, the modified immune effector cell BCORReduced expression and / or function of and CBLB Includes reduced expression and / or function of. IKZF1, IKZF3, GATA3, BCL3, TNIP1, TNFAIP3, NFKBIA, SMAD2, TGFBR1, TGFBR2, TANK, FOXP3, RC3H1, TRAF6, IKZF2, CBLB, PPP2R2D, NRP1, HAVCR2, LAG3, TIGIT, CTLA4, PTPN6, PDCD1 , and / or BCOR Although exemplary methods for modifying the expression of are described herein, the expression of these endogenous target genes may also be modified by methods known in the art. For example, PD1 ( PDCD1 Inhibitory antibodies against (encoded by), NRP1, HACR2, LAG3, TIGIT, and CTLA4 are known in the art, and some are FDA approved for oncological indications (e.g., nivolumab and pembrolizumab for PD1).

[0204] In some embodiments, modified immune effector cells are BCL2L11 , FLI1 , CALM2 , DHODH , UMPS , RBM39 , SEMA7A , CHIC2 , PCBP1 , PBRM1 , WDR6 , E2F8 , SERPINA3 , GNAS, SOCS1 and ANKRD11 It includes reduced expression and / or function of one or more genes among (e.g., one or more endogenous target genes selected from Table 3).

[0205] In some embodiments, the modified effector cells described herein are Semaphorin 7A, also known as CD108 ( SEMA7A ) includes reduced expression and / or function of the gene. In some embodiments, the modified effector cells described herein are SEMA7A Includes inactivated mutations in genes.

[0206] In some embodiments, the modified effector cells described herein are RNA-binding protein 39 ( RBM39 ) It includes reduced expression and / or function of genes. The RBM39 protein is found in the nucleus, where it colocalizes with spliceosomal proteins. Studies of mouse proteins having high sequence similarity to these proteins suggest that these proteins may act as transcriptional co-activators for JUN / AP-1 and estrogen receptors. In some embodiments, the modified effector cells described herein are RBM39 Includes inactivated mutations in genes.

[0207] In some embodiments, the modified effector cells described herein are Bcl-2-like protein 11, also commonly referred to as BIM ( BCL2L11 ) It includes reduced expression and / or function of genes. In some embodiments, the modified effector cells described herein are BCL2L11 Includes inactivated mutations in genes.

[0208] In some embodiments, the pre-end leukemia integration 1 transcription factor (Friend leukemia integration 1 transcription factor), also known as the modified effector cell transcription factor ERGB described herein: FLI1 ) includes reduced expression and / or function of the gene. In some embodiments, the modified effector cells described herein are FLI1 Includes inactivated mutations in genes.

[0209] In some embodiments, the modified effector cells described herein are calmodulin 2 ( CALM2 ) It includes reduced expression and / or function of genes. In some embodiments, the modified effector cells described herein are CALM2 Includes inactivated mutations in genes.

[0210] In some embodiments, the modified effector cells described herein are dihydroorotate dehydrogenase gene: DHODH ) includes reduced expression and / or function of the gene. The DHODH protein is a mitochondrial protein located on the outer surface of the inner mitochondrial membrane and catalyzes the ubiquinone-mediated oxidation of dihydroorotate to orotate in neopyrimidine biosynthesis. In some embodiments, the modified effector cell described herein is DHODH Includes inactivated mutations in genes.

[0211] In some embodiments, the modified effector cell described herein is a uridine monophosphate synthase, also referred to as orotate phosphoribosyl transferase or orotidin-5'-decarboxylase ( UMPS Includes reduced expression and / or function of ). UMPS proteins catalyze the formation of uridine monophosphate (UMP), an energy-retaining molecule, in a number of important biosynthetic pathways. In some embodiments, the modified effector cells described herein are UMPS Includes inactivated mutations in genes.

[0212] In some embodiments, the modified effector cell described herein has a cysteine-rich hydrophobic domain 2 (cysteine ​​rich hydrophobic domain 2: CHIC2 ) It includes reduced expression and / or function of the gene. The encoded CHIC2 protein contains a cysteine-rich hydrophobic (CHIC) motif, is localized to vesicular structures and the plasma membrane, and is associated with some cases of acute myeloid leukemia. In some embodiments, the modified effector cells described herein are CHIC2 Includes inactivated mutations in genes.

[0213] In some embodiments, the modified effector cells described herein are poly(rC)-binding protein 1 ( PCBP1 ) It includes reduced expression and / or function of genes. In some embodiments, the modified effector cells described herein are PCBP1 Includes inactivated mutations in genes.

[0214] In some embodiments, the modified effector cells described herein contain Protein polybromo-1, also known as BRG1-associated factor 180 (BAF180): PBRM1 )) Includes reduced expression and / or function of the gene. PBRM1 is a component of the SWI / SNF-B chromatin-remodeling complex and is a tumor suppressor gene in many cancer subtypes. Mutations are dominant, particularly in clear cell renal cell carcinoma. In some embodiments, the modified effector cells described herein are PBRM1 Includes inactivated mutations in genes.

[0215] In some embodiments, the modified effector cell described herein is a WD repeat-containing protein 6, a member of the WD repeat protein family that is expressed anywhere in adult and fetal tissues ( WDR6 ) It includes reduced expression and / or function of genes. The WD repeat is a minimally conserved region of approximately 40 amino acids, typically surrounded by gly-his and trp-asp (GH-WD), which can facilitate the formation of heterotrimers or multiprotein complexes. Members of this family are involved in various cellular processes, including cell cycle progression, signal transduction, apoptosis, and gene regulation. In some embodiments, the modified effector cells described herein are WDR6 Includes inactivated mutations in genes.

[0216] In some embodiments, the modified effector cells described herein are E2F transcription factor 8 ( E2F8 ) It includes reduced expression and / or function of a gene. The encoded E2F8 protein regulates the function from the G1 phase to the S phase by ensuring that the nucleus differentiates at the appropriate time. In some embodiments, the modified effector cells described herein are E2F8 Includes inactivated mutations in genes.

[0217] In some embodiments, the modified effector cell described herein is a member of the Serpin family A 3 ( SERPINA3 ) Includes reduced expression and / or function of the gene. SERPINA3 It encodes the alpha 1-antichymotrypsin (α1AC, A1AC, or a1ACT) protein, which inhibits the activity of certain proteases, such as cathepsin G and chymase. In some embodiments, the modified effector cells described herein are SERPINA3 Includes inactivated mutations in genes.

[0218] In some embodiments, the modified effector cells described herein have GNAS complex loci ( GNAS ) It includes reduced expression and / or function of a gene. It is the stimulating G-protein alpha subunit (Gs-α), which is a major component of a number of signal transduction pathways. In some embodiments, the modified effector cell described herein is GNAS Includes inactivated mutations in genes.

[0219] In some embodiments, the modified effector cells described herein are ANKRD11 It includes reduced expression of the gene. The ANKRD11 protein is an ankyrin repeat domain-containing protein thought to inhibit the ligand-dependent activation of transcripts by an unknown mechanism. The ANKRD11 protein is thought to be associated with KBG syndrome.

[0220] In some embodiments, the modified effector cell described herein is a suppressor of cytokine signaling SOCS 1 ( SOCS1 It includes reduced expression of the ) gene. The SOCS1 protein contains a C-terminal SOCS box motif, an SH2-domain, an ESS domain, and an N-terminal KIR domain. A 12-amino acid residue called the kinase inhibitory region (KIR) has been shown to be important for SOCS1's ability to negatively regulate the function of JAK1, TYK2, and JAK2 tyrosine kinases.

[0221] In some embodiments, modified immune effector cells are BCL2L11 , FLI1 , CALM2 , DHODH , UMPS , RBM39 , SEMA7A , CHIC2 , PCBP1 , PBRM1 , WDR6 , E2F8 , SERPINA3 , GNAS, SOCS1 and ANKRD11It includes reduced expression and / or function of at least two genes selected from (e.g., two or more genes selected from Table 3). For example, in some embodiments, the modified immune effector cell includes reduced expression and / or function of at least two genes selected from combination numbers 1001 to 1240 as presented in FIG. 3a and 3b. In some embodiments, the modified immune effector cell includes reduced expression and / or function of at least two genes selected from combination numbers 1001 to 1210 as presented in FIG. 3a. In some embodiments, the modified immune effector cell includes reduced expression and / or function of at least two genes selected from combination numbers 1211 to 1240 as presented in FIG. 3b. In some embodiments, the modified immune effector cell includes reduced expression and / or function of at least two genes selected from combination numbers 1211 to 1225 as presented in FIG. 3b. In some embodiments, the modified immune effector cell comprises reduced expression and / or function of at least two genes selected from combination numbers 1226 to 1240 as shown in FIG. 3b. In some embodiments, the modified immune effector cell SOCS1 and ANKRD11 Reduced expression and / or function of Includes.

[0222] In some embodiments, the modified effector cell is BCL2L11 , FLI1 , CALM2 , DHODH , UMPS , RBM39 , SEMA7A , CHIC2 , PCBP1 , PBRM1 , WDR6 , E2F8 , SERPINA3 , GNAS, SOCS1 and ANKRD11 One or more of (e.g., one or more genes selected from Table 3) andIKZF1, IKZF3, GATA3, BCL3, TNIP1, TNFAIP3, NFKBIA, SMAD2, TGFBR1, TGFBR2, TANK, FOXP3, RC3H1 , TRAF6 , IKZF2, CBLB, PPP2R2D, NRP1, HAVCR2, LAG3, TIGIT, CTLA4, PTPN6, PDCD1 , or BCOR It includes reduced expression and / or function of one or more of (e.g., one or more genes selected from Table 2). For example, the modified immune effector cell may include reduced expression and / or function of a combination of endogenous target genes selected from combination numbers 601 to 1000. In some embodiments, the modified immune effector cell may include reduced expression and / or function of a combination of two endogenous target genes selected from combination numbers 601 to 950 (as shown in FIG. 2a). In some embodiments, the modified immune effector cell may include reduced expression and / or function of a combination of two endogenous target genes selected from combination numbers 951 to 1000 (as shown in FIG. 2b). In some embodiments, the modified immune effector cell may include reduced expression and / or function of a combination of two endogenous target genes selected from combination numbers 951 to 975 (as shown in FIG. 2b). In some embodiments, the modified immune effector cell may include reduced expression and / or function of a combination of two endogenous target genes selected from combination numbers 951 to 1000 (as shown in FIG. 2b).

[0223] In some embodiments, the modified effector cell is BCL2L11 , FLI1 , CALM2 , DHODH , UMPS , RBM39 , SEMA7A , CHIC2 , PCBP1 , PBRM1 , WDR6 , E2F8 , SERPINA3 and GNAS Reduced expression and / or function of at least one gene selected from and TNFAIP3, CBLB or BCORIt includes reduced expression and / or function of at least one gene selected from. In some embodiments, the modified effector cell is SOCS1 and TNFAIP3, CBLB or BCOR It includes reduced expression and / or function of at least one gene selected from. In some embodiments, the modified effector cell is SOCS1 and NFAIP3, CBLB or BCOR It includes an inactivation mutation in at least one gene selected from. In some embodiments, the modified effector cell is ANKRD11 and TNFAIP3, CBLB or BCOR It includes reduced expression and / or function of at least one gene selected from. In some embodiments, the modified effector cell is ANKRD11 and TNFAIP3, CBLB or BCOR It includes an inactivation mutation in at least one gene selected from.

[0224] In some embodiments, the modified effector cell is BCL2L11 , FLI1 , CALM2 , DHODH , UMPS , RBM39 , SEMA7A , CHIC2 , PCBP1 , PBRM1 , WDR6 , E2F8 , SERPINA3 , GNAS, SOCS1 , and ANKRD11 Reduced expression and / or function of at least one gene selected from and CBLB It includes reduced expression and / or function of. In some embodiments, the modified effector cell is BCL2L11 , FLI1 , CALM2 , DHODH , UMPS , RBM39 , SEMA7A , CHIC2 , PCBP1 , PBRM1 , WDR6 , E2F8 , SERPINA3 and GNAS Reduced expression and / or function of at least one gene selected from and CBLB It includes reduced expression and / or function of. In some embodiments, the modified effector cell is SOCS1 and CBLB Reduced expression and / or function of Includes. In some embodiments, the modified effector cell is SOCS1 and CBLB In It includes inactivated mutations. In some embodiments, the modified effector cell is ANKRD11 and CBLB Reduced expression and / or function of Includes. In some embodiments, the modified effector cell is ANKRD11 and CBLB In Includes inactivated mutations.

[0225] In some embodiments, modified immune effector cells are IKZF1, IKZF3, GATA3, BCL3, TNIP1, TNFAIP3, NFKBIA, SMAD2, TGFBR1, TGFBR2, TANK, FOXP3, RC3H1, TRAF6, IKZF2, CBLB, PPP2R2D, NRP1, HAVCR2, LAG3, TIGIT, CTLA4, PTPN6, PDCD1 , or BCOR Reduced expression and / or function of a selected gene from (e.g., one or more genes selected from Table 2) and BCL2L11 , FLI1 , CALM2 , DHODH , UMPS , RBM39 , SEMA7A , CHIC2 , PCBP1 , PBRM1 , WDR6 , E2F8 , SERPINA3 , GNAS, SOCS1 and ANKRD11 It includes reduced expression and / or function of two genes selected from (e.g., one or more genes selected from Table 3). For example, in some embodiments, the modified immune effector cell comprises, in addition to reduced expression and / or function of two endogenous target gene combinations selected from combination numbers 1176 to 1681 (as shown in FIG. 3a and 3b), IKZF1, IKZF3, GATA3, BCL3, TNIP1, TNFAIP3, NFKBIA, SMAD2, TGFBR1, TGFBR2, TANK, FOXP3, RC3H1, TRAF6, IKZF2, CBLB, PPP2R2D, NRP1, HAVCR2, LAG3, TIGIT, CTLA4, PTPN6, PDCD1 and BCORIncludes reduced expression and / or function of a selected gene.

[0226] In some embodiments, modified immune effector cells are BCL2L11 , FLI1 , CALM2 , DHODH , UMPS , RBM39 , SEMA7A , CHIC2 , PCBP1 , PBRM1 , WDR6 , E2F8 , SERPINA3 , GNAS, SOCS1 and ANKRD11 Reduced expression and / or function of the selected gene (e.g., the gene selected from Table 3) and IKZF1, IKZF3, GATA3, BCL3, TNIP1, TNFAIP3, NFKBIA, SMAD2, TGFBR1, TGFBR2, TANK, FOXP3, RC3H1, TRAF6, IKZF2, CBLB, PPP2R2D, NRP1, HAVCR2, LAG3, TIGIT, CTLA4, PTPN6, PDCD1 or BCOR It comprises reduced expression and / or function of two genes selected from (e.g., one or more genes selected from Table 2). For example, in some embodiments, the modified immune effector cell comprises, in addition to reduced expression and / or function of two endogenous target gene combinations selected from combination numbers 1 to 600 presented in FIGS. 1a and 1b BCL2L11 , FLI1 , CALM2 , DHODH , UMPS , RBM39 , SEMA7A , CHIC2 , PCBP1 , PBRM1 , WDR6 , E2F8 , SERPINA3 , GNAS, SOCS1 and ANKRD11 It includes any one of the reduced expression and / or function. In some embodiments, the modified immune effector cell comprises, in addition to the reduced expression and / or function of two endogenous target gene combinations selected from combination numbers 1 to 600 presented in FIG. 1a and 1b BCL2L11 , FLI1 , CALM2 , DHODH , UMPS , RBM39 , SEMA7A, CHIC2 , PCBP1 , PBRM1 , WDR6 , E2F8 , SERPINA3 and GNAS It includes reduced expression and / or function of a gene selected from. In some embodiments, the modified immune effector cell comprises, in addition to reduced expression and / or function of two endogenous target gene combinations selected from combination numbers 1 to 600 presented in FIG. 1a and 1b SOCS1 It includes reduced expression and / or function of. In some embodiments, the modified immune effector cell includes, in addition to reduced expression and / or function of two endogenous target gene combinations selected from combination numbers 1 to 600 presented in FIG. 1a and 1b ANKRD11 Includes reduced expression and / or function of.

[0227] In some embodiments, the modified immune effector cell comprises reduced expression and / or function of a plurality of genes selected from Table 2 and reduced expression and / or function of a plurality of genes selected from Table 3. In some embodiments, the modified immune effector cell comprises reduced expression and / or function of two genes selected from Table 2 and reduced expression and / or function of two genes selected from Table 3. For example, in some embodiments, the modified immune effector cell comprises reduced expression and / or function of a combination of two genes selected from combination numbers 1176 to 1681 as presented in FIGS. 3a and 3b and a combination of two genes selected from combination numbers 1 to 600 as presented in FIGS. 1a and 1b. In some embodiments, the modified immune effector cell IKZF1, IKZF3, GATA3, BCL3, TNIP1, TNFAIP3, NFKBIA, SMAD2, TGFBR1, TGFBR2, TANK, FOXP3, RC3H1, TRAF6, IKZF2, CBLB, PPP2R2D, NRP1, HAVCR2, LAG3, TIGIT, CTLA4, PTPN6, PDCD1 , or BCOR Reduced expression and / or function of three or more of the BCL2L11 , FLI1 , CALM2 , DHODH , UMPS , RBM39 , SEMA7A , CHIC2 , PCBP1 , PBRM1 , WDR6 , E2F8 , SERPINA3 , GNAS, SOCS1 and ANKRD11 It may include three or more reduced expressions and / or functions.

[0228]

[0229]

[0230]

[0231] III. 유전자-조절 시스템

[0232] In this specification, the term “gene-regulatory system” refers to a protein, nucleic acid, or combination thereof capable of regulating the expression or function of an encoded gene product by modifying an endogenous target DNA sequence when introduced into a cell. A number of gene editing systems suitable for use in the methods of this disclosure are known in the art, including but not limited to shRNA, siRNA, zinc-finger nuclease systems, TALEN systems, and CRISPR / Cas systems.

[0233] As used herein, the term “regulates,” when used in relation to the effect of a gene-regulatory system on an endogenous target gene, includes any change in the sequence of the endogenous target gene, any change in the epigenetic state of the endogenous target gene, and / or any change in the expression or function of the protein encoded by the endogenous target gene.

[0234] In some embodiments, the gene-regulatory system may mediate a change in the sequence of an endogenous target gene, for example, by introducing one or more mutations within the endogenous target sequence, for example, by insertion or deletion of one or more nucleic acids within the endogenous target sequence. Exemplary mechanisms that may mediate a change in the endogenous target sequence include, but are not limited to, non-homologous end joining (NHEJ) (e.g., conventional or alternative), microhomology-mediated end joining (MMEJ), homology-directed repair (e.g., endogenous donor template-mediated), SDSA (synthetic-dependent strand annealing), single-strand annealing, or single-strand intrusion.

[0235] In some embodiments, the gene-regulatory system may mediate changes in the epigenetic state of an endogenous target sequence. For example, in some embodiments, the gene-regulatory system may mediate covalent modifications of endogenous target gene DNA (e.g., cytosine methylation and hydroxymethylation) or covalent modifications of associated histone proteins (e.g., lysine acetylation, lysine and arginine methylation, serine and threonine phosphorylation and lysine ubiquitination and sumoylation).

[0236] In some embodiments, the gene-regulatory system may mediate a change in the expression of a protein encoded by an endogenous target gene. In these embodiments, the gene-regulatory system may regulate the expression of the encoded protein by modifying the endogenous target DNA sequence or by acting on the mRNA product encoded by the DNA sequence. In some embodiments, the gene-regulatory system may result in the expression of a modified endogenous protein. In these embodiments, a modification to the endogenous DNA sequence mediated by the gene-regulatory system results in the expression of the endogenous protein, which demonstrates reduced function compared to the corresponding endogenous protein in unmodified immune effector cells. In these embodiments, the expression level of the modified endogenous protein may be increased or decreased and may be the same as or substantially similar to the expression level of the corresponding endogenous protein in unmodified immune cells.

[0237] A. 핵산-기반 유전자-조절 시스템

[0238] As used herein, a nucleic acid-based gene-regulatory system is a system comprising one or more nucleic acid molecules capable of regulating the expression of an endogenous target gene without the requirement of an exogenous protein. In some embodiments, the nucleic acid-based gene-regulatory system comprises an RNA interference molecule or an antisense RNA molecule complementary to a target nucleic acid sequence.

[0239] "Antisense RNA molecule" refers to a length-independent RNA molecule that is complementary to an mRNA transcript. An antisense RNA molecule refers to a single-stranded RNA molecule that, when introduced into a cell, tissue, or subject, can reduce the expression of an endogenous target gene product through a mechanism that is not dependent on the endogenous gene silencing pathway, but rather depends on the RNAeH-mediated degradation of the target mRNA transcript. In some embodiments, the antisense nucleic acid may comprise a modified backbone, e.g., phosphorothioate, phosphorodithioate, or other known in the art, or may comprise a non-natural internucleoside linkage. In some embodiments, the antisense nucleic acid may comprise a lock nucleic acid (LNA).

[0240] "RNA interference molecule" refers to an RNA polynucleotide that mediates the reduction in expression of an endogenous target gene product by the degradation of target mRNA through endogenous gene silencing pathways (e.g., Dicer and RNA-induced silencing complex (RNA-induced silencing complex: RISC)). Exemplary RNA interfering agents include microRNA (also referred to as "miRNA" herein), short hairpin RNA (shRNA), small interfering RNA (siRNA), RNA aptamers, and morpholinos.

[0241] In some embodiments, the nucleic acid-based gene-regulatory system comprises one or more miRNAs. miRNA refers to a naturally occurring, small, non-coding RNA molecule of about 21 to 25 nucleotides in length. miRNA is at least partially complementary to one or more target mRNA molecules. miRNA can downregulate (e.g., reduce) the expression of endogenous target gene products through translation repression, cleavage of mRNA, and / or deadenylation.

[0242] In some embodiments, the nucleic acid-based gene-regulatory system comprises one or more shRNAs. The shRNA is a single-stranded RNA molecule of about 50 to 70 nucleotides in length that forms a stem-loop structure and causes degradation of the complementary mRNA sequence. The shRNA can be cloned from a plasmid or a non-replicating recombinant viral vector and introduced into a cell, which may result in the integration of the shRNA-coding sequence within the genome. In this way, the shRNA can provide stable and consistent repression of endogenous target gene translation and expression.

[0243] In some embodiments, the nucleic acid-based gene-regulatory system comprises one or more siRNAs. siRNA typically refers to a double-stranded RNA molecule approximately 21 to 23 nucleotides in length. The siRNA associates with a multi-protein complex called the RNA-induced silencing complex (RISC), during which the "passenger" sense strand is cleaved by an enzyme. Subsequently, due to sequence homology, the antisense "guide" strand contained in the activated RISC guides the RISC to the corresponding mRNA, and the same nuclease cleaves the target mRNA, resulting in specific gene silencing. Optimally, the siRNA is 18, 19, 20, 21, 22, 23, or 24 nucleotides in length and has a 2-base overhang at the 3' end. The siRNA can be introduced into individual cells and / or culture systems to cause degradation of the target mRNA sequence. siRNA and shRNA are [Fire et al [Nature, 391:19, 1998] and further described in U.S. Patents No. 7,732,417; No. 8,202,846; and No. 8,383,599.

[0244] In some embodiments, the nucleic acid-based gene-regulatory system comprises one or more morpholinos. "Morpolino" refers to a modified nucleic acid oligomer as used herein, wherein a standard nucleic acid base is bound to a morpholino ring and linked via a phosphorodiamidate linkage. Similar to siRNA and shRNA, the morpholinos bind to a complementary mRNA sequence. However, the morpholinos function by altering mRNA translation and mRNA splicing rather than targeting the complementary mRNA sequence for degradation.

[0245] In some embodiments, nucleic acid-based gene-regulatory systems IKZF1, IKZF3, GATA3, BCL3, TNIP1, TNFAIP3, NFKBIA, SMAD2, TGFBR1, TGFBR2, TANK, FOXP3, RC3H1, TRAF6, IKZF2, CBLB, PPP2R2D, NRP1, HAVCR2, LAG3, TIGIT, CTLA4, PTPN6, PDCD1 or BCORIt comprises a nucleic acid molecule (e.g., siRNA, shRNA, RNA aptamer, or morpholino) that binds to a target RNA sequence that is at least 90%, 95%, 96%, 97%, 98%, or 99% identical or 100% identical to the RNA encoded by the DNA sequence of a target gene selected from (i.e., those listed in Table 2). In some embodiments, the nucleic acid-based gene-regulation system comprises a nucleic acid molecule (e.g., siRNA, shRNA, RNA aptamer, or morpholino) that binds to a target RNA sequence that is at least 90%, 95%, 96%, 97%, 98%, or 99% identical or 100% identical to the RNA sequence encoded by the DNA sequence defined by the set of genomic coordinates presented in Table 5A or Table 5B. Throughout this application, the referred genome coordinates are based on the genome annotations of the GRCh38 (also referred to as hg38) assembly of the human genome from the Genome Reference Consortium, which are available on the website of the National Center for Biotechnology Information. Tools and methods for converting genome coordinates between one assembly and another are known in the art and can be used to convert the genome coordinates provided herein to corresponding coordinates in another assembly of the human genome, including conversions to an initial assembly generated by the same institution or using the same algorithm (e.g., from GRCh38 to GRCh37), and conversions to assemblies generated by different institutions or algorithms (e.g., from GRCh38 to NCBI33, generated by the International Human Genome Sequencing Consortium).Available methods and tools known in the art include, but are not limited to, the NCBI Genome Remapping Service (available from the National Center for Biotechnology Information), UCSC LiftOver (available from the UCSC Genome Browser website), and Assembly Converter (available from the Ensembl.org website).

[0246] In some embodiments, the nucleic acid-based gene-regulation system comprises a nucleic acid molecule (e.g., siRNA, shRNA, RNA aptamer, or morpholino) that binds to a target RNA sequence that is at least 90%, 95%, 96%, 97%, 98%, or 99% identical or 100% identical to the RNA sequence encoded by any one of SEQ ID NOs 154 to 498 or SEQ ID NOs 499 to 813. In some embodiments, the nucleic acid-based gene-regulation system CBLB It comprises a nucleic acid molecule (e.g., siRNA, shRNA, RNA aptamer, or morpholino) that can reduce the expression and / or function of and binds to a target RNA sequence that is at least 90%, 95%, 96%, 97%, 98%, or 99% identical or 100% identical to the RNA sequence encoded by any one of SEQ ID NOs 499 to 524. In some embodiments, the nucleic acid-based gene-regulation system comprises BCOR It comprises a nucleic acid molecule (e.g., siRNA, shRNA, RNA aptamer, or morpholino) that can reduce the expression and / or function of and binds to a target RNA sequence that is at least 90%, 95%, 96%, 97%, 98%, or 99% identical or 100% identical to the RNA sequence encoded by any one of SEQ NOs 708 to 772 or SEQ NOs 708 to 764. In some embodiments, the nucleic acid-based gene-regulation system comprises TNFAIP3It comprises a nucleic acid molecule (e.g., siRNA, shRNA, RNA aptamer, or morpholino) that can reduce the expression and / or function of and binds to a target RNA sequence that is at least 90%, 95%, 96%, 97%, 98%, or 99% identical or 100% identical to the RNA sequence encoded by any one of SEQ ID NOs 348 to 396 or SEQ ID NOs 348 to 386. In some embodiments, the nucleic acid-based gene-regulation system comprises siRNA molecules or shRNA molecules selected from those known in the art, such as siRNA and shRNA constructs available from commercial sources, such as Sigma Aldrich, Dharmacon, ThermoFisher, etc.

[0247] In some embodiments, the endogenous target gene is CBLB And, the nucleic acid molecule is an shRNA encoded by a nucleic acid sequence selected from SEQ ID NOs 41 to 44 (see International PCT Publication No. 2018156886) or selected from SEQ ID NOs 45 to 53 (International PCT Publication No. WO 2017120998). In some embodiments, the endogenous target gene is CBLB And, the nucleic acid molecule is an siRNA comprising a nucleic acid sequence selected from SEQ ID NOs 54 to 63 (see International PCT Publication No. WO 2018006880) or selected from SEQ ID NOs 64 to 73 (International PCT Publication No. WO 2018120998 and No. WO 2018137293).

[0248] In some embodiments, the endogenous target gene is TNFAIP3 And, the nucleic acid molecule is shRNA encoded by a nucleic acid sequence selected from SEQ ID NOs 74 to 95 (see U.S. Patent No. 8,324,369). In some embodiments, the endogenous target gene is TNFAIP3And, the nucleic acid molecule is siRNA comprising a nucleic acid sequence selected from SEQ ID NOs 96 to 105 (see International PCT Publication No. WO 2018006880).

[0249] In some embodiments, the endogenous target gene is CTLA4 And, the nucleic acid molecule is an shRNA encoded by a nucleic acid sequence selected from SEQ ID NOs 128 to 133 (see International PCT Publication No. WO 2017120996). In some embodiments, the endogenous target gene is CTLA4 And, the nucleic acid molecule is an siRNA comprising a nucleic acid sequence selected from SEQ ID NOs 134 to 143 (see International PCT Publications No. WO2017120996, WO2017120998, WO2018137295 and WO2018137293) or selected from SEQ ID NOs 144 to 153 (International PCT Publication No. WO2018006880).

[0250] In some embodiments, the endogenous target gene is PDCD1 And, the nucleic acid molecule is an shRNA encoded by a nucleic acid sequence selected from SEQ ID NOs. 106 to 107 (see International PCT Publication No. WO 2017120996). In some embodiments, the endogenous target gene is PDCD1 And, the nucleic acid molecule is an siRNA comprising a nucleic acid sequence selected from SEQ ID NOs 108 to 117 (see International PCT Publications No. WO2017120996, WO201712998, WO2018137295 and WO2018137293) or selected from SEQ ID NOs 118 to 127 (International PCT Publication No. WO2018006880).

[0251] In some embodiments, nucleic acid-based gene-regulatory systems BCL2L11 , FLI1 , CALM2 , DHODH , UMPS , RBM39 , SEMA7A , CHIC2 , PCBP1 , PBRM1 , WDR6 , E2F8 , SERPINA3 , GNAS, SOCS1 and ANKRD11 It comprises a nucleic acid molecule (e.g., siRNA, shRNA, RNA aptamer, or morpholino) that binds to a target RNA sequence that is at least 90%, 95%, 96%, 97%, 98%, or 99% identical or 100% identical to the RNA sequence encoded by the DNA sequence of a target gene selected from (i.e., those listed in Table 3). In some embodiments, the nucleic acid-based gene-regulation system comprises a nucleic acid molecule (e.g., siRNA, shRNA, RNA aptamer, or morpholino) that binds to a target RNA sequence that is at least 90%, 95%, 96%, 97%, 98%, or 99% identical or 100% identical to the RNA sequence encoded by the DNA sequence defined by the set of genomic coordinates presented in Tables 6A through 6F. In some embodiments, the nucleic acid-based gene-regulatory system comprises a nucleic acid molecule (e.g., siRNA, shRNA, RNA aptamer, or morpholino) that binds to a target RNA sequence that is at least 90%, 95%, 96%, 97%, 98%, or 99% identical to or 100% identical to the RNA sequence encoded by any one of SEQ ID NOs 814 to 1232.

[0252] In some embodiments, nucleic acid-based gene-regulatory systems BCL2L11 , FLI1 , CALM2 , DHODH , UMPS , RBM39 , SEMA7A , CHIC2 , PCBP1 , PBRM1 , WDR6 , E2F8 , SERPINA3 and GNAS It can reduce the expression and / or function of selected target genes. .In some embodiments, the nucleic acid-based gene-regulation system comprises a nucleic acid molecule (e.g., siRNA, shRNA, RNA aptamer, or morpholino) that binds to a target RNA sequence that is at least 90%, 95%, 96%, 97%, 98%, or 99% identical or 100% identical to an RNA sequence encoded by a set of genomic coordinates presented in either Table 6A or Table 6B. In some embodiments, the nucleic acid-based gene-regulation system comprises a nucleic acid molecule (e.g., siRNA, shRNA, RNA aptamer, or morpholino) that binds to a target RNA sequence that is at least 90%, 95%, 96%, 97%, 98%, or 99% identical or 100% identical to an RNA sequence encoded by any one of SEQ ID NOs 814 to 1064.

[0253] In some embodiments, nucleic acid-based gene-regulatory systems SOCS1 It may reduce the expression and / or function of .In some embodiments, the nucleic acid-based gene-regulation system comprises a nucleic acid molecule (e.g., siRNA, shRNA, RNA aptamer, or morpholino) that binds to a target RNA sequence that is at least 90%, 95%, 96%, 97%, 98%, or 99% identical or 100% identical to an RNA sequence encoded by a set of genomic coordinates presented in either Table 6C or Table 6D. In some embodiments, the nucleic acid-based gene-regulation system comprises a nucleic acid molecule (e.g., siRNA, shRNA, RNA aptamer, or morpholino) that binds to a target RNA sequence that is at least 90%, 95%, 96%, 97%, 98%, or 99% identical or 100% identical to an RNA sequence encoded by any one of SEQ ID NOs 1088 to 1232. In some embodiments, the nucleic acid-based gene-regulation system comprises a nucleic acid molecule (e.g., siRNA, shRNA, RNA aptamer, or morpholino) that binds to a target RNA sequence that is at least 90%, 95%, 96%, 97%, 98%, or 99% identical or 100% identical to an RNA sequence encoded by one of SEQ ID NOs 1088 to 1200. In some embodiments, the nucleic acid-based gene-regulation system comprises a nucleic acid molecule (e.g., siRNA, shRNA, RNA aptamer, or morpholino) that binds to a target RNA sequence that is at least 90%, 95%, 96%, 97%, 98%, or 99% identical or 100% identical to an RNA sequence encoded by one of SEQ ID NOs 1088 to 1140. In some embodiments, the nucleic acid-based gene-regulatory system comprises a nucleic acid molecule (e.g., siRNA, shRNA, RNA aptamer, or morpholino) that binds to a target RNA sequence that is at least 90%, 95%, 96%, 97%, 98%, or 99% identical to or 100% identical to the RNA sequence encoded by any one of SEQ ID NOs 1088 to 1120.In some embodiments, the nucleic acid-based gene-regulation system comprises a nucleic acid molecule (e.g., siRNA, shRNA, RNA aptamer, or morpholino) that binds to a target RNA sequence that is at least 90%, 95%, 96%, 97%, 98%, or 99% identical or 100% identical to an RNA sequence encoded by one of SEQ ID NOs 1088 to 1110. In some embodiments, the nucleic acid-based gene-regulation system comprises a nucleic acid molecule (e.g., siRNA, shRNA, RNA aptamer, or morpholino) that binds to a target RNA sequence that is at least 90%, 95%, 96%, 97%, 98%, or 99% identical or 100% identical to an RNA sequence encoded by one of SEQ ID NOs 1102, 1103, 1105 to 1108, or 1115. In some embodiments, the nucleic acid-based gene-regulatory system comprises a nucleic acid molecule (e.g., siRNA, shRNA, RNA aptamer, or morpholino) that binds to a target RNA sequence that is at least 90%, 95%, 96%, 97%, 98%, or 99% identical or 100% identical to an RNA sequence encoded by one of SEQ ID NOs 1106, 1110, 1115, 1116, 1118, 1126, 1129, 1141, 1157, or 1174.

[0254] In some embodiments, nucleic acid-based gene-regulatory systems ANKRD11 It may reduce the expression and / or function of .In some embodiments, the nucleic acid-based gene-regulation system comprises a nucleic acid molecule (e.g., siRNA, shRNA, RNA aptamer, or morpholino) that binds to a target RNA sequence that is at least 90%, 95%, 96%, 97%, 98%, or 99% identical or 100% identical to an RNA sequence encoded by a set of genomic coordinates presented in Table 6E or Table 6F. In some embodiments, the nucleic acid-based gene-regulation system comprises a nucleic acid molecule (e.g., siRNA, shRNA, RNA aptamer, or morpholino) that binds to a target RNA sequence that is at least 90%, 95%, 96%, 97%, 98%, or 99% identical or 100% identical to an RNA sequence encoded by any one of SEQ ID NOs 1065 to 1087.

[0255] In some embodiments, the endogenous target gene is SOCS1 And, the nucleic acid molecule is an shRNA that binds to a target sequence selected from SEQ ID NOs 1236 to 1255 (see U.S. Patent No. 9,944,931). In some embodiments, the endogenous target gene is SOCS1 And, the nucleic acid molecule is an shRNA encoded by a nucleic acid sequence selected from SEQ ID NOs 1258 to 1260 (see U.S. Patent No. 8,324,369). In some embodiments, the endogenous target gene is SOCS1 The nucleic acid molecule is an siRNA comprising a nucleic acid sequence selected from SEQ NOs 1261 to 1270 (see International PCT Publications WO 2017120996; WO 2018137295; WO 2017120998; and WO 2018137293).

[0256] In some embodiments, the endogenous target gene is ANKRD11 and, the nucleic acid molecule is an shRNA that binds to a target sequence selected from SEQ ID NOs 1233 to 1235 (Reference [allagher et al[Refer to Developmental Cell (2015), 32(1); 31-42]). In some embodiments, the endogenous target gene is ANKRD11 and, the nucleic acid molecule is an shRNA encoded by a nucleic acid sequence selected from SEQ ID NOs 1256 to 1257 (Reference [Zhang et al ., Biochem Biophys Res Commun (2007) 358(4): 1034-1040].

[0257] In some embodiments, the nucleic acid-based gene-regulation system comprises siRNA molecules or shRNA molecules selected from those known in the art, such as those available from commercial sources, such as Sigma-Aldrich, Damacon, Thermo Fisher, etc. Exemplary siRNA and shRNA constructs are listed in Tables 4A and 4B below. In some embodiments, the nucleic acid-based gene-regulation system comprises two or more siRNA molecules selected from those known in the art, such as the siRNA construct listed in Table 4A. In some embodiments, the nucleic acid-based gene-regulation system comprises two or more shRNA molecules selected from those known in the art, such as the shRNA construct listed in Table 4B.

[0258] [Table 4A]

[0259]

[0260]

[0261]

[0262] [Table 4B]

[0263]

[0264]

[0265] In some embodiments, the gene-regulatory system is two or more nucleic acid molecules (e.g., ,It comprises two or more types of siRNA, two or more types of shRNA, two or more types of RNA aptamers, or two or more types of morpholinos, wherein at least one of the nucleic acid molecules IKZF1, IKZF3, GATA3, BCL3, TNIP1, TNFAIP3, NFKBIA, SMAD2, TGFBR1, TGFBR2, TANK, FOXP3, RC3H1, TRAF6, IKZF2, CBLB, PPP2R2D, NRP1, HAVCR2, LAG3, TIGIT, CTLA4, PTPN6, PDCD1 or BCOR Binding to a target RNA that is at least 90%, 95%, 96%, 97%, 98%, or 99% identical or 100% identical to the RNA sequence encoded by the DNA sequence of a target gene selected from (e.g., a gene selected from Table 2), and at least one of the nucleic acid molecules BCL2L11 , FLI1 , CALM2 , DHODH , UMPS , RBM39 , SEMA7A , CHIC2 , PCBP1 , PBRM1 , WDR6 , E2F8 , SERPINA3 , GNAS, SOCS1 and ANKRD11 It binds to a target RNA that is at least 90%, 95%, 96%, 97%, 98%, or 100% identical to the RNA sequence encoded by the DNA sequence of the target gene selected from (e.g., a gene selected from Table 3).

[0266] In some embodiments, at least one of two or more nucleic acid molecules for a target RNA sequence that is at least 90%, 95%, 96%, 97%, 98%, or 99% identical or 100% identical to the RNA sequence encoded by the DNA sequence defined by the set of genome coordinates presented in Table 5A or Table 5B, and at least one of two or more nucleic acid molecules binds to a target RNA sequence that is at least 90%, 95%, 96%, 97%, 98%, or 99% identical or 100% identical to the RNA sequence encoded by the DNA sequence defined by the set of genome coordinates presented in Tables 6A to 6F. In some embodiments, at least one of two or more nucleic acid molecules binds to a target RNA sequence that is at least 90%, 95%, 96%, 97%, 98%, or 99% identical or 100% identical to an RNA sequence encoded by one of SEQ ID NOs 814 to 1232, and at least one of two or more nucleic acid molecules binds to a target RNA sequence that is at least 90%, 95%, 96%, 97%, 98%, or 99% identical or 100% identical to an RNA sequence encoded by one of SEQ ID NOs 154 to 498 or SEQ ID NOs 499 to 813.

[0267] In some embodiments, the gene-regulatory system comprises two or more nucleic acid molecules, wherein at least one of the nucleic acid molecules is IKZF1, IKZF3, GATA3, BCL3, TNIP1, TNFAIP3, NFKBIA, SMAD2, TGFBR1, TGFBR2, TANK, FOXP3, RC3H1, TRAF6, IKZF2, CBLB, PPP2R2D, NRP1, HAVCR2, LAG3, TIGIT, CTLA4, PTPN6, PDCD1 or BCOR Binds to a target RNA sequence encoded by the DNA sequence of a target gene selected from, wherein at least one of the nucleic acid molecules BCL2L11 , FLI1 , CALM2 , DHODH , UMPS , RBM39 , SEMA7A , CHIC2 , PCBP1 , PBRM1 , WDR6 , E2F8 , SERPINA3 and GNASIt binds to a target RNA sequence encoded by the DNA sequence of a target gene selected from. . In some embodiments, at least one of two or more nucleic acid molecules for a target RNA sequence that is at least 90%, 95%, 96%, 97%, 98%, or 99% identical or 100% identical to the RNA sequence encoded by the DNA sequence defined by the set of genome coordinates presented in Table 5A or Table 5B, and at least one of two or more nucleic acid molecules binds to a target RNA sequence that is at least 90%, 95%, 96%, 97%, 98%, or 99% identical or 100% identical to the RNA sequence encoded by the DNA sequence defined by the set of genome coordinates presented in Tables 6A to 6B. In some embodiments, at least one of two or more nucleic acid molecules binds to a target RNA sequence that is at least 90%, 95%, 96%, 97%, 98%, or 99% identical or 100% identical to an RNA sequence encoded by one of SEQ ID NOs 814 to 1064, and at least one of two or more nucleic acid molecules binds to a target RNA sequence that is at least 90%, 95%, 96%, 97%, 98%, or 99% identical or 100% identical to an RNA sequence encoded by one of SEQ ID NOs 154 to 498 or SEQ ID NOs 499 to 813.

[0268] In some embodiments, the gene-regulatory system comprises two or more nucleic acid molecules, wherein at least one of the nucleic acid molecules is CBLB It binds to a target RNA sequence encoded by the DNA sequence, wherein at least one of the nucleic acid molecules BCL2L11 , FLI1 , CALM2 , DHODH , UMPS , RBM39 , SEMA7A , CHIC2 , PCBP1 , PBRM1 , WDR6 , E2F8 , SERPINA3 and GNAS It binds to a target RNA sequence encoded by the DNA sequence of a target gene selected from. . In some embodiments, at least one of two or more nucleic acid molecules binds to a target RNA sequence that is at least 90%, 95%, 96%, 97%, 98%, or 99% identical or 100% identical to an RNA sequence encoded by one of SEQ ID NOs 499 to 524, and at least one of two or more nucleic acid molecules binds to a target RNA sequence that is at least 90%, 95%, 96%, 97%, 98%, or 99% identical or 100% identical to an RNA sequence encoded by one of SEQ ID NOs 814 to 1064.

[0269] In some embodiments, the gene-regulatory system comprises two or more nucleic acid molecules, wherein at least one of the nucleic acid molecules is IKZF1, IKZF3, GATA3, BCL3, TNIP1, TNFAIP3, NFKBIA, SMAD2, TGFBR1, TGFBR2, TANK, FOXP3, RC3H1, TRAF6, IKZF2, CBLB, PPP2R2D, NRP1, HAVCR2, LAG3, TIGIT, CTLA4, PTPN6, PDCD1 or BCOR Binds to a target RNA sequence encoded by the DNA sequence of a target gene selected from, wherein at least one of the nucleic acid molecules SOCS1 It binds to the target RNA sequence encoded by the DNA sequence of the gene. .In some embodiments, at least one of two or more nucleic acid molecules for a target RNA sequence that is at least 90%, 95%, 96%, 97%, 98%, or 99% identical or 100% identical to the RNA sequence encoded by the set of genome coordinates presented in Table 5A or Table 5B, and at least one of two or more nucleic acid molecules binds to a target RNA sequence that is at least 90%, 95%, 96%, 97%, 98%, or 99% identical or 100% identical to the RNA sequence encoded by the set of genome coordinates presented in Table 6C or Table 6D. In some embodiments, at least one of two or more nucleic acid molecules binds to a target RNA sequence that is at least 90%, 95%, 96%, 97%, 98%, or 99% identical or 100% identical to an RNA sequence encoded by one of SEQ ID NOs 154 to 498 or SEQ ID NOs 499 to 813, and at least one of two or more nucleic acid molecules binds to a target RNA sequence that is at least 90%, 95%, 96%, 97%, 98%, or 99% identical or 100% identical to an RNA sequence encoded by one of SEQ ID NOs 1088 to 1232.

[0270] In some embodiments, the gene-regulatory system comprises two or more nucleic acid molecules, wherein at least one of the nucleic acid molecules is CBLB It binds to a target RNA sequence encoded by a gene, wherein at least one of the nucleic acid molecules SOCS1 It binds to the target RNA sequence encoded by the DNA sequence of the gene. .In some embodiments, at least one of two or more nucleic acid molecules binds to a target RNA sequence that is at least 90%, 95%, 96%, 97%, 98%, or 99% identical or 100% identical to an RNA sequence encoded by one of SEQ ID NOs 499 to 524, and at least one of two or more nucleic acid molecules binds to a target RNA sequence that is at least 90%, 95%, 96%, 97%, 98%, or 99% identical or 100% identical to an RNA sequence encoded by one of SEQ ID NOs 1088 to 1232, SEQ ID NOs 1088 to 1200, SEQ ID NOs 1088 to 1140, or SEQ ID NOs 1088 to 1120. In some embodiments, at least one of two or more nucleic acid molecules binds to an RNA sequence that is at least 90%, 95%, 96% identical, or 97%, 98%, or 99% identical or 100% identical to an RNA sequence encoded by one of SEQ ID NOs 499 to 524. Binds to a target RNA sequence that is 97%, 98%, or 99% identical or 100% identical, and at least one of two or more nucleic acid molecules binds to a target RNA sequence that is at least 90%, 95%, 96%, 97%, 98%, or 99% identical or 100% identical to the RNA sequence encoded by one of SEQ ID NOs 1106, 1110, 1115, 1116, 1118, 1126, 1129, 1141, 1157, 1174. In some embodiments, at least one of two or more nucleic acid molecules binds to a target RNA sequence that is at least 90%, 95%, 96%, 97%, 98%, or 99% identical or 100% identical to an RNA sequence encoded by one of SEQ ID NOs 499 to 524, and at least one of two or more nucleic acid molecules binds to a target RNA sequence that is at least 90%, 95%, 96%, 97%, 98%, or 99% identical or 100% identical to an RNA sequence encoded by one of SEQ ID NOs 1102, 1103, 1105 to 1108, and 1115.

[0271] In some embodiments, the gene-regulatory system comprises two or more nucleic acid molecules, wherein at least one of the nucleic acid molecules is IKZF1, IKZF3, GATA3, BCL3, TNIP1, TNFAIP3, NFKBIA, SMAD2, TGFBR1, TGFBR2, TANK, FOXP3, RC3H1, TRAF6, IKZF2, CBLB, PPP2R2D, NRP1, HAVCR2, LAG3, TIGIT, CTLA4, PTPN6, PDCD1 or BCOR Binds to a target RNA sequence encoded by the DNA sequence of a target gene selected from, wherein at least one of the nucleic acid molecules ANKRD11 It binds to the target RNA sequence encoded by the DNA sequence of the gene. . In some embodiments, at least one of two or more nucleic acid molecules for a target RNA sequence that is at least 90%, 95%, 96%, 97%, 98%, or 99% identical or 100% identical to the RNA sequence encoded by the DNA sequence defined by the set of genome coordinates presented in Table 5A or Table 5B, and at least one of two or more nucleic acid molecules binds to a target RNA sequence that is at least 90%, 95%, 96%, 97%, 98%, or 99% identical or 100% identical to the RNA sequence encoded by the DNA sequence defined by the set of genome coordinates presented in Table 6E or Table 6F. In some embodiments, at least one of two or more nucleic acid molecules binds to a target RNA sequence that is at least 90%, 95%, 96%, 97%, 98%, or 99% identical or 100% identical to an RNA sequence encoded by one of SEQ ID NOs 154 to 498 or SEQ ID NOs 499 to 813, and at least one of two or more nucleic acid molecules binds to a target RNA sequence that is at least 90%, 95%, 96%, 97%, 98%, or 99% identical or 100% identical to an RNA sequence encoded by one of SEQ ID NOs 1065 to 1087.

[0272] In some embodiments, the gene-regulatory system comprises two or more nucleic acid molecules, wherein at least one of the nucleic acid molecules is CBLB It binds to a target RNA sequence encoded by a gene, wherein at least one of the nucleic acid molecules ANKRD11It binds to the target RNA sequence encoded by the DNA sequence of the gene. . In some embodiments, at least one of two or more nucleic acid molecules binds to a target RNA sequence that is at least 90%, 95%, 96%, 97%, 98%, or 99% identical or 100% identical to an RNA sequence encoded by one of SEQ ID NOs 499 to 524, and at least one of two or more nucleic acid molecules binds to a target RNA sequence that is at least 90%, 95%, 96%, 97%, 98%, or 99% identical or 100% identical to an RNA sequence encoded by one of SEQ ID NOs 1065 to 1087.

[0273] In some embodiments, the gene-regulatory system comprises two or more nucleic acid molecules, wherein at least one of the nucleic acid molecules is ANKRD11 It binds to a target RNA sequence encoded by a gene, wherein at least one of the nucleic acid molecules SOCS1 It binds to the target RNA sequence encoded by the DNA sequence of the gene. . In some embodiments, at least one of two or more nucleic acid molecules binds to a target RNA sequence that is at least 90%, 95%, 96%, 97%, 98%, or 99% identical or 100% identical to an RNA sequence encoded by one of SEQ ID NOs 1065 to 1087, and at least one of two or more nucleic acid molecules binds to a target RNA sequence that is at least 90%, 95%, 96%, 97%, 98%, or 99% identical or 100% identical to an RNA sequence encoded by one of SEQ ID NOs 1088 to 1232.

[0274] B. 단백질-기반 유전자-조절 시스템

[0275] In some embodiments, the protein-based gene-regulatory system is a system comprising one or more proteins capable of regulating the expression of an endogenous target gene in a sequence-specific manner without the requirement of a nucleic acid guide molecule. In some embodiments, the protein-based gene-regulatory system comprises a protein comprising one or more zinc-finger binding domains and enzyme domains. In some embodiments, the protein-based gene-regulatory system comprises a protein comprising a transcription activator-like effector nuclease (TALEN) domain and an enzyme domain. These embodiments are referred to herein as “TALEN”.

[0276] 1. 아연 핑거 시스템

[0277] A zinc finger-based system comprises a fusion protein containing two protein domains: a zinc finger DNA binding domain and an enzyme domain. The “zinc finger DNA binding domain,” “zinc finger protein,” or “ZFP” is a domain within a protein or a larger protein that binds to DNA in a sequence-specific manner through one or more zinc fingers, which is a region of amino acid sequence within the binding domain whose structure is stabilized through the coordination of zinc ions. By binding to a target DNA sequence, the zinc finger domain directs the activity of the enzyme domain in the vicinity of that sequence, thereby inducing modification of the endogenous target gene in the vicinity of the target sequence. The zinc finger domain can be engineered to bind to virtually any desired sequence. Thus, after identifying a target gene locus containing a target DNA sequence where cleavage or recombination is desired (e.g., a target locus in the target gene mentioned in Table 2 or Table 3), one or more zinc finger binding domains can be engineered to bind to one or more target DNA sequences at the target gene locus. Expression of a fusion protein containing a zinc finger binding domain and an enzyme domain in cells achieves modification at a target gene locus.

[0278] In some embodiments, the zinc finger binding domain comprises one or more zinc fingers (Miller et al. (1985) EMBO J. 4:1609-1614; Rhodes (1993) Scientific American February:56-65; U.S. Patent No. 6,453,242). Typically, a single zinc finger domain is about 30 amino acids long. Individual zinc fingers bind to a 3-nucleotide (i.e., triplicate) sequence (or a 4-nucleotide sequence that can overlap with the 4-nucleotide binding site of an adjacent zinc finger by one nucleotide). Therefore, the length of the sequence (e.g., target sequence) to which the zinc finger binding domain is engineered to bind will determine the number of zinc fingers in the engineered zinc finger binding domain. For example, in the case of a ZFP where the finger motif does not bind to the overlap sub-site, a 6-nucleotide target sequence is bound by a 2-nucleotide binding domain; A 9-nucleotide target sequence is bound by a 3-finger binding domain, and so on. Binding sites (i.e., sub-regions) for individual zinc fingers within the target site do not need to be adjacent, but may be separated by one or several nucleotides depending on the length and nature of the amino acid sequence between zinc fingers (i.e., inter-finger linkers) within the multi-finger binding domain. In some embodiments, the DNA-binding domain of an individual ZFN comprises 3 to 6 individual zinc finger repeats, each capable of recognizing 9 to 18 base pairs.

[0279] Zinc finger binding domains can be manipulated to bind to selected sequences (e.g., literature [Beerli et al. (2002) Nature Biotechnol. 20:135-141; Pabo et al. (2001) Ann. Rev. Biochem. 70:313-340; Isalan et al.(2001) Nature Biotechnol. 19:656-660; Segal et al. (2001) Curr. Opin. Biotechnology. 12:632-637; Choo et al. [Reference: (2000) Curr. Opin. Struct. Biol. 10:411-416]). Engineered zinc finger binding domains may have novel binding specificities compared to naturally occurring zinc finger proteins. Engineering methods include, but are not limited to, rational design and various types of selection.

[0280] The selection of a target DNA sequence for binding by a zinc finger domain can be achieved, for example, according to the method disclosed in U.S. Patent No. 6,453,242. It will be apparent to those skilled in the art that a simple visual inspection of the nucleotide sequence can also be used for the selection of the target DNA sequence. Accordingly, any means for selecting the target DNA sequence may be used in the method described herein. The target site is generally bound by a zinc finger binding domain that is at least 9 nucleotides long and thus comprises at least 3 zinc fingers. However, binding to a 12-nucleotide target site of a 4-finger binding domain, binding to a 15-nucleotide target site of a 5-finger binding domain, or binding to an 18-nucleotide target site of a 6-finger binding domain is also possible. It will be apparent that binding to longer target sites of larger binding domains (e.g., 7-, 8-, 9-fingers and beyond) is also possible.

[0281] In some embodiments, the zinc finger binding domain is IKZF1, IKZF3, GATA3, BCL3, TNIP1, TNFAIP3, NFKBIA, SMAD2, TGFBR1, TGFBR2, TANK, FOXP3, RC3H1, TRAF6, IKZF2, CBLB, PPP2R2D, NRP1, HAVCR2, LAG3, TIGIT, CTLA4, PTPN6, PDCD1 or BCORIt binds to a target DNA sequence that is at least 90%, 95%, 96%, 97%, 98%, or 99% identical or 100% identical to the target DNA sequence of a target gene selected from (e.g., a gene selected from Table 2). In some embodiments, the zinc finger binding domain binds to a target DNA sequence that is at least 90%, 95%, 96%, 97%, 98%, or 99% identical or 100% identical to the target DNA sequence defined by the set of genomic coordinates presented in Table 5A or Table 5B. In some embodiments, the zinc finger binding domain binds to a target DNA sequence that is at least 90%, 95%, 96%, 97%, 98%, or 99% identical or 100% identical to one of SEQ ID NOs 154 to 498 or SEQ ID NOs 499 to 813. In some embodiments, the zinc finger binding domain CBLB It binds to a target DNA sequence that is at least 90%, 95%, 96%, 97%, 98%, or 99% identical or 100% identical to the target DNA sequence of. In some embodiments, the zinc finger binding domain binds to a target DNA sequence that is at least 90%, 95%, 96%, 97%, 98%, or 99% identical or 100% identical to one of SEQ ID NOs 499 to 524. In some embodiments, the zinc finger binding domain BCOR It binds to a target DNA sequence that is at least 90%, 95%, 96%, 97%, 98%, or 99% identical or 100% identical to the target DNA sequence of. In some embodiments, the zinc finger binding domain binds to a target DNA sequence that is at least 90%, 95%, 96%, 97%, 98%, or 99% identical or 100% identical to one of SEQ ID NOs 708 to 772 or SEQ ID NOs 708 to 764. In some embodiments, the zinc finger binding domain TNFAIP3It binds to a target DNA sequence that is at least 90%, 95%, 96%, 97%, 98%, or 99% identical or 100% identical to the target DNA sequence. In some embodiments, the zinc finger binding domain binds to a target DNA sequence that is at least 90%, 95%, 96%, 97%, 98%, or 99% identical or 100% identical to one of SEQ ID NOs 348 to 396 or SEQ ID NOs 348 to 386.

[0282] In some embodiments, the zinc finger binding domain is BCL2L11 , FLI1 , CALM2 , DHODH , UMPS , RBM39 , SEMA7A , CHIC2 , PCBP1 , PBRM1 , WDR6 , E2F8 , SERPINA3 , GNAS, SOCS1, and ANKRD11 It binds to a target DNA sequence that is at least 90%, 95%, 96%, 97%, 98%, or 99% identical or 100% identical to the target DNA sequence of a target gene selected from (e.g., a gene selected from Table 3). In some embodiments, the zinc finger binding domain binds to a target DNA sequence that is at least 90%, 95%, 96%, 97%, 98%, or 99% identical or 100% identical to the target DNA sequence defined by a set of genomic coordinates presented in one of Tables 6A through 6F. In some embodiments, the zinc finger binding domain binds to a target DNA sequence that is at least 90%, 95%, 96%, 97%, 98%, or 99% identical or 100% identical to one of SEQ ID NOs 814 through 1232.

[0283] In some embodiments, the zinc finger binding domain is BCL2L11 , FLI1 , CALM2 , DHODH , UMPS , RBM39 , SEMA7A , CHIC2 , PCBP1 , PBRM1 , WDR6 , E2F8 , SERPINA3 and GNAS It binds to a target DNA sequence that is at least 90%, 95%, 96%, 97%, 98%, or 99% identical or 100% identical to the target DNA sequence of a target gene selected from. In some embodiments, the zinc finger binding domain binds to a target DNA sequence that is at least 90%, 95%, 96%, 97%, 98%, or 99% identical or 100% identical to the target DNA sequence defined by the set of genomic coordinates presented in Table 6A or Table 6B. In some embodiments, the zinc finger binding domain binds to a target DNA sequence that is at least 90%, 95%, 96%, 97%, 98%, or 99% identical or 100% identical to one of SEQ ID NOs 814 to 1064.

[0284] In some embodiments, the zinc finger binding domain is SOCS1 It binds to a target DNA sequence that is at least 90%, 95%, 96%, 97%, 98%, or 99% identical or 100% identical to the target DNA sequence of the gene. In some embodiments, the zinc finger binding domain binds to a target DNA sequence that is at least 90%, 95%, 96%, 97%, 98%, or 99% identical or 100% identical to the target DNA sequence defined by the set of genomic coordinates presented in Table 6C or Table 6D. In some embodiments, the zinc finger binding domain binds to a target DNA sequence that is at least 90%, 95%, 96%, 97%, 98%, or 99% identical or 100% identical to one of SEQ ID NOs 1088 to 1232. In some embodiments, the zinc finger binding domain ANKRD11It binds to a target DNA sequence that is at least 90%, 95%, 96%, 97%, 98%, or 99% identical or 100% identical to the target DNA sequence of the gene. In some embodiments, the zinc finger binding domain binds to a target DNA sequence that is at least 90%, 95%, 96%, 97%, 98%, or 99% identical or 100% identical to the target DNA sequence defined by the set of genomic coordinates presented in Table 6E or Table 6F. In some embodiments, the zinc finger binding domain binds to a target DNA sequence that is at least 90%, 95%, 96%, 97%, 98%, or 99% identical or 100% identical to one of SEQ ID NOs 1065 to 1087.

[0285] In some embodiments, the zinc finger system is selected from those known in the art, for example, those available from commercial sources, such as Sigma Aldrich. For example, in some embodiments, the zinc finger system is selected from those known in this specification, for example, those listed in Table 7 below.

[0286]

[0287]

[0288] In some embodiments, the gene-regulatory system comprises two or more ZFP-fusion proteins, each comprising a zinc finger binding domain, wherein at least one of the zinc finger binding domains is IKZF1, IKZF3, GATA3, BCL3, TNIP1, TNFAIP3, NFKBIA, SMAD2, TGFBR1, TGFBR2, TANK, FOXP3, RC3H1, TRAF6, IKZF2, CBLB, PPP2R2D, NRP1, HAVCR2, LAG3, TIGIT, CTLA4, PTPN6, PDCD1 or BCOR Binding to a target DNA sequence that is at least 90%, 95%, 96%, 97%, 98%, or 99% identical to the target DNA sequence of a target gene selected from, or 100% identical to the target DNA sequence, and at least one of the zinc finger binding domains BCL2L11 , FLI1 , CALM2 , DHODH , UMPS , RBM39 , SEMA7A , CHIC2 , PCBP1, PBRM1 , WDR6 , E2F8 , SERPINA3 , GNAS, SOCS1 and ANKRD11 It binds to a target DNA sequence that is at least 90%, 95%, 96%, 97%, 98%, or 99% identical or 100% identical to the target DNA sequence of the target gene selected from. In some embodiments, at least one zinc finger binding domain binds to a target DNA sequence that is at least 90%, 95%, 96%, 97%, 98%, or 99% identical or 100% identical to the target DNA sequence defined by the set of genomic coordinates presented in Table 5A or Table 5B, and at least one zinc finger binding domain binds to a target DNA sequence that is at least 90%, 95%, 96%, 97%, 98%, or 99% identical or 100% identical to the target DNA sequence defined by the set of genomic coordinates presented in any one of Tables 6A to 6F. In some embodiments, at least one of the zinc finger binding domains binds to a target DNA sequence that is at least 90%, 95%, 96%, 97%, 98%, or 99% identical or 100% identical to one of SEQ ID NOs 154 to 498 or SEQ ID NOs 499 to 813, and at least one of the zinc finger binding domains binds to a target DNA sequence that is at least 90%, 95%, 96%, 97%, 98%, or 99% identical or 100% identical to one of SEQ ID NOs 814 to 1232.

[0289] In some embodiments, the gene-regulatory system comprises two or more ZFP-fusion proteins, each comprising a zinc finger binding domain, wherein at least one of the zinc finger binding domains is IKZF1, IKZF3, GATA3, BCL3, TNIP1, TNFAIP3, NFKBIA, SMAD2, TGFBR1, TGFBR2, TANK, FOXP3, RC3H1, TRAF6, IKZF2, CBLB, PPP2R2D, NRP1, HAVCR2, LAG3, TIGIT, CTLA4, PTPN6, PDCD1 or BCOR Binds to the target DNA sequence of a target gene selected from, and at least one of the zinc finger binding domains BCL2L11 , FLI1 , CALM2 , DHODH , UMPS , RBM39 , SEMA7A , CHIC2 , PCBP1 , PBRM1 , WDR6 , E2F8 , SERPINA3 and GNAS It binds to the target DNA sequence of a target gene selected from. In some embodiments, at least one of two or more zinc finger binding domains binds to a target DNA sequence that is at least 90%, 95%, 96%, 97%, 98%, or 99% identical or 100% identical to the target DNA sequence defined by the set of genomic coordinates presented in Table 5A or Table 5B, and at least one of two or more zinc finger binding domains binds to a target DNA sequence that is at least 90%, 95%, 96%, 97%, 98%, or 99% identical or 100% identical to the target DNA sequence defined by the set of genomic coordinates presented in Table 6A or Table 6B. In some embodiments, at least one of two or more zinc finger binding domains binds to a target DNA sequence that is at least 90%, 95%, 96%, 97%, 98%, or 99% identical or 100% identical to one of SEQ ID NOs 154 to 498 or SEQ ID NOs 499 to 813, and at least one of two or more zinc finger binding domains binds to a target DNA sequence that is at least 90%, 95%, 96%, 97%, 98%, or 99% identical or 100% identical to one of SEQ ID NOs 814 to 1064.

[0290] In some embodiments, the gene-regulatory system comprises two or more ZFP-fusion proteins, each comprising a zinc finger binding domain, wherein at least one of the zinc finger binding domains is CBLB Binds to a target DNA sequence of a gene, and at least one of the zinc finger binding domains BCL2L11 , FLI1 , CALM2 , DHODH , UMPS , RBM39 , SEMA7A , CHIC2 , PCBP1 , PBRM1 , WDR6 , E2F8 , SERPINA3 and GNAS It binds to the target DNA sequence of a target gene selected from. In some embodiments, at least one of two or more zinc finger binding domains binds to a target DNA sequence that is at least 90%, 95%, 96%, 97%, 98%, or 99% identical or 100% identical to one of SEQ ID NOs 499 to 524, and at least one of two or more zinc finger binding domains binds to a target DNA sequence that is at least 90%, 95%, 96%, 97%, 98%, or 99% identical or 100% identical to one of SEQ ID NOs 814 to 1064.

[0291] In some embodiments, the gene-regulatory system comprises two or more ZFP-fusion proteins, each comprising a zinc finger binding domain, wherein at least one of the zinc finger binding domains is IKZF1, IKZF3, GATA3, BCL3, TNIP1, TNFAIP3, NFKBIA, SMAD2, TGFBR1, TGFBR2, TANK, FOXP3, RC3H1, TRAF6, IKZF2, CBLB, PPP2R2D, NRP1, HAVCR2, LAG3, TIGIT, CTLA4, PTPN6, PDCD1 or BCOR Binds to the target DNA sequence of a target gene selected from, and at least one of the zinc finger binding domains SOCS1It binds to a target DNA sequence of a gene. In some embodiments, at least one of two or more zinc finger binding domains binds to a target DNA sequence that is at least 90%, 95%, 96%, 97%, 98%, or 99% identical or 100% identical to the target DNA sequence defined by the set of genomic coordinates presented in Table 5A or Table 5B, and at least one of two or more zinc finger binding domains binds to a target DNA sequence that is at least 90%, 95%, 96%, 97%, 98%, or 99% identical or 100% identical to the target DNA sequence defined by the set of genomic coordinates presented in Table 6C or Table 6D. In some embodiments, at least one of two or more zinc finger binding domains binds to a target DNA sequence that is at least 90%, 95%, 96%, 97%, 98%, or 99% identical or 100% identical to one of SEQ ID NOs 154 to 498 or SEQ ID NOs 499 to 813, and at least one of two or more zinc finger binding domains binds to a target DNA sequence that is at least 90%, 95%, 96%, 97%, 98%, or 99% identical or 100% identical to one of SEQ ID NOs 1088 to 1232.

[0292] In some embodiments, the gene-regulatory system comprises two or more ZFP-fusion proteins, each comprising a zinc finger binding domain, wherein at least one of the zinc finger binding domains is CBLB Binds to the target DNA sequence of the target gene of the gene, and at least one of the zinc finger binding domains SOCS1It binds to a target DNA sequence of a gene. In some embodiments, at least one of two or more zinc finger binding domains binds to a target DNA sequence that is at least 90%, 95%, 96%, 97%, 98%, or 99% identical or 100% identical to one of SEQ ID NOs 499 to 524, and at least one of two or more zinc finger binding domains binds to a target DNA sequence that is at least 90%, 95%, 96%, 97%, 98%, or 99% identical or 100% identical to one of SEQ ID NOs 1088 to 1232.

[0293] In some embodiments, the gene-regulatory system comprises two or more ZFP-fusion proteins, each comprising a zinc finger binding domain, wherein at least one of the zinc finger binding domains is IKZF1, IKZF3, GATA3, BCL3, TNIP1, TNFAIP3, NFKBIA, SMAD2, TGFBR1, TGFBR2, TANK, FOXP3, RC3H1, TRAF6, IKZF2, CBLB, PPP2R2D, NRP1, HAVCR2, LAG3, TIGIT, CTLA4, PTPN6, PDCD1 or BCOR Binds to the target DNA sequence of a target gene selected from, and at least one of the zinc finger binding domains ANKRD11It binds to a target DNA sequence of a gene. In some embodiments, at least one of two or more zinc finger binding domains binds to a target DNA sequence that is at least 90%, 95%, 96%, 97%, 98%, or 99% identical or 100% identical to the target DNA sequence defined by the set of genomic coordinates presented in Table 5A or Table 5B, and at least one of two or more zinc finger binding domains binds to a target DNA sequence that is at least 90%, 95%, 96%, 97%, 98%, or 99% identical or 100% identical to the target DNA sequence defined by the set of genomic coordinates presented in Table 6D or Table 6E. In some embodiments, at least one of two or more zinc finger binding domains binds to a target DNA sequence that is at least 90%, 95%, 96%, 97%, 98%, or 99% identical or 100% identical to one of SEQ ID NOs 154 to 498 or SEQ ID NOs 499 to 813, and at least one of two or more zinc finger binding domains binds to a target DNA sequence that is at least 90%, 95%, 96%, 97%, 98%, or 99% identical or 100% identical to one of SEQ ID NOs 1065 to 1087.

[0294] In some embodiments, the gene-regulatory system comprises two or more ZFP-fusion proteins, each comprising a zinc finger binding domain, wherein at least one of the zinc finger binding domains is selected CBLB Binds to a target DNA sequence of a gene, and at least one of the zinc finger binding domains ANKRD11It binds to a target DNA sequence of a gene. In some embodiments, at least one of two or more zinc finger binding domains binds to a target DNA sequence that is at least 90%, 95%, 96%, 97%, 98%, or 99% identical or 100% identical to one of SEQ ID NOs 499 to 524, and at least one of two or more zinc finger binding domains binds to a target DNA sequence that is at least 90%, 95%, 96%, 97%, 98%, or 99% identical or 100% identical to one of SEQ ID NOs 1065 to 1087.

[0295] The enzyme domain portion of the zinc finger fusion protein can be obtained from an endo- or exonuclease. Exemplary endonucleases from which the enzyme domain may be derived include, but are not limited to, restriction endonucleases and homing endonucleases (e.g., 2002-2003 Catalogue, New England Biolabs, Beverly, Mass.; and Belfort). et al. (1997) Nucleic Acids Res. 25:3379-3388] see reference). Additional enzymes that cleave DNA are known (e.g., 51 nuclease; mung bean nuclease; pancreatic DNaseI; micrococcal nuclease; yeast HO endonuclease (also see literature [Linn et al. [See (eds.) Nucleases, Cold Spring Harbor Laboratory Press, 1993]. One or more of these enzymes (or their functional fragments) can be used as sources for cleavage domains.

[0296] In some embodiments, the gene-regulatory system comprises two or more ZFP-fusion proteins, each comprising a zinc finger binding domain, wherein at least one of the zinc finger binding domains is SOCS1 Binds to the target DNA sequence of, and at least one of the zinc finger binding domains ANKRD11 It binds to the target DNA sequence. In some embodiments, at least one of two or more zinc finger binding domains binds to a target DNA sequence that is at least 90%, 95%, 96%, 97%, 98%, or 99% identical or 100% identical to the target DNA sequence defined by the set of genomic coordinates presented in Table 6C or Table 6D, and at least one of two or more zinc finger binding domains binds to a target DNA sequence that is at least 90%, 95%, 96%, 97%, 98%, or 99% identical or 100% identical to the target DNA sequence defined by the set of genomic coordinates presented in Table 6E or Table 6F. In some embodiments, at least one of two or more zinc finger binding domains binds to a target DNA sequence that is at least 90%, 95%, 96%, 97%, 98%, or 99% identical or 100% identical to one of SEQ ID NOs 1088 to 1232, and at least one of two or more zinc finger binding domains binds to a target DNA sequence that is at least 90%, 95%, 96%, 97%, 98%, or 99% identical or 100% identical to one of SEQ ID NOs 1065 to 1087.

[0297] Exemplary restriction endonucleases (restriction enzymes) suitable for use as enzyme domains of ZFPs described herein exist in numerous species, can bind to DNA (at the recognition site) in a sequence-specific manner, and can cleave DNA at or near the binding site. Certain restriction enzymes (e.g., type IIS) cleave DNA at a site removed from the recognition site and have a separable cleavage domain and a binding domain. For example, the type IIS enzyme FokI catalyzes double-strand cleavage of DNA at 9 nucleotides from the recognition site on one strand and 13 nucleotides from the recognition site on the remaining strand (e.g., U.S. Patents No. 5,356,802; No. 5,436,150 and No. 5,487,994, as well as literature [Li et al. (1992) Proc. Natl. Acad. Sci. USA 89:4275-4279; Li et al. (1993) Proc. Natl. Acad. Sci. USA 90:2764-2768; Kim et al. (1994a) Proc. Natl. Acad. Sci. USA 91:883-887; Kim et al. (1994b) J. Biol. Chem. 269:31,978-31,982] Note). Accordingly, in one embodiment, the fusion protein comprises an enzyme domain from at least one type IIS restriction enzyme and one or more zinc finger binding domains.

[0298] An exemplary type IIS restriction enzyme in which the cleavage domain is separable from the binding domain is FokI. This particular enzyme is active as a dimer (Literature [Bitinaite] et al.[Reference: (1998) Proc. Natl. Acad. Sci. USA 95: 10,570-10,575]). Therefore, for targeted double-stranded DNA cleavage using a zinc finger-FokI fusion, a catalytically active cleavage domain can be reconfigured using two fusion proteins, each containing a FokI enzyme domain. Alternatively, a single polypeptide molecule containing a zinc finger binding domain and two FokI enzyme domains can also be used. An exemplary ZFP containing a FokI enzyme domain is described in U.S. Patent No. 9,782,437.

[0299] 2. TALEN 시스템

[0300] TALEN-based systems include proteins comprising a TAL effector DNA-binding domain and an enzyme domain. This is prepared by fusing the TAL effector DNA-binding domain to a DNA cleavage domain (a nuclease that cleaves DNA strands). The FokI restriction enzyme described above is an exemplary enzyme domain suitable for use in TALEN-based gene-regulation systems.

[0301] TAL effectors infect Xanthomonas ( XanthomonasIt is a protein secreted by bacteria. The DNA binding domain contains a repeating and highly conserved 33 to 34-amino acid sequence having branching 12th and 13th amino acids. These two positions, referred to as Repeat Variable Diresidues (RVDs), are highly variable and strongly correlated with specific nucleotide recognition. Therefore, the TAL effector domain can be engineered to bind to a specific target DNA sequence by selecting a combination of repeat segments containing appropriate RVDs. The nucleic acid specificities for the RVD combinations are as follows: HD targets cytosine, NI targets adenine, NG targets thymine, and NN targets guanine (however, in some embodiments, NN may bind to adenine with lower specificity).

[0302] In some embodiments, the TAL effector domain is IKZF1, IKZF3, GATA3, BCL3, TNIP1, TNFAIP3, NFKBIA, SMAD2, TGFBR1, TGFBR2, TANK, FOXP3, RC3H1, TRAF6, IKZF2, CBLB, PPP2R2D, NRP1, HAVCR2, LAG3, TIGIT, CTLA4, PTPN6, PDCD1 or BCOR It binds to a target DNA sequence that is at least 90%, 95%, 96%, 97%, 98%, or 99% identical or 100% identical to the target DNA sequence of a target gene selected from (e.g., a gene selected from Table 2). In some embodiments, the TAL effector domain binds to a target DNA sequence that is at least 90%, 95%, 96%, 97%, 98%, or 99% identical or 100% identical to the target DNA sequence defined by the set of genomic coordinates presented in Table 5A or Table 5B. In some embodiments, the TAL effector domain binds to a target DNA sequence that is at least 90%, 95%, 96%, 97%, 98%, or 99% identical or 100% identical to one of SEQ ID NOs 154 to 498 or SEQ ID NOs 499 to 813. In some embodiments, the TAL effector domain CBLBIt binds to a target DNA sequence that is at least 90%, 95%, 96%, 97%, 98%, or 99% identical to the target DNA sequence of the gene, or 100% identical to the target DNA sequence. In some embodiments, the TAL effector domain binds to a target DNA sequence that is at least 90%, 95%, 96%, 97%, 98%, or 99% identical to one of SEQ ID NOs 499 to 524, or 100% identical to the target DNA sequence. In some embodiments, the TAL effector domain BCOR It binds to a target DNA sequence that is at least 90%, 95%, 96%, 97%, 98%, or 99% identical to the target DNA sequence of the gene or 100% identical to it, and binds to a target DNA sequence that is at least 90%, 95%, 96%, 97%, 98%, or 99% identical to one of SEQ ID NOs 708 to 772 or SEQ ID NOs 708 to 764 or 100% identical to it. In some embodiments, the TAL effector domain is TNFAIP3 It binds to a target DNA sequence that is at least 90%, 95%, 96%, 97%, 98%, or 99% identical or 100% identical to the target DNA sequence of SEQ ID NOs 348 to 396 or SEQ ID NOs 348 to 386 and at least 90%, 95%, 96%, 97%, 98%, or 99% identical or 100% identical to the target DNA sequence of SEQ ID NOs 348 to 396 or SEQ ID NOs 348 to 386.

[0303] In some embodiments, the TAL effector domain is BCL2L11 , FLI1 , CALM2 , DHODH , UMPS , RBM39 , SEMA7A , CHIC2 , PCBP1 , PBRM1 , WDR6 , E2F8 , SERPINA3 , GNAS, SOCS1 and ANKRD11It binds to a target DNA sequence that is at least 90%, 95%, 96%, 97%, 98%, or 99% identical or 100% identical to the target DNA sequence of a target gene selected from (e.g., a gene selected from Table 3). In some embodiments, the TAL effector domain binds to a target DNA sequence that is at least 90%, 95%, 96%, 97%, 98%, or 99% identical or 100% identical to the target DNA sequence defined by a set of genomic coordinates presented in one of Tables 6A through 6F. In some embodiments, the TAL effector domain binds to a target DNA sequence that is at least 90%, 95%, 96%, 97%, 98%, or 99% identical or 100% identical to one of SEQ ID NOs 814 through 1232.

[0304] In some embodiments, the TAL effector domain is BCL2L11 , FLI1 , CALM2 , DHODH , UMPS , RBM39 , SEMA7A , CHIC2 , PCBP1 , PBRM1 , WDR6 , E2F8 , SERPINA3 and GNAS It binds to a target DNA sequence that is at least 90%, 95%, 96%, 97%, 98%, or 99% identical to or 100% identical to the target DNA sequence of a target gene selected from. In some embodiments, the TAL effector domain binds to a target DNA sequence that is at least 90%, 95%, 96%, 97%, 98%, or 99% identical or 100% identical to the target DNA sequence defined by the set of genomic coordinates presented in Table 6A or Table 6B. In some embodiments, the TAL effector domain binds to a target DNA sequence that is at least 90%, 95%, 96%, 97%, 98%, or 99% identical or 100% identical to one of SEQ ID NOs 814 to 1064.

[0305] In some embodiments, the TAL effector domain is SOCS1 It binds to a target DNA sequence that is at least 90%, 95%, 96%, 97%, 98%, or 99% identical or 100% identical to the target DNA sequence of the gene. In some embodiments, the TAL effector domain binds to a target DNA sequence that is at least 90%, 95%, 96%, 97%, 98%, or 99% identical or 100% identical to the target DNA sequence defined by the set of genomic coordinates presented in Table 6C or Table 6D. In some embodiments, the TAL effector domain binds to a target DNA sequence that is at least 90%, 95%, 96%, 97%, 98%, or 99% identical or 100% identical to one of SEQ ID NOs 1088 to 1232. In some embodiments, the TAL effector domain ANKRD11 It binds to a target DNA sequence that is at least 90%, 95%, 96%, 97%, 98%, or 99% identical or 100% identical to the target DNA sequence of the gene. In some embodiments, the TAL effector domain binds to a target DNA sequence that is at least 90%, 95%, 96%, 97%, 98%, or 99% identical or 100% identical to the target DNA sequence defined by the set of genomic coordinates presented in Table 6E or Table 6F. In some embodiments, the TAL effector domain binds to a target DNA sequence that is at least 90%, 95%, 96%, 97%, 98%, or 99% identical or 100% identical to one of SEQ ID NOs 1065 to 1087.

[0306] In some embodiments, the gene-regulatory system comprises two or more TAL effector-fusion proteins, each comprising a TAL effector domain, wherein at least one of the TAL effector domains is IKZF1, IKZF3, GATA3, BCL3, TNIP1, TNFAIP3, NFKBIA, SMAD2, TGFBR1, TGFBR2, TANK, FOXP3, RC3H1, TRAF6, IKZF2, CBLB, PPP2R2D, NRP1, HAVCR2, LAG3, TIGIT, CTLA4, PTPN6, PDCD1 or BCOR Binding to a target DNA sequence that is at least 90%, 95%, 96%, 97%, 98%, or 99% identical to the target DNA sequence of a target gene selected from, or 100% identical to the target DNA sequence, and at least one of the TAL effector domains BCL2L11 , FLI1 , CALM2 , DHODH , UMPS , RBM39 , SEMA7A , CHIC2 , PCBP1 , PBRM1 , WDR6 , E2F8 , SERPINA3 , GNAS, SOCS1, and ANKRD11It binds to a target DNA sequence that is at least 90%, 95%, 96%, 97%, 98%, or 99% identical or 100% identical to the target DNA sequence of the target gene selected from. In some embodiments, at least one of the TAL effector domains binds to a target DNA sequence that is at least 90%, 95%, 96%, 97%, 98%, or 99% identical or 100% identical to the target DNA sequence defined by the set of genomic coordinates presented in Table 5A or Table 5B, and at least one of the TAL effector domains binds to a target DNA sequence that is at least 90%, 95%, 96%, 97%, 98%, or 99% identical or 100% identical to the target DNA sequence defined by the set of genomic coordinates presented in any one of Tables 6A to 6F. In some embodiments, at least one of the TAL effector domains binds to a target DNA sequence that is at least 90%, 95%, 96%, 97%, 98%, or 99% identical or 100% identical to one of SEQ ID NOs 154 to 498 or SEQ ID NOs 499 to 813, and at least one of the TAL effector domains binds to a target DNA sequence that is at least 90%, 95%, 96%, 97%, 98%, or 99% identical or 100% identical to one of SEQ ID NOs 814 to 1232.

[0307] In some embodiments, the gene-regulatory system comprises two or more TAL effector-fusion proteins, each comprising a TAL effector domain, wherein at least one of the TAL effector domains is IKZF1, IKZF3, GATA3, BCL3, TNIP1, TNFAIP3, NFKBIA, SMAD2, TGFBR1, TGFBR2, TANK, FOXP3, RC3H1, TRAF6, IKZF2, CBLB, PPP2R2D, NRP1, HAVCR2, LAG3, TIGIT, CTLA4, PTPN6, PDCD1 or BCOR Binds to the target DNA sequence of a target gene selected from, and at least one of the TAL effector domains BCL2L11 , FLI1 , CALM2 , DHODH , UMPS , RBM39 , SEMA7A , CHIC2 , PCBP1 , PBRM1 , WDR6 , E2F8 , SERPINA3 and GNAS It binds to the target DNA sequence of a target gene selected from. In some embodiments, at least one of two or more TAL effector domains binds to a target DNA sequence that is at least 90%, 95%, 96%, 97%, 98%, or 99% identical or 100% identical to the target DNA sequence defined by the set of genomic coordinates presented in Table 5A or Table 5B, and at least one of two or more TAL effector domains binds to a target DNA sequence that is at least 90%, 95%, 96%, 97%, 98%, or 99% identical or 100% identical to the target DNA sequence defined by the set of genomic coordinates presented in Table 6A or Table 6B. In some embodiments, at least one of two or more TAL effector domains binds to a target DNA sequence that is at least 90%, 95%, 96%, 97%, 98%, or 99% identical or 100% identical to one of SEQ ID NOs 154 to 498 or SEQ ID NOs 499 to 813, and at least one of two or more TAL effector domains binds to a target DNA sequence that is at least 90%, 95%, 96%, 97%, 98%, or 99% identical or 100% identical to one of SEQ ID NOs 814 to 1064.

[0308] In some embodiments, the gene-regulatory system comprises two or more TAL effector-fusion proteins, each comprising a TAL effector domain, wherein at least one of the TAL effector domains is CBLB Binds to a target DNA sequence of a gene, and at least one of the TAL effector domains BCL2L11 , FLI1 , CALM2 , DHODH , UMPS , RBM39 , SEMA7A , CHIC2 , PCBP1 , PBRM1 , WDR6 , E2F8 , SERPINA3 and GNAS It binds to the target DNA sequence of a target gene selected from. In some embodiments, at least one of two or more TAL effector domains binds to a target DNA sequence that is at least 90%, 95%, 96%, 97%, 98%, or 99% identical or 100% identical to one of SEQ ID NOs 499 to 524, and at least one of two or more TAL effector domains binds to a target DNA sequence that is at least 90%, 95%, 96%, 97%, 98%, or 99% identical or 100% identical to one of SEQ ID NOs 814 to 1064.

[0309] In some embodiments, the gene-regulatory system comprises two or more TAL effector-fusion proteins, each comprising a TAL effector domain, wherein at least one of the TAL effector domains is IKZF1, IKZF3, GATA3, BCL3, TNIP1, TNFAIP3, NFKBIA, SMAD2, TGFBR1, TGFBR2, TANK, FOXP3, RC3H1, TRAF6, IKZF2, CBLB, PPP2R2D, NRP1, HAVCR2, LAG3, TIGIT, CTLA4, PTPN6, PDCD1 or BCOR Binds to the target DNA sequence of a target gene selected from, and at least one of the TAL effector domains SOCS1It binds to a target DNA sequence of a gene. In some embodiments, at least one of two or more TAL effector domains binds to a target DNA sequence that is at least 90%, 95%, 96%, 97%, 98%, or 99% identical or 100% identical to the target DNA sequence defined by the set of genomic coordinates presented in Table 5A or Table 5B, and at least one of two or more TAL effector domains binds to a target DNA sequence that is at least 90%, 95%, 96%, 97%, 98%, or 99% identical or 100% identical to the target DNA sequence defined by the set of genomic coordinates presented in Table 6C or Table 6D. In some embodiments, at least one of two or more TAL effector domains binds to a target DNA sequence that is at least 90%, 95%, 96%, 97%, 98%, or 99% identical or 100% identical to one of SEQ ID NOs 154 to 498 or SEQ ID NOs 499 to 813, and at least one of two or more TAL effector domains binds to a target DNA sequence that is at least 90%, 95%, 96%, 97%, 98%, or 99% identical or 100% identical to one of SEQ ID NOs 1088 to 1232.

[0310] In some embodiments, the gene-regulatory system comprises two or more TAL effector-fusion proteins, each comprising a TAL effector domain, wherein at least one of the TAL effector domains is CBLB Binds to the target DNA sequence of, and at least one of the TAL effector domains SOCS1It binds to a target DNA sequence of a gene. In some embodiments, at least one of two or more TAL effector domains binds to a target DNA sequence that is at least 90%, 95%, 96%, 97%, 98%, or 99% identical or 100% identical to one of SEQ ID NOs 499 to 524, and at least one of two or more TAL effector domains binds to a target DNA sequence that is at least 90%, 95%, 96%, 97%, 98%, or 99% identical or 100% identical to one of SEQ ID NOs 1088 to 1232.

[0311] In some embodiments, the gene-regulatory system comprises two or more TAL effector-fusion proteins, each comprising a TAL effector domain, wherein at least one of the TAL effector domains is IKZF1, IKZF3, GATA3, BCL3, TNIP1, TNFAIP3, NFKBIA, SMAD2, TGFBR1, TGFBR2, TANK, FOXP3, RC3H1, TRAF6, IKZF2, CBLB, PPP2R2D, NRP1, HAVCR2, LAG3, TIGIT, CTLA4, PTPN6, PDCD1 or BCOR Binds to the target DNA sequence of a target gene selected from, and at least one of the TAL effector domains ANKRD11It binds to a target DNA sequence of a gene. In some embodiments, at least one of two or more TAL effector domains binds to a target DNA sequence that is at least 90%, 95%, 96%, 97%, 98%, or 99% identical or 100% identical to the target DNA sequence defined by the set of genomic coordinates presented in Table 5A or Table 5B, and at least one of two or more TAL effector domains binds to a target DNA sequence that is at least 90%, 95%, 96%, 97%, 98%, or 99% identical or 100% identical to the target DNA sequence defined by the set of genomic coordinates presented in Table 6D or Table 6E. In some embodiments, at least one of two or more TAL effector domains binds to a target DNA sequence that is at least 90%, 95%, 96%, 97%, 98%, or 99% identical or 100% identical to one of SEQ ID NOs 154 to 498 or SEQ ID NOs 499 to 813, and at least one of two or more TAL effector domains binds to a target DNA sequence that is at least 90%, 95%, 96%, 97%, 98%, or 99% identical or 100% identical to one of SEQ ID NOs 1065 to 1087.

[0312] In some embodiments, the gene-regulatory system comprises two or more TAL effector-fusion proteins, each comprising a TAL effector domain, wherein at least one of the TAL effector domains is CBLB Binds to a target DNA sequence of a gene, and at least one of the TAL effector domains ANKRD11It binds to a target DNA sequence of a gene. In some embodiments, at least one of two or more TAL effector domains binds to a target DNA sequence that is at least 90%, 95%, 96%, 97%, 98%, or 99% identical or 100% identical to one of SEQ ID NOs 499 to 524, and at least one of two or more TAL effector domains binds to a target DNA sequence that is at least 90%, 95%, 96%, 97%, 98%, or 99% identical or 100% identical to one of SEQ ID NOs 1065 to 1087.

[0313] In some embodiments, the gene-regulatory system comprises two or more TAL effector-fusion proteins, each comprising a TAL effector domain, wherein at least one of the TAL effector domains is SOCS1 Binds to a target DNA sequence of a gene, and at least one of the TAL effector domains ANKRD11 It binds to the target DNA sequence of the gene. In some embodiments, at least one of two or more TAL effector domains binds to a target DNA sequence that is at least 90%, 95%, 96%, 97%, 98%, or 99% identical or 100% identical to the target DNA sequence defined by the set of genomic coordinates presented in Table 6C or Table 6D, and at least one of two or more TAL effector domains binds to a target DNA sequence that is at least 90%, 95%, 96%, 97%, 98%, or 99% identical or 100% identical to the target DNA sequence defined by the set of genomic coordinates presented in Table 6E or Table 6F. In some embodiments, at least one of two or more TAL effector domains binds to a target DNA sequence that is at least 90%, 95%, 96%, 97%, 98%, or 99% identical or 100% identical to one of SEQ ID NOs 1088 to 1232, and at least one of two or more TAL effector domains binds to a target DNA sequence that is at least 90%, 95%, 96%, 97%, 98%, or 99% identical or 100% identical to one of SEQ ID NOs 1065 to 1087.

[0314] Methods and compositions for assembling repeating sections of a TAL-effector are known in the art (e.g., literature [Cermak and others [Refer to Nucleic Acids Research, 39:12, 2011, e82]. Plasmids for constructing the TAL-effect repeat are commercially available from Addgene.

[0315] C. 상이한산 / 단백임-기반 가이-조전 system

[0316] A combination gene-regulatory system comprises a site-directed modification polypeptide and a nucleic acid guide molecule. As specified herein, “site-directed modification polypeptide” refers to a polypeptide that binds to a nucleic acid guide molecule, is targeted by the bound nucleic acid guide molecule to a target nucleic acid sequence (e.g., endogenous target DNA or RNA sequence), and modifies the target nucleic acid sequence (e.g., by cleavage, mutation, or methylation of the target nucleic acid sequence).

[0317] The site-directed modified polypeptide comprises two parts: a part that binds to a nucleic acid guide and an active part. In some embodiments, the site-directed modified polypeptide comprises an active part that exhibits site-directed enzymatic activity (e.g., DNA methylation, DNA or RNA cleavage, histone acetylation, histone methylation, etc.), wherein the enzymatic active site is determined by the guide nucleic acid. In some cases, site-directed modified polypeptides include an active portion having enzymatic activity that modifies an endogenous target nucleic acid sequence (e.g., nuclease activity, methyltransferase activity, demethylase activity, DNA repair activity, DNA damage activity, deamination activity, dismutase activity, alkylation activity, depurination activity, oxidation activity, pyrimidine dimer formation activity, integrase activity, transposase activity, recombinase activity, polymerase activity, ligase activity, helicase activity, photolyase activity, or glycosylase activity). In other cases, the site-directed modified polypeptide comprises an active portion having enzymatic activity (e.g., methyltransferase activity, demethylase activity, acetyltransferase activity, deacetylase activity, kinase activity, phosphatase activity, ubiquitin ligase activity, deubiquitination activity, adenylation activity, deadenylation activity, hydromylation activity, dehydromolation activity, ribosylation activity, deribosylation activity, myristoylation activity, or demyristoylation activity) that modifies a polypeptide (e.g., deribosylation activity) associated with an endogenous target nucleic acid sequence. In some embodiments, the site-directed modified polypeptide comprises an active portion that regulates the transcription of a target DNA sequence (e.g., increasing or decreasing transcription). In some embodiments, the site-directed modified polypeptide comprises an active portion that regulates the expression or translation of a target RNA sequence (e.g., increasing or decreasing transcription).

[0318] The nucleic acid guide comprises two parts: a first part (referred to herein as the “nucleic acid-binding segment”) that is complementary to and capable of binding to an endogenous target nucleic acid sequence, and a second part (referred to herein as the “protein-binding segment”) that can interact with a site-directed modified polypeptide. In some embodiments, the nucleic acid-binding segment and the protein-binding segment of the nucleic acid guide are contained within a single polynucleotide molecule. In some embodiments, the nucleic acid-binding segment and the protein-binding segment of the nucleic acid guide are each contained within separate polynucleotide molecules, so that the nucleic acid guide comprises two polynucleotide molecules that associate with each other to form a functional guide.

[0319] A nucleic acid guide mediates the target specificity of a combined protein / nucleic acid gene-regulatory system by specifically hybridizing with a target nucleic acid sequence. In some embodiments, the target nucleic acid sequence is an RNA sequence, e.g., an RNA sequence contained within the mRNA transcript of a target gene. In some embodiments, the target nucleic acid sequence is a DNA sequence contained within the DNA sequence of a target gene. References to a target gene herein include a full-length DNA sequence for a specific gene comprising a plurality of target gene loci (i.e., portions of a specific target gene sequence (e.g., exons or introns)). Within each target gene loci, there exists a shorter stretch of DNA sequence referred to herein as the “target DNA sequence” which can be modified by the gene-regulatory system described herein. Additionally, each target gene loci includes a “target modification site,” which refers to the precise location of a modification induced by the gene-regulatory system (e.g., a location of an insertion, deletion, or mutation, a location of a DNA break, or a location of an epigenetic modification).

[0320] The gene-regulatory system described herein may include a single nucleic acid guide or a plurality of nucleic acid guides (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10 or more nucleic acid guides).

[0321] In some embodiments, the combined protein / nucleic acid gene-regulatory system is an Argonaute (Ago) protein (e.g., T. thermophiles ( T. thermophilus It includes a site-directed modified polypeptide derived from ) Ago or TtAgo). In these embodiments, the site-directed modified polypeptide is T. thermophiles Ago DNA endonuclease, and the nucleic acid guide is guide DNA (gDNA) (Reference [Swarts and others [Reference: Nature 507 (2014), 258-261]). In some embodiments, the present disclosure provides a polynucleotide encoding gDNA. In some embodiments, the gDNA-coding nucleic acid is included in an expression vector, e.g., a recombinant expression vector. In some embodiments, the present disclosure provides a polynucleotide encoding a TtAgo site-directed modified polypeptide or a variant thereof. In some embodiments, the polynucleotide encoding a TtAgo site-directed modified polypeptide is included in an expression vector, e.g., a recombinant expression vector.

[0322] In some embodiments, the gene editing system described herein is a CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) / Cas (CRISPR-associated) nuclease system. In some embodiments, the CRISPR / Cas system is a Class 2 system. Class 2 CRISPR / Cas systems are divided into three types: Type II, Type V, and Type VI systems. In some embodiments, the CRISPR / Cas system is a Class 2 Type II system using a Cas9 protein. In these embodiments, the site-directed modified polypeptide is a Cas9 DNA endonuclease (or a variant thereof), and the nucleic acid guide molecule is a guide RNA (gRNA). In some embodiments, the CRISPR / Cas system is a Class 2 Type V system using Cas12 proteins (e.g., Cas12a (also known as Cpf1), Cas12b (also known as C2c1), Cas12c (also known as C2c3), Cas12d (also known as CasY), and Cas12e (also known as CasX)). In these embodiments, the site-directed modification polypeptide is a Cas12 DNA endonuclease (or a variant thereof), and the nucleic acid guide molecule is gRNA. In some embodiments, the CRISPR / Cas system is a Class 2 and Type VI system using Cas13 proteins (e.g., Cas13a (also known as C2c2), Cas13b, and Cas13c) (Reference [Pyzocha et al. [See ACS Chemical Biology, 13(2), 347-356]). In these embodiments, the site-directed modification polypeptide is Cas13 RNA riboendonuclease, and the nucleic acid guide molecule is gRNA.

[0323] Cas polypeptides refer to polypeptides that can interact with or cooperate with gRNA molecules to hom to or localize to target DNA or target RNA sequences. Cas polypeptides include naturally occurring Cas proteins and engineered, altered, or otherwise modified Cas proteins in which one or more amino acid residues differ from the naturally occurring Cas sequence.

[0324] Guide RNA (gRNA) comprises two segments, a DNA-binding segment and a protein-binding segment. In some embodiments, the protein-binding segment of the gRNA is contained in one RNA molecule, and the DNA-binding segment is contained in another separate RNA molecule. These embodiments are referred herein as “double-molecule gRNA” or “two-molecule gRNA” or “double gRNA”. In some embodiments, the gRNA is a single RNA molecule and is referred herein as “single-guide RNA” or “sgRNA”. The term “guide RNA” or “gRNA” includes both what are referred to as two-molecule guide RNA and sgRNA.

[0325] The protein-binding segment of the gRNA partially contains two complementary stretches of nucleotides that hybridize to form a double-stranded RNA duplex (dsRNA duplex), which facilitates binding to the Cas protein. The nucleic acid-binding segment of the gRNA (or "nucleic acid-binding sequence") contains a nucleotide sequence that is complementary to a specific target nucleic acid sequence and capable of binding to it. The protein-binding segment of the gRNA interacts with the Cas polypeptide, and the interaction between the gRNA molecule and the site-directed modification polypeptide causes Cas binding to the endogenous nucleic acid sequence, generating one or more modifications within or around the target nucleic acid sequence. The precise location of the target modification site is (i) base-coupled complementarity between the gRNA and the target nucleic acid sequence; and (ii) is determined by the position of a short motif, referred to as a protospacer adjacent motif (PAM) within the target DNA sequence (referred to as a protospacer flanking sequence (PFS) within the target RNA sequence). The PAM / PFS sequence is required for Cas binding to the target nucleic acid sequence. Various PAM / PFS sequences are known in the art and are suitable for use with specific Cas endonucleases (e.g., Cas9 endonucleases) (see, for example, [Nat Methods. 2013 Nov; 10(11): 1116-1121 and Sci Rep. 2014; 4: 5405]). In some embodiments, the PAM sequence is located within 50 base pairs of the target modification site within the target DNA sequence. In some embodiments, the PAM sequence is located within 10 base pairs of the target modification site within the target DNA sequence.The DNA sequences that can be targeted by this method are limited only by the relative distance of the PAM sequence to the target modification site and the presence of a unique 20-base pair sequence mediating sequence-specific, gRNA-mediated Cas binding. In some embodiments, the PFS sequence is located at the 3' end of the target RNA sequence. In some embodiments, the target modification site is located at the 5' end of the target locus. In some embodiments, the target modification site is located at the 3' end of the target locus. In some embodiments, the target modification site is located within an intron or exon of the target locus.

[0326] In some embodiments, the present disclosure provides a polynucleotide encoding gRNA. In some embodiments, the gRNA-coding nucleic acid is included in an expression vector, e.g., a recombinant expression vector. In some embodiments, the present disclosure provides a polynucleotide encoding a site-directed modified polypeptide. In some embodiments, the polynucleotide encoding the site-directed modified polypeptide is included in an expression vector, e.g., a recombinant expression vector.

[0327] 1. Case protein

[0328] In some embodiments, the site-directed modified polypeptide is a Cas protein. Various species of Cas molecules may be used in the methods and compositions described herein, and S. pyogenes ( S. pyogenes ), Es. Aureus( S. aureus ), N. Meningitidis( Meningitis N. ), S. Thermophiles( S. thermophilus ), Acidoborax avenae( Acidovorax oat ), Actinobacillus pleuropymoniae( Actinobacillus pleuropneumoniae ), Actinobacillus sucinogenes( Actinobacillus succinogenes ), Actinobacillus suis( Actinobacillus suis ), Actinomyces species ( Actinomyces sp. ), Cycliphilus denitripicans( Cycliphilusdenitrificans), Aminomonas fauciborans ( Aminomonas paucivorans ), Bacillus cereus( Bacillus cereus ), Bacillus smittyi( Bacillus smithii ), Bacillus thuringiensis( Bacillus thuringiensis ), Bacteroides species ( Bacteroides sp. .), Blastopirelula Marina( Marine blastopirellula ), Bradirizobium species ( Bradyrhizobium sp. ), Brevibacillus laterospoxus( Brevibacillus laterospoxus ), Campylobacter coli ( Campylobacter coli ), Campylobacter jejuni( Campylobacter jejuni ), Campylobacter lari( Campylobacter lari ), Candidatus funiseisspirilum( Candidatus Puniceispirillum ), Clostridium celluloticum ( Clostridium cellulolyticum ), Clostridium perfringens( Clostridium perfringens ), Corynebacterium accolens( Corynebacterium accolens ), Corynebacterium diphtheria( Corynebacterium diphtheria ), Corynebacterium matrucoti( Corynebacterium matruchotii ), Dino Roseobacter chibae( Dinoroseobacter shibae ), Eubacterium dolly dance( Eubacterium dolichum ), Gamma Proteobacterium ( Gamma proteobacterium ), Gluconacetobacter diazotropicus ( Gluconacetobacter diazotrophicus ), Haemophilus parainfluenzae( Haemophilus parainfluenzae ), Haemophilus sputrum( Haemophilus sputum ), Helicobacter canadensis ( Helicobacter canadensis ), Helicobacter pylori ( Helicobacter pylori ), Helicobacter mustela ( Helicobacter mustelid ), Iliobacter polytropus( Ilyobacter polytropus ), Kingella Kingae( Kingella kingae ), Lactobacillus crispatus( Lactobacillus crispatus ), Listeria Ivanovi( Listeria ivanovii ), Listeria monocytogenes( Listeria monocytogenes ), Listeriaceae bacterium ( Listeria bacteria ), Methylocystis species ( Methylocystis sp. ) Methylosinus tricosporium( Methylosinus trichosporium ), Mobilunkus Mulieris( Woman's mobile phone), Neisseria basiliformis( Neisseria bacilliformis ), Neiceria Cinerea( Neisseria cinerea ), Neisseria flavescens( Neisseria flavuscens ), Neiceria lactamica, Neiceria meningitidis, Neiceria species( Neisseria sp. ) Neiceria Wardworthy( Neisseria wadsworthii ), Nitrosomonas species ( Nitrosomonas sp. ), Parvivaculum lavamentivorans( Small wash-eating stick ), Pasteurella water citron ( Pasteurella multocida ), Pascolactobacterium succinatutens( Phascolarctobacterium succinatutens ), Ralstonia Sizigi( Ralstonia syzygii ) , Rhodochmonas palustris ( Rhodopseudomonas palustris ), Rhodobloum species ( Rhodovulum sp. ), Simonciela Muelleri( Simonsiella muelleri ), Sphingomonas species ( Sphingomonas sp. ), Sporolactobacillus viniae ( Sporolactobacillus vineae ), Staphilococcus aureus( Staphylococcus aureus ), Staphylococcus lugdunensis( Staphylococcus lugdunensis ), Streptococcus species ( Streptococcus sp. ), subdoligranulum species ( Subdoligranulum sp. ), Tistellar Mobilis( Mobile Tistrella ), Treponema species ( Treponema sp. ) or Vermineprobacter aisenia ( Verminephrobacter eiseniae It contains Cas molecules derived from ).

[0329] In some embodiments, the Cas protein is a naturally occurring Cas protein. In some embodiments, Cas endonucleases are C2C1, C2C3, Cpf1 (also referred to as Cas12a), Cas12b, Cas12c, Cas12d, Cas12e, Cas13a, Cas13b, Cas13c, Cas13d, Casl, CaslB, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9 (also known as Csnl and Csx12), Cas10, Csyl, Csy2, Csy3, Csel, Cse2, Cscl, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmrl, Cmr3, Cmr4, Cmr5, Cmr6, Csbl, Csb2, Csb3, It is selected from the group consisting of Csxl7, Csxl4, Csx10, Csx16, CsaX, Csx3, Csxl, Csxl5, Csfl, Csf2, Csf3, and Csf4.

[0330] In some embodiments, the Cas protein is an endoribonuclease, e.g., the Cas13 protein. In some embodiments, the Cas13 protein is Cas13a (Abudayyeh and others ., Nature 550 (2017), 280-284), Cas13b (Cox and others ., Science (2017) 358:6336, 1019-1027), Cas13c (Cox and others ., Science (2017) 358:6336, 1019-1027) or Cas13d(Zhang and others ., Cell 175 (2018), 212-223) is a protein.

[0331] In some embodiments, the Cas protein is a wild-type or naturally occurring Cas9 protein or a Cas9 ortholog. Wild-type Cas9 is a multi-domain enzyme that cleaves the target strand of DNA using an HNH nuclease domain and cleaves the non-target strand using a RuvC-like domain. Binding of WT Cas9 to DNA based on gRNA specificity results in a double-stranded DNA break that can be repaired by non-homologous end junctions (NHEJ) or homology-induced repairs (HDR). An exemplary naturally occurring Cas9 molecule is [Chylinski et al.It is described in RNA Biology 2013 10:5, 727-737, and additional Cas9 orthographs are described in International PCT Publication No. WO 2015 / 071474. These Cas9 molecules are Cluster 1 bacterial family, Cluster 2 bacterial family, Cluster 3 bacterial family, Cluster 4 bacterial family, Cluster 5 bacterial family, Cluster 6 bacterial family, Cluster 7 bacterial family, Cluster 8 bacterial family, Cluster 9 bacterial family, Cluster 10 bacterial family, Cluster 11 bacterial family, Cluster 12 bacterial family, Cluster 13 bacterial family, Cluster 14 bacterial family, Cluster 15 bacterial family, Cluster 16 bacterial family, Cluster 17 bacterial family, Cluster 18 bacterial family, Cluster 19 bacterial family, Cluster 20 bacterial family, Cluster 21 bacterial family, Cluster 22 bacterial family, Cluster 23 bacterial family, Cluster 24 bacterial family, Cluster 25 bacterial family, Cluster 26 bacterial family, Cluster 27 bacterial family, Cluster 28 bacterial family, Cluster 29 bacterial family, Cluster 30 bacterial family, Cluster 31 bacterial family, Cluster 32 bacterial family, Cluster 33 bacterial family, Cluster 34 bacterial family, Cluster 35 bacterial family, Cluster 36 Bacteria family, Cluster 37 Bacteria family, Cluster 38 Bacteria family, Cluster 39 Bacteria family, Cluster 40 Bacteria family, Cluster 41 Bacteria family, Cluster 42 Bacteria family, Cluster 43 Bacteria family, Cluster 44 Bacteria family, Cluster 45 Bacteria family, Cluster 46 Bacteria family, Cluster 47 Bacteria family, Cluster 48 Bacteria family, Cluster 49 Bacteria family, Cluster 50 Bacteria family,Includes Cas9 molecules of the bacterial family Cluster 51, Cluster 52, Cluster 53, Cluster 54, Cluster 55, Cluster 56, Cluster 57, Cluster 58, Cluster 59, Cluster 60, Cluster 61, Cluster 62, Cluster 63, Cluster 64, Cluster 65, Cluster 66, Cluster 67, Cluster 68, Cluster 69, Cluster 70, Cluster 71, Cluster 72, Cluster 73, Cluster 74, Cluster 75, Cluster 76, Cluster 77, or Cluster 78.

[0332] In some embodiments, the naturally occurring Cas9 polypeptide is selected from the group consisting of SpCas9, SpCas9-HF1, SpCas9-HF2, SpCas9-HF3, SpCas9-HF4, SaCas9, FnCpf, FnCas9, eSpCas9, and NmeCas9. In some embodiments, the Cas9 protein is from the literature [Chylinski et al. , RNA Biology 2013 10:5, 727-737; Hou et al. It includes an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the Cas9 amino acid sequence described in [PNAS Early Edition 2013, 1-6].

[0333] In some embodiments, the Cas polypeptide comprises one or more of the following activities:

[0334] (a) Nikcase activity, i.e., the ability to cleave a single strand of a nucleic acid molecule, e.g., a non-complementary strand or a complementary strand;

[0335] (b) double-stranded nuclease activity, i.e., the ability to cleave both strands of double-stranded nucleic acid and generate double-stranded breaks (this is the presence of two nikase activities in the embodiments);

[0336] (c) Endonuclease activity;

[0337] (d) exonuclease activity; and / or

[0338] (e) Helicase activity, i.e., the ability to unwind the helical structure of double-stranded nucleic acids.

[0339] In some embodiments, the Cas polypeptide is fused to a heterologous protein that recruits a DNA-damage signaling protein, exonuclease, or phosphatase to further increase the possibility or rate of repair of a target sequence by one repair mechanism or another. In some embodiments, the WT Cas polypeptide is co-expressed with a nucleic acid repair template to facilitate the incorporation of an exogenous nucleic acid sequence by homology-induced repair.

[0340] In some embodiments, different Cas proteins (i.e., Cas9 proteins from different species) may be advantageous to use in various provided methods to utilize different enzymatic characteristics of different Cas proteins (e.g., for different PAM sequence preferences; for increased or decreased enzymatic activity; for increased or decreased levels of cytotoxicity; for changing the balance between NHEJ, homology-induced repair, single-strand break, double-strand break, etc.).

[0341] In some embodiments, the Cas protein is a Cas9 protein derived from S. pyogenes and recognizes the PAM sequence motifs NGG, NAG, and NGA (Mali et al, Science 2013; 339(6121): 823-826). In some embodiments, the Cas protein is a Cas9 protein derived from S. thermophiles and recognizes the PAM sequence motifs NGGNG and / or NNAGAAW (where W = A or T) (e.g., Horvath et al, Science, 2010; 327(5962): 167-170 and Deveau and others [ , J Bacteriol 2008; 190(4): 1390-1400] see reference). In some embodiments, the Cas protein is S. mutans ( S. mutansIt is a Cas9 protein derived from ) and recognizes the PAM sequence motif NGG and / or NAAR (where R = A or G) (see, for example, [Deveau et al, J BACTERIOL 2008; 190(4): 1390-1400]). In some embodiments, the Cas protein is a Cas9 protein derived from S. aureus and recognizes the PAM sequence motif NNGRR (where R = A or G). In some embodiments, the Cas protein is a Cas9 protein derived from S. aureus and recognizes the PAM sequence motif N GRRT (where R = A or G). In some embodiments, the Cas protein is a Cas9 protein derived from S. aureus and recognizes the PAM sequence motif N GRRV (where R = A or G). In some embodiments, the Cas protein is N. It is a Cas9 protein derived from Meningitidis and recognizes the PAM sequence motif N GATT or N GCTT (where R = A or G, V = A, G, or C) (see, for example, [Hou et ah, PNAS 2013, 1-6]). In the embodiments mentioned above, N may be any nucleotide residue, for example, any of A, G, C, or T. In some embodiments, the Cas protein is Leptotricia sahii ( Leptotrichia shahii It is a Cas13a protein derived from ) and recognizes a single 3' A, U, or C PFS sequence motif.

[0342] In some embodiments, a polynucleotide encoding a Cas protein is provided. In some embodiments, the polynucleotide is International PCT Publication No. WO 2015 / 071474 or literature [Chylinski et al. It encodes a Cas protein that is at least 90% identical to the Cas protein described in RNA Biology 2013 10:5, 727-737. In some embodiments, the polynucleotide is International PCT Publication No. WO 2015 / 071474 or literature [Chylinski et al. It encodes a Cas protein that is at least 95%, 96%, 97%, 98%, or 99% identical to the Cas protein described in [RNA Biology 2013 10:5, 727-737]. In some embodiments, the polynucleotide is International PCT Publication No. WO 2015 / 071474 or literature [Chylinski et al. It encodes a Cas protein that is at least 100% identical to the Cas protein described in RNA Biology 2013 10:5, 727-737.

[0343] 2. The case of mutation

[0344] In some embodiments, the Cas polypeptide is engineered to alter one or more properties of the Cas polypeptide. For example, in some embodiments, the Cas polypeptide includes altered enzymatic properties, for example, altered nuclease activity (compared to naturally occurring or other reference Cas molecules) or altered helicase activity. In some embodiments, the engineered Cas polypeptide may have modifications that alter size, for example, deletions of amino acid sequences that reduce size without having a significant effect on other properties of the Cas polypeptide. In some embodiments, the engineered Cas polypeptide includes modifications that affect PAM recognition. For example, the engineered Cas polypeptide may be modified to recognize a PAM sequence other than the PAM sequence recognized by the corresponding wild-type Cas protein.

[0345] A Cas polypeptide having desired characteristics may be prepared in a number of ways, including modification of a naturally occurring Cas polypeptide or a parent Cas polypeptide, to provide a mutant or modified Cas polypeptide having desired characteristics. For example, one or more mutations may be introduced into the sequence of a parent Cas polypeptide (e.g., a naturally occurring or engineered Cas polypeptide). Such mutations and differences may include substitutions (e.g., conservative substitutions or substitutions of non-essential amino acids); insertions; or deletions. In some embodiments, the mutant Cas polypeptide comprises one or more mutations (e.g., at least 1, 2, 3, 4, 5, 10, 15, 20, 30, 40, or 50 mutations) compared to the parent Cas polypeptide.

[0346] In an embodiment, the mutant Cas polypeptide possesses cleavage properties different from those of the naturally occurring Cas polypeptide. In some embodiments, Cas is an inactivated Cas(dCas) mutant. In these embodiments, the Cas polypeptide does not possess any inherent enzymatic activity and cannot mediate target nucleic acid cleavage. In these embodiments, dCas may be fused with a heterologous protein capable of modifying the target nucleic acid in a non-cleavage-based manner. For example, in some embodiments, the dCas protein is fused to a transcription activator or transcription repressor domain (e.g., Krupel-associated box (KRAB or SKD); Mad mSIN3 interaction domain (SID or SID4X); ERF repressor domain (ERD); MAX-interacting protein 1 (MXI1); methyl-CpG binding protein 2 (MECP2); etc.). In some of these cases, the dCas fusion protein is targeted by ggRNA to a specific location (i.e., sequence) within the target nucleic acid, exerts locus-specific regulation, for example, by blocking RNA polymerase binding to the promoter (which selectively inhibits transcriptional activator function) and / or modifying the local chromatin state (e.g., modifying the target DNA or the polypeptide associated with the target DNA when the fusion sequence is used). In some cases, the change is transient (e.g., transcriptional repression or activation). In some cases, the change is heritable (e.g., when epigenetic modification occurs in the target DNA or the protein associated with the target DNA, e.g., nucleosomal histone).

[0347] In some embodiments, dCas is a dCas13 mutant (Konermann and others., Cell 173 (2018), 665-676). Subsequently, these dCas13 mutants can be fused to RNA-modifying enzymes, including adenosine deaminases (e.g., ADAR1 and ADAR2). Adenosine deaminases convert adenine to inosine, where a translation machine similarly processes guanine to generate a functional A→G in the RNA sequence. In some embodiments, dCas is a dCas9 mutant.

[0348] In some embodiments, the mutant Cas9 is a Cas9 nickase mutant. The Cas9 nickase mutant comprises only one catalytically active domain (an HNH domain or a RuvC domain). The Cas9 nickase mutant possesses DNA binding based on gRNA specificity but cuts only one strand of DNA to produce a single-strand break (e.g., a "nick"). In some embodiments, two complementary Cas9 nickase mutants (e.g., one Cas9 nickase mutant having an inactivated RuvC domain, and one Cas9 nickase mutant having an inactivated HNH domain) are expressed in the same cell having two gRNAs corresponding to two respective target sequences; one target sequence is expressed on the sense DNA strand and one on the antisense DNA strand. These double-nikase systems can generate staggered double-strand breaks and increase target specificity because two non-target niks are unlikely to be generated close enough to generate a double-strand break. In some embodiments, a Cas9 nikase mutant is co-expressed with a nucleic acid repair template to facilitate the incorporation of exogenous nucleic acid sequences by homology-induced repair.

[0349] In some embodiments, the Cas polypeptide described herein may be engineered to alter the PAM / PFS specificity of the Cas polypeptide. In some embodiments, the mutant Cas polypeptide has a PAM / PFS specificity different from that of the parent Cas polypeptide. For example, the naturally occurring Cas protein may be modified to alter the PAM / PFS sequence that the mutant Cas polypeptide recognizes so that the target site is reduced, specificity is improved, or the PAM / PFS recognition requirement is eliminated. In some embodiments, the Cas protein may be modified to increase the length of the PAM / PFS recognition sequence. In some embodiments, the length of the PAM recognition sequence is at least 4, 5, 6, 7, 8, 9, 10, or 15 amino acids. Cas polypeptides that recognize different PAM / PFS sequences and / or have reduced off-target activity may be generated using directed evolution. Exemplary methods and systems that can be used for the induced differentiation of Cas polypeptides are, for example, in the literature [Esvelt et al. It is described in [Nature 2011, 472(7344): 499-503].

[0350] An exemplary Cas mutant is International PCT Publication No. WO 2015 / 161276 and literature [Konermann and others [., Cell 173 (2018), 665-676] is described, the full text of which is incorporated herein by reference.

[0351] 3. gRNA

[0352] The present disclosure provides a guide RNA (gRNA) that guides a site-directed modified polypeptide to a specific target nucleic acid sequence. The gRNA comprises a “nucleic acid-targeting domain” or “targeting domain” and a protein-binding segment. The targeting domain may also be referred to as a “spacer” sequence and comprises a nucleotide sequence complementary to the target nucleic acid sequence. Thus, the targeting domain segment of the gRNA interacts with the target nucleic acid in a sequence-specific manner through hybridization (i.e., base coupling) and determines the location within the target nucleic acid to which the gRNA binds. The targeting domain segment of the gRNA may be modified (e.g., by genetic engineering) to hybridize to a desired sequence within the target nucleic acid sequence. In some embodiments, the targeting domain sequence is about 13 to about 22 nucleotides long. In some embodiments, the targeting domain sequence is about 13, 14, 15, 16, 17, 18, 19, 20, 21, or 22 nucleotide lengths. In some embodiments, the targeting domain sequence is about 20 nucleotide lengths.

[0353] The protein-binding segment of the gRNA interacts with a site-directed modification polypeptide (e.g., Cas protein) to form a ribonucleoprotein (RNP) complex comprising the gRNA and the site-directed modification polypeptide. Subsequently, the targeting domain segment of the gRNA guides the bound site-directed modification polypeptide to a specific nucleotide sequence within the target nucleic acid via the spacer sequence described above. The protein-binding segment of the gRNA comprises at least two stretches of nucleotides that are complementary to each other and form a double-stranded RNA duplex. The protein-binding segment of the gRNA may also be referred to as a "scaffold" segment or "tracr RNA." In some embodiments, the tracr RNA sequence is about 30 to about 180 nucleotides long. In some embodiments, the tracr RNA sequence is of length about 40 to about 90 nucleotides, about 50 to about 90 nucleotides, about 60 to about 90 nucleotides, about 65 to about 85 nucleotides, about 70 to about 80 nucleotides, about 65 to about 75 nucleotides, or about 75 to about 85 nucleotides. In some embodiments, the tracr RNA sequence is of length about 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, or about 90 nucleotides. In some embodiments, the tracr RNA is SEQ ID NO. 34 (Reference [Mali and others [Refer to ., Science (2013) 339(6121):823-826]), Sequence Nos. 35 to 36 (Refer to PCT Publication No. WO 2016 / 106236), Sequence Nos. 37 to 39 (Reference [Deltcheva and others [Refer to ., Nature. 2011 Mar 31; 471(7340): 602-607] or Sequence No. 40 (Reference [Chen and othersIt includes a nucleic acid sequence encoded by the DNA sequence of [., Cell. 2013; 155(7); 1479-1491]. Any of the above tracr sequences is suitable for use in combination with any of the gRNA targeting domain embodiments described herein.

[0354] In some embodiments, the gRNA comprises two distinct RNA molecules (i.e., "double gRNA"). In some embodiments, the gRNA comprises a single RNA molecule (i.e., "single guide RNA" or "sgRNA"). In this specification, the use of the terms "guide RNA" or "gRNA" includes both double gRNA and sgRNA. The double gRNA comprises two distinct RNA molecules: "crispr RNA" (or "crRNA") and "tracr RNA". The crRNA molecule comprises a spacer sequence covalently linked to a "tracr mate" sequence. The tracer mate sequence comprises a stretch of nucleotides complementary to the corresponding sequence within the tracr RNA molecule. The crRNA molecule and the tracr RNA molecule hybridize to each other through the complementarity of the tracr and tracer mate sequences.

[0355] In some embodiments, the gRNA is sgRNA. In these embodiments, the nucleic acid-targeting sequence and the protein-binding sequence are present in a single RNA molecule by the fusion of a spacer sequence to the tracr RNA sequence. In some embodiments, the sgRNA is about 50 to about 200 nucleotide long. In some embodiments, the sgRNA is about 75 to about 150 or about 100 to about 125 nucleotide long. In some embodiments, the sgRNA is about 100 nucleotide long.

[0356] In some embodiments, the gRNA of the present disclosure comprises a targeting domain sequence that is at least 90%, 95%, 96%, 97%, 98%, or 99% complementary, or 100% complementary, to a target nucleic acid sequence within a target locus. In some embodiments, the target nucleic acid sequence is an RNA target sequence. In some embodiments, the target nucleic acid sequence is a DNA target sequence.

[0357] In some embodiments, the gRNA provided herein is IKZF1, IKZF3, GATA3, BCL3, TNIP1, TNFAIP3, NFKBIA, SMAD2, TGFBR1, TGFBR2, TANK, FOXP3, RC3H1, TRAF6, IKZF2, CBLB, PPP2R2D, NRP1, HAVCR2, LAG3, TIGIT, CTLA4, PTPN6, PDCD1 or BCOR It comprises a targeting domain sequence that binds to a target DNA sequence that is at least 90%, 95%, 96%, 97%, 98%, or 99% identical or 100% identical to the sequence of a target gene selected from (e.g., a gene selected from Table 2). In some embodiments, the targeting domain sequence binds to a target DNA sequence that is at least 90%, 95%, 96%, 97%, 98%, or 99% identical or 100% identical to the target DNA sequence defined by the set of genomic coordinates presented in Table 5A or Table 5B. In some embodiments, the targeting domain sequence binds to a target DNA sequence that is at least 90%, 95%, 96%, 97%, 98%, or 99% identical or 100% identical to one of SEQ ID NOs 154 to 498 or SEQ ID NOs 499 to 813. In some embodiments, the targeting domain sequence is encoded by a DNA sequence that is at least 90%, 95%, 96%, 97%, 98%, or 99% identical or 100% identical to one of SEQ ID NOs 154 to 498 or SEQ ID NOs 499 to 813. In some embodiments, the gRNA provided herein is CBLBIt includes a targeting domain sequence that binds to a target DNA sequence that is at least 90%, 95%, 96%, 97%, 98%, or 99% identical to or 100% identical to the target DNA sequence of the gene. In some embodiments, the nucleotide-binding segment of the gRNA sequence binds to a target DNA sequence that is at least 90%, 95%, 96%, 97%, 98%, or 99% identical to or 100% identical to one of SEQ ID NOs 499 to 524. In some embodiments, the nucleotide-binding segment of the gRNA sequence is encoded by a DNA sequence that is at least 90%, 95%, 96%, 97%, 98%, or 99% identical to or 100% identical to one of SEQ ID NOs 499 to 524. Additional gRNAs suitable for targeting CBLB are described in U.S. Patent Application Publication No. 2017 / 0175128.

[0358] In some embodiments, the gRNA provided herein is TNFAIP3 It comprises a targeting domain sequence that binds to a target DNA sequence that is at least 90%, 95%, 96%, 97%, 98%, or 99% identical to, or 100% identical to, the target DNA sequence of the gene. In some embodiments, a nucleo-binding segment of the gRNA sequence binds to a target DNA sequence that is at least 90%, 95%, 96%, 97%, 98%, or 99% identical to, or 100% identical to, one of SEQ ID NOs 348 to 396 or SEQ ID NOs 348 to 386. In some embodiments, the gRNA provided herein is BCORIt includes a targeting domain sequence that binds to a target DNA sequence that is at least 90%, 95%, 96%, 97%, 98%, or 99% identical to or 100% identical to the target DNA sequence of the gene. In some embodiments, the nucleic acid-binding segment of the gRNA sequence binds to a target DNA sequence that is at least 90%, 95%, 96%, 97%, 98%, or 99% identical to or 100% identical to one of SEQ ID NOs 708 to 772 or SEQ ID NOs 708 to 764.

[0359] In some embodiments, the gRNA provided herein is BCL2L11 , CHILD1 , CALM2 , DHODH , UMPS , RBM39 , SEMA7A , CHIC2 , PCBP1 , PBRM1 , WDR6 , E2F8 , SERPINA3 , GNAS, SOCS1 and ANKRD11It comprises a targeting domain sequence that binds to a target DNA sequence that is at least 90%, 95%, 96%, 97%, 98%, or 99% identical or 100% identical to the sequence of a target gene selected from (e.g., a gene selected from Table 3). In some embodiments, the targeting domain sequence binds to a target DNA sequence that is at least 90%, 95%, 96%, 97%, 98%, or 99% identical or 100% identical to the target DNA sequence defined by the set of genomic coordinates presented in Tables 6A through 6F. In some embodiments, the targeting domain sequence binds to a target DNA sequence that is at least 90%, 95%, 96%, 97%, 98%, or 99% identical or 100% identical to one of SEQ ID NOs 814 through 1232. In some embodiments, the targeting domain sequence is encoded by a DNA sequence that is at least 90%, 95%, 96%, 97%, 98%, or 99% identical or 100% identical to one of SEQ ID NOs 814 to 1232.

[0360] In some embodiments, the gRNA provided herein is BCL2L11 , FLI1 , CALM2 , DHODH , UMPS , RBM39 , SEMA7A , CHIC2 , PCBP1 , PBRM1 , WDR6 , E2F8 , SERPINA3 and GNASIt comprises a targeting domain sequence that binds to a target DNA sequence that is at least 90%, 95%, 96%, 97%, 98%, or 99% identical or 100% identical to the sequence of a target gene selected from. In some embodiments, the targeting domain sequence binds to a target DNA sequence that is at least 90%, 95%, 96%, 97%, 98%, or 99% identical or 100% identical to the target DNA sequence defined by the set of genomic coordinates presented in Table 6A or Table 6B. In some embodiments, the targeting domain sequence binds to a target DNA sequence that is at least 90%, 95%, 96%, 97%, 98%, or 99% identical or 100% identical to one of SEQ ID NOs 814 to 1064. In some embodiments, the targeting domain sequence is encoded by a DNA sequence that is at least 90%, 95%, 96%, 97%, 98%, or 99% identical or 100% identical to one of SEQ ID NOs 814 to 1064.

[0361] In some embodiments, the gRNA provided herein is SOCS1It comprises a targeting domain sequence that binds to a target DNA sequence that is at least 90%, 95%, 96%, 97%, 98%, or 99% identical or 100% identical to the sequence of the gene. In some embodiments, the targeting domain sequence binds to a target DNA sequence that is at least 90%, 95%, 96%, 97%, 98%, or 99% identical or 100% identical to the target DNA sequence defined by the set of genomic coordinates presented in Table 6C or Table 6D. In some embodiments, the targeting domain sequence binds to a target DNA sequence that is at least 90%, 95%, 96%, 97%, 98%, or 99% identical or 100% identical to one of SEQ ID NOs 1088 to 1232. In some embodiments, the targeting domain sequence is encoded by a DNA sequence that is at least 90%, 95%, 96%, 97%, 98%, or 99% identical or 100% identical to one of SEQ ID NOs 1088 to 1232. In some embodiments, the targeting domain sequence binds to a target DNA sequence that is at least 90%, 95%, 96%, 97%, 98%, or 99% identical or 100% identical to one of SEQ ID NOs 1088 to 1200. In some embodiments, the targeting domain sequence is encoded by a DNA sequence that is at least 90%, 95%, 96%, 97%, 98%, or 99% identical or 100% identical to one of SEQ ID NOs 1088 to 1200.

[0362] In some embodiments, the gRNA provided herein is SOCS1It comprises a targeting domain that binds to a target DNA sequence within a gene, wherein the targeting domain sequence binds to a target DNA sequence that is at least 90%, 95%, 96%, 97%, 98%, or 99% identical or 100% identical to one of SEQ ID NOs 1088 to 1140. In some embodiments, the targeting domain sequence is encoded by a DNA sequence that is at least 90%, 95%, 96%, 97%, 98%, or 99% identical or 100% identical to one of SEQ ID NOs 1088 to 1140. In some embodiments, the targeting domain sequence binds to a target DNA sequence that is at least 90%, 95%, 96%, 97%, 98%, or 99% identical or 100% identical to one of SEQ ID NOs 1088 to 1120. In some embodiments, the targeting domain sequence is encoded by a DNA sequence that is at least 90%, 95%, 96%, 97%, 98%, or 99% identical or 100% identical to one of SEQ ID NOs 1088 to 1120. In some embodiments, the targeting domain sequence binds to a target DNA sequence that is at least 90%, 95%, 96%, 97%, 98%, or 99% identical or 100% identical to one of SEQ ID NOs 1106, 1110, 1115, 1116, 1118, 1126, 1129, 1141, 1157, 1174. In some embodiments, the targeting domain sequence is encoded by a DNA sequence that is at least 90%, 95%, 96%, 97%, 98%, or 99% identical or 100% identical to one of SEQ ID NOs 1106, 1110, 1115, 1116, 1118, 1126, 1129, 1141, 1157, 1174. In some embodiments, the targeting domain sequence binds to a target DNA sequence that is at least 90%, 95%, 96%, 97%, 98%, or 99% identical or 100% identical to one of SEQ ID NOs 1102, 1103, 1105 to 1108, 1115.In some embodiments, the targeting domain sequence is encoded by a DNA sequence that is at least 90%, 95%, 96%, 97%, 98%, or 99% identical or 100% identical to one of SEQ ID NOs 1102, 1103, 1105 to 1108, and 1115.

[0363] In some embodiments, the gRNA provided herein is ANKRD11 It comprises a targeting domain sequence that binds to a target DNA sequence that is at least 90%, 95%, 96%, 97%, 98%, or 99% identical or 100% identical to the sequence of the gene. In some embodiments, the targeting domain sequence binds to a target DNA sequence that is at least 90%, 95%, 96%, 97%, 98%, or 99% identical or 100% identical to the target DNA sequence defined by the set of genomic coordinates presented in Table 6E or Table 6F. In some embodiments, the targeting domain sequence binds to a target DNA sequence that is at least 90%, 95%, 96%, 97%, 98%, or 99% identical or 100% identical to one of SEQ ID NOs 1065 to 1087. In some embodiments, the targeting domain sequence is encoded by a DNA sequence that is at least 90%, 95%, 96%, 97%, 98%, or 99% identical to one of SEQ ID NOs 1065 to 1087, or 100% identical.

[0364] In some embodiments, the gene-regulatory system comprises two or more gRNA molecules, wherein at least one of the gRNAs is IKZF1, IKZF3, GATA3, BCL3, TNIP1, TNFAIP3, NFKBIA, SMAD2, TGFBR1, TGFBR2, TANK, FOXP3, RC3H1, TRAF6, IKZF2, CBLB, PPP2R2D, NRP1, HAVCR2, LAG3, TIGIT, CTLA4, PTPN6, PDCD1 or BCOR The gRNA molecule comprises a targeting domain that binds to a target DNA sequence that is at least 90%, 95%, 96%, 97%, 98%, or 99% identical to, or 100% identical to, the target DNA sequence of a target gene selected from (e.g., a gene selected from Table 2), and the gRNA molecule BCL2L11 , FLI1 , CALM2 , DHODH , UMPS, RBM39 , SEMA7A , CHIC2 , PCBP1 , PBRM1 , WDR6 , E2F8 , SERPINA3 , GNAS, SOCS1 and ANKRD11 It includes a targeting domain that binds to a target DNA sequence that is at least 90%, 95%, 96%, 97%, 98%, or 99% identical or 100% identical to the target DNA sequence of a target gene selected from (genes selected from Table 3).

[0365] In some embodiments, at least one of the gRNAs comprises a targeting domain that binds to a target DNA sequence that is at least 90%, 95%, 96%, 97%, 98%, or 99% identical or 100% identical to a target DNA sequence defined by a set of genomic coordinates presented in Table 5A or Table 5B, and at least one of the gRNAs comprises a targeting domain that binds to a target DNA sequence that is at least 90%, 95%, 96%, 97%, 98%, or 99% identical or 100% identical to a target DNA sequence defined by a set of genomic coordinates presented in any one of Tables 6A to 6F. In some embodiments, at least one of the gRNAs comprises a targeting domain that binds to a target DNA sequence that is at least 90%, 95%, 96%, 97%, 98%, or 99% identical or 100% identical to one of SEQ ID NOs 154 to 498 or SEQ ID NOs 499 to 813, and at least one of the gRNAs comprises a targeting domain that binds to a target DNA sequence that is at least 90%, 95%, 96%, 97%, 98%, or 99% identical or 100% identical to one of SEQ ID NOs 814 to 1232. In some embodiments, at least one of the gRNAs comprises a targeting domain encoded by a nucleic acid sequence that is at least 90%, 95%, 96%, 97%, 98%, or 99% identical or 100% identical to one of SEQ ID NOs 154 to 498 or SEQ ID NOs 499 to 813, and at least one of the gRNAs comprises a targeting domain encoded by a nucleic acid sequence that is at least 90%, 95%, 96%, 97%, 98%, or 99% identical or 100% identical to one of SEQ ID NOs 814 to 1232.

[0366] In some embodiments, the gene-regulatory system comprises two or more gRNA molecules, wherein at least one of the gRNAs is IKZF1, IKZF3, GATA3, BCL3, TNIP1, TNFAIP3, NFKBIA, SMAD2, TGFBR1, TGFBR2, TANK, FOXP3, RC3H1, TRAF6, IKZF2, CBLB, PPP2R2D, NRP1, HAVCR2, LAG3, TIGIT, CTLA4, PTPN6, PDCD1 or BCORThe gRNA molecule comprises a targeting domain that binds to a target DNA sequence that is at least 90%, 95%, 96%, 97%, 98%, or 99% identical to, or 100% identical to, the target DNA sequence of a target gene selected from, and BCL2L11 , FLI1 , CALM2 , DHODH , UMPS , RBM39 , SEMA7A , CHIC2 , PCBP1 , PBRM1 , WDR6 , E2F8 , SERPINA3 and GNASIt comprises a targeting domain that binds to a target DNA sequence that is at least 90%, 95%, 96%, 97%, 98%, or 99% identical or 100% identical to the target DNA sequence of a target gene selected from. In some embodiments, at least one of the gRNAs comprises a targeting domain that binds to a target DNA sequence that is at least 90%, 95%, 96%, 97%, 98%, or 99% identical or 100% identical to the target DNA sequence defined by the set of genomic coordinates presented in Table 5A or Table 5B, and at least one of the gRNAs comprises a targeting domain that binds to a target DNA sequence that is at least 90%, 95%, 96%, 97%, 98%, or 99% identical or 100% identical to the target DNA sequence defined by the set of genomic coordinates presented in either Table 6A or Table 6B. In some embodiments, at least one of the gRNAs comprises a targeting domain that binds to a target DNA sequence that is at least 90%, 95%, 96%, 97%, 98%, or 99% identical or 100% identical to one of SEQ ID NOs 154 to 498 or SEQ ID NOs 499 to 813, and at least one of the gRNAs comprises a targeting domain that binds to a target DNA sequence that is at least 90%, 95%, 96%, 97%, 98%, or 99% identical or 100% identical to one of SEQ ID NOs 814 to 1064. In some embodiments, at least one of the gRNAs comprises a targeting domain encoded by a nucleic acid sequence that is at least 90%, 95%, 96%, 97%, 98%, or 99% identical or 100% identical to one of SEQ ID NOs 154 to 498 or SEQ ID NOs 499 to 813, and at least one of the gRNAs comprises a targeting domain encoded by a nucleic acid sequence that is at least 90%, 95%, 96%, 97%, 98%, or 99% identical or 100% identical to one of SEQ ID NOs 814 to 1064.

[0367] In some embodiments, the gene-regulatory system comprises two or more gRNA molecules, wherein at least one of the gRNAs is CBLB The gRNA molecule includes a targeting domain that binds to a target DNA sequence that is at least 90%, 95%, 96%, 97%, 98%, or 99% identical to, or 100% identical to, the target DNA sequence, and BCL2L11 , FLI1 , CALM2 , DHODH , UMPS , RBM39 , SEMA7A , CHIC2 , PCBP1 , PBRM1 , WDR6 , E2F8 , SERPINA3 and GNAS It comprises a targeting domain that binds to a target DNA sequence that is at least 90%, 95%, 96%, 97%, 98%, or 99% identical or 100% identical to a target DNA sequence of a target gene selected from. In some embodiments, at least one of the gRNAs comprises a targeting domain that binds to a target DNA sequence that is at least 90%, 95%, 96%, 97%, 98%, or 99% identical or 100% identical to one of SEQ ID NOs 499 to 524, and at least one of the gRNAs comprises a targeting domain that binds to a target DNA sequence that is at least 90%, 95%, 96%, 97%, 98%, or 99% identical or 100% identical to one of SEQ ID NOs 814 to 1064. In some embodiments, at least one of the gRNAs comprises a targeting domain encoded by a nucleic acid sequence that is at least 90%, 95%, 96%, 97%, 98%, or 99% identical or 100% identical to one of SEQ ID NOs 499 to 524, and at least one of the gRNAs comprises a targeting domain encoded by a nucleic acid sequence that is at least 90%, 95%, 96%, 97%, 98%, or 99% identical or 100% identical to one of SEQ ID NOs 814 to 1064.

[0368] In some embodiments, the gene-regulatory system comprises two or more gRNA molecules, wherein at least one of the gRNAs is IKZF1, IKZF3, GATA3, BCL3, TNIP1, TNFAIP3, NFKBIA, SMAD2, TGFBR1, TGFBR2, TANK, FOXP3, RC3H1, TRAF6, IKZF2, CBLB, PPP2R2D, NRP1, HAVCR2, LAG3, TIGIT, CTLA4, PTPN6, PDCD1 or BCOR The gRNA molecule comprises a targeting domain that binds to a target DNA sequence that is at least 90%, 95%, 96%, 97%, 98%, or 99% identical to, or 100% identical to, the target DNA sequence of a target gene selected from, and SOCS1It comprises a targeting domain that binds to a target DNA sequence that is at least 90%, 95%, 96%, 97%, 98%, or 99% identical or 100% identical to the target DNA sequence of the gene. In some embodiments, at least one of the gRNAs comprises a targeting domain that binds to a target DNA sequence that is at least 90%, 95%, 96%, 97%, 98%, or 99% identical or 100% identical to the target DNA sequence defined by the set of genomic coordinates presented in Table 5A or Table 5B, and at least one of the gRNAs comprises a targeting domain that binds to a target DNA sequence that is at least 90%, 95%, 96%, 97%, 98%, or 99% identical or 100% identical to the target DNA sequence defined by the set of genomic coordinates presented in either Table 6C or Table 6D. In some embodiments, at least one of the gRNAs comprises a targeting domain that binds to a target DNA sequence that is at least 90%, 95%, 96%, 97%, 98%, or 99% identical or 100% identical to one of SEQ ID NOs 154 to 498 or SEQ ID NOs 499 to 813, and at least one of the gRNAs comprises a targeting domain that binds to a target DNA sequence that is at least 90%, 95%, 96%, 97%, 98%, or 99% identical or 100% identical to one of SEQ ID NOs 1088 to 1232. In some embodiments, at least one of the gRNAs comprises a targeting domain encoded by a nucleic acid sequence that is at least 90%, 95%, 96%, 97%, 98%, or 99% identical or 100% identical to one of SEQ ID NOs 154 to 498 or SEQ ID NOs 499 to 813, and at least one of the gRNAs comprises a targeting domain encoded by a nucleic acid sequence that is at least 90%, 95%, 96%, 97%, 98%, or 99% identical or 100% identical to one of SEQ ID NOs 1088 to 1232.

[0369] In some embodiments, the gene-regulatory system comprises two or more gRNA molecules, wherein at least one of the gRNAs is CBLB The gRNA molecule includes a targeting domain that binds to a target DNA sequence that is at least 90%, 95%, 96%, 97%, 98%, or 99% identical to the target DNA sequence of the gene, or 100% identical to the target DNA sequence, and SOCS1 It comprises a targeting domain that binds to a target DNA sequence that is at least 90%, 95%, 96%, 97%, 98%, or 99% identical or 100% identical to the target DNA sequence. In some embodiments, at least one of the gRNAs comprises a targeting domain that binds to a target DNA sequence that is at least 90%, 95%, 96%, 97%, 98%, or 99% identical or 100% identical to one of SEQ ID NOs 499 to 524, and at least one of the gRNAs comprises a targeting domain that binds to a target DNA sequence that is at least 90%, 95%, 96%, 97%, 98%, or 99% identical or 100% identical to one of SEQ ID NOs 1088 to 1232. In some embodiments, at least one of the gRNAs comprises a targeting domain encoded by a nucleic acid sequence that is at least 90%, 95%, 96%, 97%, 98%, or 99% identical or 100% identical to one of SEQ ID NOs 499 to 524, and at least one of the gRNAs comprises a targeting domain encoded by a nucleic acid sequence that is at least 90%, 95%, 96%, 97%, 98%, or 99% identical or 100% identical to one of SEQ ID NOs 1088 to 1232.

[0370] In some embodiments, the gene-regulatory system comprises two or more gRNA molecules, wherein at least one of the gRNAs is IKZF1, IKZF3, GATA3, BCL3, TNIP1, TNFAIP3, NFKBIA, SMAD2, TGFBR1, TGFBR2, TANK, FOXP3, RC3H1, TRAF6, IKZF2, CBLB, PPP2R2D, NRP1, HAVCR2, LAG3, TIGIT, CTLA4, PTPN6, PDCD1 or BCORThe gRNA molecule comprises a targeting domain that binds to a target DNA sequence that is at least 90%, 95%, 96%, 97%, 98%, or 99% identical to, or 100% identical to, the target DNA sequence of a target gene selected from, and ANKRD11It comprises a targeting domain that binds to a target DNA sequence that is at least 90%, 95%, 96%, 97%, 98%, or 99% identical or 100% identical to the target DNA sequence of the gene. In some embodiments, at least one of the gRNAs comprises a targeting domain that binds to a target DNA sequence that is at least 90%, 95%, 96%, 97%, 98%, or 99% identical or 100% identical to the target DNA sequence defined by the set of genomic coordinates presented in Table 5A or Table 5B, and at least one of the gRNAs comprises a targeting domain that binds to a target DNA sequence that is at least 90%, 95%, 96%, 97%, 98%, or 99% identical or 100% identical to the target DNA sequence defined by the set of genomic coordinates presented in Table 6E or Table 6F. In some embodiments, at least one of the gRNAs comprises a targeting domain that binds to a target DNA sequence that is at least 90%, 95%, 96%, 97%, 98%, or 99% identical or 100% identical to one of SEQ ID NOs 154 to 498 or SEQ ID NOs 499 to 813, and at least one of the gRNAs comprises a targeting domain that binds to a target DNA sequence that is at least 90%, 95%, 96%, 97%, 98%, or 99% identical or 100% identical to one of SEQ ID NOs 1065 to 1087. In some embodiments, at least one of the gRNAs comprises a targeting domain encoded by a nucleic acid sequence that is at least 90%, 95%, 96%, 97%, 98%, or 99% identical or 100% identical to one of SEQ ID NOs 154 to 498 or SEQ ID NOs 499 to 813, and at least one of the gRNAs comprises a targeting domain encoded by a nucleic acid sequence that is at least 90%, 95%, 96%, 97%, 98%, or 99% identical or 100% identical to one of SEQ ID NOs 1065 to 1087.

[0371] In some embodiments, the gene-regulatory system comprises two or more gRNA molecules, wherein at least one of the gRNAs is CBLB The gRNA molecule includes a targeting domain that binds to a target DNA sequence that is at lea...

Claims

Claim 1 Endogenous SOCS1 As a modified T cell containing an inactivation mutation in the SH2 domain of a gene, wherein the inactivation mutation comprises a deletion, substitution, or insertion of one or more nucleotide(s), and endogenous SOCS1 Gene expression is reduced in modified T cells, the antitumor efficacy of modified T cells is enhanced compared to non-modified T cells, and inactivated mutations Cas endonuclease and endogenous SOCS1 Modified T cells introduced using a guide RNA (gRNA) containing a targeting domain sequence that specifically binds to a target sequence within the SH2 domain of a gene. Claim 2 In claim 1, a modified T cell in which the modified T cell is a tumor infiltrating lymphocyte (TIL). Claim 3 In claim 1, the modified T cell has one or more of the following feature(s) compared to the non-modified T cell: increased infiltration or migration into the tumor, increased proliferation, increased or prolonged cell survival, increased resistance to inhibitory factors in the surrounding microenvironment that cause the activated state of the modified T cell to be prolonged or increased, increased production of pro-inflammatory immune factors, increased cytotoxicity, and increased resistance to consumption. Claim 4 A modified T cell, wherein the targeting domain sequence is: (a) specifically binds to a target DNA sequence selected from SEQ ID NOs 1106, 1110, 1115, 1116, 1118, 1126, 1129, 1141, 1157, and 1174, or (b) encoded by a nucleic acid sequence selected from SEQ ID NOs 1106, 1110, 1115, 1116, 1118, 1126, 1129, 1141, 1157, and 1174. Claim 5 In claim 1, the Cas endonuclease comprises (a) a wild-type Cas protein having two enzymatic active domains; (b) a Cas nikcase mutant having one enzymatic active domain; (c) a deactivated Cas protein (dCas) associated with a heterologous protein, or (d) a Cas orthologous, modified T cell. Claim 6 In paragraph 5, a modified T cell in which the Cas endonuclease is the Cas9 protein. Claim 7 In paragraph 1, the fruit is endogenous SOCS1 Modified T cells that have a partial or complete deletion of a gene. Claim 8 In paragraph 1, a modified T cell in which the inactivating mutation is a frame shift mutation. Claim 9 In paragraph 1, endogenous ANKRD11, IKZF1, IKZF3, GATA3, BCL3, TNIP1, TNFAIP3, NFKBIA, SMAD2, TGFBR1, TGFBR2, TANK, FOXP3, RC3H1, TRAF6, IKZF2, CBLB, PPP2R2D, NRP1, HAVCR2, LAG3, TIGIT, CTLA4, PTPN6, PDCD1, BCOR, BCL2L11, FLI1, CALM2, DHODH, UMPS, RBM39, SEMA7A, CHIC2, PCBP1, PBRM1, WDR6, E2F8, SERPINA3, or GNAS Modified T cells in which the expression and / or activity of are reduced compared to non-modified T cells. Claim 10 In claim 1, the modified T cell expresses a modified antigen receptor presented on the surface of the modified T cell, and the modified antigen receptor is a chimeric antigen receptor (CAR) and a T cell receptor (TCR). Claim 11 A modified T cell according to claim 1, further comprising an exogenous transgene expressing an immune-activating molecule, wherein the immune-activating molecule is selected from the group consisting of cytokines, chemokines, co-stimulating molecules, activating peptides, and antibodies or antigen-binding fragments thereof. Claim 12 A pharmaceutical composition for treating a cell proliferative disorder, an inflammatory disorder, or an infectious disease, comprising a modified T cell according to any one of claims 1 to 11. Claim 13 (a) Endogenous SOCS1 (b) an enzyme protein that specifically binds to the target sequence or endogenous SOCS1 A gene-regulatory system comprising a guide RNA (gRNA) containing a targeting domain sequence that specifically binds to a target sequence and a Cas endonuclease, wherein endogenous SOCS1 A gene-regulatory system comprising (a) a target nucleic acid sequence defined by a set of genomic coordinates selected from those listed in Table 6C, and / or (b) a target DNA sequence selected from SEQ ID NOs 1088-1232, and / or (c) a targeting domain sequence encoded by a nucleic acid sequence selected from SEQ ID NOs 1088-1232. Claim 14 A guide RNA (gRNA) comprising (a) a target nucleic acid sequence defined by a set of genomic coordinates selected from those listed in Table 6C and / or (b) a target DNA sequence selected from SEQ ID NOs 1088-1232 and / or (c) a targeting domain sequence encoded by a nucleic acid sequence selected from SEQ ID NOs 1088-1232. Claim 15 A method for producing modified T cells in vitro, comprising the step of introducing the gene-regulatory system of claim 13 into the T cell. Claim 16 A method of in vitro production according to claim 15, comprising: (a) a step of expanding T cells in a culture; and (b) a step of introducing a gene-regulatory system into the T cells. Claim 17 A method of in vitro production according to claim 15, comprising: (a) obtaining T cells from a human subject; and (b) introducing the gene-regulatory system of claim 13 into the T cells. Claim 18 A method of in vitro production according to claim 15, comprising: (a) obtaining T cells from a human subject; (b) expanding the T cells in a culture to produce an expanded population of T cells; and (c) introducing a gene-regulatory system into the expanded population of T cells. Claim 19 In paragraph 15, endogenous compared to non-modified T cells by culturing T cells SOCS1 A method for in vitro production comprising a step of further reducing the expression of Claim 20 In Paragraph 19, endogenous SOCS1 An in vitro production method in which reduced expression of increases the expansion of modified T cells compared to non-modified T cells. Claim 21 A method of in vitro production according to claim 20, further comprising the step of obtaining a population of T cells from a human subject. Claim 22 In paragraph 16, an in vitro production method in which a gene-regulatory system is introduced into a population of T cells before, during, or after expansion of (a). Claim 23 In paragraph 15, an in vitro production method in which a gene-regulatory system is introduced into a T cell by transfusion, transduction, electroporation, or physical disruption of the cell membrane by a microfluidic device. Claim 24 In paragraph 15, a method of in vitro production in which the T cells are tumor-infiltrating lymphocytes (TILs). Claim 25 A pharmaceutical composition comprising a population of cells comprising modified T cells according to any one of claims 1 to 11 for treating a disease or disorder in a subject requiring such treatment by administering the population of cells comprising modified T cells to the subject, wherein the disease or disorder is a cell proliferative disorder, an inflammatory disorder, or an infectious disorder. Claim 26 A pharmaceutical composition in which the disease or disorder in paragraph 25 is cancer or a viral infection. Claim 27 A pharmaceutical composition according to claim 26, wherein the cancer is leukemia, lymphoma, or a solid tumor. Claim 28 A pharmaceutical composition according to claim 27, wherein the solid tumor is melanoma, pancreatic tumor, bladder tumor, lung tumor or metastasis, colorectal cancer or head and neck cancer. Claim 29 A pharmaceutical composition according to claim 26, wherein the cancer is a PD1-resistant cancer or a PD1-insensitive cancer. Claim 30 A pharmaceutical composition according to claim 25, wherein the subject has previously been treated with a PD1 inhibitor or a PDL1 inhibitor. Claim 31 In paragraph 25, the treatment to the subject NRP1, HAVCR2, LAG3, TIGIT, CTLA4 , or PDCD1 A pharmaceutical composition comprising the step of administering an antibody or a binding fragment thereof that specifically binds to a protein encoded by and inhibits its function. Claim 32 A pharmaceutical composition in which, in paragraph 25, the population of cells is autogenous with respect to the subject. Claim 33 A pharmaceutical composition according to claim 25, wherein the population of cells is homologous to the subject. Claim 34 A pharmaceutical composition comprising a modified T cell according to any one of claims 1 to 11 for killing cancer cells by exposing cancer cells to a modified T cell. Claim 35 A pharmaceutical composition in which the exposure occurs in vitro, as described in paragraph 34. Claim 36 A pharmaceutical composition in which exposure occurs in vivo, as described in paragraph 34. Claim 37 A pharmaceutical composition in which exposure occurs in vitro, as described in paragraph 34. Claim 38 In paragraph 1, a modified T cell in which an inactivated mutation is introduced in vitro. 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