Genetic composition and gene modification method for improving immunotherapy

Abstract

To provide gene-modifying compositions and gene-modifying methods for improving immunotherapy. [Solution] This disclosure provides methods and compositions relating to modifying immune effector cells to improve therapeutic efficacy. In some embodiments, immune effector cells are provided that are modified to enhance the effector function of immune cells by reducing the expression of one or more endogenous target genes or reducing the function of one or more endogenous proteins. In some embodiments, immune effector cells are provided that are further modified by introducing a transgene that confers antigen specificity, such as an exogenous T cell receptor (TCR) or a chimeric antigen receptor (CAR). Methods for treating cell proliferation disorders such as cancer using the modified immune effector cells described herein are also provided.

Description

[Technical Field]

[0001] Cross-reference of related applications This application is a compilation of the following U.S. Patent Provisional Applications: 62 / 643,578 filed on March 15, 2018; 62 / 692,010 filed on June 29, 2018; 62 / 768,428 filed on November 16, 2018; 62 / 643,587 filed on March 15, 2018; 62 / 692,019 filed on June 29, 2018; 62 / 768,443 filed on November 16, 2018; 62 / 804,265 filed on February 12, 2019; and March 2018. These applications claim priority based on U.S. Patent Provisional Application No. 62 / 643,597 filed on 15, U.S. Patent Provisional Application No. 62 / 692,100 filed on 29 June 2018, U.S. Patent Provisional Application No. 62 / 768,448 filed on 16 November 2018, U.S. Patent Provisional Application No. 62 / 643,598 filed on 15 March 2018, U.S. Patent Provisional Application No. 62 / 692,110 filed on 29 June 2018, and U.S. Patent Provisional Application No. 62 / 768,458 filed on 16 November 2018, each of which is incorporated herein by reference in its entirety.

[0002] Description of the electronically submitted text file The contents of the text file submitted electronically with this specification, namely a computer-readable copy of the sequence listing (filename: KSQT_007_04WO_SeqList_ST25.txt, date of submission: March 14, 2019, file size: 308 kilobytes), are incorporated herein by reference in their entirety.

[0003] field This disclosure relates to methods, compositions, and components for editing or modulating the expression of target nucleic acid sequences, as well as immunotherapy-related applications thereof, including use in conjunction with receptor-transformed immune effector cells in the treatment of proliferative disorders, inflammatory diseases, and / or infections. [Background technology]

[0004] Adoptive cell transfer using genetically modified T cells, particularly CAR-T cells, is in clinical trials as a therapy for solid and hematological malignancies. Results to date have been mixed. In hematological malignancies (especially lymphoma, CLL, and ALL), at least partial responses were observed in the majority of patients in several phase 1 and phase 2 trials, and complete responses were observed in some (Kochenderfer et al., 2012 Blood 1 19, 2709-2720). In 2017, the FDA approved two CAR-T therapies, Kymriah® and Yescarta®, both for the treatment of hematological cancers. However, in most types of tumors (including melanoma, renal cell carcinoma, and colorectal cancer), lower responses were observed than in hematological malignancies (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). Therefore, since the results are largely limited to CAR-T cell approaches targeting B-cell hematological malignancies, there is considerable room for improvement in adoptive T-cell therapy. [Prior art documents] [Non-patent literature]

[0005] [Non-Patent Document 1] Kochenderfer et al.,2012 Blood 1 19,2709-2720 [Non-Patent Document 2] Johnson et al.,2009 Blood 1 14,535-546 [Non-Patent Document 3] Lamers et al.,2013 Mol.Ther.21,904-912 [Non-Patent Document 4] Warren et al.,1998 Cancer Gene Ther.5,S1-S2 [Overview of the Initiative] [Means for solving the problem]

[0006] In the above-mentioned types of tumors (melanoma, renal cell carcinoma, and colorectal cancer, Yong, 2017, Imm Cell Biol., 95:356-363), a decrease in response was observed, indicating a need to improve the effectiveness of adoptive cell transfer of modified immune cells in cancer treatment, particularly in adoptive cell therapy for solid malignancies. In addition, even in hematological malignancies where the benefits of adoptive cell transfer were observed, not all patients responded, and recurrence occurred at a higher frequency than desired, likely due to reduced function of the adopted T cells. Factors that limit the effectiveness of genetically modified immune cells as cancer treatments include (1) cell proliferation, e.g., limited proliferation of T cells after adoptive transfer; (2) cell viability, e.g., induction of T cell apoptosis by factors in the tumor environment; and (3) cell function, e.g., inhibition of cytotoxic T cell function by inhibitory factors secreted by host immune cells and cancer cells, and exhaustion of immune cells during the manufacturing process and / or after transfer.

[0007] Specific characteristics that are thought to enhance the antitumor activity of immune cells include: 1) the ability to proliferate in the host after adoptive transfer, 2) the ability to infiltrate tumors, 3) the ability to survive in the host and / or exhibit resistance to immune cell exhaustion, and 4) the ability to function in a way that can kill tumor cells. This disclosure provides immune cells in which the expression and / or function of one or more endogenous target genes are reduced, and these modified immune cells exhibit enhancement of one or more effector functions, including improved proliferation, improved tumor infiltration, immune cell survival in the target, and / or improved resistance to immune cell exhaustion. This disclosure also provides methods and compositions for modifying immune effector cells to induce enhanced immune cell activity against tumor cells, as well as methods and compositions suitable for use in connection with adoptive immune cell transfer therapy.

[0008] In some embodiments, the Disclosure provides modified immunoeffector cells in which the expression and / or function of the ZC3H12A nucleic acid or ZC3H12A protein (also known as Regnase-1) is reduced. The Disclosure describes and demonstrates that ZC3H12A is inhibited by several methods, including CRISPR / Cas systems, zinc finger systems and siRNA / shRNA systems. In some embodiments, the reduction in ZC3H12A expression / function is mediated by an antibody, small molecule or peptide. In some embodiments, the Disclosure provides a method for killing cancer cells, comprising exposing the cancer cells to a ZC3H12A protein inhibitor, wherein the inhibitor is an antibody, small molecule or peptide that binds to ZC3H12A and reduces the function of ZC3H12A, and the amount of the inhibitor is effective in killing the cancer cells. In some embodiments, the exposure is in vitro, in vivo or ex vivo.

[0009] In some embodiments, this disclosure includes (a) BCL2L11, FLI1, CALM2, DHODH, UMPS, RBM39, SEMA7A, CHIC2, PCBP1, PBRM The present invention provides modified immunoeffector cells comprising 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 (b) WDR6, E2F8, SERPINA3, and GNAS, (b) ZC3H12A, (c) MAP4K1, or (d) NR4A3. In some embodiments, the effector function of the immunoeffector cells is enhanced by reducing the expression and / or function of one or more endogenous genes.

[0010] In some embodiments, the disclosure provides modified immunoeffector cells comprising 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 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 effector function of the immunoeffector cells is enhanced by the reduction in the expression and / or function of one or more endogenous genes. In some embodiments, the gene regulatory system can reduce the expression and / or function of two or more endogenous target genes selected from the group consisting 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 BCOR. In some embodiments, at least one of the endogenous target genes is selected from the group consisting of IKZF1, IKZF3, GATA3, BCL3, TNIP1, TNFAIP3, NFKBIA, SMAD2, TGFBR1, TGFBR2, TANK, FOXP3, RC3H1, TRAF6, and IKZF2, and at least one of the endogenous target genes is selected from the group consisting of CBLB, PPP2R2D, NRP1, HAVCR2, LAG3, TIGIT, CTLA4, PTPN6, PDCD1, and BCOR.

[0011] In some embodiments, the gene regulatory system can reduce the expression and / or function of at least one endogenous target gene selected from the group consisting of BCL2L11, FLI1, CALM2, DHODH, UMPS, RBM39, SEMA7A, CHIC2, PCBP1, PBRM1, WDR6, E2F8, SERPINA3, and GNAS, as well as at least one endogenous target gene selected from the group consisting 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 BCOR.

[0012] In some embodiments, the gene regulatory system can reduce the expression and / or function of ZC3H12A and at least one endogenous target gene selected from the group consisting 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 BCOR. In some embodiments, the gene regulatory system can reduce the expression and / or function of ZC3H12A and CBLB. In some embodiments, the gene regulatory system can reduce the expression and / or function of ZC3H12A and BCOR. In some embodiments, the gene regulatory system can reduce the expression and / or function of ZC3H12A and TNFAIP3.

[0013] In some embodiments, the gene regulatory system can reduce the expression and / or function of MAP4K1 and at least one endogenous target gene selected from the group consisting 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 BCOR. In some embodiments, the gene regulatory system can reduce the expression and / or function of MAP4K1 and CBLB. In some embodiments, the gene regulatory system can reduce the expression and / or function of MAP4K1 and BCOR. In some embodiments, the gene regulatory system can reduce the expression and / or function of MAP4K1 and TNFAPI3.

[0014] In some embodiments, the gene regulatory system can reduce the expression and / or function of NR4A3 and at least one endogenous target gene selected from the group consisting 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 BCOR. In some embodiments, the gene regulatory system can reduce the expression and / or function of NR4A3 and CBLB. In some embodiments, the gene regulatory system can reduce the expression and / or function of NR4A3 and BCOR. In some embodiments, the gene regulatory system can reduce the expression and / or function of NR4A3 and TNFAPI3.

[0015] In some embodiments, the disclosure provides modified immunoeffector cells comprising a gene regulatory system, wherein 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, the one or more nucleic acid molecules are selected from siRNA, shRNA, microRNA (miR), antagonist miR, or antisense RNA. In some embodiments, the gene regulatory system comprises an siRNA nucleic acid molecule or an shRNA nucleic acid molecule.

[0016] In some embodiments, the disclosure provides modified immune effector cells comprising a gene regulatory system, wherein the gene regulatory system comprises an siRNA nucleic acid molecule or an shRNA nucleic acid molecule, and the siRNA molecule or shRNA molecule comprises about 19 to 30 nucleotides that bind to an RNA sequence encoded by a DNA sequence defined by a set of genomic coordinates shown 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. In some embodiments, the gene regulatory system comprises an siRNA nucleic acid molecule or an shRNA nucleic acid molecule, and the siRNA molecule or shRNA molecule comprises about 19 to 30 nucleotides that bind to an RNA sequence encoded by a DNA sequence defined by a set of genomic coordinates shown 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.

[0017] In some embodiments, the gene regulatory system comprises an siRNA nucleic acid molecule or an shRNA nucleic acid molecule, which is encoded by a DNA sequence defined by a set of genomic coordinates shown in Tables 6C and 6D. The siRNA or shRNA contains approximately 19 to 30 nucleotides that bind to the NA sequence. In some embodiments, the siRNA or shRNA contains approximately 19 to 30 nucleotides that bind to the RNA sequence encoded by a DNA sequence selected from the group consisting of SEQ ID NOs: 1065 to 1509. In some embodiments, the gene regulatory system comprises an siRNA nucleic acid molecule or an shRNA nucleic acid molecule, the siRNA or shRNA molecule containing approximately 19 to 30 nucleotides that bind to the RNA sequence encoded by a DNA sequence defined by a set of genomic coordinates shown in Tables 6E and 6F. In some embodiments, the siRNA or shRNA contains approximately 19 to 30 nucleotides that bind to the RNA sequence encoded by a DNA sequence selected from the group consisting of SEQ ID NOs: 510 to 1538. In some embodiments, the gene regulatory system comprises an siRNA nucleic acid molecule or an shRNA nucleic acid molecule, the siRNA or shRNA molecule containing approximately 19 to 30 nucleotides that bind to the RNA sequence encoded by a DNA sequence defined by a set of genomic coordinates shown in Tables 6G and 6H. In some embodiments, the siRNA or shRNA contains approximately 19 to 30 nucleotides bound to an RNA sequence encoded by a DNA sequence selected from the group consisting of SEQ ID NOs: 1539 to 1566.

[0018] In some embodiments, the gene regulatory system includes multiple siRNA or shRNA molecules and can reduce the expression and / or function of two or more endogenous target genes. In some embodiments, at least one of the endogenous target genes is selected from the group consisting of BCL2L11, FLI1, CALM2, DHODH, UMPS, RBM39, SEMA7A, CHIC2, PCBP1, PBRM1, WDR6, E2F8, SERPINA3, and GNAS, and at least one of the endogenous target genes is selected from the group consisting 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 BCOR. In some embodiments, at least one of the multiple siRNA molecules or shRNA molecules contains about 19 to 30 nucleotides that bind to an RNA sequence encoded by a DNA sequence defined by a set of genomic coordinates shown in Tables 6A and 6B, and at least one of the multiple siRNA molecules or shRNA molecules contains about 19 to 30 nucleotides that bind to an RNA sequence encoded by a DNA sequence defined by a set of genomic coordinates shown in Tables 5A and 5B. In some embodiments, at least one of the multiple siRNA molecules or shRNA molecules contains 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 multiple siRNA molecules or shRNA molecules contains 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.In some embodiments, at least one of the endogenous target genes is selected from the group consisting of BCL2L11, FLI1, CALM2, DHODH, UMPS, RBM39, SEMA7A, CHIC2, PCBP1, PBRM1, WDR6, E2F8, SERPINA3, and GNAS, and at least one of the endogenous target genes is selected from the group consisting of TNFAIP3, CBLB, and BCOR.

[0019] In some embodiments, the gene regulatory system includes multiple siRNA or shRNA molecules and can reduce the expression and / or function of two or more endogenous target genes. In some embodiments, at least one of the endogenous target genes is ZC3H12A, and at least one of the endogenous target genes is IKZF1, IKZF3, GATA3, BCL3, TNIP1, TNFAIP3, NFK The group is selected from BIA, SMAD2, TGFBR1, TGFBR2, TANK, FOXP3, RC3H1, TRAF6, IKZF2, CBLB, PPP2R2D, NRP1, HAVCR2, LAG3, TIGIT, CTLA4, PTPN6, PDCD1, and BCOR. In some embodiments, at least one of the multiple siRNA molecules or shRNA molecules contains about 19 to 30 nucleotides that bind to an RNA sequence encoded by a DNA sequence defined by a set of genomic coordinates shown in Tables 6C and 6D, and at least one of the multiple siRNA molecules or shRNA molecules contains about 19 to 30 nucleotides that bind to an RNA sequence encoded by a DNA sequence defined by a set of genomic coordinates shown in Tables 5A and 5B. In some embodiments, at least one of the multiple siRNA molecules or shRNA molecules contains 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 1509, and at least one of the multiple siRNA molecules or shRNA molecules contains 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. In some embodiments, at least one of the endogenous target genes is selected from the group consisting of ZC3H12A, and at least one of the endogenous target genes is selected from the group consisting of TNFAIP3, CBLB, and BCOR.

[0020] In some embodiments, the gene regulatory system includes multiple siRNA or shRNA molecules and can reduce the expression and / or function of two or more endogenous target genes. In some embodiments, at least one of the endogenous target genes is MAP4K1, and at least one of the endogenous target genes is selected from the group consisting 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 BCOR. In some embodiments, at least one of the multiple siRNA molecules or shRNA molecules contains about 19 to 30 nucleotides that bind to an RNA sequence encoded by a DNA sequence defined by a set of genomic coordinates shown in Tables 6E and 6F, and at least one of the multiple siRNA molecules or shRNA molecules contains about 19 to 30 nucleotides that bind to an RNA sequence encoded by a DNA sequence defined by a set of genomic coordinates shown in Tables 5A and 5B. In some embodiments, at least one of the multiple siRNA molecules or shRNA molecules contains 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: 510 to 1538, and at least one of the multiple siRNA molecules or shRNA molecules contains 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. In some embodiments, at least one of the endogenous target genes is MAP4K1, and at least one of the endogenous target genes is selected from the group consisting of TNFAIP3, CBLB, and BCOR.

[0021] In some embodiments, the gene regulatory system includes multiple siRNA molecules or shRNA molecules and can reduce the expression and / or function of two or more endogenous target genes. In some embodiments, at least one of the endogenous target genes is NR4A3, and at least one of the endogenous target genes is selected from the group consisting 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 BCOR. In some embodiments, at least one of the multiple siRNA molecules or shRNA molecules contains about 19 to 30 nucleotides that bind to an RNA sequence encoded by a DNA sequence defined by a set of genomic coordinates shown in Tables 6G and 6H, and at least one of the multiple siRNA molecules or shRNA molecules contains about 19 to 30 nucleotides that bind to an RNA sequence encoded by a DNA sequence defined by a set of genomic coordinates shown in Tables 5A and 5B. In some embodiments, at least one of the multiple siRNA molecules or shRNA molecules contains 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: 1539 to 1566, and at least one of the multiple siRNA molecules or shRNA molecules contains 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. In some embodiments, at least one of the endogenous target genes is NR4A3, and at least one of the endogenous target genes is selected from the group consisting of TNFAIP3, CBLB, and BCOR.

[0022] In some embodiments, the disclosure provides modified immunoeffector cells comprising a gene regulatory system, wherein the gene regulatory system comprises an enzyme protein, which is engineered to specifically bind to a target sequence in one or more of its endogenous genes. In some embodiments, the protein is a transcriptional activator-like effector nuclease (TALEN), a zinc finger nuclease, or a meganuclease.

[0023] In some embodiments, the present disclosure provides modified immunoeffector cells comprising a gene regulatory system, wherein the gene regulatory system comprises a guide nucleic acid molecule and an enzyme protein, the nucleic acid molecule being a guide RNA (gRNA) molecule and the enzyme protein being a Cas protein or a Cas orthologue.

[0024] In some embodiments, the disclosure provides modified immune effector cells comprising a gene regulatory system capable of reducing the expression and / or function of at least one endogenous target gene, wherein the gene regulatory system comprises a guide RNA (gRNA) molecule and a Cas protein or Cas orthologue, and one or more endogenous target genes are selected from the group consisting 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 BCOR, and the gRNA molecule comprises a targeting domain sequence that binds to a nucleic acid sequence defined by a set of genomic coordinates shown in Tables 5A and 5B. In some embodiments, the gRNA molecule includes a targeting domain sequence that binds to a target DNA sequence selected from the group consisting of SEQ ID NOs: 154-813.

[0025] In some embodiments, the present disclosure relates to modified immune effector cells comprising a gene regulatory system capable of reducing the expression and / or function of at least one endogenous target gene, wherein the gene regulatory system comprises a guide RNA (gRNA) molecule and a Cas protein or Cas orthologue, and one or more endogenous target genes are selected from the group consisting of BCL2L11, FLI1, CALM2, DHODH, UMPS, RBM39, SEMA7A, CHIC2, PCBP1, PBRM1, WDR6, E2F8, SERPINA3, and GNAS, and the gRNA molecule is a targeting domain sequence that binds to a nucleic acid sequence defined by a set of genomic coordinates shown in Tables 6A and 6B. The present invention provides modified immune effector cells containing the following: In some embodiments, the gRNA molecule contains a targeting domain sequence that binds to a target DNA sequence selected from the group consisting of SEQ ID NOs: 814-1064. In some embodiments, the gRNA molecule contains a targeting domain sequence encoded by a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 814-1064.

[0026] In some embodiments, the disclosure provides modified immune effector cells comprising a gene regulatory system capable of reducing the expression and / or function of at least one endogenous target gene, wherein the gene regulatory system comprises a guide RNA (gRNA) molecule and a Cas protein or Cas orthologue, wherein one or more endogenous target genes are ZC3H12A, and the gRNA molecule comprises a targeting domain sequence that binds to a nucleic acid sequence defined by a set of genomic coordinates shown in Tables 6C and 6D. In some embodiments, the gRNA molecule comprises a targeting domain sequence that binds to a nucleic acid sequence selected from the group consisting of SEQ ID NOs. 1065-1509.

[0027] In some embodiments, the disclosure provides modified immune effector cells comprising a gene regulatory system capable of reducing the expression and / or function of at least one endogenous target gene, wherein the gene regulatory system comprises a guide RNA (gRNA) molecule and a Cas protein or Cas orthologue, wherein one or more endogenous target genes are MAP4K1, and the gRNA molecule comprises a targeting domain sequence that binds to a nucleic acid sequence defined by a set of genomic coordinates shown 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. 510–1538. 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. 510–1538.

[0028] In some embodiments, the disclosure provides modified immune effector cells comprising a gene regulatory system capable of reducing the expression and / or function of at least one endogenous target gene, wherein the gene regulatory system comprises a guide RNA (gRNA) molecule and a Cas protein or Cas orthologue, wherein one or more endogenous target genes are NR4A3, and the gRNA molecule comprises a targeting domain sequence that binds to a nucleic acid sequence defined by a set of genomic coordinates shown in Tables 6G and 6H. 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. 1539-1566.

[0029] In some embodiments, the disclosure provides modified immune effector cells comprising a gene regulatory system capable of reducing the expression and / or function of at least two endogenous target genes, wherein the gene regulatory system comprises a plurality of gRNAs and Cas proteins or Cas orthologues. The invention provides modified immunoeffector cells in which at least one of the endogenous target genes is selected from the group consisting of 2, DHODH, UMPS, RBM39, SEMA7A, CHIC2, PCBP1, PBRM1, WDR6, E2F8, SERPINA3, and GNAS, and at least one of the endogenous target genes is selected from the group consisting 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 BCOR. In some embodiments, at least one of the multiple gRNA molecules includes a targeting domain sequence that binds to a nucleic acid sequence defined by a set of genomic coordinates shown in Tables 6A and 6B, and at least one of the multiple gRNA molecules includes a targeting domain sequence that binds to a nucleic acid sequence defined by a set of genomic coordinates shown in Tables 5A and 5B. In some embodiments, at least one of the multiple gRNA molecules includes 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 multiple gRNA molecules includes 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 multiple gRNA molecules includes 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 multiple gRNA molecules includes a targeting domain sequence encoded by a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 154 to 813. In some embodiments, at least one of the endogenous target genes is selected from the group consisting of BCL2L11, FLI1, CALM2, DHODH, UMPS, RBM39, SEMA7A, CHIC2, PCBP1, PBRM1, WDR6, E2F8, SERPINA3, and GNAS, and at least one of the endogenous target genes is selected from the group consisting of TNFAIP3, CBLB, and BCOR.

[0030] In some embodiments, the present disclosure provides modified immunoeffector cells comprising a gene regulatory system capable of reducing the expression and / or function of at least two endogenous target genes, wherein the gene regulatory system comprises a plurality of gRNAs and Cas proteins or Cas orthologues, at least one of the endogenous target genes being ZC3H12A, and at least one of the endogenous target genes being selected from the group consisting 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 BCOR. In some embodiments, at least one of the multiple gRNA molecules includes a targeting domain sequence that binds to a nucleic acid sequence defined by a set of genomic coordinates shown in Tables 6C and 6D, and at least one of the multiple gRNA molecules includes a targeting domain sequence that binds to a nucleic acid sequence defined by a set of genomic coordinates shown in Tables 5A and 5B. In some embodiments, at least one of the multiple gRNA molecules includes a targeting domain sequence that binds to a target DNA sequence selected from the group consisting of SEQ ID NOs: 1065 to 1509, and at least one of the multiple gRNA molecules includes 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 multiple gRNA molecules includes a targeting domain sequence encoded by a DNA sequence selected from the group consisting of SEQ ID NOs: 1065 to 1509, and at least one of the multiple gRNA molecules includes a targeting domain sequence encoded by a DNA sequence selected from the group consisting of SEQ ID NOs: 154 to 813. In some embodiments, at least one of the endogenous target genes is ZC3H12A, and at least one of the endogenous target genes is TNFAIP3, The group is selected from those consisting of CBLB and BCOR.

[0031] In some embodiments, the disclosure provides modified immune effector cells comprising a gene regulatory system capable of reducing the expression and / or function of at least two endogenous target genes, wherein the gene regulatory system comprises a plurality of gRNAs and Cas proteins or Cas orthologues, at least one of the endogenous target genes is MAP4K1, and at least one of the endogenous target genes is selected from the group consisting 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 BCOR. In some embodiments, at least one of the multiple gRNA molecules includes a targeting domain sequence that binds to a nucleic acid sequence defined by a set of genomic coordinates shown in Tables 6E and 6F, and at least one of the multiple gRNA molecules includes a targeting domain sequence that binds to a nucleic acid sequence defined by a set of genomic coordinates shown in Tables 5A and 5B. In some embodiments, at least one of the multiple gRNA molecules includes a targeting domain sequence that binds to a target DNA sequence selected from the group consisting of SEQ ID NOs. 510 to 1538, and at least one of the multiple gRNA molecules includes 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 multiple gRNA molecules includes a targeting domain sequence encoded by a nucleic acid sequence selected from the group consisting of SEQ ID NOs. 510 to 1538, and at least one of the multiple gRNA molecules includes a targeting domain sequence encoded by a DNA sequence selected from the group consisting of SEQ ID NOs. 154 to 813. In some embodiments, at least one of the endogenous target genes is MAP4K1, and at least one of the endogenous target genes is selected from the group consisting of TNFAIP3, CBLB, and BCOR.

[0032] In some embodiments, the disclosure provides modified immune effector cells comprising a gene regulatory system capable of reducing the expression and / or function of at least two endogenous target genes, wherein the gene regulatory system comprises a plurality of gRNAs and Cas proteins or Cas orthologues, at least one of the endogenous target genes being NR4A3, and at least one of the endogenous target genes being selected from the group consisting 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 BCOR. In some embodiments, at least one of the multiple gRNA molecules includes a targeting domain sequence that binds to a nucleic acid sequence defined by a set of genomic coordinates shown in Tables 6G and 6H, and at least one of the multiple gRNA molecules includes a targeting domain sequence that binds to a nucleic acid sequence defined by a set of genomic coordinates shown in Tables 5A and 5B. In some embodiments, at least one of the multiple gRNA molecules includes a targeting domain sequence that binds to a target DNA sequence selected from the group consisting of SEQ ID NOs: 1539 to 1566, and at least one of the multiple gRNA molecules includes 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 multiple gRNA molecules includes a targeting domain sequence encoded by a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 1539 to 1566, and at least one of the multiple gRNA molecules includes a targeting domain sequence encoded by a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 154 to 813. In some embodiments, at least one of the endogenous target genes is NR4A3, and at least one of the endogenous target genes is selected from the group consisting of TNFAIP3, CBLB, and BCOR.

[0033] In some embodiments, the disclosure provides modified immunoeffector cells comprising a Cas protein or Cas orthologue, wherein (a) the Cas protein is a wild-type Cas protein comprising two enzymatic activity domains capable of inducing double-strand DNA breaks, (b) the Cas protein is a Cas nickase mutant comprising one enzymatic activity domain capable of inducing single-strand DNA breaks, or (c) the Cas protein is an inactive Cas protein (dCas) bound to 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), Kruppel-associated box (KRAB) domain, methyl-CpG binding protein 2 (MECP2), and mSin3 4-linking domain (SID4X).

[0034] In some embodiments, the gene regulatory system introduces an inactivating mutation into one or more endogenous target genes. In some embodiments, the inactivating mutation includes a deletion, substitution, or insertion of one or more nucleotides in the genomic sequence of two or more endogenous genes. In some embodiments, the deletion is a partial or complete deletion of the two or more endogenous target genes. In some embodiments, the inactivating mutation is a frameshift mutation. In some embodiments, the inactivating mutation reduces the expression and / or function of the two or more endogenous target genes.

[0035] In some embodiments, the gene regulatory system is introduced into immunoeffector cells by transfection, transduction, electroporation, or physical disruption of the cell membrane using a microfluidic device. In some embodiments, the gene regulatory system is introduced as a polynucleotide, protein, or ribonucleoprotein (RNP) complex encoding one or more components of the system.

[0036] In some embodiments, the disclosure provides modified immunoeffector cells in which the expression and / or function of one or more endogenous genes selected from the group consisting 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 BCOR is reduced, and the effector function of the immunoeffector cells is enhanced by the reduction in the expression and / or function of the one or more endogenous genes. In some embodiments, the disclosure provides modified immunoeffector cells in which the expression and / or function of one or more endogenous genes selected from (a) the group consisting of IKZF1, IKZF3, GATA3, BCL3, TNIP1, TNFAIP3, NFKBIA, SMAD2, TGFBR1, TGFBR2, TANK, FOXP3, RC3H1, TRAF6 and IKZF2, or (b) the group consisting of CBLB, PPP2R2D, NRP1, HAVCR2, LAG3, TIGIT, CTLA4, PTPN6, PDCD1 and BCOR is reduced, wherein the effector function of the immunoeffector cells is enhanced by the reduction in the expression and / or function of the one or more endogenous genes.

[0037] In some embodiments, this disclosure includes (a) BCL2L11, FLI1, CALM2, The present invention provides modified immunoeffector cells in which the expression and / or function of one or more endogenous genes selected from the group consisting of DHODH, UMPS, RBM39, SEMA7A, CHIC2, PCBP1, PBRM1, WDR6, E2F8, SERPINA3, and GNAS is reduced, and the effector function of the modified immunoeffector cells is enhanced by the reduction in the expression and / or function of the one or more endogenous genes.

[0038] In some embodiments, the disclosure provides modified immunoeffector cells in which the expression and / or function of 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 is reduced, and the effector function of the modified immunoeffector cells is enhanced by the reduction in the expression and / or function of the two or more endogenous genes. In some embodiments, the modified immunoeffector has reduced expression and / or function of CBLB and BCOR.

[0039] In some embodiments, the disclosure relates to modified immunoeffector cells in which the expression and / or function of two or more target genes are reduced, wherein at least one target gene is selected from the group consisting of BCL2L11, FLI1, CALM2, DHODH, UMPS, RBM39, SEMA7A, CHIC2, PCBP1, PBRM1, WDR6, E2F8, SERPINA3, and GNAS, and at least one target gene is IKZF1, IKZF3, GATA3, BCL3, and TNIP The present invention provides modified immune effector cells selected from the group consisting of TNFAIP3, NFKBIA, SMAD2, TGFBR1, TGFBR2, TANK, FOXP3, RC3H1, TRAF6, IKZF2, CBLB, PPP2R2D, NRP1, HAVCR2, LAG3, TIGIT, CTLA4, PTPN6, PDCD1, and BCOR, in which the effector function of the modified immune effector cells is enhanced by the reduction of expression and / or function of two or more endogenous genes.

[0040] In some embodiments, the disclosure provides modified immunoeffector cells in which the expression and / or function of two or more target genes is reduced, wherein at least one target gene is ZC3H12A, and at least one target gene is selected from the group consisting 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 BCOR, and the effector function of the modified immunoeffector cells is enhanced by the reduction in the expression and / or function of the two or more endogenous genes. In some embodiments, the modified immunoeffector has reduced expression and / or function of ZC3H12A and CBLB. In some embodiments, the modified immunoeffector has reduced expression and / or function of ZC3H12A and BCOR. In some embodiments, the modified immunoeffector has reduced expression and / or function of ZC3H12A and TNFAIP3.

[0041] In some embodiments, the disclosure relates to modified immunoeffector cells in which the expression and / or function of two or more target genes are reduced, wherein at least one target gene is MAP4K1, and at least one 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, PTP The present invention provides modified immunoeffector cells selected from the group consisting of N6, PDCD1, and BCOR, in which the effector function of the modified immunoeffector cells is enhanced by the reduction of the expression and / or function of two or more endogenous genes. In some embodiments, the modified immunoeffector has reduced expression and / or function of MAP4K1 and CBLB. In some embodiments, the modified immunoeffector has reduced expression and / or function of MAP4K1 and BCOR. In some embodiments, the modified immunoeffector has reduced expression and / or function of MAP4K1 and TNFAIP3.

[0042] In some embodiments, the disclosure provides modified immune effector cells in which the expression and / or function of two or more target genes is reduced, wherein at least one target gene is NR4A3, and at least one target gene is selected from the group consisting 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 BCOR, and the effector function of the modified immune effector cells is enhanced by the reduction in the expression and / or function of the two or more endogenous genes. In some embodiments, the modified immune effector cells have reduced expression and / or function of NR4A3 and CBLB. In some embodiments, the modified immunoeffector has reduced expression and / or function of NR4A3 and BCOR. In some embodiments, the modified immunoeffector has reduced expression and / or function of NR4A3 and TNFAIP3.

[0043] In some embodiments, the disclosure provides modified immunoeffector cells containing inactivating mutations in one or more endogenous genes selected from the group consisting 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 BCOR. In some embodiments, the disclosure provides modified immunoeffector cells containing inactivating mutations in one or more endogenous genes selected from (a) the group consisting of IKZF1, IKZF3, GATA3, BCL3, TNIP1, TNFAIP3, NFKBIA, SMAD2, TGFBR1, TGFBR2, TANK, FOXP3, RC3H1, TRAF6, and IKZF2, or (b) the group consisting of CBLB, PPP2R2D, NRP1, HAVCR2, LAG3, TIGIT, CTLA4, PTPN6, PDCD1, and BCOR.

[0044] In some embodiments, the disclosure provides modified immunoeffector cells containing inactivating mutations in one or more endogenous genes selected from (a) BCL2L11, FLI1, CALM2, DHODH, UMPS, RBM39, SEMA7A, CHIC2, PCBP1, PBRM1, WDR6, E2F8, SERPINA3, and GNAS, (b) ZC3H12A, (c) MAP4K1, or (d) NR4A3.

[0045] In some embodiments, the disclosure provides modified immunoeffector cells containing inactivating mutations in 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 immunoeffector contains inactivating mutations in the CBLB gene and the BCOR gene.

[0046] In some embodiments, the disclosure provides modified immunoeffector cells comprising inactivating mutations in two or more target genes, wherein at least one target gene is selected from the group consisting of BCL2L11, FLI1, CALM2, DHODH, UMPS, RBM39, SEMA7A, CHIC2, PCBP1, PBRM1, WDR6, E2F8, SERPINA3, and GNAS, and at least one target gene is selected from the group consisting 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 BCOR.

[0047] In some embodiments, the disclosure provides modified immunoeffector cells comprising inactivating mutations in two or more target genes, wherein at least one target gene is ZC3H12A, and at least one target gene is selected from the group consisting 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 BCOR. In some embodiments, the modified immunoeffector comprises inactivating mutations in the ZC3H12A gene and the CBLB gene. In some embodiments, the modified immunoeffector comprises inactivating mutations in the ZC3H12A gene and the BCOR gene. In some embodiments, the modified immunoeffector includes inactivating mutations in the ZC3H12A gene and the TNFAIP3 gene.

[0048] In some embodiments, the disclosure provides modified immunoeffector cells comprising inactivating mutations in two or more target genes, wherein at least one target gene is MAP4K1, and at least one target gene is selected from the group consisting 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 BCOR. In some embodiments, the modified immunoeffector comprises inactivating mutations in the MAP4K1 gene and the CBLB gene. In some embodiments, the modified immunoeffector comprises inactivating mutations in the MAP4K1 gene and the BCOR gene. In some embodiments, the modified immunoeffector includes inactivating mutations in the MAP4K1 gene and the TNFAIP3 gene.

[0049] In some embodiments, the disclosure provides modified immunoeffector cells comprising inactivating mutations in two or more target genes, wherein at least one target gene is NR4A3, and at least one target gene is selected from the group consisting 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 BCOR. In some embodiments, the modified immunoeffector comprises inactivating mutations in the NR4A3 gene and the CBLB gene. In some embodiments, the modified immunoeffector comprises inactivating mutations in the NR4A3 gene and the BCOR gene. In some embodiments, the modified immune effector cells contain inactivating mutations in the NR4A3 and TNFAIP3 genes.

[0050] In some embodiments, the inactivating mutation comprises a deletion, substitution, or insertion of one or more nucleotides in the genomic sequence 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 inactivating mutation is a frameshift mutation. In some embodiments, the inactivating mutation reduces the expression and / or function of two or more endogenous target genes.

[0051] 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 immune effector cells 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 immune effector cells or control immune effector cells.

[0052] In some embodiments, the modified immune effector cells further include a modified immune receptor presented on their 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, the antigen being a tumor-associated antigen.

[0053] In some embodiments, the modified immune effector cells further include exogenous transgenes expressing immune activation molecules. In some embodiments, the immune activation molecules are selected from the group consisting of cytokines, chemokines, costimulatory molecules, activating peptides, antibodies, or antigen-binding fragments thereof. In some embodiments, the antibody or its binding fragment specifically binds to proteins encoded by NRP1, HAVCR2, LAG3, TIGIT, CTLA4, or PDCD1, thereby inhibiting the function of those proteins.

[0054] In some embodiments, the immune effector cells are lymphocytes selected from T cells, natural killer (NK) cells, and NKT cells. In some embodiments, the lymphocytes are tumor-infiltrating lymphocytes (TILs).

[0055] In some embodiments, the effector function is selected from cell proliferation, cell viability, tumor infiltration, cytotoxicity, antitumor immune response, and / or exhaustion resistance.

[0056] In some embodiments, this disclosure provides compositions comprising modified immunoeffector cells as described herein. In some embodiments, the composition further comprises a pharmaceutically acceptable carrier or diluent. In some embodiments, the composition comprises modified immunoeffector cells comprising at least 1 × 10⁶ cells. 4 pieces, 1×10 5 pieces, 1×10 6 pieces, 1×10 7 pieces, 1×10 8 pieces, 1×10 9 pieces, 1×10 10 individual or 1 x 10 11 Contains [number] [number]. In some embodiments, the composition is suitable for administration to a subject requiring the composition. In some embodiments, the composition comprises autoimmune effector cells derived from a subject requiring the composition. In some embodiments, the composition comprises allogeneic immune effector cells derived from a donor subject.

[0057] In some embodiments, the present disclosure includes (a) the group consisting of IKZF1, IKZF3, GATA3, BCL3, TNIP1, TNFAIP3, NFKBIA, SMAD2, TGFBR1, TGFBR2, TANK, FOXP3, RC3H1, TRAF6 and IKZF2, Or (b) a gene regulatory system that can reduce the expression and / or function of one or more endogenous target genes in a cell, selected from the group consisting of CBLB, PPP2R2D, NRP1, HAVCR2, LAG3, TIGIT, CTLA4, PTPN6, PDCD1, and BCOR, comprising (i) a nucleic acid molecule, (ii) an enzyme molecule, or (iii) a guide nucleic acid molecule and an enzyme protein.

[0058] In some embodiments, the present disclosure provides a gene regulatory system capable of reducing the expression of one or more endogenous target genes in a cell, selected from (a) the group consisting of BCL2L11, FLI1, CALM2, DHODH, UMPS, RBM39, SEMA7A, CHIC2, PCBP1, PBRM1, WDR6, E2F8, SERPINA3 and GNAS, (b) ZC3H12A, (c) MAP4K1, or (d) NR4A3, comprising (i) a nucleic acid molecule, (ii) an enzyme molecule, or (iii) a guide nucleic acid molecule and an enzyme protein.

[0059] In some embodiments, the present disclosure provides a gene regulatory system capable of reducing the expression of one or more endogenous target genes within a cell, the gene regulatory system comprising a guide RNA (gRNA) nucleic acid molecule and a Cas endonuclease.

[0060] In some embodiments, the present disclosure provides a gene regulatory system capable of reducing the expression of one or more endogenous target genes in a cell, comprising a guide RNA (gRNA) nucleic acid molecule and a Cas endonuclease, wherein the one or more endogenous target genes are selected from IKZF1, IKZF3, GATA3, BCL3, TNIP1, TNFAIP3, NFKBIA, SMAD2, TGFBR1, TGFBR2, TANK, FOXP3, RC3H1, TRAF6 and IKZF2, or selected from CBLB, PPP2R2D, NRP1, HAVCR2, LAG3, TIGIT, CTLA4, PTPN6, PDCD1 and BCOR, and the gRNA molecule comprises a targeting domain sequence that binds to a target DNA sequence defined by a set of genomic coordinates shown in Tables 5A and 5B. In some embodiments, the gene regulatory system comprises a gRNA molecule and a Cas endonuclease and can reduce the expression of one or more endogenous target genes, the one or more endogenous target genes being selected from IKZF1, IKZF3, GATA3, BCL3, TNIP1, TNFAIP3, NFKBIA, SMAD2, TGFBR1, TGFBR2, TANK, FOXP3, RC3H1, TRAF6, and IKZF2, and the gRNA molecule comprises a targeting domain sequence that binds to a target DNA sequence selected from the group consisting of SEQ ID NOs.154-498. 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.154-498.

[0061] In some embodiments, the present disclosure provides a gene regulatory system capable of reducing the expression of one or more endogenous target genes in a cell, comprising a guide RNA (gRNA) nucleic acid molecule and a Cas endonuclease, wherein the one or more endogenous target genes are selected from CBLB, PPP2R2D, NRP1, HAVCR2, LAG3, TIGIT, CTLA4, PTPN6, PDCD1, and BCOR, and the gRNA molecule comprises a targeting domain sequence that binds to a target DNA sequence selected from the group consisting of SEQ ID NOs. 499-813. 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. 499-813.

[0062] In some embodiments, the present disclosure is a gene regulatory system capable of reducing the expression of one or more endogenous target genes in a cell, comprising a guide RNA (gRNA) nucleic acid molecule and a Cas endonuclease, wherein one or more endogenous target genes are BCL2L The gRNA molecule is selected from 11, FLI1, CALM2, DHODH, UMPS, RBM39, SEMA7A, CHIC2, PCBP1, PBRM1, WDR6, E2F8, SERPINA3, and GNAS, and provides a gene regulation system in which the gRNA molecule includes a targeting domain sequence that binds to a target DNA sequence defined by a set of genomic coordinates shown in Tables 6A and 6B. In some embodiments, the gRNA molecule includes a targeting domain sequence that binds to a target DNA sequence selected from the group consisting of SEQ ID NOs.

[0063] In some embodiments, the disclosure provides a gene regulatory system capable of reducing the expression of one or more endogenous target genes in a cell, comprising a guide RNA (gRNA) nucleic acid molecule and a Cas endonuclease, wherein the one or more endogenous target genes are ZC3H12A, and the gRNA molecule comprises a targeting domain sequence that binds to a target DNA sequence defined by a set of genomic coordinates shown 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: 1065-1509. 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: 1065-1509. 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: 1065-1509.

[0064] In some embodiments, the present disclosure provides a gene regulatory system capable of reducing the expression of one or more endogenous target genes in a cell, comprising a guide RNA (gRNA) nucleic acid molecule and a Cas endonuclease, wherein the one or more endogenous target genes include MAP4K1, and the gRNA molecule comprises a targeting domain sequence that binds to a target DNA sequence defined by a set of genomic coordinates shown 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. 510–1538. 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. 510–1538.

[0065] In some embodiments, the present disclosure provides a gene regulatory system capable of reducing the expression of one or more endogenous target genes in a cell, comprising a guide RNA (gRNA) nucleic acid molecule and a Cas endonuclease, wherein the one or more endogenous target genes include NR4A3, and the gRNA molecule comprises a targeting domain sequence that binds to a target DNA sequence defined by a set of genomic coordinates shown in Tables 6G and 6H. 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: 1539-1566. 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: 1539-1566.

[0066] In some embodiments, the gene regulatory system includes an siRNA nucleic acid molecule or an shRNA nucleic acid molecule.

[0067] In some embodiments, the present disclosure is a gene regulatory system capable of reducing the expression of one or more endogenous target genes in a cell, comprising an siRNA nucleic acid molecule or an shRNA nucleic acid molecule, wherein one or more endogenous target genes are IKZF1, IKZF3, GATA3, BCL3, TNIP1, TNFAIP3, NFKBIA, SMAD2, TGFB The siRNA or shRNA molecule is selected from R1, TGFBR2, TANK, FOXP3, RC3H1, TRAF6, and IKZF2, or selected from CBLB, PPP2R2D, NRP1, HAVCR2, LAG3, TIGIT, CTLA4, PTPN6, PDCD1, and BCOR, and provides a gene regulatory system containing approximately 19 to 30 nucleotides that bind to an RNA sequence encoded by a DNA sequence defined by a set of genomic coordinates shown in Tables 5A and 5B. In some embodiments, the one or more endogenous target genes are selected from IKZF1, IKZF3, GATA3, BCL3, TNIP1, TNFAIP3, NFKBIA, SMAD2, TGFBR1, TGFBR2, TANK, FOXP3, RC3H1, TRAF6, and IKZF2, and the siRNA or shRNA molecule contains 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, the one or more endogenous target genes are selected from CBLB, PPP2R2D, NRP1, HAVCR2, LAG3, TIGIT, CTLA4, PTPN6, PDCD1, and BCOR, and the siRNA or shRNA molecule contains about 19 to 30 nucleotides that bind to an RNA sequence encoded by a DNA sequence selected from SEQ ID NOs. 499 to 813.

[0068] In some embodiments, the disclosure provides a gene regulatory system capable of reducing the expression of one or more endogenous target genes in a cell, comprising an siRNA nucleic acid molecule or an shRNA nucleic acid molecule, wherein the one or more endogenous target genes are selected from BCL2L11, FLI1, CALM2, DHODH, UMPS, RBM39, SEMA7A, CHIC2, PCBP1, PBRM1, WDR6, E2F8, SERPINA3, and GNAS, and the siRNA molecule or shRNA molecule comprises about 19 to 30 nucleotides that bind to an RNA sequence encoded by a set of genomic coordinates shown in Tables 6A and 6B. In some embodiments, the siRNA molecule 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 81064.

[0069] In some embodiments, the present disclosure provides a gene regulatory system capable of reducing the expression of one or more endogenous target genes in a cell, comprising an siRNA nucleic acid molecule or an shRNA nucleic acid molecule, wherein the one or more endogenous target genes are ZC3H12A, and the siRNA molecule or shRNA molecule comprises about 19 to 30 nucleotides bound to an RNA sequence encoded by a set of genomic coordinates shown in Tables 6C and 6D. In some embodiments, the siRNA molecule or shRNA molecule comprises about 19 to 30 nucleotides bound to an RNA sequence encoded by a DNA sequence selected from SEQ ID NOs: 1065 to 1509.

[0070] In some embodiments, the present disclosure provides a gene regulatory system capable of reducing the expression of one or more endogenous target genes in a cell, comprising an siRNA nucleic acid molecule or an shRNA nucleic acid molecule, wherein the one or more endogenous target genes comprise MAP4K1, and the siRNA molecule or shRNA molecule comprises about 19 to 30 nucleotides bound to an RNA sequence encoded by a set of genomic coordinates shown in Tables 6E and 6F. In some embodiments, the siRNA molecule or shRNA molecule comprises about 19 to 30 nucleotides bound to an RNA sequence encoded by a DNA sequence selected from SEQ ID NOs. 510 to 1538.

[0071] In some embodiments, the present disclosure relates to a gene regulatory system capable of reducing the expression of one or more endogenous target genes within a cell, wherein the expression of an siRNA nucleic acid molecule or shRN The present invention provides a gene regulatory system comprising a nucleic acid molecule, one or more of which contain NR4A3, and whose siRNA or shRNA molecule comprises approximately 19 to 30 nucleotides that bind to an RNA sequence encoded by a set of genomic coordinates defined in Tables 6G and 6H. In some embodiments, the siRNA or shRNA molecule comprises approximately 19 to 30 nucleotides that bind to an RNA sequence encoded by a DNA sequence selected from SEQ ID NOs. 1539 to 1566.

[0072] In some embodiments, the present disclosure relates to 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 selected from (a) the group consisting of BCL2L11, FLI1, CALM2, DHODH, UMPS, RBM39, SEMA7A, CHIC2, PCBP1, PBRM1, WDR6, E2F8, SERPINA3 and GNAS, (b) ZC3H12A, (c) MAP4K1, or (d) NR4A3, and at least one of the endogenous target genes However, the system is selected from (e) the group consisting of IKZF1, IKZF3, GATA3, BCL3, TNIP1, TNFAIP3, NFKBIA, SMAD2, TGFBR1, TGFBR2, TANK, FOXP3, RC3H1, TRAF6 and IKZF2, or (f) the group consisting of CBLB, PPP2R2D, NRP1, HAVCR2, LAG3, TIGIT, CTLA4, PTPN6, PDCD1 and BCOR, and the system provides a gene regulation system comprising (i) a nucleic acid molecule, (ii) an enzyme molecule, or (iii) a guide nucleic acid molecule and an enzyme protein.

[0073] 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 within a cell, comprising a plurality of guide RNA (gRNA) nucleic acid molecules and a Cas endonuclease.

[0074] In some embodiments, the present disclosure relates to a gene regulatory system capable of reducing the expression and / or function of two or more endogenous target genes in a cell, comprising a plurality of guide RNA (gRNA) nucleic acid molecules and a Cas endonuclease, wherein at least one of the endogenous target genes is BCL2L11, FLI1, CALM2, DHODH, UMPS, RBM39, SEMA7A, CHIC2, PCBP1, PBRM1, WDR6, E2F8, SERPINA3, and GNAS. The present invention provides a gene regulatory system in which at least one of its endogenous target genes is selected from the group consisting 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 BCOR. In some embodiments, at least one of its multiple gRNAs binds to a target DNA sequence defined by a set of genomic coordinates shown in Tables 6A and 6B, and at least one of its multiple gRNAs binds to a target DNA sequence defined by a set of genomic coordinates shown in Tables 5A and 5B. In some embodiments, at least one of the multiple gRNA molecules includes a targeting domain sequence that binds to a target DNA sequence selected from the group consisting of SEQ ID NOs: 814-1064, and at least one of the multiple gRNA molecules includes a targeting domain sequence that binds to a target DNA sequence selected from the group consisting of SEQ ID NOs: 154-498 or SEQ ID NOs: 499-813. In some embodiments, at least one of the multiple gRNA molecules includes a targeting domain sequence encoded by a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 814-1064, and at least one of the multiple gRNA molecules includes a targeting domain sequence encoded by a nucleic acid sequence selected from SEQ ID NOs: 154-498 or SEQ ID NOs: 499-813.

[0075] 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, comprising a plurality of guide RNA (gRNA) nucleic acid molecules and a Cas endonuclease, wherein at least one of the endogenous target genes is ZC3H12A, and at least one of the endogenous target genes is selected from the group consisting 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 BCOR. In some embodiments, at least one of the multiple gRNAs binds to a target DNA sequence defined by a set of genomic coordinates shown in Tables 6C and 6D, and at least one of the multiple gRNAs binds to a target DNA sequence defined by a set of genomic coordinates shown in Tables 5A and 5B. In some embodiments, at least one of the multiple gRNA molecules includes a targeting domain sequence that binds to a target DNA sequence selected from the group consisting of SEQ ID NOs: 1065-1509, and at least one of the multiple gRNA molecules includes a targeting domain sequence that binds to a target DNA sequence selected from the group consisting of SEQ ID NOs: 154-498 or SEQ ID NOs: 499-813. In some embodiments, at least one of the multiple gRNA molecules includes a targeting domain sequence that binds to a target DNA sequence selected from the group consisting of SEQ ID NOs: 1065-1509, and at least one of the multiple gRNA molecules includes a targeting domain sequence that binds to a target DNA sequence selected from the group consisting of SEQ ID NOs: 499-524.In some embodiments, at least one of the multiple gRNA molecules includes a targeting domain sequence encoded by a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 1065-1509, and at least one of the multiple gRNA molecules includes a targeting domain sequence encoded by a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 154-498 or SEQ ID NOs: 499-813. In some embodiments, at least one of the multiple gRNA molecules includes a targeting domain sequence encoded by a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 1065-1509, and at least one of the multiple gRNA molecules includes a targeting domain sequence encoded by a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 499-524.

[0076] 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, comprising a plurality of guide RNA (gRNA) nucleic acid molecules and a Cas endonuclease, wherein at least one of the endogenous target genes is MAP4K1, and at least one of the endogenous target genes is selected from the group consisting of IKZF1, IKZF3, GATA3, BCL3, TNIP1, TNFAIP3, NFKBIA, SMAD2, TGFBR1, TGFBR2, TANK, FOXP3, RC3H1, TRAF6, IKZF2, CBLB, PPP2R2R, NRP1, HAVCR2, LAG3, TIGIT, CTLA4, PTPN6, PDCD1, and BCOR. In some embodiments, at least one of the multiple gRNAs binds to a target DNA sequence defined by a set of genomic coordinates shown in Tables 6E and 6F, and at least one of the multiple gRNAs binds to a target DNA sequence defined by a set of genomic coordinates shown in Tables 5A and 5B. In some embodiments, at least one of the multiple gRNA molecules includes a targeting domain sequence that binds to a target DNA sequence selected from the group consisting of SEQ ID NOs: 510-1538, and at least one of the multiple gRNA molecules includes a targeting domain sequence that binds to a target DNA sequence selected from the group consisting of SEQ ID NOs: 154-498 or SEQ ID NOs: 499-813. In some embodiments, at least one of the multiple gRNA molecules includes a targeting domain sequence that binds to a target DNA sequence selected from the group consisting of SEQ ID NOs: 510-1538 The gRNA molecules include a targeting domain sequence that binds to a target DNA sequence selected from a group, and at least one of the gRNA molecules includes 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 gRNA molecules includes a targeting domain sequence encoded by a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 510 to 1538, and at least one of the gRNA molecules includes 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. In some embodiments, at least one of the gRNA molecules includes a targeting domain sequence encoded by a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 510 to 1538, and at least one of the gRNA molecules includes a targeting domain sequence encoded by a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 499 to 524.

[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, comprising a plurality of guide RNA (gRNA) nucleic acid molecules and a Cas endonuclease, wherein at least one of the endogenous target genes is NR4A3, and at least one of the endogenous target genes is selected from the group consisting 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 BCOR. In some embodiments, at least one of the multiple gRNAs binds to a target DNA sequence defined by a set of genomic coordinates shown in Tables 6G and 6H, and at least one of the multiple gRNAs binds to a target DNA sequence defined by a set of genomic coordinates shown in Tables 5A and 5B. In some embodiments, at least one of the multiple gRNA molecules includes a targeting domain sequence that binds to a target DNA sequence selected from the group consisting of SEQ ID NOs: 1539-1566, and at least one of the multiple gRNA molecules includes a targeting domain sequence that binds to a target DNA sequence selected from the group consisting of SEQ ID NOs: 154-498 or SEQ ID NOs: 499-813. In some embodiments, at least one of the multiple gRNA molecules includes a targeting domain sequence that binds to a target DNA sequence selected from the group consisting of SEQ ID NOs: 1539-1566, and at least one of the multiple gRNA molecules includes a targeting domain sequence that binds to a target DNA sequence selected from the group consisting of SEQ ID NOs: 499-524.In some embodiments, at least one of the multiple gRNA molecules includes a targeting domain sequence encoded by a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 1539-1566, and at least one of the multiple gRNA molecules includes a targeting domain sequence encoded by a nucleic acid sequence selected from SEQ ID NOs: 154-498 or SEQ ID NOs: 499-813. In some embodiments, at least one of the multiple gRNA molecules includes a targeting domain sequence encoded by a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 1539-1566, and at least one of the multiple gRNA molecules includes a targeting domain sequence encoded by a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 499-524.

[0078] In some embodiments, the gene regulatory system comprises a Cas protein, which is (a) a wild-type Cas protein containing two enzyme-active domains capable of inducing double-strand DNA breaks, (b) a Cas nickase variant containing one enzyme-active domain capable of inducing single-strand DNA breaks, or (c) an inactive Cas protein (dCas) bound to a heterologous protein capable of regulating the expression of one or more endogenous target genes. In some embodiments, the heterologous protein is MAX The Cas protein is selected from the group consisting of interaction protein 1 (MXI1), a Kruppel-associated box (KRAB) domain, and an mSin3 4-linked domain (SID4X). In some embodiments, the Cas protein is the Cas9 protein.

[0079] In some embodiments, the gene regulatory system comprises nucleic acid molecules, which are siRNA, shRNA, microRNA (miR), antagonist miR, or antisense RNA. In some embodiments, the gene regulatory system comprises multiple shRNA molecules or siRNA molecules.

[0080] In some embodiments, the present disclosure relates to a gene regulatory system capable of reducing the expression and / or function of two or more endogenous target genes in a cell, comprising a plurality of shRNA molecules or siRNA molecules, wherein at least one of the endogenous target genes is selected from the group consisting of BCL2L11, FLI1, CALM2, DHODH, UMPS, RBM39, SEMA7A, CHIC2, PCBP1, PBRM1, WDR6, E2F8, SERPINA3, and GNAS. The present invention provides a gene regulatory system in which at least one of its endogenous target genes is selected from the group consisting 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 BCOR. In some embodiments, at least one of the multiple siRNA molecules or shRNA molecules contains about 19 to 30 nucleotides that bind to an RNA sequence encoded by a DNA sequence defined by a set of genomic coordinates shown in Tables 6A and 6B, and at least one of the multiple siRNA molecules or shRNA molecules contains about 19 to 30 nucleotides that bind to an RNA sequence encoded by a DNA sequence defined by a set of genomic coordinates shown in Tables 5A and 5B. In some embodiments, at least one of the multiple siRNA molecules or shRNA molecules contains 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 multiple siRNA molecules or shRNA molecules contains 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.

[0081] 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, comprising a plurality of shRNA molecules or siRNA molecules, wherein at least one of the endogenous target genes is ZC3H12A, and at least one of the endogenous target genes is selected from the group consisting 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 BCOR. In some embodiments, at least one of the multiple siRNA molecules or shRNA molecules contains about 19 to 30 nucleotides that bind to an RNA sequence encoded by a DNA sequence defined by a set of genomic coordinates shown in Tables 6C and 6D, and at least one of the multiple siRNA molecules or shRNA molecules contains about 19 to 30 nucleotides that bind to an RNA sequence encoded by a DNA sequence defined by a set of genomic coordinates shown in Tables 5A and 5B. In some embodiments, at least one of the multiple siRNA molecules or shRNA molecules contains 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 1509. At least one of the multiple siRNA molecules or shRNA molecules contains 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: 154-498 or SEQ ID NOs: 499-813. In some embodiments, at least one of the multiple siRNA molecules or shRNA molecules contains 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: 1065-1509, and at least one of the multiple siRNA molecules or shRNA molecules contains 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: 499-524.

[0082] 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, comprising a plurality of shRNA molecules or siRNA molecules, wherein at least one of the endogenous target genes is MAP4K1, and at least one of the endogenous target genes is selected from the group consisting 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 BCOR. In some embodiments, at least one of the multiple siRNA molecules or shRNA molecules contains about 19 to 30 nucleotides that bind to an RNA sequence encoded by a DNA sequence defined by a set of genomic coordinates shown in Tables 6E and 6F, and at least one of the multiple siRNA molecules or shRNA molecules contains about 19 to 30 nucleotides that bind to an RNA sequence encoded by a DNA sequence defined by a set of genomic coordinates shown in Tables 5A and 5B. In some embodiments, at least one of the multiple siRNA molecules or shRNA molecules contains 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: 510 to 1538, and at least one of the multiple siRNA molecules or shRNA molecules contains 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 multiple siRNA molecules or shRNA molecules contains 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: 510 to 1538, and at least one of the multiple siRNA molecules or shRNA molecules contains 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.

[0083] 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, comprising a plurality of shRNA molecules or siRNA molecules, wherein at least one of the endogenous target genes is NR4A3, and at least one of the endogenous target genes is selected from the group consisting 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 BCOR. In some embodiments, at least one of the multiple siRNA molecules or shRNA molecules contains about 19 to 30 nucleotides that bind to an RNA sequence encoded by a DNA sequence defined by a set of genomic coordinates shown in Tables 6G and 6H, and at least one of the multiple siRNA molecules or shRNA molecules is defined by a set of genomic coordinates shown in Tables 5A and 5B. It contains about 19 to 30 nucleotides that bind to an RNA sequence encoded by a DNA sequence. In some embodiments, at least one of the multiple siRNA molecules or shRNA molecules contains 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: 1539 to 1566, and at least one of the multiple siRNA molecules or shRNA molecules contains 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 multiple siRNA molecules or shRNA molecules contains 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: 1539 to 1566, and at least one of the multiple siRNA molecules or shRNA molecules contains 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.

[0084] In some embodiments, the gene regulatory system comprises a protein containing a DNA-binding domain and an enzyme domain, the protein being selected from zinc finger nucleases and transcription activator-like effector nucleases (TALENs).

[0085] 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 the one or more gRNAs comprises a targeting domain sequence encoded by nucleic acid sequences selected from SEQ ID NOs: 814-1064, SEQ ID NOs: 1065-1509, SEQ ID NOs: 510-1538, SEQ ID NOs: 1539-1566, SEQ ID NOs: 154-498, or SEQ ID NOs: 499-813.

[0086] 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. 814-1064, SEQ ID NOs. 1065-1509, SEQ ID NOs. 510-1538 or SEQ ID NOs. 1539-1566, 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. 154-498 or SEQ ID NOs. 499-813.

[0087] 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 the one or more gRNAs comprises a targeting domain sequence encoded by nucleic acid sequences selected from SEQ ID NOs. 814-1064, SEQ ID NOs. 1065-1509, SEQ ID NOs. 510-1538, SEQ ID NOs. 1539-1566, SEQ ID NOs. 154-498, or SEQ ID NOs. 499-813.

[0088] 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. 814-1064, SEQ ID NOs. 1065-1509, SEQ ID NOs. 510-1538, or SEQ ID NOs. 1539-1566, 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. 154-498, or SEQ ID NOs. 499-813.

[0089] In some embodiments, the present disclosure provides a gene regulatory system comprising one or more gRNAs and Cas endonuclease proteins, wherein one or more gRNAs include a targeting domain sequence encoded by a nucleic acid sequence selected from SEQ ID NOs. 814-1064, SEQ ID NOs. 1065-1509, SEQ ID NOs. 510-1538, SEQ ID NOs. 1539-1566, SEQ ID NOs. 154-498, or SEQ ID NOs. 499-813, and the one or more gRNAs and their Cas endonuclease proteins complex to form a ribonucleoprotein (RNP) complex.

[0090] In some embodiments, the present disclosure provides a gene regulatory system comprising a plurality of gRNAs and Cas endonuclease proteins, 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. 814-1064, SEQ ID NOs. 1065-1509, SEQ ID NOs. 510-1538, or SEQ ID NOs. 1539-1566, 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. 154-498 or SEQ ID NOs. 499-813, and one or more of the gRNAs and their Cas endonuclease proteins complex to form a ribonucleoprotein (RNP) complex.

[0091] In some embodiments, the present disclosure provides a kit comprising a gene regulation system described herein.

[0092] In some embodiments, the disclosure provides gRNA nucleic acid molecules comprising a targeting domain nucleic acid sequence that binds to a target sequence in an endogenous target gene selected from (a) the group consisting of BCL2L11, FLI1, CALM2, DHODH, UMPS, RBM39, SEMA7A, CHIC2, PCBP1, PBRM1, WDR6, E2F8, SERPINA3, and GNAS, (b) ZC3H12A, (c) MAP4K1, (d) NR4A3, (e) IKZF1, IKZF3, GATA3, BCL3, TNIP1, TNFAIP3, NFKBIA, SMAD2, TGFBR1, TGFBR2, TANK, FOXP3, RC3H1, TRAF6, and IKZF2, or (f) CBLB, PPP2R2D, NRP1, HAVCR2, LAG3, TIGIT, CTLA4, PTPN6, PDCD1, and BCOR.In some embodiments, (a) the endogenous gene is selected from the group consisting of BCL2L11, FLI1, CALM2, DHODH, UMPS, RBM39, SEMA7A, CHIC2, PCBP1, PBRM1, WDR6, E2F8, SERPINA3, and GNAS, and its gRNA includes a targeting domain sequence that binds to a target DNA sequence located at a genomic coordinate selected from the genomic coordinates shown in Tables 6A and 6B; (b) the endogenous gene is ZC3H12A, and its gRNA includes a targeting domain sequence that binds to a target DNA sequence located at a genomic coordinate selected from the genomic coordinates shown in Tables 6C and 6D; (c) the endogenous gene is MAP4K1, and its gRNA includes a targeting domain sequence that binds to a target DNA sequence located at a genomic coordinate selected from the genomic coordinates shown in Tables 6E and 6F; (d) the endogenous (e) The sex gene is NR4A3, and its gRNA contains a targeting domain sequence that binds to a target DNA sequence located at a genomic coordinate selected from the genomic coordinates shown in Tables 6G and 6H, or (f) the endogenous gene is selected from the group consisting of IKZF1, IKZF3, GATA3, BCL3, TNIP1, TNFAIP3, NFKBIA, SMAD2, TGFBR1, TGFBR2, TANK, FOXP3, RC3H1, TRAF6 and IKZF2, and its gRNA contains a targeting domain sequence that binds to a target DNA sequence located at a genomic coordinate selected from the genomic coordinates shown in Tables 5A and 5B, or (f) the endogenous gene is selected from the group consisting of CBLB, PPP2R2D, NRP1, HAVCR2, LAG3, TIGIT, CTLA4, PTPN6, PDCD1 and BCOR, and its gRNA contains a targeting domain sequence that binds to a target DNA sequence located at a genomic coordinate selected from the genomic coordinates shown in Tables 5A and 5B. It contains a targeting domain sequence that binds to a target DNA sequence located at a genomic coordinate selected from the genomic coordinates.

[0093] In some embodiments, the gRNA includes a targeting domain sequence that binds to a target DNA sequence selected from SEQ ID NOs: 814-1064, 1065-1509, 510-1538, 1539-1566, 154-498, or 499-813. In some embodiments, the gRNA includes a targeting domain sequence encoded by a sequence selected from SEQ ID NOs: 814-1064, 1065-1509, 510-1538, 1539-1566, 154-498, or 499-813. In some embodiments, the target sequence includes a PAM sequence.

[0094] In some embodiments, the gRNA is a modular gRNA molecule. In some embodiments, the gRNA is a dual gRNA molecule. In some embodiments, the targeting domain is 16 nucleotides long, 17 nucleotides long, 18 nucleotides long, 19 nucleotides long, 20 nucleotides long, 21 nucleotides long, 22 nucleotides long, 23 nucleotides long, 24 nucleotides long, 25 nucleotides long, 26 nucleotides long, or longer. In some embodiments, the gRNA molecule includes modifications at its 5' end or near its 5' end (e.g., within 1 to 10 nucleotides, 1 to 5 nucleotides, or 1 to 2 nucleotides from its 5' end) and / or at its 3' end or near its 3' end (e.g., within 1 to 10 nucleotides, 1 to 5 nucleotides, or 1 to 2 nucleotides from its 3' end). In some embodiments, when the modified gRNA is introduced into T cells, it improves stability against nucleases. In some embodiments, when the modified gRNA is introduced into T cells, it reduces the innate immune response.

[0095] In some embodiments, this disclosure provides polynucleotide molecules encoding the gRNA molecules described herein. In some embodiments, this disclosure provides compositions comprising one or more gRNA molecules described herein, or polynucleotides encoding those gRNA molecules. In some embodiments, this disclosure provides kits comprising one or more gRNA molecules described herein, or polynucleotides encoding those gRNA molecules.

[0096] In some embodiments, the present disclosure provides a method for producing modified immune effector cells, comprising: (a) collecting immune effector cells from a subject; (b) introducing a gene regulatory system according to any one of claims 156 to 239 into the immune effector cells; and (c) culturing the immune effector cells so that the expression and / or function of one or more endogenous target genes are reduced compared to unmodified immune effector cells.

[0097] In some embodiments, the present disclosure provides a method for producing modified immunoeffector cells, comprising introducing a gene regulatory system described herein into immunoeffector cells. In some embodiments, the method further comprises introducing a polynucleotide sequence encoding a modified immune receptor selected from CARs and TCRs. In some embodiments, the gene regulatory system and / or the polynucleotide encoding its modified immune receptor are introduced into immunoeffector cells by transfection, 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.

[0098] In some embodiments, the Disclosure provides a method for producing modified immunoeffector cells, comprising (a) growing an immunoeffector cell population in a culture medium, and (b) introducing a gene regulatory system according to any one of claims 156 to 239 into the immunoeffector cell population. In some embodiments, the method further comprises harvesting the immunoeffector cell population from a subject. In some embodiments, the gene regulatory system is introduced into the immunoeffector cell population before, during, or after proliferation. In some embodiments, the proliferation of the immunoeffector cell population comprises a first round of proliferation and a second round of proliferation. In some embodiments, the gene regulatory system is introduced into the immunoeffector cell population before, during, or after the first round of proliferation. In some embodiments, the gene regulatory system is introduced into the immunoeffector cell population before, during, or after the second round of proliferation. In some embodiments, the gene regulatory system is introduced into the immunoeffector cell population before the first and second rounds of proliferation. In some embodiments, the gene regulatory system is introduced into the immune effector cell population after the first and second rounds of proliferation. In some embodiments, the gene regulatory system is introduced into the immune effector cell population after the first round of proliferation and before the second round of proliferation.

[0099] In some embodiments, the Disclosure provides a method for treating a disease or disorder of a subject in need of treatment, comprising administering an effective amount of a modified immunoeffector or composition thereof described herein. In some embodiments, the disease or disorder is a cell proliferation disorder, an inflammatory disorder, or an infection. In some embodiments, the disease or disorder is cancer or a viral infection. In some embodiments, the cancer is selected from leukemia, lymphoma, or solid tumors. In some embodiments, the solid tumor is melanoma, pancreatic tumor, bladder tumor, lung tumor or lung metastasis, colorectal cancer, or head and neck cancer. In some embodiments, the cancer is PD1-resistant or PD1-insensitive cancer. In some embodiments, the subject has previously been treated with a PD1 inhibitor or a PD1 inhibitor. In some embodiments, the method further comprises administering to the subject an antibody or a conjugated fragment that specifically binds to a protein encoded by NRP1, HAVCR2, LAG3, TIGIT, CTLA4, or PDCD1 and inhibits the function of that protein. In some embodiments, the modified immunoeffector cells are autologous to the subject. In some embodiments, the modified immune effector cells are allogeneic to the subject. In some embodiments, the subject has not undergone lymphocyte depletion prior to administration of the modified immune effector cells or their composition. In some embodiments, the subject does not receive high-dose IL-2 therapy with or after administration of the modified immune effector cells or their composition. In some embodiments, the subject receives low-dose IL-2 therapy with or after administration of the modified immune effector cells or their composition. In some embodiments, the subject does not receive IL-2 therapy with or after administration of the modified immune effector cells or their composition.

[0100] In some embodiments, this disclosure provides a method for killing cancer cells, comprising exposing the cancer cells to modified immunoeffector cells or compositions thereof as described herein. In some embodiments, the exposure is in vitro, in vivo, or ex vivo.

[0101] In some embodiments, the Disclosure provides a method for enhancing one or more effector functions of an immune effector cell, comprising introducing a gene regulatory system described herein into the immune effector cell. In some embodiments, the Disclosure provides The present invention provides a method for enhancing one or more effector functions of an immune effector cell, comprising introducing a gene regulatory system described herein into the immune effector cell, wherein in the modified immune effector cell, one or more effector functions are enhanced compared to the unmodified immune effector cell. In some embodiments, the one or more effector functions are selected from cell proliferation, cell viability, cytotoxicity, tumor infiltration, increased cytokine production, antitumor immune responsiveness, and / or exhaustion tolerance. The present invention provides, for example, the following items: (Item 1) Modified immunoeffector cells comprising a gene regulatory system capable of reducing the expression and / or function of one or more endogenous target genes selected from (a) the group consisting of BCL2L11, FLI1, CALM2, DHODH, UMPS, RBM39, SEMA7A, CHIC2, PCBP1, PBRM1, WDR6, E2F8, SERPINA3 and GNAS, (b) ZC3H12A, (c) MAP4K1, or (d) NR4A3, The modified immune effector cells, wherein the effector function of the immune effector cells is enhanced by the reduction of the expression and / or function of one or more endogenous genes. (Item 2) Modified immunoeffector cells as described in item 1, wherein the gene regulatory system can reduce the expression and / or function of two or more endogenous target genes selected from (a) the group consisting of BCL2L11, FLI1, CALM2, DHODH, UMPS, RBM39, SEMA7A, CHIC2, PCBP1, PBRM1, WDR6, E2F8, SERPINA3, and GNAS, (b) ZC3H12A, (c) MAP4K1, or (d) NR4A3. (Item 3) Modified immune effector cells comprising 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 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, wherein the effector function is enhanced by the reduction in the expression and / or function of the one or more endogenous target genes. (Item 4) Modified immunoeffector cells as described in item 3, wherein the gene regulatory system can reduce the expression and / or function of two or more endogenous target genes selected from the group consisting 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 BCOR. (Item 5) Modified immunoeffector cells as described in item 4, wherein at least one of the endogenous target genes is selected from the group consisting of IKZF1, IKZF3, GATA3, BCL3, TNIP1, TNFAIP3, NFKBIA, SMAD2, TGFBR1, TGFBR2, TANK, FOXP3, RC3H1, TRAF6, and IKZF2, and at least one of the endogenous target genes is selected from the group consisting of CBLB, PPP2R2D, NRP1, HAVCR2, LAG3, TIGIT, CTLA4, PTPN6, PDCD1, and BCOR. (Item 6) The aforementioned gene regulatory systems further include IKZF1, IKZF3, GATA3, BCL3, TNIP1, TNFAIP3, NFKBIA, SMAD2, TGFBR1, and TGFBR2 Modified immunoeffector cells as described in item 1 or 2, which can reduce the expression and / or function of one or more endogenous target genes selected from TANK, FOXP3, RC3H1, TRAF6, IKZF2, CBLB, PPP2R2D, NRP1, HAVCR2, LAG3, TIGIT, CTLA4, PTPN6, PDCD1, and BCOR. (Item 7) Modified immunoeffector cells as described in item 6, wherein the gene regulatory system can reduce the expression and / or function of at least one endogenous target gene selected from the group consisting of BCL2L11, FLI1, CALM2, DHODH, UMPS, RBM39, SEMA7A, CHIC2, PCBP1, PBRM1, WDR6, E2F8, SERPINA3, and GNAS, as well as at least one endogenous target gene selected from the group consisting 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 BCOR. (Item 8) Modified immunoeffector cells as described in item 6, wherein the gene regulatory system can reduce the expression and / or function of ZC3H12A and at least one endogenous target gene selected from the group consisting 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 BCOR. (Item 9) Modified immunoeffector cells as described in item 8, wherein the gene regulatory system can reduce the expression and / or function of ZC3H12A and CBLB. (Item 10) Modified immunoeffector cells as described in item 8, wherein the gene regulatory system can reduce the expression and / or function of ZC3H12A and BCOR. (Item 11) Modified immunoeffector cells as described in item 8, wherein the gene regulatory system can reduce the expression and / or function of ZC3H12A and TNFAIP3. (Item 12) Modified immunoeffector cells as described in item 6, wherein the gene regulatory system can reduce the expression and / or function of MAP4K1 and at least one endogenous target gene selected from the group consisting 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 BCOR. (Item 13) Modified immunoeffector cells as described in item 12, wherein the gene regulatory system can reduce the expression and / or function of MAP4K1 and CBLB. (Item 14) Modified immunoeffector cells as described in item 12, wherein the gene regulatory system can reduce the expression and / or function of MAP4K1 and BCOR. (Item 15) Modified immunoeffector cells as described in item 12, wherein the gene regulatory system can reduce the expression and / or function of MAP4K1 and TNFAPI3. (Item 16) The aforementioned gene regulatory system includes NR4A3, as well as IKZF1, IKZF3, and GATA Modified immunoeffector cells as described in item 6, capable of reducing the expression and / or function of at least one endogenous target gene selected from the group consisting of 3, BCL3, TNIP1, TNFAIP3, NFKBIA, SMAD2, TGFBR1, TGFBR2, TANK, FOXP3, RC3H1, TRAF6, IKZF2, CBLB, PPP2R2D, NRP1, HAVCR2, LAG3, TIGIT, CTLA4, PTPN6, PDCD1, and BCOR. (Item 17) Modified immunoeffector cells as described in item 16, wherein the gene regulatory system can reduce the expression and / or function of NR4A3 and CBLB. (Item 18) Modified immunoeffector cells as described in item 16, wherein the gene regulatory system can reduce the expression and / or function of NR4A3 and BCOR. (Item 19) Modified immunoeffector cells as described in item 16, wherein the gene regulatory system can reduce the expression and / or function of NR4A3 and TNFAPI3. (Item 20) Modified immunoeffector cells according to any one of items 1 to 19, wherein 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. (Item 21) Modified immunoeffector cells as described in item 20, wherein one or more nucleic acid molecules are selected from siRNA, shRNA, microRNA (miR), antagonist miR, or antisense RNA. (Item 22) The modified immunoeffector cells described in item 20, wherein the gene regulatory system comprises an siRNA nucleic acid molecule or an shRNA nucleic acid molecule. (Item 23) The modified immunoeffector cells described in item 22, wherein one or more endogenous target genes are selected from the group consisting 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 BCOR, and the siRNA molecule or shRNA molecule contains approximately 19 to 30 nucleotides that bind to an RNA sequence encoded by a DNA sequence defined by a set of genomic coordinates shown in Tables 5A and 5B. (Item 24) The modified immunoeffector cell described in item 23, wherein the siRNA or shRNA contains approximately 19 to 30 nucleotides bound to an RNA sequence encoded by a DNA sequence selected from the group consisting of sequence numbers 154 to 813. (Item 25) The modified immunoeffector cells described in item 22, wherein one or more endogenous target genes are selected from the group consisting of BCL2L11, FLI1, CALM2, DHODH, UMPS, RBM39, SEMA7A, CHIC2, PCBP1, PBRM1, WDR6, E2F8, SERPINA3, and GNAS, and the siRNA molecule or shRNA molecule contains approximately 19 to 30 nucleotides that bind to an RNA sequence encoded by a set of genomic coordinates shown in Tables 6A and 6B. (Item 26) The modified immunoeffector cell described in item 25, wherein the siRNA or shRNA contains approximately 19 to 30 nucleotides bound to an RNA sequence encoded by a DNA sequence selected from the group consisting of sequence numbers 814 to 1064. (Item 27) The modified immunoeffector cells described in item 22, wherein the one or more endogenous target genes are ZC3H12A, and the siRNA or shRNA molecule contains about 19 to 30 nucleotides that bind to an RNA sequence encoded by a set of genomic coordinates shown in Tables 6C and 6D. (Item 28) The modified immunoeffector cell described in item 27, wherein the siRNA or shRNA contains approximately 19 to 30 nucleotides bound to an RNA sequence encoded by a DNA sequence selected from the group consisting of sequence numbers 1065 to 1509. (Item 29) The modified immunoeffector cells described in item 22, wherein the one or more endogenous target genes is MAP4K1, and the siRNA or shRNA molecule contains about 19 to 30 nucleotides that bind to an RNA sequence encoded by a set of genomic coordinates shown in Tables 6E and 6F. (Item 30) The modified immunoeffector cells described in item 29, wherein the siRNA or shRNA contains approximately 19 to 30 nucleotides bound to an RNA sequence encoded by a DNA sequence selected from the group consisting of sequence numbers 510 to 1538. (Item 31) The modified immunoeffector cells described in item 22, wherein the one or more endogenous target genes are NR4A3, and the siRNA or shRNA molecule contains about 19 to 30 nucleotides that bind to an RNA sequence encoded by a set of genomic coordinates shown in Tables 6G and 6H. (Item 32) The modified immunoeffector cell described in item 31, wherein the siRNA or shRNA contains approximately 19 to 30 nucleotides bound to an RNA sequence encoded by a DNA sequence selected from the group consisting of sequence numbers 1539 to 1566. (Item 33) The modified immunoeffector cell described in item 20, wherein the gene regulatory system comprises multiple siRNA molecules or shRNA molecules and can reduce the expression and / or function of two or more endogenous target genes. (Item 34) Modified immunoeffector cells as described in item 33, wherein at least one of the endogenous target genes is selected from the group consisting of BCL2L11, FLI1, CALM2, DHODH, UMPS, RBM39, SEMA7A, CHIC2, PCBP1, PBRM1, WDR6, E2F8, SERPINA3, and GNAS, and at least one of the endogenous target genes is selected from the group consisting 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 BCOR. (Item 35) Modified immune effector cells as described in item 34, wherein at least one of the plurality of siRNA molecules or shRNA molecules contains about 19 to 30 nucleotides that bind to an RNA sequence encoded by a DNA sequence defined by a set of genomic coordinates shown in Tables 6A and 6B, and at least one of the plurality of siRNA molecules or shRNA molecules contains about 19 to 30 nucleotides that bind to an RNA sequence encoded by a DNA sequence defined by a set of genomic coordinates shown in Tables 5A and 5B. (Item 36) Modified immune effector cells according to item 34 or 35, wherein at least one of the plurality of siRNA molecules or shRNA molecules contains 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 molecules or shRNA molecules contains 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. (Item 37) Modified immunoeffector cells as described in item 33, wherein at least one of the endogenous target genes is selected from the group consisting of BCL2L11, FLI1, CALM2, DHODH, UMPS, RBM39, SEMA7A, CHIC2, PCBP1, PBRM1, WDR6, E2F8, SERPINA3, and GNAS, and at least one of the endogenous target genes is selected from the group consisting of TNFAIP3, CBLB, and BCOR. (Item 38) Modified immunoeffector cells as described in item 33, wherein at least one of the endogenous target genes is selected from the group consisting of ZC3H12A, and at least one of the endogenous target genes is selected from the group consisting 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 BCOR. (Item 39) Modified immune effector cells as described in item 38, wherein at least one of the plurality of siRNA molecules or shRNA molecules contains about 19 to 30 nucleotides that bind to an RNA sequence encoded by a DNA sequence defined by a set of genomic coordinates shown in Tables 6C and 6D, and at least one of the plurality of siRNA molecules or shRNA molecules contains about 19 to 30 nucleotides that bind to an RNA sequence encoded by a DNA sequence defined by a set of genomic coordinates shown in Tables 5A and 5B. (Item 40) The modified immune effector cell according to item 39, wherein at least one of the plurality of siRNA molecules or shRNA molecules contains 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 1509, and at least one of the plurality of siRNA molecules or shRNA molecules contains 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. (Item 41) Modified immune effector cells as described in item 33, wherein at least one of the endogenous target genes is selected from the group consisting of ZC3H12A, and at least one of the endogenous target genes is selected from the group consisting of TNFAIP3, CBLB, and BCOR. (Item 42) Modified immunoeffector cells as described in item 33, wherein at least one of the endogenous target genes is MAP4K1, and at least one of the endogenous target genes is selected from the group consisting 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 BCOR. (Item 43) Modified immune effector cells as described in item 42, wherein at least one of the plurality of siRNA molecules or shRNA molecules contains about 19 to 30 nucleotides that bind to an RNA sequence encoded by a DNA sequence defined by a set of genomic coordinates shown in Tables 6E and 6F, and at least one of the plurality of siRNA molecules or shRNA molecules contains about 19 to 30 nucleotides that bind to an RNA sequence encoded by a DNA sequence defined by a set of genomic coordinates shown in Tables 5A and 5B. (Item 44) Modified immune effector cells according to item 42 or 43, wherein at least one of the plurality of siRNA molecules or shRNA molecules contains 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: 510 to 1538, and at least one of the plurality of siRNA molecules or shRNA molecules contains 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. (Item 45) Modified immune effector cells as described in item 33, wherein at least one of the endogenous target genes is MAP4K1, and at least one of the endogenous target genes is selected from the group consisting of TNFAIP3, CBLB, and BCOR. (Item 46) Modified immunoeffector cells as described in item 33, wherein at least one of the endogenous target genes is NR4A3, and at least one of the endogenous target genes is selected from the group consisting 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 BCOR. (Item 47) Modified immune effector cells as described in item 46, wherein at least one of the plurality of siRNA molecules or shRNA molecules contains about 19 to 30 nucleotides that bind to an RNA sequence encoded by a DNA sequence defined by a set of genomic coordinates shown in Tables 6G and 6H, and at least one of the plurality of siRNA molecules or shRNA molecules contains about 19 to 30 nucleotides that bind to an RNA sequence encoded by a DNA sequence defined by a set of genomic coordinates shown in Tables 5A and 5B. (Item 48) Modified immune effector cells according to item 46 or item 47, wherein at least one of the plurality of siRNA molecules or shRNA molecules contains 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: 1539 to 1566, and at least one of the plurality of siRNA molecules or shRNA molecules contains 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. (Item 49) Modified immune effector cells as described in item 33, wherein at least one of the endogenous target genes is NR4A3, and at least one of the endogenous target genes is selected from the group consisting of TNFAIP3, CBLB, and BCOR. (Item 50) The modified immunoeffector cell according to item 20, wherein the gene regulatory system comprises an enzyme protein, and the enzyme protein is engineered to specifically bind to a target sequence in one or more of the endogenous genes. (Item 51) Modified immunoeffector cells as described in item 50, wherein the protein is a transcription activator-like effector nuclease (TALEN), a zinc finger nuclease, or a meganuclease. (Item 52) The modified immunoeffector cell according to item 20, wherein the gene regulatory system comprises a guide nucleic acid molecule and an enzyme protein, the nucleic acid molecule being a guide RNA (gRNA) molecule and the enzyme protein being a Cas protein or a Cas orthologue. (Item 53) The modified immunoeffector cells described in item 52, wherein one or more endogenous target genes are selected from the group consisting 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 BCOR, and the gRNA molecule contains a targeting domain sequence that binds to a nucleic acid sequence defined by a series of genomic coordinates shown in Tables 5A and 5B. (Item 54) The modified immunoeffector cell described in item 53, wherein the gRNA molecule includes a targeting domain sequence that binds to a target DNA sequence selected from the group consisting of SEQ ID NOs: 154-813. (Item 55) The modified immunoeffector cell described in item 53, wherein the gRNA molecule comprises a targeting domain sequence encoded by a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 154-813. (Item 56) The modified immune effector cells described in item 52, wherein one or more endogenous target genes are selected from the group consisting of BCL2L11, FLI1, CALM2, DHODH, UMPS, RBM39, SEMA7A, CHIC2, PCBP1, PBRM1, WDR6, E2F8, SERPINA3, and GNAS, and the gRNA molecule contains a targeting domain sequence that binds to a nucleic acid sequence defined by a series of genomic coordinates shown in Tables 6A and 6B. (Item 57) The modified immunoeffector cell described in item 56, wherein the gRNA molecule includes a targeting domain sequence that binds to a target DNA sequence selected from the group consisting of SEQ ID NOs. 814 to 1064. (Item 58) The modified immunoeffector cell described in item 56, wherein 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. (Item 59) The modified immune effector cell described in item 52, wherein one or more endogenous target genes are selected from the group consisting of ZC3H12A, and the gRNA molecule includes a targeting domain sequence that binds to a nucleic acid sequence defined by a series of genomic coordinates shown in Tables 6C and 6D. (Item 60) The modified immunoeffector cell according to item 59, wherein 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 1509. (Item 61) The one or more endogenous target genes are MAP4K1, and the gRNA molecule binds to a nucleic acid sequence defined by a series of genomic coordinates shown in Tables 6E and 6F. Modified immunoeffector cells as described in item 52, containing a phagocytic domain sequence. (Item 62) The modified immunoeffector cell according to item 61, wherein the gRNA molecule includes a targeting domain sequence that binds to a target DNA sequence selected from the group consisting of SEQ ID NOs. 510 to 1538. (Item 63) The modified immunoeffector cell described in item 61, wherein the gRNA molecule comprises a targeting domain sequence encoded by a nucleic acid sequence selected from the group consisting of SEQ ID NOs. 510 to 1538. (Item 64) The modified immunoeffector cell described in item 52, wherein one or more endogenous target genes are NR4A3, and the gRNA molecule comprises a targeting domain sequence that binds to a nucleic acid sequence defined by a set of genomic coordinates shown in Tables 6G and 6H. (Item 65) The modified immunoeffector cell described in item 64, wherein the gRNA molecule includes a targeting domain sequence that binds to a target DNA sequence selected from the group consisting of SEQ ID NOs. 1539 to 1566. (Item 66) The modified immunoeffector cell according to item 64, wherein the gRNA molecule comprises a targeting domain sequence encoded by a nucleic acid sequence selected from the group consisting of SEQ ID NOs. 1539 to 1566. (Item 67) The modified immunoeffector cell according to item 52, wherein the gene regulatory system comprises multiple gRNA molecules and can reduce the expression and / or function of two or more endogenous target genes. (Item 68) Modified immunoeffector cells as described in item 67, wherein at least one of the endogenous target genes is selected from the group consisting of BCL2L11, FLI1, CALM2, DHODH, UMPS, RBM39, SEMA7A, CHIC2, PCB1, PBRM1, WDR6, E2F8, SERPINA3, and GNAS, and at least one of the endogenous target genes is selected from the group consisting 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 BCOR. (Item 69) Modified immune effector cells as described in item 68, wherein at least one of the plurality of gRNA molecules includes a targeting domain sequence that binds to a nucleic acid sequence defined by a series of genomic coordinates shown in Tables 6A and 6B, and at least one of the plurality of gRNA molecules includes a targeting domain sequence that binds to a nucleic acid sequence defined by a series of genomic coordinates shown in Tables 5A and 5B. (Item 70) Modified immune effector cells according to item 69, wherein at least one of the plurality of gRNA molecules includes 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 includes a targeting domain sequence that binds to a target DNA sequence selected from the group consisting of SEQ ID NOs. 154 to 813. (Item 71) At least one of the aforementioned multiple gRNA molecules includes a targeting domain sequence encoded by a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 814-1064, Modified immune effector cells as described in item 69, wherein at least one of several gRNA molecules contains a targeting domain sequence encoded by a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 154-813. (Item 72) Modified immunoeffector cells as described in item 67, wherein at least one of the endogenous target genes is selected from the group consisting of BCL2L11, FLI1, CALM2, DHODH, UMPS, RBM39, SEMA7A, CHIC2, PCBP1, PBRM1, WDR6, E2F8, SERPINA3, and GNAS, and at least one of the endogenous target genes is selected from the group consisting of TNFAIP3, CBLB, and BCOR. (Item 73) Modified immunoeffector cells as described in item 67, wherein at least one of the endogenous target genes is selected from the group consisting of ZC3H12A, and at least one of the endogenous target genes is selected from the group consisting 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 BCOR. (Item 74) Modified immune effector cells according to item 73, wherein at least one of the plurality of gRNA molecules includes a targeting domain sequence that binds to a nucleic acid sequence defined by a series of genomic coordinates shown in Tables 6C and 6D, and at least one of the plurality of gRNA molecules includes a targeting domain sequence that binds to a nucleic acid sequence defined by a series of genomic coordinates shown in Tables 5A and 5B. (Item 75) Modified immune effector cells according to item 74, wherein at least one of the plurality of gRNA molecules includes a targeting domain sequence that binds to a target DNA sequence selected from the group consisting of SEQ ID NOs: 1065 to 1509, and at least one of the plurality of gRNA molecules includes a targeting domain sequence that binds to a target DNA sequence selected from the group consisting of SEQ ID NOs: 154 to 813. (Item 76) Modified immune effector cells according to item 74, wherein at least one of the plurality of gRNA molecules includes a targeting domain sequence encoded by a target DNA sequence selected from the group consisting of SEQ ID NOs: 1065 to 1509, and at least one of the plurality of gRNA molecules includes a targeting domain sequence encoded by a target DNA sequence selected from the group consisting of SEQ ID NOs: 154 to 813. (Item 77) Modified immunoeffector cells as described in item 67, wherein at least one of the endogenous target genes is ZC3H12A, and at least one of the endogenous target genes is selected from the group consisting of TNFAIP3, CBLB, and BCOR. (Item 78) Modified immunoeffector cells as described in item 67, wherein at least one of the endogenous target genes is MAP4K1, and at least one of the endogenous target genes is selected from the group consisting 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 BCOR. (Item 79) At least one of the aforementioned gRNA molecules is shown in Tables 6E and 6F. A modified immune effector cell according to item 78, comprising a targeting domain sequence that binds to a nucleic acid sequence defined by a series of genomic coordinates, wherein at least one of the plurality of gRNA molecules comprises a targeting domain sequence that binds to a nucleic acid sequence defined by a series of genomic coordinates shown in Tables 5A and 5B. (Item 80) Modified immune effector cells according to item 79, wherein at least one of the plurality of gRNA molecules includes a targeting domain sequence that binds to a target DNA sequence selected from the group consisting of SEQ ID NOs. 510 to 1538, and at least one of the plurality of gRNA molecules includes a targeting domain sequence that binds to a target DNA sequence selected from the group consisting of SEQ ID NOs. 154 to 813. (Item 81) Modified immune effector cells according to item 79, wherein at least one of the plurality of gRNA molecules includes a targeting domain sequence encoded by a nucleic acid sequence selected from the group consisting of SEQ ID NOs. 510 to 1538, and at least one of the plurality of gRNA molecules includes a targeting domain sequence encoded by a DNA sequence selected from the group consisting of SEQ ID NOs. 154 to 813. (Item 82) Modified immune effector cells as described in item 67, wherein at least one of the endogenous target genes is MAP4K1, and at least one of the endogenous target genes is selected from the group consisting of TNFAIP3, CBLB, and BCOR. (Item 83) Modified immunoeffector cells as described in item 67, wherein at least one of the endogenous target genes is NR4A3, and at least one of the endogenous target genes is selected from the group consisting 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 BCOR. (Item 84) Modified immune effector cells as described in item 83, wherein at least one of the plurality of gRNA molecules includes a targeting domain sequence that binds to a nucleic acid sequence defined by a series of genomic coordinates shown in Tables 6G and 6H, and at least one of the plurality of gRNA molecules includes a targeting domain sequence that binds to a nucleic acid sequence defined by a series of genomic coordinates shown in Tables 5A and 5B. (Item 85) Modified immune effector cells according to item 84, wherein at least one of the plurality of gRNA molecules includes a targeting domain sequence that binds to a target DNA sequence selected from the group consisting of SEQ ID NOs. 1539 to 1566, and at least one of the plurality of gRNA molecules includes a targeting domain sequence that binds to a target DNA sequence selected from the group consisting of SEQ ID NOs. 154 to 813. (Item 86) Modified immune effector cells according to item 84, wherein at least one of the plurality of gRNA molecules includes a targeting domain sequence encoded by a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 1539 to 1566, and at least one of the plurality of gRNA molecules includes a targeting domain sequence encoded by a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 154 to 813. (Item 87) Modified immunoeffector cells as described in item 67, wherein at least one of the endogenous target genes is NR4A3, and at least one of the endogenous target genes is selected from the group consisting of TNFAIP3, CBLB, and BCOR. (Item 88) a. Is the Cas protein a wild-type Cas protein that contains two enzyme activity domains and is capable of inducing double-strand DNA breaks? b. The Cas protein is a Cas nickase mutant that contains one enzyme activity domain and is capable of inducing single-strand DNA breaks, or c. The Cas protein is an inactive Cas protein (dCas) and is bound to a heterologous protein capable of regulating the expression of one or more endogenous target genes. Modified immunoeffector cells as described in any one of items 52-87. (Item 89) Modified immunoeffector cells as described in any one of items 52 to 88, wherein the Cas protein is the Cas9 protein. (Item 90) Modified immunoeffector cells as described in item 88, wherein the heterologous protein is selected from the group consisting of MAX interacting protein 1 (MXI1), Kruppel-associated box (KRAB) domain, methyl-CpG binding protein 2 (MECP2), and mSin3 4-linking domain (SID4X). (Item 91) Modified immunoeffector cells according to any one of items 50 to 90, wherein the gene regulatory system introduces an inactivating mutation into one or more endogenous target genes. (Item 92) The modified immunoeffector cell according to item 91, wherein the inactivating mutation comprises the deletion, substitution, or insertion of one or more nucleotides in the genomic sequence of the two or more endogenous genes. (Item 93) Modified immunoeffector cells as described in item 92, wherein the deletion is a partial or complete deletion of two or more endogenous target genes. (Item 94) The modified immunoeffector cells described in item 92, wherein the inactivating mutation is a frameshift mutation. (Item 95) Modified immunoeffector cells according to any one of items 91 to 94, wherein the inactivating mutation reduces the expression and / or function of the two or more endogenous target genes. (Item 96) Modified immunoeffector cells according to any one of items 1 to 95, wherein the gene regulatory system is introduced into the immunoeffector cells by transfection, transduction, electroporation, or physical disruption of the cell membrane by a microfluidic device. (Item 97) Modified immunoeffector cells as described in item 96, wherein the gene regulatory system is introduced as a polynucleotide, protein, or ribonucleoprotein (RNP) complex encoding one or more components of the system. (Item 98) Modified immune effector cells in which the expression and / or function of one or more endogenous genes selected from the group consisting 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 BCOR is reduced, wherein the effector function is enhanced by the reduction in the expression and / or function of the one or more endogenous genes. (Item 99) (a) Modified immunoeffector cells in which the expression and / or function of one or more endogenous genes selected from the group consisting of IKZF1, IKZF3, GATA3, BCL3, TNIP1, TNFAIP3, NFKBIA, SMAD2, TGFBR1, TGFBR2, TANK, FOXP3, RC3H1, TRAF6 and IKZF2, or (b) Modified immunoeffector cells in which the expression and / or function of one or more endogenous genes selected from the group consisting of CBLB, PPP2R2D, NRP1, HAVCR2, LAG3, TIGIT, CTLA4, PTPN6, PDCD1 and BCOR are reduced. The modified immunoeffector cells, wherein effector function is enhanced by the reduction of expression and / or function of one or more endogenous genes. (Item 100) (a) Modified immunoeffector cells in which the expression and / or function of one or more endogenous genes selected from the group consisting of BCL2L11, FLI1, CALM2, DHODH, UMPS, RBM39, SEMA7A, CHIC2, PCBP1, PBRM1, WDR6, E2F8, SERPINA3 and GNAS is reduced, (b) ZC3H12A, (c) MAP4K1, or (d) NR4A3 is reduced. The modified immunoeffector cells, wherein effector function is enhanced by the reduction of expression and / or function of one or more endogenous genes. (Item 101) Modified immune effector cells in which the expression and / or function of 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 is reduced, wherein the effector function is enhanced by the reduction in the expression and / or function of the two or more endogenous genes. (Item 102) Modified immunoeffector cells as described in item 101, in which the expression and / or function of CBLB and BCOR are reduced. (Item 103) Modified immune effector cells in which the expression and / or function of two or more target genes are reduced, wherein at least one target gene is selected from the group consisting of BCL2L11, FLI1, CALM2, DHODH, UMPS, RBM39, SEMA7A, CHIC2, PCBP1, PBRM1, WDR6, E2F8, SERPINA3, and GNAS, and at least one target gene is IKZF1, IKZF3, GATA3, BCL3, and TNIP. Modified immunoeffector cells selected from the group consisting of 1, TNFAIP3, NFKBIA, SMAD2, TGFBR1, TGFBR2, TANK, FOXP3, RC3H1, TRAF6, IKZF2, CBLB, PPP2R2D, NRP1, HAVCR2, LAG3, TIGIT, CTLA4, PTPN6, PDCD1, and BCOR, wherein effector function is enhanced by the reduction of expression and / or function of two or more of the above endogenous genes. (Item 104) Modified immune effector cells in which the expression and / or function of two or more target genes are reduced, wherein at least one target gene is ZC3H12A, and at least one target gene is selected from the group consisting 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 BCOR, and the effector function is enhanced by the reduction in the expression and / or function of the two or more endogenous genes. (Item 105) Reduced expression and / or function of ZC3H12A and CBLB, as described in item 104. Modified immune effector cells. (Item 106) Modified immunoeffector cells as described in item 104, in which the expression and / or function of ZC3H12A and BCOR are reduced. (Item 107) Modified immunoeffector cells as described in item 104, in which the expression and / or function of ZC3H12A and TNFAIP3 are reduced. (Item 108) Modified immune effector cells in which the expression and / or function of two or more target genes are reduced, wherein at least one target gene is MAP4K1, and at least one target gene is selected from the group consisting 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 BCOR, and the effector function is enhanced by the reduction in the expression and / or function of the two or more endogenous genes. (Item 109) Modified immunoeffector cells as described in item 108, in which the expression and / or function of MAP4K1 and CBLB are reduced. (Item 110) Modified immunoeffector cells as described in item 108, in which the expression and / or function of MAP4K1 and BCOR are reduced. (Item 111) Modified immunoeffector cells as described in item 108, in which the expression and / or function of MAP4K1 and TNFAIP3 are reduced. (Item 112) Modified immune effector cells in which the expression and / or function of two or more target genes are reduced, wherein at least one target gene is NR4A3, and at least one target gene is selected from the group consisting 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 BCOR, and the effector function is enhanced by the reduction in the expression and / or function of the two or more endogenous genes. (Item 113) Modified immunoeffector cells as described in item 112, in which the expression and / or function of NR4A3 and CBLB are reduced. (Item 114) Modified immunoeffector cells as described in item 112, in which the expression and / or function of NR4A3 and BCOR are reduced. (Item 115) Modified immunoeffector cells as described in item 112, in which the expression and / or function of NR4A3 and TNFAIP3 are reduced. (Item 116) Modified immunoeffector cells containing inactivating mutations in one or more endogenous genes selected from the group consisting 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 BCOR. (Item 117) Modified immunoeffector cells containing inactivating mutations in one or more endogenous genes selected from (a) the group consisting of IKZF1, IKZF3, GATA3, BCL3, TNIP1, TNFAIP3, NFKBIA, SMAD2, TGFBR1, TGFBR2, TANK, FOXP3, RC3H1, TRAF6, and IKZF2, or (b) the group consisting of CBLB, PPP2R2D, NRP1, HAVCR2, LAG3, TIGIT, CTLA4, PTPN6, PDCD1, and BCOR. (Item 118) Modified immunoeffector cells containing inactivating mutations in one or more endogenous genes selected from (a) the group consisting of BCL2L11, FLI1, CALM2, DHODH, UMPS, RBM39, SEMA7A, CHIC2, PCBP1, PBRM1, WDR6, E2F8, SERPINA3, and GNAS, (b) ZC3H12A, (c) MAP4K1, or (d) NR4A3. (Item 119) Modified immunoeffector cells containing inactivating mutations in 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. (Item 120) Modified immune effector cells as described in item 119, comprising inactivating mutations in the CBLB gene and BCOR gene. (Item 121) Modified immune effector cells comprising inactivating mutations in two or more target genes, wherein at least one target gene is selected from the group consisting of BCL2L11, FLI1, CALM2, DHODH, UMPS, RBM39, SEMA7A, CHIC2, PCBP1, PBRM1, WDR6, E2F8, SERPINA3, and GNAS, and at least one target gene is selected from the group consisting 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 BCOR. (Item 122) Modified immune effector cells comprising inactivating mutations in two or more target genes, wherein at least one target gene is ZC3H12A, and at least one target gene is selected from the group consisting 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 BCOR. (Item 123) Modified immunoeffector cells as described in item 122, comprising inactivating mutations in the ZC3H12A gene and the CBLB gene. (Item 124) Modified immunoeffector cells as described in item 122, comprising inactivating mutations in the ZC3H12A gene and the BCOR gene. (Item 125) Modified immunoeffector cells as described in item 122, comprising inactivating mutations in the ZC3H12A gene and the TNFAIP3 gene. (Item 126) Modified immune effector cells containing inactivating mutations in two or more target genes, and fewer The modified immunoeffector cells wherein at least one target gene is MAP4K1, and at least one target gene is selected from the group consisting 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 BCOR. (Item 127) Modified immunoeffector cells as described in item 126, comprising inactivating mutations in the MAP4K1 gene and the CBLB gene. (Item 128) Modified immunoeffector cells as described in item 126, comprising inactivating mutations in the MAP4K1 gene and the BCOR gene. (Item 129) Modified immunoeffector cells as described in item 126, comprising inactivating mutations in the MAP4K1 gene and the TNFAIP3 gene. (Item 130) Modified immune effector cells comprising inactivating mutations in two or more target genes, wherein at least one target gene is NR4A3, and at least one target gene is selected from the group consisting 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 BCOR. (Item 131) Modified immunoeffector cells as described in item 130, comprising inactivating mutations in the NR4A3 gene and the CBLB gene. (Item 132) Modified immunoeffector cells as described in item 130, comprising inactivating mutations in the NR4A3 gene and the BCOR gene. (Item 133) Modified immunoeffector cells as described in item 130, comprising inactivating mutations in the NR4A3 gene and the TNFAIP3 gene. (Item 134) Modified immunoeffector cells according to any one of items 116 to 133, wherein the inactivating mutation comprises the deletion, substitution, or insertion of one or more nucleotides in the genomic sequence of the two or more endogenous genes. (Item 135) Modified immunoeffector cells as described in item 134, wherein the deletion is a partial or complete deletion of two or more endogenous target genes. (Item 136) The modified immunoeffector cells described in item 134, wherein the inactivating mutation is a frameshift mutation. (Item 137) Modified immunoeffector cells as described in any one of items 116 to 136, wherein the inactivating mutation reduces the expression and / or function of two or more endogenous target genes. (Item 138) Modified immune effector cells according to any one of items 1 to 137, wherein 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 immune effector cells or control immune effector cells. (Item 139) Modified immune effector cells according to any one of items 1 to 137, wherein 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 immune effector cells or control immune effector cells. (Item 140) Modified immune effector cells as described in any one of items 1 to 139, further comprising manipulated immune receptors presented on the cell surface. (Item 141) The modified immune effector cell described in item 140, wherein the modified immune receptor is a CAR comprising an antigen-binding domain, a transmembrane domain, and an intracellular signaling domain. (Item 142) Modified immune effector cells as described in item 140, wherein the aforementioned modified immune receptor is a modified TCR. (Item 143) The modified immune effector cell according to any one of items 140 to 142, wherein the modified immune receptor specifically binds to an antigen expressed on a target cell, and the antigen is a tumor-associated antigen. (Item 144) Modified immune effector cells as described in any one of items 1 to 143, further comprising exogenous transgenes expressing immune-activating molecules. (Item 145) The modified immune effector cells described in item 144, wherein the immune-activating molecule is selected from the group consisting of cytokines, chemokines, costimulatory molecules, activating peptides, antibodies or their antigen-binding fragments. (Item 146) Modified immunoeffector cells as described in item 145, wherein the antibody or its binding fragment specifically binds to a protein encoded by NRP1, HAVCR2, LAG3, TIGIT, CTLA4, or PDCD1, and inhibits the function of the protein. (Item 147) The modified immune effector cells described in any one of items 1 to 146, wherein the immune effector cells are lymphocytes selected from T cells, natural killer (NK) cells, and NKT cells. (Item 148) The modified immune effector cells described in item 147, wherein the lymphocytes are tumor-infiltrating lymphocytes (TILs). (Item 149) Modified immune effector cells according to any one of items 1 to 148, wherein the effector function is selected from cell proliferation, cell viability, tumor infiltration, cytotoxicity, antitumor immune response, and / or exhaustion resistance. (Item 150) A composition comprising modified immunoeffector cells as described in any one of items 1 to 149. (Item 151) The composition according to item 150, further comprising a pharmaceutically acceptable carrier or diluent. (Item 152) At least 1×10 4 cells, 1×10 5 cells, 1×10 6 cells, 1×10 7 cells, 1×10 8 cells, 1×10 9 cells, 1×10 10 cells or 1×10 11 cells of modified immune effector cells, the composition according to item 150 or 151. (Item 153) The composition according to any one of items 150 to 152, which is suitable for administration to a subject in need thereof. The composition as described. (Item 154) The composition according to any one of items 150 to 153, which comprises autoreactive effector cells derived from the subject in need thereof. (Item 155) The composition according to any one of items 150 to 153, which comprises allogeneic immune effector cells derived from a donor subject. (Item 156) (a) A gene regulatory system capable of reducing the expression and / or function of one or more endogenous target genes in cells, selected from the group consisting of IKZF1, IKZF3, GATA3, BCL3, TNIP1, TNFAIP3, NFKBIA, SMAD2, TGFBR1, TGFBR2, TANK, FOXP3, RC3H1, TRAF6 and IKZF2, or (b) selected from the group consisting of CBLB, PPP2R2D, NRP1, HAVCR2, LAG3, TIGIT, CTLA4, PTPN6, PDCD1 and BCOR, (i) A nucleic acid molecule, (ii) An enzyme molecule, or (iii) The system comprising a guide nucleic acid molecule and an enzyme protein. (Item 157) (a) A gene regulatory system that can reduce the expression of one or more endogenous target genes selected from the group consisting of BCL2L11, FLI1, CALM2, DHODH, UMPS, RBM39, SEMA7A, CHIC2, PCBP1, PBRM1, WDR6, E2F8, SERPINA3, and GNAS; (b) ZC3H12A; (c) MAP4K1; or (d) NR4A3, said system comprising (i) a nucleic acid molecule, (ii) an enzyme molecule, or (iii) a guide nucleic acid molecule and an enzyme protein. (Item 158) The gene regulatory system according to item 156 or item 157, comprising a guide RNA (gRNA) nucleic acid molecule and a Cas endonuclease. (Item 159) where the one or more endogenous target genes are selected from IKZF1, IKZF3, GATA3, BCL3, TNIP1, TNFAIP3, NFKBIA, SMAD2, TGFBR1, TGFBR2, TANK, FOXP3, RC3H1, TRAF6, and IKZF2, or are selected from CBLB, PPP2R2D, NRP1, HAVCR2, LAG3, TIGIT, CTLA4, PTPN6, PDCD1, and BCOR, and the gRNA molecule comprises a targeting domain sequence that binds to a target DNA sequence defined by a series of genomic coordinates shown in Table 5A and Table 5B. The gene regulatory system according to item 158. (Item 160) where the one or more endogenous target genes are selected from IKZF1, IKZF3, GATA3, BCL3, TNIP1, TNFAIP3, NFKBIA, SMAD2, TGFBR1, TGFBR2, TANK, FOXP3, RC3H1, TRAF6, and IKZF2, and 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. The gene regulatory system according to item 159. (Item 161) The gene regulation system according to item 159 or item 160, wherein 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. (Item 162) The one or more endogenous target genes are selected from CBLB, PPP2R2D, NRP1, HAVCR2, LAG3, TIGIT, CTLA4, PTPN6, PDCD1 and BCOR, and 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. The gene regulation system according to item 159. (Item 163) The gene regulation system according to item 159 or item 162, wherein 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. (Item 164) The one or more endogenous target genes are selected from BCL2L11, FLI1, CALM2, DHODH, UMPS, RBM39, SEMA7A, CHIC2, PCBP1, PBRM1, WDR6, E2F8, SERPINA3 and GNAS, and the gRNA molecule comprises a targeting domain sequence that binds to a target DNA sequence defined by a series of genomic coordinates shown in Table 6A and Table 6B. The gene regulation system according to item 158. (Item 165) The gene regulation system according to item 164, 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: 814 to 1064. (Item 166) The gene regulation system according to item 164 or item 165, wherein 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. (Item 167) The gene regulatory system according to item 158, wherein the one or more endogenous target genes are ZC3H12A, and the gRNA molecule comprises a targeting domain sequence that binds to a target DNA sequence defined by a set of genomic coordinates shown in Tables 6C and 6D. (Item 168) The gene regulatory system according to item 167, wherein the gRNA molecule includes a targeting domain sequence that binds to a target DNA sequence selected from the group consisting of sequence numbers 1065 to 1509. (Item 169) The gene regulatory system according to item 167 or item 168, wherein the gRNA molecule includes a targeting domain sequence encoded by a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 1065 to 1509. (Item 170) The gene regulatory system according to item 169, wherein the gRNA molecule includes a targeting domain sequence encoded by a nucleic acid sequence selected from the group consisting of sequence numbers 1065 to 1509. (Item 171) The gene regulatory system according to item 158, wherein one or more endogenous target genes include MAP4K1, and the gRNA molecule includes a targeting domain sequence that binds to a target DNA sequence defined by a series of genomic coordinates shown in Tables 6E and 6F. (Item 172) The gene regulatory system according to item 171, wherein the gRNA molecule includes a targeting domain sequence that binds to a target DNA sequence selected from the group consisting of SEQ ID NOs. 510 to 1538. (Item 173) The gene regulatory system according to item 171 or item 172, wherein the gRNA molecule comprises a targeting domain sequence encoded by a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 510 to 1538. (Item 174) The gene regulatory system according to item 158, wherein one or more endogenous target genes include NR4A3, and the gRNA molecule includes a targeting domain sequence that binds to a target DNA sequence defined by a series of genomic coordinates shown in Tables 6G and 6H. (Item 175) The gene regulatory system according to item 174, wherein the gRNA molecule includes a targeting domain sequence that binds to a target DNA sequence selected from the group consisting of sequence numbers 1539 to 1566. (Item 176) The gene regulatory system according to item 174 or item 175, wherein the gRNA molecule comprises a targeting domain sequence encoded by a nucleic acid sequence selected from the group consisting of sequence numbers 1539 to 1566. (Item 177) A gene regulatory system as described in item 156 or item 157, comprising an siRNA nucleic acid molecule or an shRNA nucleic acid molecule. (Item 178) The gene regulatory system described in item 177, wherein one or more endogenous target genes are selected from IKZF1, IKZF3, GATA3, BCL3, TNIP1, TNFAIP3, NFKBIA, SMAD2, TGFBR1, TGFBR2, TANK, FOXP3, RC3H1, TRAF6 and IKZF2, or selected from CBLB, PPP2R2D, NRP1, HAVCR2, LAG3, TIGIT, CTLA4, PTPN6, PDCD1 and BCOR, and the siRNA molecule or shRNA molecule contains approximately 19 to 30 nucleotides that bind to an RNA sequence encoded by a set of genomic coordinates shown in Tables 5A and 5B. (Item 179) The gene regulatory system according to item 178, wherein one or more endogenous target genes are selected from IKZF1, IKZF3, GATA3, BCL3, TNIP1, TNFAIP3, NFKBIA, SMAD2, TGFBR1, TGFBR2, TANK, FOXP3, RC3H1, TRAF6, and IKZF2, and the siRNA molecule or shRNA molecule contains about 19 to 30 nucleotides that bind to an RNA sequence encoded by a DNA sequence selected from sequence numbers 154 to 498. (Item 180) The gene regulatory system according to item 178, wherein one or more endogenous target genes are selected from CBLB, PPP2R2D, NRP1, HAVCR2, LAG3, TIGIT, CTLA4, PTPN6, PDCD1, and BCOR, and the siRNA molecule or shRNA molecule contains about 19 to 30 nucleotides that bind to an RNA sequence encoded by a DNA sequence selected from sequence numbers 499 to 813. (Item 181) The gene regulatory system described in item 177, wherein one or more endogenous target genes are selected from BCL2L11, FLI1, CALM2, DHODH, UMPS, RBM39, SEMA7A, CHIC2, PCBP1, PBRM1, WDR6, E2F8, SERPINA3, and GNAS, and the siRNA molecule or shRNA molecule contains approximately 19 to 30 nucleotides that bind to an RNA sequence encoded by a set of genomic coordinates shown in Tables 6A and 6B. (Item 182) The gene regulatory system according to item 181, wherein the siRNA molecule or shRNA molecule contains approximately 19 to 30 nucleotides bound to an RNA sequence encoded by a DNA sequence selected from sequence numbers 814 to 1064. (Item 183) The one or more endogenous target genes are ZC3H12A, and the siRNA molecule and The gene regulatory system described in item 177, in which an shRNA molecule contains approximately 19 to 30 nucleotides that bind to an RNA sequence encoded by a DNA sequence defined by a set of genomic coordinates shown in Tables 6C and 6D. (Item 184) The gene regulatory system according to item 183, wherein the siRNA molecule or shRNA molecule contains approximately 19 to 30 nucleotides bound to an RNA sequence encoded by a DNA sequence selected from sequence numbers 1065 to 1509. (Item 185) The gene regulatory system according to item 177, wherein the one or more endogenous target genes include MAP4K1, and the siRNA molecule or shRNA molecule contains about 19 to 30 nucleotides that bind to an RNA sequence encoded by a series of genomic coordinates shown in Tables 6E and 6F. (Item 186) The gene regulatory system according to item 185, wherein the siRNA molecule or shRNA molecule contains approximately 19 to 30 nucleotides bound to an RNA sequence encoded by a DNA sequence selected from sequence numbers 510 to 1538. (Item 187) The gene regulatory system according to item 177, wherein one or more endogenous target genes include NR4A3, and the siRNA molecule or shRNA molecule contains about 19 to 30 nucleotides that bind to an RNA sequence encoded by a series of genomic coordinates shown in Tables 6G and 6H. (Item 188) The gene regulatory system according to item 187, wherein the siRNA molecule or shRNA molecule contains approximately 19 to 30 nucleotides bound to an RNA sequence encoded by a DNA sequence selected from sequence numbers 1539 to 1566. (Item 189) A gene regulatory system capable of reducing the expression and / or function of two or more endogenous target genes within a cell, At least one of the aforementioned endogenous target genes is selected from (a) the group consisting of BCL2L11, FLI1, CALM2, DHODH, UMPS, RBM39, SEMA7A, CHIC2, PCBP1, PBRM1, WDR6, E2F8, SERPINA3, and GNAS, (b) ZC3H12A, (c) MAP4K1, or (d) NR4A3. At least one of the aforementioned endogenous target genes is selected from (e) the group consisting of IKZF1, IKZF3, GATA3, BCL3, TNIP1, TNFAIP3, NFKBIA, SMAD2, TGFBR1, TGFBR2, TANK, FOXP3, RC3H1, TRAF6, and IKZF2, or (f) the group consisting of CBLB, PPP2R2D, NRP1, HAVCR2, LAG3, TIGIT, CTLA4, PTPN6, PDCD1, and BCOR. The system comprises (i) a nucleic acid molecule, (ii) an enzyme molecule, or (iii) a guide nucleic acid molecule and an enzyme protein. (Item 190) A gene regulatory system described in item 189, comprising multiple guide RNA (gRNA) nucleic acid molecules and Cas endonucleases. (Item 191) At least one of the aforementioned endogenous target genes is selected from the group consisting of BCL2L11, FLI1, CALM2, DHODH, UMPS, RBM39, SEMA7A, CHIC2, PCBP1, PBRM1, WDR6, E2F8, SERPINA3, and GNAS, and at least one of the aforementioned endogenous target genes is IKZF1, IKZF3, GATA3, BCL3, TNIP1, TNFAIP3, NFKBIA, SMAD2, TGFBR1, TGFBR2, TANK, FOXP3, RC3H1, TRAF6, IKZF2, A gene regulatory system as described in item 190, selected from the group consisting of CBLB, PPP2R2D, NRP1, HAVCR2, LAG3, TIGIT, CTLA4, PTPN6, PDCD1, and BCOR. (Item 192) At least one of the plurality of gRNAs binds to a target DNA sequence defined by a series of genomic coordinates shown in Table 6A and Table 6B, and at least one of the plurality of gRNAs binds to a target DNA sequence defined by a series of genomic coordinates shown in Table 5A and Table 5B. The gene regulation system according to item 191. (Item 193) At least one of the plurality of gRNA molecules includes 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 includes 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. The gene regulation system according to item 191 or item 192. (Item 194) At least one of the plurality of gRNA molecules includes 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 includes 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. The gene regulation system according to item 191 or item 192. (Item 195) At least one of the endogenous target genes is ZC3H12A, and at least one of the endogenous target genes is selected from the group consisting 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 BCOR. The gene regulation system according to item 190. (Item 196) The gene regulatory system according to item 195, wherein at least one of the plurality of gRNAs binds to a target DNA sequence defined by a series of genomic coordinates shown in Tables 6C and 6D, and at least one of the plurality of gRNAs binds to a target DNA sequence defined by a series of genomic coordinates shown in Tables 5A and 5B. (Item 197) The gene regulation system according to item 195 or item 196, wherein at least one of the plurality of gRNA molecules includes a targeting domain sequence that binds to a target DNA sequence selected from the group consisting of SEQ ID NOs: 1065 to 1509, and at least one of the plurality of gRNA molecules includes 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. (Item 198) The gene regulation system according to item 195 or item 196, wherein at least one of the plurality of gRNA molecules includes a targeting domain sequence that binds to a target DNA sequence selected from the group consisting of SEQ ID NOs: 1065 to 1509, and at least one of the plurality of gRNA molecules includes a targeting domain sequence that binds to a target DNA sequence selected from the group consisting of SEQ ID NOs: 499 to 524. (Item 199) At least one of the plurality of gRNA molecules includes a targeting domain sequence encoded by a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 1065-1509, and at least one of the plurality of gRNA molecules includes a targeting domain sequence encoded by a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 154-498 or SEQ ID NOs: 499-813. A gene regulatory system as described in item 195 or item 196, including a gene domain sequence. (Item 200) The gene regulation system according to item 195 or item 196, wherein at least one of the plurality of gRNA molecules includes a targeting domain sequence encoded by a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 1065 to 1509, and at least one of the plurality of gRNA molecules includes a targeting domain sequence encoded by a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 499 to 524. (Item 201) The gene regulatory system described in item 190, wherein at least one of the endogenous target genes is MAP4K1, and at least one of the endogenous target genes is selected from the group consisting 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 BCOR. (Item 202) The gene regulatory system according to item 201, wherein at least one of the plurality of gRNAs binds to a target DNA sequence defined by a series of genomic coordinates shown in Tables 6E and 6F, and at least one of the plurality of gRNAs binds to a target DNA sequence defined by a series of genomic coordinates shown in Tables 5A and 5B. (Item 203) The gene regulation system according to item 201 or item 202, wherein at least one of the plurality of gRNA molecules includes a targeting domain sequence that binds to a target DNA sequence selected from the group consisting of SEQ ID NOs. 510 to 1538, and at least one of the plurality of gRNA molecules includes 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. (Item 204) The gene regulation system according to item 201 or item 202, wherein at least one of the plurality of gRNA molecules includes a targeting domain sequence that binds to a target DNA sequence selected from the group consisting of SEQ ID NOs. 510 to 1538, and at least one of the plurality of gRNA molecules includes a targeting domain sequence that binds to a target DNA sequence selected from the group consisting of SEQ ID NOs. 499 to 524. (Item 205) The gene regulation system according to item 201 or item 202, wherein at least one of the plurality of gRNA molecules includes a targeting domain sequence encoded by a nucleic acid sequence selected from the group consisting of SEQ ID NOs. 510 to 1538, and at least one of the plurality of gRNA molecules includes 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. (Item 206) The gene regulation system according to item 201 or item 202, wherein at least one of the plurality of gRNA molecules includes a targeting domain sequence encoded by a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 510 to 1538, and at least one of the plurality of gRNA molecules includes a targeting domain sequence encoded by a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 499 to 524. (Item 207) At least one of the endogenous target genes is NR4A3, 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, PDC A gene regulatory system described in item 190, selected from the group consisting of D1 and BCOR. (Item 208) The gene regulatory system according to item 207, wherein at least one of the plurality of gRNAs binds to a target DNA sequence defined by a series of genomic coordinates shown in Tables 6G and 6H, and at least one of the plurality of gRNAs binds to a target DNA sequence defined by a series of genomic coordinates shown in Tables 5A and 5B. (Item 209) The gene regulation system according to item 207 or item 208, wherein at least one of the plurality of gRNA molecules includes a targeting domain sequence that binds to a target DNA sequence selected from the group consisting of SEQ ID NOs: 1539 to 1566, and at least one of the plurality of gRNA molecules includes 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. (Item 210) The gene regulation system according to item 207 or item 208, wherein at least one of the plurality of gRNA molecules includes a targeting domain sequence that binds to a target DNA sequence selected from the group consisting of SEQ ID NOs: 1539 to 1566, and at least one of the plurality of gRNA molecules includes a targeting domain sequence that binds to a target DNA sequence selected from the group consisting of SEQ ID NOs: 499 to 524. (Item 211) The gene regulatory system according to item 207 or item 208, wherein at least one of the plurality of gRNA molecules includes a targeting domain sequence encoded by a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 1539 to 1566, and at least one of the plurality of gRNA molecules includes 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. (Item 212) The gene regulatory system according to item 207 or item 208, wherein at least one of the plurality of gRNA molecules includes a targeting domain sequence encoded by a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 1539 to 1566, and at least one of the plurality of gRNA molecules includes a targeting domain sequence encoded by a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 499 to 524. (Item 213) The aforementioned Cas tomato a. A wild-type Cas protein containing two enzyme activity domains that can induce double-strand DNA breaks. b. A Cas niccas mutant containing one enzyme activity domain that can induce single-strand DNA breaks. c. An inactive Cas protein (dCas) bound to a heterologous protein capable of regulating the expression of one or more endogenous target genes. A gene regulatory system described in any one of items 156-212. (Item 214) The gene regulatory system described in item 213, wherein the heterogeneous protein is selected from the group consisting of MAX interaction protein 1 (MXI1), Kruppel-associated box (KRAB) domain, and mSin3 4-linked domain (SID4X). (Item 215) The gene regulatory system described in item 213 or 214, wherein the Cas protein is the Cas9 protein. (Item 216) The system includes a nucleic acid molecule, wherein the nucleic acid molecule is siRNA, shRNA, microRNA (miR), antagonist miR, or antisense RNA, as described in item 189. The gene regulatory system. (Item 217) A gene regulatory system as described in item 216, comprising multiple shRNA molecules or siRNA molecules. (Item 218) The gene regulatory system described in item 217, wherein at least one of the endogenous target genes is selected from the group consisting of BCL2L11, FLI1, CALM2, DHODH, UMPS, RBM39, SEMA7A, CHIC2, PCBP1, PBRM1, WDR6, E2F8, SERPINA3, and GNAS, and at least one of the endogenous target genes is selected from the group consisting 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 BCOR. (Item 219) The gene regulatory system according to item 218, wherein at least one of the plurality of siRNA molecules or shRNA molecules contains about 19 to 30 nucleotides that bind to an RNA sequence encoded by a DNA sequence defined by a set of genomic coordinates shown in Tables 6A and 6B, and at least one of the plurality of siRNA molecules or shRNA molecules contains about 19 to 30 nucleotides that bind to an RNA sequence encoded by a DNA sequence defined by a set of genomic coordinates shown in Tables 5A and 5B. (Item 220) The gene regulation system according to item 218 or 219, wherein at least one of the plurality of siRNA molecules or shRNA molecules contains 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 molecules or shRNA molecules contains 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. (Item 221) The gene regulatory system described in item 217, wherein at least one of the endogenous target genes is ZC3H12A, and at least one of the endogenous target genes is selected from the group consisting 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 BCOR. (Item 222) The gene regulatory system described in item 221, wherein at least one of the plurality of siRNA molecules or shRNA molecules contains about 19 to 30 nucleotides that bind to an RNA sequence encoded by a DNA sequence defined by a series of genomic coordinates shown in Tables 6C and 6D, and at least one of the plurality of siRNA molecules or shRNA molecules contains about 19 to 30 nucleotides that bind to an RNA sequence encoded by a DNA sequence defined by a series of genomic coordinates shown in Tables 5A and 5B. (Item 223) At least one of the plurality of siRNA molecules or shRNA molecules contains approximately 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 1509, and at least one of the plurality of siRNA molecules or shRNA molecules contains approximately 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 A gene regulatory system described in item 221 or item 222, comprising 19 to 30 nucleotides. (Item 224) The gene regulation system according to item 221 or 222, wherein at least one of the plurality of siRNA molecules or shRNA molecules contains 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 1509, and at least one of the plurality of siRNA molecules or shRNA molecules contains 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. (Item 225) The gene regulatory system according to item 217, wherein at least one of the endogenous target genes is MAP4K1, and at least one of the endogenous target genes is selected from the group consisting 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 BCOR. (Item 226) The gene regulatory system according to item 225, wherein at least one of the plurality of siRNA molecules or shRNA molecules contains about 19 to 30 nucleotides that bind to an RNA sequence encoded by a DNA sequence defined by a series of genomic coordinates shown in Tables 6E and 6F, and at least one of the plurality of siRNA molecules or shRNA molecules contains about 19 to 30 nucleotides that bind to an RNA sequence encoded by a DNA sequence defined by a series of genomic coordinates shown in Tables 5A and 5B. (Item 227) The gene regulation system according to item 225 or item 226, wherein at least one of the plurality of siRNA molecules or shRNA molecules contains 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: 510 to 1538, and at least one of the plurality of siRNA molecules or shRNA molecules contains 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. (Item 228) The gene regulation system according to item 225 or 226, wherein at least one of the plurality of siRNA molecules or shRNA molecules contains 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: 510 to 1538, and at least one of the plurality of siRNA molecules or shRNA molecules contains 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. (Item 229) The gene regulatory system described in item 217, wherein at least one of the endogenous target genes is NR4A3, and at least one of the endogenous target genes is selected from the group consisting 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 BCOR. (Item 230) At least one of the aforementioned siRNA molecules or shRNA molecules is encoded by a DNA sequence defined by a series of genomic coordinates shown in Tables 6G and 6H. A gene regulatory system according to item 229, comprising approximately 19 to 30 nucleotides that bind to an RNA sequence, wherein at least one of the plurality of siRNA molecules or shRNA molecules comprises approximately 19 to 30 nucleotides that bind to an RNA sequence encoded by a set of genomic coordinates shown in Tables 5A and 5B. (Item 231) The gene regulation system according to item 229 or 230, wherein at least one of the plurality of siRNA molecules or shRNA molecules contains 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: 1539 to 1566, and at least one of the plurality of siRNA molecules or shRNA molecules contains 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. (Item 232) The gene regulation system according to item 229 or 230, wherein at least one of the plurality of siRNA molecules or shRNA molecules contains 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: 1539 to 1566, and at least one of the plurality of siRNA molecules or shRNA molecules contains 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. (Item 233) The gene regulatory system according to item 156, 157, or 189, wherein 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 (TALENs). (Item 234) A gene regulatory system comprising a vector encoding one or more gRNAs and a vector encoding a Cas endonuclease protein, wherein the one or more gRNAs include a targeting domain sequence encoded by a nucleic acid sequence selected from SEQ ID NOs: 814-1064, SEQ ID NOs: 1065-1509, SEQ ID NOs: 510-1538, SEQ ID NOs: 1539-1566, SEQ ID NOs: 154-498, or SEQ ID NOs: 499-813. (Item 235) A gene regulatory system comprising a vector encoding multiple gRNAs and a vector encoding a Cas endonuclease protein, At least one of the aforementioned multiple gRNAs includes a targeting domain sequence encoded by a nucleic acid sequence selected from SEQ ID NOs. 814-1064, SEQ ID NOs. 1065-1509, SEQ ID NOs. 510-1538, or SEQ ID NOs. 1539-1566. The gene regulatory system wherein at least one of the plurality of gRNAs includes a targeting domain sequence encoded by a nucleic acid sequence selected from SEQ ID NOs: 154-498 or SEQ ID NOs: 499-813. (Item 236) A gene regulatory system comprising a vector encoding one or more gRNAs and an mRNA molecule encoding a Cas endonuclease protein, wherein the one or more gRNAs include a targeting domain sequence encoded by a nucleic acid sequence selected from SEQ ID NOs: 814-1064, SEQ ID NOs: 1065-1509, SEQ ID NOs: 510-1538, SEQ ID NOs: 1539-1566, SEQ ID NOs: 154-498, or SEQ ID NOs: 499-813. (Item 237) A gene regulatory system comprising a vector encoding multiple gRNAs and an mRNA molecule encoding a Cas endonuclease protein, At least one of the aforementioned multiple gRNAs includes a targeting domain sequence encoded by a nucleic acid sequence selected from SEQ ID NOs. 814-1064, SEQ ID NOs. 1065-1509, SEQ ID NOs. 510-1538, or SEQ ID NOs. 1539-1566. The gene regulatory system wherein at least one of the plurality of gRNAs includes a targeting domain sequence encoded by a nucleic acid sequence selected from SEQ ID NOs: 154-498 or SEQ ID NOs: 499-813. (Item 238) A gene regulatory system comprising one or more gRNAs and Cas endonuclease proteins, The one or more gRNAs include a targeting domain sequence encoded by a nucleic acid sequence selected from SEQ ID NOs: 814-1064, 1065-1509, 510-1538, 1539-1566, 154-498, or 499-813. The gene regulatory system comprising one or more gRNAs and the Cas endonuclease protein, which complex together to form a ribonucleoprotein (RNP) complex. (Item 239) A gene regulatory system comprising multiple gRNAs and Cas endonuclease proteins, At least one of the aforementioned multiple gRNAs includes a targeting domain sequence encoded by a nucleic acid sequence selected from SEQ ID NOs. 814-1064, SEQ ID NOs. 1065-1509, SEQ ID NOs. 510-1538, or SEQ ID NOs. 1539-1566. At least one of the aforementioned multiple gRNAs includes a targeting domain sequence encoded by a nucleic acid sequence selected from SEQ ID NOs. 154-498 or SEQ ID NOs. 499-813. The gene regulatory system comprising one or more gRNAs and the Cas endonuclease protein, which complex together to form a ribonucleoprotein (RNP) complex. (Item 240) A kit containing a gene regulatory system as described in any one of items 156-239. (Item 241) gRNA nucleic acid molecules containing a targeting domain nucleic acid sequence that binds to a target sequence within an endogenous target gene selected from (a) the group consisting of BCL2L11, FLI1, CALM2, DHODH, UMPS, RBM39, SEMA7A, CHIC2, PCBP1, PBRM1, WDR6, E2F8, SERPINA3, and GNAS, (b) ZC3H12A, (c) MAP4K1, (d) NR4A3, (e) IKZF1, IKZF3, GATA3, BCL3, TNIP1, TNFAIP3, NFKBIA, SMAD2, TGFBR1, TGFBR2, TANK, FOXP3, RC3H1, TRAF6, and IKZF2, or (f) CBLB, PPP2R2D, NRP1, HAVCR2, LAG3, TIGIT, CTLA4, PTPN6, PDCD1, and BCOR. (Item 242) a. The endogenous gene is selected from the group consisting of BCL2L11, FLI1, CALM2, DHODH, UMPS, RBM39, SEMA7A, CHIC2, PCBP1, PBRM1, WDR6, E2F8, SERPINA3, and GNAS, and the gRNA contains a targeting domain sequence that binds to a target DNA sequence located at a genomic coordinate selected from the genomic coordinates shown in Tables 6A and 6B, b. The endogenous gene is ZC3H12A, and the gRNA contains a targeting domain sequence that binds to a target DNA sequence located at a genomic coordinate selected from the genomic coordinates shown in Tables 6C and 6D, c. The endogenous gene is MAP4K1, and the gRNA contains a targeting domain sequence that binds to a target DNA sequence located at a genomic coordinate selected from the genomic coordinates shown in Tables 6E and 6F. d. The endogenous gene is NR4A3, and the gRNA contains a targeting domain sequence that binds to a target DNA sequence located at a genomic coordinate selected from the genomic coordinates shown in Tables 6G and 6H, e. The endogenous gene is selected from the group consisting of IKZF1, IKZF3, GATA3, BCL3, TNIP1, TNFAIP3, NFKBIA, SMAD2, TGFBR1, TGFBR2, TANK, FOXP3, RC3H1, TRAF6, and IKZF2, and the gRNA contains a targeting domain sequence that binds to a target DNA sequence located at a genomic coordinate selected from the genomic coordinates shown in Tables 5A and 5B, or f. The endogenous gene is selected from the group consisting of CBLB, PPP2R2D, NRP1, HAVCR2, LAG3, TIGIT, CTLA4, PTPN6, PDCD1, and BCOR, and the gRNA includes a targeting domain sequence that binds to a target DNA sequence located at a genomic coordinate selected from the genomic coordinates shown in Tables 5A and 5B. A gRNA molecule listed in any one of items 241-247. (Item 243) The gRNA molecule described in any one of items 241 to 247, wherein the gRNA includes a targeting domain sequence that binds to a target DNA sequence selected from SEQ ID NOs. 814 to 1064, SEQ ID NOs. 1065 to 1509, SEQ ID NOs. 510 to 1538, SEQ ID NOs. 1539 to 1566, SEQ ID NOs. 154 to 498, or SEQ ID NOs. 499 to 813. (Item 244) The gRNA molecule described in any one of items 241 to 247, wherein the gRNA includes a targeting domain sequence encoded by a sequence selected from sequence numbers 814 to 1064, 1065 to 1509, 510 to 1538, 1539 to 1566, 154 to 498, or 499 to 813. (Item 245) The target sequence is a gRNA molecule described in any one of items 241 to 244, which includes a PAM sequence. (Item 246) The gRNA is a modular gRNA molecule, as described in any one of items 241 to 245. (Item 247) The gRNA is a dual gRNA molecule, as described in any one of items 241-245. (Item 248) A gRNA molecule as described in any one of items 241 to 247, wherein the targeting domain has a nucleotide length of 16 nucleotides, 17 nucleotides, 18 nucleotides, 19 nucleotides, 20 nucleotides, 21 nucleotides, 22 nucleotides, 23 nucleotides, 24 nucleotides, 25 nucleotides, 26 nucleotides, or longer. (Item 249) A gRNA molecule as described in any one of items 241 to 248, including modifications to its 5' end or the vicinity of its 5' end (e.g., within 1 to 10 nucleotides, 1 to 5 nucleotides, or 1 to 2 nucleotides from its 5' end), and / or modifications to its 3' end or the vicinity of its 3' end (e.g., within 1 to 10 nucleotides, 1 to 5 nucleotides, or 1 to 2 nucleotides from its 3' end). (Item 250) The gRNA molecule described in item 249, which, when introduced into T cells, exhibits improved stability against nucleases. (Item 251) The modified gRNA molecule described in item 249 or item 250, which, when introduced into T cells, reduces the innate immune response. (Item 252) A polynucleotide molecule encoding a gRNA molecule as described in any one of items 241-251. (Item 253) A composition comprising one or more gRNA molecules as described in any one of items 241 to 251, or a polynucleotide as described in item 252. (Item 254) A kit containing a gRNA molecule as described in any one of items 241-251, or a polynucleotide as described in item 252. (Item 255) A method for producing modified immunoeffector cells, a. Collecting immune effector cells from the subject. b. Introducing the gene regulatory system described in any one of items 156 to 239 into the immune effector cells, c. Culturing the immune effector cells so that the expression and / or function of one or more endogenous target genes are reduced compared to unmodified immune effector cells. The method, including the method described above. (Item 256) A method for producing modified immune effector cells, comprising introducing a gene regulatory system described in any one of items 156 to 239 into the immune effector cells. (Item 257) The method according to item 255 or 256, further comprising introducing a polynucleotide sequence encoding a modified immune receptor selected from CARs and TCRs. (Item 258) The method according to item 257, wherein the polynucleotide encoding the gene regulatory system and / or the manipulated immune receptor is introduced into the immune effector cells by transfection, transduction, electroporation, or physical disruption of the cell membrane by a microfluidic device. (Item 259) The method according to any one of items 255 to 258, comprising introducing the gene regulatory system as a polynucleotide sequence encoding one or more components of the gene regulatory system, as a protein, or as a ribonucleoprotein (RNP) complex. (Item 260) A method for producing modified immunoeffector cells, a. Proliferating a population of immune effector cells in a culture medium. b. Introducing the gene regulatory system described in any one of items 156-239 into the immunoeffector cell population. The method, including the method described above. (Item 261) The method according to item 260, further comprising collecting the aforementioned immunoeffector cell population from a subject. (Item 262) The method according to item 260 or 261, wherein the gene regulatory system is introduced into the immunoeffector cell population before, during, or after proliferation. (Item 263) The method according to item 260 or item 261, wherein the proliferation of the immunoeffector cell population comprises a first round of proliferation and a second round of proliferation. (Item 264) The gene regulatory system is configured before the first round of proliferation and during the first round of proliferation. Alternatively, the method according to item 263, wherein the immunoeffector cell population is introduced after the first round of proliferation. (Item 265) The method according to item 263, wherein the gene regulatory system is introduced into the immunoeffector cell population before, during, or after the second round of proliferation. (Item 266) The method according to item 263, wherein the gene regulatory system is introduced into the immunoeffector cell population before the first and second rounds of proliferation. (Item 267) The method according to item 263, wherein the gene regulatory system is introduced into the immunoeffector cell population after the proliferation of the first and second rounds. (Item 268) The method according to item 263, wherein the gene regulatory system is introduced into the immunoeffector cell population after the first round of proliferation and before the second round of proliferation. (Item 269) A method for treating a disease or disorder requiring treatment, comprising administering an effective amount of a modified immunoeffector cell described in any one of items 1 to 149, or a composition described in any one of items 150 to 155. (Item 270) The method according to item 269, wherein the disease or disorder is a cell proliferation disorder, an inflammatory disorder, or an infection. (Item 271) The method described in item 269, wherein the disease or disorder is cancer or a viral infection. (Item 272) The method according to item 271, wherein the cancer is selected from leukemia, lymphoma, or solid tumor. (Item 273) The method according to item 272, wherein the solid tumor is melanoma, pancreatic tumor, bladder tumor, lung tumor or lung metastasis, colorectal cancer or head and neck cancer. (Item 274) The method according to any one of items 271 to 273, wherein the cancer is PD1-resistant or PD1-insensitive. (Item 275) The method described in any one of items 271 to 274, wherein the subject has previously received treatment with a PD1 inhibitor or a PDL1 inhibitor. (Item 276) The method according to any one of items 271 to 275, further comprising administering to the subject an antibody or a conjugated fragment that specifically binds to a protein encoded by NRP1, HAVCR2, LAG3, TIGIT, CTLA4, or PDCD1 and inhibits the function of that protein. (Item 277) The method according to any one of items 269 to 276, wherein the modified immunoeffector cells are autologous to the subject. (Item 278) The method according to any one of items 269 to 276, wherein the modified immunoeffector cells are allogeneic with respect to the subject. (Item 279) A method for killing cancer cells, wherein the cancer cells are subjected to the modified immune effector cells described in any one of items 1 to 149, or the composition described in any one of items 150 to 155. The method, which includes exposing the following. (Item 280) The method according to item 279, wherein the exposure is in vitro, in vivo, or ex vivo exposure. (Item 281) A method for enhancing one or more effector functions of immune effector cells, comprising introducing a gene regulatory system described in any one of items 156 to 239 into the immune effector cells. (Item 282) A method for enhancing one or more effector functions of immune effector cells, comprising introducing a gene regulatory system described in any one of items 156 to 239 into the immune effector cells, wherein one or more effector functions are enhanced in the modified immune effector cells compared to the unmodified immune effector cells. (Item 283) The method according to item 282, wherein one or more effector functions are selected from cell proliferation, cell viability, cytotoxicity, tumor infiltration, increased cytokine production, antitumor immune response, and / or exhaustion resistance. [Brief explanation of the drawing]

[0102] [Figure 1A] This specification shows combinations of endogenous target genes that can be modified by the methods described herein. [Figure 1B] This specification shows combinations of endogenous target genes that can be modified by the methods described herein. [Figure 2A] This specification shows combinations of endogenous target genes that can be modified by the methods described herein. [Figure 2B] This specification shows combinations of endogenous target genes that can be modified by the methods described herein. [Figure 3A] This specification shows combinations of endogenous target genes that can be modified by the methods described herein. [Figure 3B] This specification shows combinations of endogenous target genes that can be modified by the methods described herein. [Figure 4] Figures A through D show the results of editing the TRAC gene and the B2M gene using the methods described herein. [Figure 5]A shows TIDE analysis data regarding CBLB editing in primary human T cells. B shows TIDE analysis data regarding CBLB editing in primary human T cells. [Figure 6] This shows the Western blot results of CBLB protein in primary human T cells edited with CBLB gRNA (D6551-CBLB) compared with an unedited control (D6551-WT). [Figure 7A] This shows tumor growth over time in a mouse B16 / Ova syngeneic tumor model. It compares tumor growth in mice treated with CBLB-edited OT1 T cells to that of mice treated with unedited OT1 T cells. [Figure 7B] This shows tumor growth over time in a mouse B16 / Ova syngeneic tumor model. It compares tumor growth in mice treated with ZC3H12A-edited OT1 T cells with that of mice treated with unedited OT1 T cells. [Figure 7C] This shows tumor growth over time in a mouse B16 / Ova syngeneic tumor model. It compares tumor growth in mice treated with ZC3H12A-edited OT1 T cells with that of mice treated with unedited OT1 T cells. [Figure 7D] This shows tumor growth over time in a mouse B16 / Ova syngeneic tumor model. It compares tumor growth in mice treated with MAP4K1-edited OT1 T cells to that of mice treated with unedited OT1 T cells. [Figure 7E] This shows tumor growth over time in a mouse B16 / Ova syngeneic tumor model. It compares tumor growth in mice treated with NR4A3-edited OT1 T cells to that of mice treated with unedited OT1 T cells. [Figure 8]Figures A and B show the time course of tumor growth in mice treated with single-edited T cells and / or in combination with anti-PD1 therapy in a mouse B16 / Ova syngeneic tumor model. Figure A shows the tumor growth in mice treated with MAP4K1-edited OT1 T cells in combination with anti-PD1 therapy compared to control OT1 cells and MAP4K1-edited OT1 cells alone. Figure B shows the tumor growth in mice treated with NR4A3-edited OT1 T cells in combination with anti-PD1 therapy compared to control OT1 cells and NR4A3-edited OT1 cells alone. [Figure 9] This shows the time course of tumor growth in mice treated with Zc3h12a-edited PMEL T cells in a mouse MC38 / gp100 syngeneic tumor model. [Figure 10] The survival curves for mice treated with Zc3h12a-edited PMEL T cells and PD1-edited PMEL T cells in the B16F10 lung metastasis model are shown. [Figure 11] This shows the time course of tumor growth in mice treated with Zc3h12a-edited T cells in a mouse Eg7 Ova syngeneic tumor model. [Figure 12] Figures A and B show the time course of tumor growth in the mouse A375 xenograft model for mice. Figure A shows the time course of tumor growth in the mouse A375 xenograft model for mice treated with CBLB-edited NY-ESO-1-specific TCR transgenic T cells compared to that of unedited NY-ESO-1-specific TCR transgenic T cells. Figure B shows the time course of tumor growth in the mouse A375 xenograft model for mice treated with ZC3H12A-edited NY-ESO-1-specific TCR transgenic T cells compared to that of control edited T cells. [Figure 13]Figures A and B show tumor growth after treatment with single-edited CD19 CAR T cells in a subcutaneous model of Burkitt lymphoma using Raji cells. Figure A compares tumor growth after treatment with MAP4K1-edited CD19 CAR T cells to that of unedited CD19 CAR T cells. Figure B compares tumor growth after treatment with NR4A3-edited CD19 CAR T cells to that of unedited CD19 CAR T cells. [Figure 14] This shows the time course of tumor growth in mice treated with BCOR-edited anti-CD19 CAR T cells, CBLB-edited anti-CD19 CAR T cells, or BCOR / CBLB dual-edited anti-CD19 CAR T cells. Tumor growth is compared to that of mice treated with non-CAR T cells or unedited anti-CD19 CAR T cells. [Figure 15] This shows the accumulation of BCOR-edited CD19 CAR T cells or BCOR / CBLB-edited CD19 CAR T cells in an in vitro culture system. [Figure 16] This shows IL-2 production by BCOR-edited CD19 CAR T cells or BCOR / CBLB-edited CD19 CAR T cells in an in vitro culture system. [Figure 17] This shows IFNγ production by BCOR-edited CD19 CAR T cells or BCOR / CBLB-edited CD19 CAR T cells in an in vitro culture system. [Figure 18] This shows the time course of tumor growth in mice treated with Zc3h12a / Cblb dual-edited OT1 T cells in a mouse B16 / Ova syngeneic tumor model. [Figure 19] This shows the time course of tumor growth in mice treated with Pd1 / Lag3 dual-edited OT1 T cells in a mouse B16 / Ova syngeneic tumor model. [Figure 20] This paper presents the results of validation of Zc3h12a as a target that induces antitumor memory and epitope expansion. [Figure 21]This shows the production of IFNγ, IL-2, and TNFα in MAP4K1-edited PBMCs. [Figure 22] This shows the expression of Icos, Il6, Il2, Ifng, and Nfkbiz mRNA in Zc3h12a-edited mouse CD8 T cells. [Figure 23] This shows the cell surface expression of ICOS in Zc3h12a-edited mouse CD8 T cells. [Figure 24] This shows the production of IL-2 and IFNγ by Zc3h12a-edited mouse CD8 T cells after stimulation with anti-CD3 / CD28. [Figure 25] Images A and B show increased IL-6 expression in ZC3H12A-edited primary human T cells. Image A shows the production of IL-6 protein from ZC3H12A-edited PBMCs (personal biomicroorganisms) collected from donors. Image B shows the expression of IL-6 mRNA in ZC3H12A-edited PBMCs. [Modes for carrying out the invention]

[0103] This disclosure provides methods and compositions relating to modifying immune effector cells to improve the therapeutic efficacy of those cells in relation to immunotherapy. In some embodiments, immune effector cells are modified by the methods of this disclosure to reduce the expression of one or more endogenous target genes or to reduce the function of one or more endogenous proteins so that one or more effector functions of those immune cells are enhanced. In some embodiments, the immune effector cells are further modified by introducing an antigen-constituting transgene, such as the introduction of a T cell receptor (TCR) expression construct or a chimeric antigen receptor (CAR) expression construct. In some embodiments, this disclosure provides compositions and methods for modifying immune effector cells, such as a gene regulatory system composition. In some embodiments, this disclosure provides a method for treating a cell proliferation disorder such as cancer, which comprises administering the modified immune effector cells described herein to a subject in need of treatment.

[0104] I. Definition As used herein and in the appended claims, the singular forms "a," "an," and "the" include plural references unless the context clearly indicates otherwise.

[0105] As used herein, the term "and / or" is used in this disclosure to mean either "and" or "or," unless otherwise indicated.

[0106] Throughout this specification, unless otherwise specified in the context, the word “comprise,” or variations such as “comprises” or “comprising,” shall be understood to mean that the elements or completes, or groups of elements or completes, shown are included, but no other elements or completes, or groups of elements or completes, are excluded.

[0107] In use in this Application, the terms “about” and “approximately” are to be used as synonyms. Any numbers used in this Application, whether marked with “about” or not, are intended to cover all ordinary variations recognized by a person skilled in the art. In certain embodiments, unless otherwise stated or evident from the context, the terms “approximately” or “about” refer to a range of values ​​that, in either direction (above or below the indicated value), fall within 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less than those (except where the number exceeds 100% of the possible values).

[0108] "To decrease" or "to decline" means that a particular value decreases or declines by at least 5%, for example, by 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% compared to a reference value. A decrease or decline in a particular value may also be expressed as a percentage change compared to a reference value, for example, a decrease of at least 1 / 1.1, 1 / 1.2, 1 / 1.3, 1 / 1.4, 1 / 1.5, 1 / 1.6, 1 / 1.7, 1 / 1.8, 1 / 1.9, 1 / 2, 1 / 3, 1 / 4, 1 / 5, 1 / 6, 1 / 7, 1 / 8, 1 / 9, 1 / 10, 1 / 15, 1 / 20, 1 / 30, 1 / 40, 1 / 50, 1 / 60, 1 / 70, 1 / 80, 1 / 90, 1 / 100, 1 / 200, 1 / 500, 1 / 1000 or more compared to the reference value.

[0109] "Increase" means that a particular value increases by at least 5%, for example, by 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 compared to the reference value. An increase in a particular value may also be expressed as a multiplier of change compared to a reference value, for example, an increase of at least 1.1 times, 1.2 times, 1.3 times, 1.4 times, 1.5 times, 1.6 times, 1.7 times, 1.8 times, 1.9 times, 2 times, 3 times, 4 times, 5 times, 6 times, 7 times, 8 times, 9 times, 10 times, 15 times, 20 times, 30 times, 40 times, 50 times, 60 times, 70 times, 80 times, 90 times, 100 times, 200 times, 500 times, 1000 times, or more, compared to the level of the reference value.

[0110] The terms “peptide,” “polypeptide,” and “protein” are used synonymously herein and refer to polymeric forms of amino acids of any length, including coding amino acids, non-coding amino acids, chemically or biochemically modified or derivatized amino acids, and polypeptides having a modified peptide backbone.

[0111] The terms “polynucleotide” and “nucleic acid” are used synonymously herein and refer to polymeric forms of nucleotides (either ribonucleotides or deoxyribonucleotides) of any length. Therefore, the terms include, but are not limited to, single-stranded, double-stranded, or multi-stranded DNA or RNA, genomic DNA, cDNA, DNA-RNA hybrids, or polymers containing purine and pyrimidine bases, other natural bases, chemically modified bases, biochemically modified bases, unnatural bases, or derivatized nucleotide bases. “Oligonilocyte” generally refers to a polynucleotide consisting of approximately 5 to approximately 100 nucleotides of single-stranded or double-stranded DNA. However, for the purposes of this disclosure, there is no upper limit to the length of oligonucleotides. Oligonucleotides are also known as “oligomers” or “oligos” and may be isolated from genes or chemically synthesized by methods known in the art. The terms “polynucleotide” and “nucleic acid” should be understood to include single-stranded polynucleotides (such as sense strands or antisense strands) and double-stranded polynucleotides, as applicable to the embodiments described.

[0112] A “fragment” refers to a portion of a polypeptide or polynucleotide molecule that contains fewer sequences than the entire polypeptide or polynucleotide sequence. In some embodiments, a polypeptide or polynucleotide fragment contains 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 reference polynucleotide. In some embodiments, the polypeptide or polynucleotide fragment contains 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, or 80 It may contain nucleotides or amino acids in quantities of 1, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, or more.

[0113] The term "sequence identity" refers to the percentage (%) of bases or amino acids that are identical and in the same relative positions between two polynucleotide or polypeptide sequences. Therefore, one polynucleotide or polypeptide sequence has a certain percentage (%) of sequence identity with respect to another polynucleotide or polypeptide sequence. In sequence comparisons, typically one sequence serves as a reference sequence compared to the test sequence. The term "reference sequence" refers to the molecule compared to the test sequence.

[0114] "Complementary" refers to the ability of two sequences, including native or non-native bases, or analogs thereof, to pair through base stacking and specific hydrogen bonding. For example, if a base at one position in a nucleic acid can hydrogen bond with a base at a corresponding position in a target, those bases are considered complementary at that position. Nucleic acids may contain universal bases or inert, base-free spacers that do not contribute positively or negatively to hydrogen bonding. Base pairing may include both canonical Watson-Crick base pairing and non-Watson-Crick base pairing (e.g., fluctuation base pairing and Hoogsteen base pairing). In complementary base pairing, adenosine-type bases (A) are complementary to thymidine-type bases (T) or uracil-type bases (U), cytosine-type bases (C) are complementary to guanosine-type bases (G), and universal bases such as 3-nitropyrrole or 5-nitroindole can hybridize with any of A, C, U, or T and can be considered complementary to them. (Nichols et al., Nature, 1994;369:492-493 and Loakes et al.) al., Nucleic Acids Res., 1994;22:4039-4043. Inosine (I) has also come to be considered a universal base in the art and is considered complementary to A, C, U, or T. See Watkins and SantaLucia, Nucl. Acids Research, 2005;33(19):6258-6267.

[0115] Where used herein, “complementary nucleic acid sequence” is a nucleic acid sequence comprising a nucleotide sequence that can be sequence-specific and antiparallel in which it can non-covalently bind to another nucleic acid (i.e., the nucleic acid specifically binds to the complementary nucleic acid) under appropriate temperature and solution ionic strength conditions in vitro and / or in vivo.

[0116] Methods for sequence alignment to compare sequences and determine the percentage of sequence identity and complementarity are well known in the art. For comparison, optimal alignment of sequences can be achieved, for example, by the homology alignment algorithm of Needleman and Wunsch, (1970) J.Mol.Biol.48:443, by the similarity search method of Pearson and Lipman, (1988) Proc.Nat'l.Acad.Sci.USA 85:2444, by computer implementation of these algorithms (GAP, BESTFIT, FASTA, and TFAST in the Wisconsin Genetics Software Package (Genetics Computer Group, 575 Science Dr., Madison, WI)), by manual alignment and visual verification (see, for example, Brent et al., (2003) Current Protocols in Molecular Biology), by BLAST as described in Altschul et al., (1977) Nuc.Acids Res.25:3389-3402, and Altschul et al. This includes the BLAST2.0 algorithm described in al., (1990) J.Mol.Biol.215:403-410. Therefore, this can be done by using algorithms known in the relevant technical field. Software for performing BLAST analysis is publicly available through the National Center for Biotechnology Information.

[0117] In this specification, the term “hybridize” refers to the pairing of complementary nucleotide bases (for example, adenine (A) base-pairs with thymine (T) in DNA molecules and with uracil (U) in RNA molecules, and guanine (G) base-pairs with cytosine (C) in both DNA and RNA molecules) to form a double-stranded nucleic acid molecule (see, e.g., Wahl and Berger (1987) Methods Enzymol. 152:399, Kimmel, (1987) Methods Enzymol. 152:507). In addition, in the art, it is also known that in the hybridization of two RNA molecules (e.g., dsRNA), guanine (G) base-pairs with uracil (U). For example, the formation of the G / U base pair is a partial cause of genetic code degeneracy (i.e., redundancy) with respect to the base pairing of the tRNA anticodon with the codon in mRNA. In relation to this disclosure, guanine (G) in the protein-binding segment (double-stranded dsRNA) of a guide RNA molecule is considered complementary to uracil (U), and U is considered complementary to its G. Therefore, when a G / U base pair can be formed at a given nucleotide position in the protein-binding segment (double-stranded dsRNA) of a guide RNA molecule, that position is not considered complementary, but rather complementary. In the art, it is understood that the sequence of a polynucleotide does not need to be 100% complementary to the sequence of its target nucleic acid in order to be specifically hybridizable. Furthermore, a polynucleotide may hybridize across one or more segments so that an intervening segment or adjacent segment does not participate in the hybridization event (e.g., a loop structure or a hairpin structure). The sequence complementarity of a polynucleotide with a target region in its target nucleic acid sequence can be at least 70%, at least 80%, at least 90%, at least 95%, at least 99%, or 100%.

[0118] The term "modified" refers to a substance or compound (e.g., cells, polynucleotide sequences, and / or polypeptide sequences) that has been altered or changed compared to the corresponding unmodified substance or compound.

[0119] When used herein, the term “natural” refers to nucleic acids, polypeptides, cells, or organisms that are found in nature, when applied to nucleic acids, polypeptides, cells, or organisms. For example, a polypeptide sequence or polynucleotide sequence present in an organism (including a virus) that can be isolated from a natural source and that has not been intentionally modified by humans in a laboratory is considered natural.

[0120] "Isolation" refers to a substance that, in its natural state, is typically freed to varying degrees from its associated components.

[0121] An "expression cassette" or "expression construct" refers to a DNA polynucleotide sequence functionally ligated to a promoter. "Functionally ligated" means that the components described as functionally ligated are in a juxtaposed state where they function in the intended manner. For example, if a promoter influences the transcription or expression of a polynucleotide sequence, then that promoter is functionally ligated to that polynucleotide sequence.

[0122] The term “recombinant vector” as used herein means that the vector has been inserted into it. This refers to a polynucleotide molecule into which another polynucleotide can be inserted or transported. 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).

[0123] The term “sample” refers to a biological composition (e.g., a cell or tissue) that is to be analyzed and / or genetically modified. In some embodiments, the sample is a “primary sample” in that it is taken directly from the subject, and in some embodiments, the “sample” is obtained by processing the primary sample for the purpose of removing certain components and / or isolating or purifying certain components of interest.

[0124] The term “subject” includes animals such as mammals. In some embodiments, the mammal is a primate. In some embodiments, the mammal is a human. In some embodiments, the subject is a domestic animal such as a cow, sheep, goat, dairy cow, or pig, or a domestic animal such as a dog or cat. In some embodiments (e.g., particularly in a research setting), the subject is a rodent (e.g., mouse, rat, hamster), a rabbit, a primate, or a pig such as an inbred pig. The terms “subject” and “patient” are used synonymously herein.

[0125] In this specification, "administration" refers to the introduction of a drug or composition into a target.

[0126] As used herein, "treatment" means delivering a drug or composition to a target in order to affect a physiological outcome.

[0127] As used herein, the term “effective dose” refers to the minimum amount of drug or composition required to produce a particular physiological effect. The effective dose of a particular drug may be expressed in various ways, depending on the properties of the drug, such as mass / volume, cell count / volume, particle / volume, (drug mass) / (target mass), cell count / (target mass), or particle / (target mass). The effective dose of a particular drug is the half-maximal effective concentration (EC2). 50 It may also be expressed as ), and this value refers to a drug concentration that elicits a specific physiological response, which is intermediate between the reference level and the maximum response level.

[0128] A "group" of cells refers to any number of cells, two or more, but preferably at least 1 × 10⁻⁶ 3 A single cell, at least 1 × 10⁶ 4 A single cell, at least 1 × 10⁶ 5 A single cell, at least 1 × 10⁶ 6 A single cell, at least 1 × 10⁶ 7 A single cell, at least 1 × 10⁶ 8 A single cell, at least 1 × 10⁶ 9 A single cell, at least 1 × 10⁶ 10 A single cell, at least 1 × 10⁶ 11 A cell population refers to one or more cells. A cell population may refer to an in vitro population (e.g., a population of cells in a culture medium) or an in vivo population (e.g., a population of cells present in a specific tissue).

[0129] Common methods in molecular and cellular biochemistry are described in 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), and Protein Methods (Bollag et al., John Wiley). & Sons 1996), Nonviral Vectors for Gene Therapy (Wagner et al. eds., Academic Press 1999), Viral Vectors (Kaplift & Loewy eds., Academic Press 1995), Immunology Methods Manual (I. Lefkovits ed., Academic Press This can be found in standard textbooks such as *Cell and Tissue Culture: Laboratory Procedures in Biotechnology* (Doyle & Griffiths, John Wiley & Sons 1997) and *Cell and Tissue Culture: Laboratory Procedures in Biotechnology* (Doyle & Griffiths, John Wiley & Sons 1998), the disclosures thereof are incorporated herein by reference.

[0130] II. Modified Immunoeffector Cells In some embodiments, this disclosure provides modified immune effector cells. Herein, the term “modified immune effector cells” includes immune effector cells comprising one or more genomic modifications that reduce the expression and / or function of one or more endogenous target genes, as well as immune effector cells comprising gene regulatory systems that can reduce the expression and / or function of one or more endogenous target genes. Herein, “unmodified immune effector cells” or “control immune effector cells” refers to cells or cell populations whose genomes are not modified and which either do not contain gene regulatory systems or contain control gene regulatory systems (e.g., empty vector controls, non-targeting gRNAs, scrambled siRNAs, etc.).

[0131] The term "immune effector cells" refers to cells involved in inducing innate and adaptive immune responses, including, but not limited to, lymphocytes (such as T cells (including 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 cells are T cells such as CD4+ T cells, CD8+ T cells (also known as cytotoxic T cells or CTLs), regulatory T cells (Treg), Th1 cells, Th2 cells, or Th17 cells.

[0132] In some embodiments, the immune effector cells are T cells isolated from a tumor sample (hereinafter referred to herein as “tumor-infiltrating lymphocytes” or “TILs”). While we do not wish to be bound by theory, TILs are thought to have enhanced specificity to tumor antigens (Radvanyi et al., 2012 Clin Canc Res 18:6758-6770) and therefore can destroy cancer cells by mediating a tumor antigen-specific immune response (e.g., activation, proliferation, and cytotoxic activity against cancer cells) without the introduction of exogenous manipulated receptors (Brudno et al., 2018 Nat Rev Clin Onc 15:31-46). That is, in some embodiments, TILs are isolated from the tumor of interest, grown ex vivo, and reinjected into the subject. In some embodiments, TILs are modified to express one or more exogenous receptors specific to autologous tumor antigens, grown ex vivo, and reinjected into the subject. Such embodiments can be modeled using an in vivo mouse model in which a cancer cell line expressing a cancer antigen (e.g., CD19) is transplanted, and the mouse is treated with modified T cells expressing an exogenous receptor specific to that cancer antigen (see, for example, Examples 10 and 11).

[0133] In some embodiments, the immune effector cells are animal cells or derived from the cells of animals including invertebrates and vertebrates (e.g., fish, amphibians, reptiles, birds, or mammals). In some embodiments, the immune effector cells are mammalian cells or 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 derived from rodent cells (e.g., rats or mice). In some embodiments, the modified immune effector cells are human cells or derived from human cells.

[0134] In some embodiments, the modified immune effector cells are gene-free of endogenous target genes. The DNA sequence includes one or more modifications (e.g., one or more nucleic acid insertions, deletions, or mutations) that reduce the expression and / or function of the endogenous gene. Such modifications are referred to herein as “inactivating mutations,” and the endogenous gene containing the inactivating mutation is referred to as the “modified endogenous target gene.” In some embodiments, the inactivating mutation reduces the expression levels of the encoded mRNA transcript and protein by reducing or inhibiting mRNA transcription. In some embodiments, the inactivating mutation reduces the expression level of the encoded protein by reducing or inhibiting mRNA translation. In some embodiments, the inactivating mutation encodes a modified endogenous protein (e.g., a dominant-negative variant described below) whose function is reduced or altered compared to the unmodified (i.e., wild-type) endogenous protein.

[0135] In some embodiments, the modified immune effector cells include one or more genomic modifications that reduce the expression and / or function of endogenous target genes or express modified endogenous proteins at genomic locations other than the endogenous target genes. For example, in some embodiments, the expression and / or function of endogenous target genes are reduced when the gene regulatory system is expressed by inserting polynucleotide sequences encoding a gene regulatory system at one or more locations in the genome. In some embodiments, polynucleotide sequences encoding modified endogenous proteins are inserted at one or more locations in the genome, and the function of the modified protein is reduced compared to the unmodified or wild-type protein (e.g., dominant-negative mutants described below).

[0136] In some embodiments, the modified immunoeffector cells described herein contain one or more modified endogenous target genes, and the 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 unmodified immunoeffector cells. For example, in some embodiments, modified immunoeffector cells have reduced mRNA transcript expression and / or protein expression. In some embodiments, the expression of the gene product in modified immunoeffector cells is reduced by at least 5% compared to the expression of the gene product in unmodified immunoeffector cells. In some embodiments, the expression of the gene product in modified immunoeffector 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 unmodified immunoeffector cells. In some embodiments, the modified immunoeffector cells described herein exhibit reduced expression and / or function of gene products encoded by multiple (e.g., two or more) endogenous target genes compared to the expression of those gene products in unmodified immunoeffector cells. For example, in some embodiments, the modified immunoeffector cells exhibit reduced expression and / or function of gene products derived from two, three, four, five, six, seven, eight, nine, ten, or more endogenous target genes compared to the expression of those gene products in unmodified immunoeffector cells.

[0137] In some embodiments, the disclosure provides modified immunoeffector cells in which one or more endogenous target genes or a portion thereof are deleted (i.e., “knocked out”) and do not express their mRNA transcript or protein. In some embodiments, the modified immunoeffector cells include deletions of multiple endogenous target genes or a portion thereof. In some embodiments, the modified immunoeffector cells include deletions of two, three, four, five, six, seven, eight, nine, ten or more endogenous target genes.

[0138] In some embodiments, the modified immunoeffector cells described herein include one or more modified endogenous target genes, and one or more modifications to the target DNA sequence thereof In this configuration, modified immunoeffector cells express proteins with reduced or altered function (e.g., "modified endogenous proteins") compared to the function of the corresponding proteins expressed in non-modified immunoeffector cells (e.g., "non-modified endogenous proteins"). In some embodiments, the modified immunoeffector cells described herein include 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 proteins have reduced or altered binding affinity to other proteins expressed by the modified immunoeffector cells or by other cells, reduced or altered signaling ability, reduced or altered enzyme activity, reduced or altered DNA binding activity, or reduced or altered ability to function as scaffold proteins.

[0139] In some embodiments, the modified endogenous target gene contains one or more dominant-negative mutations. As used herein, “dominant-negative mutation” means that one or more nucleotides in the target gene are substituted, deleted, or inserted so that the encoded protein acts antagonistically to the protein encoded by the unmodified target gene. The mutation is dominant-negative because this negative phenotype is genetically dominant over the positive phenotype of the corresponding unmodified gene. A gene containing one or more dominant-negative mutations, and the protein encoded by that gene, are referred to as a “dominant-negative mutant,” e.g., a dominant-negative gene and a dominant-negative protein. In some embodiments, the dominant-negative mutant protein is encoded by an exogenous transgene inserted at one or more locations in the genome of the immune effector cell.

[0140] Various mechanisms of dominant-negative mutations are known. Typically, the gene product of a dominant-negative mutant retains some functions of the unmodified gene product but lacks one or more other important functions of the unmodified gene product. This allows the dominant-negative mutant to antagonistically inhibit the unmodified gene product. For example, in an exemplary embodiment, a dominant-negative mutant of a transcription factor may lack the functional activation domain but retain the functional DNA-binding domain. In this example, the dominant-negative transcription factor cannot activate DNA transcription like the unmodified transcription factor, and can indirectly inhibit gene expression by preventing the unmodified transcription factor from binding to its transcription factor-binding site. Another exemplary embodiment is the dominant-negative mutation of a protein that functions as a dimer. Such a dominant-negative mutant of a dimeric protein may retain the ability to form a dimer with the unmodified protein but may otherwise be non-functional. Dominant-negative monomers form heterodimers by dimerizing with unmodified monomers, thereby preventing the formation of functional homodimers of the unmodified monomers.

[0141] In some embodiments, the modified immunoeffector cells of the present invention include a gene regulatory system capable of reducing the expression or function of one or more endogenous target genes. The gene regulatory system can reduce the expression and / or function of the modified endogenous target gene by various mechanisms, including modifying the genomic DNA sequence of the endogenous target gene (e.g., by insertion, deletion, or mutation of one or more nucleic acids in the genomic DNA sequence), regulating the transcription of the endogenous target gene (e.g., by inhibition or repression of mRNA transcription), and / or regulating the translation of the endogenous target gene (e.g., by degradation of mRNA).

[0142] In some embodiments, the modified immune effector cells described herein are genetically modified by gene regulatory systems (e.g., nucleic acid-based gene regulatory systems, protein-based genetic systems). This includes a gene regulatory system (or a protein / nucleic acid complex-based gene regulatory system). In such embodiments, the gene regulatory system contained in the modified immune effector cell can modify one or more endogenous target genes. In some embodiments, the modified immune effector cells described herein are (a) One or more nucleic acid molecules that can reduce the expression of or modify the function of a gene product encoded by one or more endogenous target genes. (b) One or more polynucleotides encoding nucleic acid molecules that can reduce the expression of or modify the function of a gene product encoded by one or more endogenous target genes. (c) One or more proteins that can reduce the expression of or modify the function of a gene product encoded by one or more endogenous target genes. (d) One or more polynucleotides encoding a protein that can reduce the expression of a gene product encoded by one or more endogenous target genes or modify its function. (e) One or more guide RNAs (gRNAs) that can bind to a target DNA sequence within an endogenous gene, (f) One or more polynucleotides encoding one or more gRNAs that can bind to a target DNA sequence within an endogenous gene, (g) One or more site-directed modified polypeptides that interact with gRNA and can modify target DNA sequences within endogenous genes, (h) One or more polynucleotides encoding a site-directed modification polypeptide that can interact with gRNA and modify a target DNA sequence within an endogenous gene, (i) One or more guide DNAs (gDNAs) that can bind to a target DNA sequence within an endogenous gene, (j) One or more polynucleotides encoding one or more gDNAs that can bind to a target DNA sequence within an endogenous gene, One or more site-directed modification polypeptides that interact with (k)gDNA to modify target DNA sequences within endogenous genes, (1) One or more polynucleotides encoding a site-directed modification polypeptide that can interact with gDNA and modify a target DNA sequence within an endogenous gene, (m) One or more gRNAs capable of binding to a target mRNA sequence encoded by an endogenous gene, (n) One or more polynucleotides encoding one or more gRNAs that can bind to a target mRNA sequence encoded by an endogenous gene, (o) One or more site-directed modified polypeptides that interact with gRNA and modify a target mRNA sequence encoded by an endogenous gene, One or more polynucleotides encoding a site-directed modification polypeptide that interacts with (p)gRNA and can modify a target mRNA sequence encoded by an endogenous gene, or (q) A gene regulatory system that includes any combination of the above, Includes.

[0143] In some embodiments, one or more polynucleotides encoding the gene regulatory system are inserted into the genome of the immune effector cell. In some embodiments, one or more polynucleotides encoding the gene regulatory system are episomal expressed and are not inserted into the genome of the immune effector cell.

[0144] In some embodiments, the modified immune effector cells described herein have reduced expression and / or function of one or more endogenous target genes and further include one or more exogenous transgenes inserted into one or more genomic loci (e.g., gene "knock"). In some embodiments, one or more exogenous transgenes encode a detectable tag, a safety switch system, a chimeric switch receptor, and / or a manipulated antigen-specific receptor.

[0145] In some embodiments, the modified immunoeffector cells described herein further include exogenous transgenes encoding detectable tags. Examples of detectable tags include, but are not limited to, FLAG tags, polyhistidine tags (e.g., 6×His), 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 (BFP, TagBFP, mTagBFP2, Azurite, EBFP2, mKalama1, Sirius, Sapphire, and T-Sapphire, etc.), a cyan protein (CFP, eCFP, Cerulean, SCFP3A, mTurquoise, mTurquoise2, Monomeric Midoriishi-Cyan, TagCFP, and mTFP1, etc.), a green protein (GFP, eGFP, meGFP (A208K mutation), Emerald, Superfolder GFP, Monomeric Azami Green, TagGFP2, mUKG, mWasabi, Clover, and mNeonGreen, etc.), a yellow protein (YFP, eYFP, Citrine, Venus, SYFP2, and TagYFP, etc.), or an orange protein (Monomeric Proteins such as Kusabira-Orange, mKOκ, mKO2, mOrange, and mOrange2; red proteins (RFP, mRaspberry, mCherry, mStrawberry, mTangerine, tdTomato, TagRFP, TagRFP-T, mApple, mRuby, and mRuby2); far-red proteins (mPlum, HcRed-Tandem, mKate2, mNeptune, and NirFP); near-infrared fluorescent proteins (TagRFP657, IFP1.4, and iRFP); and proteins with long Stokes shifts (mKeima The proteins selected are from a group consisting of Red, LSS-mKate1, LSS-mKate2, and mBeRFP, photoactivatable proteins (PA-GFP, PAmCherry1, and PATagRFP, etc.), photoconvertible proteins (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, etc.), and light-switchable proteins (Dronpa, etc.).In some embodiments, the detectable tags can be selected from AmCyan, AsRed, DsRed2, DsRed Express, E2-Crimson, HcRed, ZsGreen, ZsYellow, mCherry, mStrawberry, mOrange, mBanana, mPlum, mRaspberry, tdTomato, DsRed Monomer, and / or AcGFP, all of which are available from Clontech.

[0146] In some embodiments, the modified immunoeffector cells described herein further include exogenous transgenes encoding a safety switch system. A safety switch system (also known in the art as a suicide gene system) includes exogenous transgenes encoding one or more proteins that make modified immunoeffector cells eliminateable after administration to a target. Examples of safety switch systems are known in the art. For example, a safety switch system includes genes encoding proteins that convert non-harmful prodrugs into harmful compounds, such as herpesthymidine kinase (Hsv-tk) and the ganciclovir (GCV) system (Hsv-tk / GCV). Hsv-tk converts non-harmful GCV into a harmful compound that leads to cell apoptosis. Therefore, modified immunoeffector cells containing a transgene encoding the Hsv-tk protein... When GCV is administered to subjects treated with cells, the modified immune effector cells can be selectively eliminated, while endogenous immune effector cells remain unaffected. (e.g., Bonini) See et al., Science, 1997, 276(5319):1719-1724, Ciceri et al., Blood, 2007, 109(11):1828-1836, and Bondanza et al., Blood 2006, 107(5):1828-1836.

[0147] An additional safety switch system includes a gene encoding a cell surface marker, allowing modified immunoeffector cells to be eliminated via ADCC by administration of a monoclonal antibody specific to that cell surface marker. In some embodiments, the cell surface marker is CD20, and the modified immunoeffector cells can be eliminated by administration of an anti-CD20 monoclonal antibody such as rituximab (see, e.g., 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., Gene Ther, 2006, 13, 789-797). A similar system using EGF-R and cetuximab or panitumumab is described in International PCT Publication WO2018006880. An additional safety switch system includes a transgene encoding a pro-apoptotic molecule containing one or more binding sites for a chemical inducer (CID) of dimerization, allowing for the elimination of modified immune effector cells by administration of a CID that induces oligomerization of the pro-apoptotic molecule and activation of the apoptotic 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).

[0148] In some embodiments, the modified immune effector cells described herein further include exogenous transgenes encoding chimeric switch receptors. Chimeric switch receptors are manipulated cell surface receptors comprising an extracellular domain derived from an endogenous cell surface receptor and a heterogeneous intracellular signaling domain, wherein the extracellular domain, upon recognition of a ligand, activates a signaling cascade different from that activated by the wild-type 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, the intracellular domain transmitting an activating signal rather than the inhibitory signal normally transmitted by the inhibitory cell surface receptor. In certain embodiments, an extracellular domain derived from a cell surface receptor known to inhibit the activation of immune effector cells can be fused to an intracellular activating domain. Then, upon binding of the corresponding ligand, a signaling cascade is activated that enhances, rather than inhibits, the activation of the immune effector cell. For example, in some embodiments, the modified immune effector cells described herein include a transgene encoding a PD1-CD28 switch receptor, in which the extracellular domain of PD1 is fused to the intracellular signaling domain of CD28 (e.g., Liu et al., Cancer Res 76:6 (2016), 1578-1590 and Moon et al.). See al., Molecular Therapy 22(2014), S201. In some embodiments, the modified immunoeffector cells described herein include transgenes encoding the extracellular domain of CD200R and the intracellular signaling domain of CD28 (see Oda et al., Blood 130:22(2017), 2410-2419).

[0149] In some embodiments, the modified immunoeffector cells described herein further comprise a manipulated antigen-specific receptor that recognizes a protein target expressed by a target cell such as a tumor cell or antigen-presenting cell (APC) (hereinafter referred to as “modified receptor-transformed cells” or “modified RE cells”). The term “manipulated antigen receptor” refers to a non-native antigen-specific receptor such as a chimeric antigen receptor (CAR) or recombinant T cell receptor (TCR). In some embodiments, the manipulated antigen receptor is a CAR comprising an extracellular antigen-binding domain fused to a cytoplasmic domain containing a signaling domain via a hinge domain and a transmembrane domain. In some embodiments, the extracellular domain of the CAR binds to an antigen expressed by a target cell in an MHC-independent manner, activating and proliferating the RE cells. In some embodiments, the extracellular domain of the CAR recognizes a tag fused to an antibody or its antigen-binding fragment. In such embodiments, the antigen specificity of the CAR depends on the antigen specificity of the labeled antibody, and as a result, multiple different antigens can be targeted using a single CAR construct by replacing one antibody with another (see, for example, U.S. Patent Nos. 9,233,125 and 9,624,279, and U.S. Patent Application Publications 20150238631 and 20180104354). In some embodiments, the extracellular domain of the CAR may include an antigen-binding fragment derived from the antibody. Useful antigen-binding domains in this disclosure include, for example, scFv, antibodies, antigen-binding regions of antibodies, heavy / light chain variable regions, and single-chain antibodies.

[0150] In some embodiments, the intracellular signaling domain of CAR may originate from a TCR complex ζ-chain domain (such as a CD3ξ signaling domain), an FcγRIII domain, an FcεRI domain, or a T lymphocyte activation domain. In some embodiments, the intracellular signaling domain of CAR further comprises a co-stimulatory domain, e.g., a 4-1BB domain, a CD28 domain, a CD40 domain, a MyD88 domain, or a CD70 domain. In some embodiments, the intracellular signaling domain of CAR comprises two co-stimulatory domains, e.g., any two of the 4-1BB domain, a CD28 domain, a CD40 domain, a MyD88 domain, or a CD70 domain. Exemplary CAR structures and intracellular signaling domains are known in the art (see, for example, WO2009 / 091826, US20130287748, WO2015 / 142675, WO2014 / 055657 and WO2015 / 090229 (incorporated herein by reference)).

[0151] Various tumor antigen-specific CARs are known in this field, for example, CD171-specific CARs (Park et al., Mol Ther (2007) 15(4):825-833), EGFRvIII-specific CARs (Morgan et al., Hum Gene Ther (2012) 23(10):1043-1053), EGF-R-specific CARs (Kobold et al., J Natl Cancer Inst (2014) 107(1):364), and carbonic anhydrase K-specific CARs (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 CARs (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 These include Clin Immunol (2012) 32(5):1059-1070, VEGFF-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), and CD19 specific CAR (axicabutadine silolucel (Yescarta®) and tisagenlecleucel (Kymriah®)). Li et al., J Hematol and have commented on clinical trials of tumor-specific CARs. See also Oncol(2018)11(22).

[0152] In some embodiments, the engineered antigen receptor is an engineered TCR. The engineered TCR comprises a TCRα chain and / or a TCRβ chain isolated and cloned from a T cell population that recognizes a specific target antigen. For example, the TCRα gene and / or TCRβ gene (i.e., TRAC and TRBC) can be cloned from a T cell population isolated from an individual with a specific malignancy, or from a T cell population isolated from a humanized mouse immunized with a given tumor antigen or tumor cells. The engineered TCR recognizes the antigen through the same mechanism as its endogenous counterpart (for example, by recognizing its alloantigen presented in association with major histocompatibility complex (MHC) protein expressed on the surface of the target cell). Binding to this antigen stimulates an endogenous signaling pathway, thereby activating and proliferating the engineered TCR cells.

[0153] Tumor antigen-specific modified TCRs are known in the art, for example, WT1-specific TCRs (JTCR016 (Juno Therapeutics), WT1-TCRc4 described in U.S. Patent Application Publication No. 20160083449), MART-1-specific TCRs (including the DMF4T clone described in Morgan et al., Science 314 (2006) 126-129, the DMF5T clone described in Johnson et al., Blood 114 (2009) 535-546, and the ID3T clone described in van den Berg et al., Mol. Ther. 23 (2015) 1541-1550), gp100-specific TCRs (Johnson et al., Blood 114 (2009) 535-546), and CEA-specific TCRs (Parkhurst et al., Mol. Ther. 23 (2015) 1541-1550). Ther. 19 (2011) 620-626), NY-ESO and LAGE-1 specific TCR (Robbins These include the 1G4T clone described in et al., J Clin Oncol 26(2011)917-924, Robbins et al., Clin Cancer Res 21(2015)1019-1027, and Rapoport et al., Nature Medicine 21(2015)914-921, as well as MAGE-A3 specific TCRs (Morgan et al., J Immunother 36(2013)133-151 and Linette et al., Blood 122(2013)227-242). (See also Debets et al., Seminars in Immunology 23(2016)10-21.)

[0154] In some embodiments, the manipulated antigen receptors are CD3, CD4, CD8, CD16, CD24, CD25, CD33, CD34, CD45, CD64, CD71, CD78 Differentiation cluster molecules such as CD80 (also known as B7-1), CD86 (also known as B7-2), CD96, CD116, CD117, CD123, CD133, CD138, CD371 (also known as CLL1), 5T4, BCMA (also known as CD269 and TNFRSF17).UniProt number Q02223), carcinoembryonic antigen (CEA), carbonic anhydrase 9 (CAIX or MN / CAIX), CD19, CD20, CD22, CD30, CD40, disialoganglioside (GD2, etc.), ELF2M, ductal epithelial mucin, ephrin B2, epithelial cell adhesion molecule (EpCAM), ErbB2 (HER2 / neu), FCRL5 (UniProt number Q68SN8), FKBP11 (UniProt number Q9NYL4), glioma-associated antigen, sphingoglycolipid, gp36, GPRC5D (UniProt number Q9NZD1), mut Tumor-associated surface antigens such as hsp70-2, intestinal carboxylesterase, IGF-I receptor, ITGA8 (UniProt number P53708), KAMP3, LAGE-1a, MAGE, mesoserine, neutrophil elastase, NKG2D, Nkp30, NY-ESO-1, PAP, prostase, prostate cancer tumor antigen-1 (PCTA-1), prostate-specific antigen (PSA), PSMA, prostain, RAGE-1, ROR1, RU1 (SFMBT1), RU2 (DCDC2), SLAMF7 (UniProt number Q9NQ25), Survivin, Tag-72 and telomerase, major histocompatibility complex (MHC) molecules presenting tumor-specific peptide epitopes, tumor stromal antigens such as extradomain A (EDA) and extradomain B (EDB) of fibronectin, and the A1 domain (TnC) of tenascin-C A1) and target antigens selected from fibroblast-related proteins (FAP), cytokine receptors such as epidermal growth factor receptor (EGFR), EGFR variant III (EGFRvIII), TFGβ-R or its components (such as endoglin), major histocompatibility complex (MHC) molecules, virus-specific surface antigens such as HIV-specific antigens (such as HIV gp120), EBV-specific antigens, CMV-specific antigens, HPV-specific antigens, Lassa virus-specific antigens, influenza virus-specific antigens, and derivatives or variants of any of these surface antigens.

[0155] A. Effects function In some embodiments, the modified immune effector cells described herein exhibit enhanced immune cell effector function. Hereinafter, the term “effector function” refers to immune cell functions related to the generation, maintenance, and / or enhancement of an immune response to a target cell or target antigen. In some embodiments, the modified immune effector cells described herein exhibit, compared to unmodified immune effector cells, one or more of the following characteristics: improved tumor infiltration or migration, improved proliferation, improved or prolonged cell viability, improved resistance to inhibitory factors in the surrounding microenvironment leading to prolonged or increased activation of the cells, increased production of pro-inflammatory immune factors (e.g., pro-inflammatory cytokines, chemokines, and / or enzymes), improved cytotoxicity, and / or improved exhaustion resistance.

[0156] In some embodiments, the modified immune effector cells described herein exhibit improved tumor infiltration compared to unmodified immune effector cells. In some embodiments, improved tumor infiltration by modified immune effector cells means that the number of modified immune effector cells infiltrating the tumor over a given period is increased compared to the number of unmodified immune effector cells infiltrating the tumor over the same period. In some embodiments, the modified immune effector cells exhibit tumor infiltration capabilities that are 1.1 times, 1.2 times, 1.3 times, 1.4 times, 1.5 times, 1.6 times, 1.7 times, 1.8 times, 1.9 times, 2 times, 2.5 times, 3 times, 3.5 times, 4 times, 4.5 times, 5 times, 6 times, 7 times, 8 times, 9 times, 10 times, 15 times, 20 times, 25 times, 30 times, 35 times, 40 times, 45 times, 50 times, 60 times, 70 times, 80 times, 90 times, 100 times or more compared to unmodified immune cells. Tumor infiltration capabilities are determined by isolating one or more tumors from a subject and performing flow cytometry, immunohistochemistry and / or immunoassay. This can be measured by evaluating the number of modified immune cells in the sample using fluorescence imaging.

[0157] In some embodiments, the modified immunoeffector cells described herein exhibit improved cell proliferation compared to unmodified immunoeffector cells. In these embodiments, the result is that, after a predetermined period, the number of modified immunoeffector cells present increases compared to unmodified immunoeffector cells. For example, in some embodiments, the modified immunoeffector cells have a higher proliferation rate compared to unmodified immunoeffector cells, in which case the modified immunoeffector cells divide at a faster rate than the unmodified immunoeffector cells. In some embodiments, the modified immune effector cells proliferate at a rate 1.1 times, 1.2 times, 1.3 times, 1.4 times, 1.5 times, 1.6 times, 1.7 times, 1.8 times, 1.9 times, 2 times, 2.5 times, 3 times, 3.5 times, 4 times, 4.5 times, 5 times, 6 times, 7 times, 8 times, 9 times, 10 times, 15 times, 20 times, 25 times, 30 times, 35 times, 40 times, 45 times, 50 times, 60 times, 70 times, 80 times, 90 times, 100 times, or more, compared to unmodified immune cells. In some embodiments, the modified immune effector cells proliferate for a longer period compared to unmodified immune effector cells, in which case the modified and unmodified immune effector cells divide at the same rate, but the modified immune effector cells maintain their proliferative state for a longer period. In some embodiments, the modified immune effector cells maintain a proliferative state for 1.1 times, 1.2 times, 1.3 times, 1.4 times, 1.5 times, 1.6 times, 1.7 times, 1.8 times, 1.9 times, 2 times, 2.5 times, 3 times, 3.5 times, 4 times, 4.5 times, 5 times, 6 times, 7 times, 8 times, 9 times, 10 times, 15 times, 20 times, 25 times, 30 times, 35 times, 40 times, 45 times, 50 times, 60 times, 70 times, 80 times, 90 times, 100 times, or more, longer than unmodified immune cells.

[0158] In some embodiments, the modified immune effector cells described herein have improved or extended cell viability compared to unmodified immune effector cells. In such embodiments, the result is that, after a predetermined period, the number of modified immune effector cells present is increased compared to unmodified immune effector cells. For example, in some embodiments, the modified immune effector cells described herein maintain and survive for 1.1 times, 1.2 times, 1.3 times, 1.4 times, 1.5 times, 1.6 times, 1.7 times, 1.8 times, 1.9 times, 2 times, 2.5 times, 3 times, 3.5 times, 4 times, 4.5 times, 5 times, 6 times, 7 times, 8 times, 9 times, 10 times, 15 times, 20 times, 25 times, 30 times, 35 times, 40 times, 45 times, 50 times, 60 times, 70 times, 80 times, 90 times, 100 times or more.

[0159] In some embodiments, the modified immune effector cells described herein exhibit enhanced resistance to inhibitors compared to unmodified immune effector cells. Exemplary inhibitors include signaling by immune checkpoint molecules (e.g., PD1, PDL1, CTLA4, LAG3, IDO) and / or inhibitory cytokines (e.g., IL-10, TGFβ).

[0160] In some embodiments, the modified T cells described herein exhibit enhanced resistance to T cell exhaustion compared to unmodified T cells. T cell exhaustion is a state of antigen-specific T cell dysfunction characterized by reduced effector function followed by depletion of antigen-specific T cells. In some embodiments, exhausted T cells lack the ability to proliferate in response to antigens, exhibit reduced cytokine production, and / or reduced cytotoxicity against target cells such as tumor cells. In some embodiments, exhausted T cells are identified by altered expression of cell surface markers and transcription factors, such as decreased expression of CD122 and CD127 on the cell surface, increased expression of repressive cell surface markers such as PD1, Lag3, CD244, CD160, TIM3, and / or CTLA4, and / or increased expression of transcription factors such as Blimp1, NFAT, and / or BATF. Exhausted T cells exhibit altered sensitivity to cytokine signaling, such as increased sensitivity to TGFβ signaling and / or decreased sensitivity to IL-7 and IL-15 signaling. T cell exhaustion can be determined, for example, by co-culturing the T cells with a target cell population and measuring T cell proliferation, cytokine production, and / or target cell lysis. In some embodiments, modified immune effector cells described herein are co-culturned with a population of target cells (e.g., autologous tumor cells or autologous tumor cell lines engineered to express a target tumor antigen), and effector cell proliferation, cytokine production, and / or target cell lysis are measured. These results are then compared with those obtained by co-culturing the target cells with a control immune cell population (unmodified immune effector cells, or a control of modified immune effector cells).

[0161] In some embodiments, resistance to T cell exhaustion is indicated by increased production of one or more cytokines (e.g., IFNγ, TNFα, or IL-2) by modified immune effector cells compared to cytokine production observed in a control immune cell population. In some embodiments, improved resistance to T cell exhaustion is indicated by an increase in cytokine production by modified immune effector cells 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 compared to cytokine production by a control immune cell population. In some embodiments, resistance to T cell exhaustion is demonstrated by an improvement in the proliferation of modified immune effector cells compared to the proliferation observed in the control immune cell population. In some embodiments, improved resistance to T cell exhaustion is demonstrated by an improvement in the proliferation of modified immune effector cells by 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 compared to the proliferation of the control immune cell population. In some embodiments, resistance to T cell exhaustion is demonstrated by increased lysis of target cells by modified immune effector cells compared to the lysis of target cells observed in a control immune cell population.In some embodiments, improved resistance to T cell exhaustion is demonstrated by the lysis of target cells by modified immune effector cells being increased by 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 times or more compared to the lysis of target cells by a control immune cell population.

[0162] In some embodiments, the exhaustion of modified immune effector cells compared to a control immune cell population is measured during the manufacturing process in vitro or ex vivo. For example, in some embodiments, TILs isolated from tumor fragments are modified according to the method described herein, and then grown for one or more rounds to produce a modified TIL population. In such embodiments, the exhaustion of the modified TILs can be determined immediately after harvesting and before the first round of growth, after the first round of growth and before the second round of growth, and / or after the first and second rounds of growth. In some embodiments, the exhaustion 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 have been transferred to a subject. For example, in some embodiments, the modified cells are prepared according to the method described herein and administered to a subject. Subsequently, at various time points after transfer, samples are taken from the subject in viv This allows us to assess the time-dependent exhaustion of modified immune effector cells in o.

[0163] In some embodiments, the modified immune effector cells described herein exhibit increased expression or production of pro-inflammatory immune factors compared to unmodified immune effector cells. Examples of pro-inflammatory immune factors include cytolytic factors such as granzyme B, perforin, and granulysin, as well as pro-inflammatory cytokines such as interferons (IFNα, IFNβ, IFNγ), TNFα, IL-1β, IL-12, IL-2, IL-17, CXCL8, and / or IL-6.

[0164] In some embodiments, the modified immune effector cells described herein exhibit increased cytotoxicity against target cells compared to unmodified immune effector cells. In some embodiments, the modified immune effector cells exhibit increased cytotoxicity against target cells by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100 times or more compared to unmodified immune cells.

[0165] Assays for measuring immunoeffector function are known in the art. For example, tumor invasiveness 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 blotting, and / or qPCR. Cytokine and chemokine expression and production can be measured by flow cytometry, immunohistochemistry, immunofluorescence, Western blotting, ELISA, and / or qPCR. Responsiveness or sensitivity to extracellular stimuli (e.g., cytokines, inhibitory ligands, or antigens) can be measured by assaying cell proliferation and / or activation of downstream signaling pathways (e.g., phosphorylation of downstream signaling intermediates) in response to the stimulus. Cytotoxicity can be measured by targeted cell lysis assays known in the art, including in vitro or ex vivo co-culture of modified immunoeffector cells and target cells and in vivo mouse tumor models, as described throughout the examples.

[0166] B. Endogenous pathways and gene regulation In some embodiments, the modified immune effector cells described herein include a gene regulatory system that can reduce the expression or function of one or more endogenous target genes and / or reduce the expression and / or function of one or more endogenous target genes (listed below). In some embodiments, the one or more endogenous target genes are located in pathways related to the activation and regulation of effector cell responses. In such embodiments, the reduction in the expression or function of one or more endogenous target genes enhances the effector function of one or more of the immune cells.

[0167] Exemplary pathways suitable for modulation by the methods described herein are shown in Table 1. In some embodiments, the expression of endogenous target genes in a particular pathway is reduced in the modified immune effector cells. In some embodiments, the expression of multiple (e.g., two or more) endogenous target genes in a particular pathway is reduced in the modified immune effector cells. For example, the expression of two, three, four, five, six, seven, eight, nine, ten or more endogenous target genes in a particular 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 is reduced in the 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 is reduced in the modified immune effector cells. For example The expression of two, three, four, five, six, seven, eight, nine, ten or more endogenous target genes in one pathway may be reduced, and the expression of two, three, four, five, six, seven, eight, nine, ten or more endogenous target genes in another specific pathway may be reduced.

[0168] In some embodiments, the expression of multiple endogenous target genes within multiple pathways is reduced. For example, one endogenous gene originating from each of multiple pathways (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10 or more pathways) may be reduced. In additional embodiments, multiple endogenous genes originating from each of multiple pathways (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10 or more pathways) (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10 or more genes) may be reduced. Table 1: Exemplary endogenous pathways [Table 1]

[0169] Exemplary endogenous target genes are shown in Tables 2 and 3 below.

[0170] In some embodiments, the modified effector cells of the present invention have reduced expression and / or function of one or more of the following (e.g., one or more endogenous target genes selected from Table 2): 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. In some embodiments, the modified effector cells have reduced expression and / or function of one or more of the following: TNFAIP3, CBLB, or BCOR.

[0171] In some embodiments, the modified immunoeffector cells have reduced expression and / or function of at least two 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 (for example, at least two genes selected from Table 2). For example, in some embodiments, the modified immunoeffector cells have reduced expression and / or function of at least two genes selected from combinations 1 to 600 as shown in Figures 1A to 1B. In some embodiments, the modified immunoeffector cells have reduced expression and / or function of BCOR, as well as reduced expression and / or function of CBLB. Exemplary methods for modifying the expression 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 are described herein, but the expression of these endogenous target genes may also be modified by methods known in the art. For example, inhibitory antibodies against PD1 (encoded by PDCD1), NRP1, HACR2, LAG3, TIGIT and CTLA4 are known in the art, some of which have received FDA approval for tumor indications (e.g., nivolumab and pembrolizumab against PD1).

[0172] In some embodiments, the modified immunoeffector cells have reduced expression and / or function of one or more of the following (e.g., one or more endogenous target genes selected from Table 3): BCL2L11, FLI1, CALM2, DHODH, UMPS, RBM39, SEMA7A, CHIC2, PCBP1, PBRM1, WDR6, E2F8, SERPINA3, GNAS, ZC3H12A, MAP4K1, and NR4A3.

[0173] In some embodiments, the modified effector cells described herein have reduced expression and / or function of the semaphorin 7A (SEMA7A) gene, also known as CD108. In some embodiments, the modified effector cells described herein contain an inactivating mutation in the SEMA7A gene.

[0174] In some embodiments, the modified effector cells described herein have reduced expression and / or function of the RNA-binding protein 39 (RBM39) gene. The RBM39 protein is located in the nucleus and co-localizes with core spliceosome proteins within the nucleus. Studies of mouse proteins with high sequence similarity to this protein suggest that this protein may function as a transcriptional coactivator for JUN / AP-1 and estrogen receptors. In some embodiments, as described herein... These modified effector cells contain an inactivating mutation in the RBM39 gene.

[0175] In some embodiments, the modified effector cells described herein have reduced expression and / or function of the Bcl-2-like protein 11 (BCL2L11) gene, also commonly known as BIM. In some embodiments, the modified effector cells described herein contain an inactivating mutation in the BCL2L11 gene.

[0176] In some embodiments, the modified effector cells described herein have reduced expression and / or function of the Friend leukemia integration 1 transcription factor (FLI1) gene, also known as the transcription factor ERGB. In some embodiments, the modified effector cells described herein contain an inactivating mutation in the FLI1 gene.

[0177] In some embodiments, the modified effector cells described herein have reduced expression and / or function of the calmodulin 2 (CALM2) gene. In some embodiments, the modified effector cells described herein contain an inactivating mutation in the CALM2 gene.

[0178] In some embodiments, the modified effector cells described herein have reduced expression and / or function of the dihydroorotate dehydrogenase gene (DHODH). The DHODH protein is a mitochondrial protein located on the outer surface of the inner mitochondrial membrane that catalyzes the ubiquinone-mediated oxidation of dihydroorotates to orotates in de novo pyrimidine biosynthesis. In some embodiments, the modified effector cells described herein contain an inactivating mutation in the DHODH gene.

[0179] In some embodiments, the modified effector cells described herein have reduced expression and / or function of the uridine monophosphate synthase (UMPS) gene, also known as orotidine phosphoribosyltransferase or orotidine-5'-decarboxylase. The UMPS protein catalyzes the formation of uridine monophosphate (UMP), an energy-carrying molecule, in many important biosynthetic pathways. In some embodiments, the modified effector cells described herein contain an inactivating mutation in the UMPS gene.

[0180] In some embodiments, the modified effector cells described herein have reduced expression and / or function of the cysteine-rich hydrophobic domain 2 (CHIC2) gene. The encoded CHIC2 protein contains a cysteine-rich hydrophobic (CHIC) motif, is localized to vesicular structures and cell membranes, and has been associated with certain cases of acute myeloid leukemia. In some embodiments, the modified effector cells described herein contain an inactivating mutation in the CHIC2 gene.

[0181] In some embodiments, the modified effector cells described herein have reduced expression and / or function of the poly(rC)-binding protein 1 (PCBP1) gene. In some embodiments, the modified effector cells described herein contain an inactivating mutation in the PCBP1 gene.

[0182] In some embodiments, the modified effector cells described herein have reduced expression and / or function of the protein polybromo-1 (PBRM1) gene, also known as BRG1-related factor 180 (BAF180). PBRM1 is a component of the chromatin remodeling complex SWI / SNF-B and is a tumor suppressor gene in many subtypes of cancer. Mutations are particularly common in clear cell renal cell carcinoma. In some embodiments, the modified effector cells described herein have inactivated PBRM1 gene. Includes sex-changing mutations.

[0183] In some embodiments, the modified effector cells described herein have reduced expression and / or function of the WD repeat-containing protein 6 (WDR6) gene, a member of the WD repeat protein family that is ubiquitously expressed in adult and fetal tissues. WD repeats are minimally conserved regions, typically consisting of gly-his and trp-asp (GH-WD), approximately 40 amino acids in length, that can promote the formation of heterotrimeric complexes or multi-protein complexes. Members of this family are involved in a variety of cellular processes, including cell cycle progression, signal transduction, apoptosis, and gene regulation. In some embodiments, the modified effector cells described herein contain an inactivating mutation in the WDR6 gene.

[0184] In some embodiments, the modified effector cells described herein have reduced expression and / or function of the E2F transcription factor 8 (E2F8) gene. The encoded E2F8 protein regulates progression from G1 phase to S phase by causing the nucleus to divide at the appropriate time. In some embodiments, the modified effector cells described herein contain an inactivating mutation in the E2F8 gene.

[0185] In some embodiments, the modified effector cells described herein have reduced expression and / or function of the serpin family A member 3 (SERPINA3) gene. SERPINA3 encodes the α1-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 contain an inactivating mutation in the SERPINA3 gene.

[0186] In some embodiments, the modified effector cells described herein have reduced expression and / or function of the GNAS complex locus (GNAS) gene, which is a stimulant G protein α subunit (Gs-α), a key component of many signaling pathways. In some embodiments, the modified effector cells described herein contain an inactivating mutation in the GNAS gene.

[0187] In some embodiments, the modified effector cells described herein have reduced expression and / or function of the ZC3H12A gene. ZC3H12A (also referred to as MCPIP1 and Regnase-1) is an RNase having an RNase domain immediately upstream of a CCCH-type zinc finger motif. ZC3H12A destabilizes mRNA of transcripts such as IL-6 by targeting them through its nuclease activity, by binding to a conserved stem-loop structure within the 3'UTR of the transcript gene. In T cells, ZC3H12A regulates the transcription levels of several pro-inflammatory genes, including c-Rel, Ox40, and IL-2. In monocytes, ZC3H12A downregulates mRNA of IL-6 and IL-12B, thereby mitigating inflammation. In cancer cells, Zc3h12a promotes apoptosis by inhibiting anti-apoptotic genes, including Bcl2L1, Bcl2A1, RelB, and Bcl3. Activation of Zc3h12a is transient and subject to negative feedback mechanisms, including proteasome-mediated degradation or mucosa-associated lymphoid tissue 1 (MALT1)-mediated cleavage. In some embodiments, the modified effector cells described herein contain inactivating mutations in the ZC3H12A gene.

[0188] In some embodiments, the modified effector cells described herein have reduced expression and / or function of the MAP4K1 gene. MAP4K1 (also referred to as HPK1) is a member of the MAP4K family of mammalian Ste20-associated protein kinases. MAP4K1 is a serine-threonine kinase, a protein primarily expressed in hematopoietic organs. The MAP4K1 protein contains four proline-rich motifs following its N-terminal kinase domain, which can bind to proteins containing three Src homologous domains. When phosphorylated by kinases such as Lck and Zap70, and in the presence of ATP, MAP4K1 exhibits catalytic activity, mediating autophosphorylation as well as phosphorylation of downstream substrate proteins such as SLP-76 and MAP3K proteins. When SLP-76 is phosphorylated at Ser-376, both ubiquitination and proteasome degradation of SLP-76 occur, along with increased interaction between SLP-76 and 14-3-3τ, which negatively regulates TCR signaling. In some embodiments, the modified effector cells described herein contain inactivating mutations in the MAP4K1 gene.

[0189] In some embodiments, the modified effector cells described herein have reduced expression and / or function of the NR4A3 gene. NR4A3 is an inducible orphan receptor, a member of the steroid-thyroid hormone-retinoid receptor superfamily, and a transcription activator. When induced by various stimuli, NR4A3 translocates from the cytoplasm to the nucleus and drives ligand-independent gene expression by binding as a monomer to the NR4A1 response element (NBRE) and as a homodimer to the Nur response element (NurRE). Subsequently, NR4A3 drives the transcription of a distinct set of genes that control cell survival and differentiation both inside and outside the immune system. In some embodiments, the modified effector cells described herein contain an inactivating mutation in the NR4A3 gene.

[0190] In some embodiments, the modified immunoeffector cells of the present invention have reduced expression and / or function of at least two genes selected from BCL2L11, FLI1, CALM2, DHODH, UMPS, RBM39, SEMA7A, CHIC2, PCBP1, PBRM1, WDR6, E2F8, SERPINA3, GNAS, ZC3H12A, MAP4K1, and NR4A3 (for example, two or more genes selected from Table 3). For example, in some embodiments, the modified immunoeffector cells have reduced expression and / or function of at least two genes selected from combinations 1176 to 1681 as shown in Figures 3A to 3B. In some embodiments, the modified immunoeffector cells have reduced expression and / or function of at least two genes selected from combinations 1176 to 1483 as shown in Figure 3A. In some embodiments, the modified immune effector cells have reduced expression and / or function of at least two genes selected from combinations 1250–1265, as shown in Figure 3B. In some embodiments, the modified immune effector cells have reduced expression and / or function of at least two genes selected from combinations 1266–1281, as shown in Figure 3B. In some embodiments, the modified immune effector cells have reduced expression and / or function of at least two genes selected from combinations 1282–1297, as shown in Figure 3B.

[0191] In some embodiments, the modified effector cells have reduced expression and / or function of one or more of the following genes: BCL2L11, FLI1, CALM2, DHODH, UMPS, RBM39, SEMA7A, CHIC2, PCBP1, PBRM1, WDR6, E2F8, SERPINA3, GNAS, ZC3H12A, MAP4K1, and NR4A3 (e.g., one or more genes selected from Table 3), as well as one or more of the following 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, or BCOR (e.g., one or more genes selected from Table 2). For example, the modified immune effector cells are numbered 601-1025. The expression and / or function of endogenous target genes in combinations selected from the above combinations may be reduced. In some embodiments, the modified immune effector cells may have reduced expression and / or function of two endogenous target genes in combinations selected from combinations 601 to 950 (combinations as shown in Figure 2A). In some embodiments, the modified immune effector cells may have reduced expression and / or function of two endogenous target genes in combinations selected from combinations 951 to 975 (combinations as shown in Figure 2B). In some embodiments, the modified immune effector cells may have reduced expression and / or function of two endogenous target genes in combinations selected from combinations 976 to 1000 (combinations as shown in Figure 2B). In some embodiments, the modified immune effector cells may have reduced expression and / or function of two endogenous target genes in combinations selected from combinations 1001 to 1025 (combinations as shown in Figure 2B).

[0192] In some embodiments, the modified effector cells have reduced expression and / or function of at least one gene selected from BCL2L11, FLI1, CALM2, DHODH, UMPS, RBM39, SEMA7A, CHIC2, PCBP1, PBRM1, WDR6, E2F8, SERPINA3, and GNAS, and reduced expression and / or function of at least one gene selected from TNFAIP3, CBLB, or BCOR. In some embodiments, the modified effector cells have reduced expression and / or function of ZC3H12A and at least one gene selected from TNFAIP3, CBLB, or BCOR. In some embodiments, the modified effector cells contain inactivating mutations in ZC3H12A and at least one gene selected from TNFAIP3, CBLB, or BCOR. In some embodiments, the modified effector cells have reduced expression and / or function of MAP4K1 and at least one gene selected from TNFAIP3, CBLB, or BCOR. In some embodiments, the modified effector cells contain inactivating mutations in MAP4K1 and at least one gene selected from TNFAIP3, CBLB, or BCOR. In some embodiments, the modified effector cells have reduced expression and / or function of NR4A3 and at least one gene selected from TNFAIP3, CBLB, or BCOR. In some embodiments, the modified effector cells contain inactivating mutations in NR4A3 and at least one gene selected from TNFAIP3, CBLB, or BCOR.

[0193] In some embodiments, the modified effector cells have reduced expression and / or function of at least one gene selected from BCL2L11, FLI1, CALM2, DHODH, UMPS, RBM39, SEMA7A, CHIC2, PCBP1, PBRM1, WDR6, E2F8, SERPINA3, GNAS, ZC3H12A, MAP4K1, and NR4A3, and reduced expression and / or function of CBLB. In some embodiments, the modified effector cells have reduced expression and / or function of at least one gene selected from BCL2L11, FLI1, CALM2, DHODH, UMPS, RBM39, SEMA7A, CHIC2, PCBP1, PBRM1, WDR6, E2F8, SERPINA3, and GNAS, and reduced expression and / or function of CBLB. In some embodiments, the modified effector cells have reduced expression and / or function of ZC3H12A and CBLB. In some embodiments, the modified effector cells contain inactivating mutations in ZC3H12A and CBLB. In some embodiments, the modified effector cells have reduced expression and / or function of MAP4K1 and CBLB. In some embodiments, the modified effector cells contain inactivating mutations in MAP4K1 and CBLB. In some embodiments, the modified effector cells have reduced expression and / or function of NR4A3 and CBLB. In some embodiments, the modified effector cells contain inactivating mutations in NR4A3 and CBLB.

[0194] In some embodiments, the modified immunoeffector cells are selected genes 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, or BCOR (e.g., table The expression and / or function of one or more genes selected from 2 is reduced, as is the expression and / or function of two genes selected from BCL2L11, FLI1, CALM2, DHODH, UMPS, RBM39, SEMA7A, CHIC2, PCBP1, PBRM1, WDR6, E2F8, SERPINA3, GNAS, ZC3H12A, MAP4K1, and NR4A3 (for example, one or more genes selected from Table 3). For example, in some embodiments, the modified immunoeffector cells exhibit reduced expression and / or function of two endogenous target genes selected from combinations 1026-1297 (as shown in Figures 3A-3B), in addition to reduced expression and / or function of 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.

[0195] In some embodiments, the modified immunoeffector cells have reduced expression and / or function of genes selected from BCL2L11, FLI1, CALM2, DHODH, UMPS, RBM39, SEMA7A, CHIC2, PCBP1, PBRM1, WDR6, E2F8, SERPINA3, GNAS, ZC3H12A, MAP4K1, and NR4A3 (e.g., genes selected from Table 3), and reduced expression and / or function of two 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, or BCOR (e.g., one or more genes selected from Table 2). For example, in some embodiments, the modified immunoeffector cells have reduced expression and / or function of one of the following: BCL2L11, FLI1, CALM2, DHODH, UMPS, RBM39, SEMA7A, CHIC2, PCBP1, PBRM1, WDR6, E2F8, SERPINA3, GNAS, ZC3H12A, MAP4K1, and NR4A3, in addition to reduced expression and / or function of a combination of two endogenous target genes selected from combinations 1 to 600 shown in Figures 1A to 1B. In some embodiments, the modified immune effector cells exhibit reduced expression and / or function of a combination of two endogenous target genes selected from combinations 1 to 600 shown in Figures 1A to 1B, in addition to reduced expression and / or function of a gene selected from BCL2L11, FLI1, CALM2, DHODH, UMPS, RBM39, SEMA7A, CHIC2, PCBP1, PBRM1, WDR6, E2F8, SERPINA3, and GNAS. In some embodiments, the modified immune effector cells exhibit reduced expression and / or function of ZC3H12A, in addition to reduced expression and / or function of a combination of two endogenous target genes selected from combinations 1 to 600 shown in Figures 1A to 1B.In some embodiments, the modified immune effector cells exhibit reduced expression and / or function of MAP4K1 in addition to reduced expression and / or function of a combination of two endogenous target genes selected from combinations 1 to 600 shown in Figures 1A to 1B. In some embodiments, the modified immune effector cells exhibit reduced expression and / or function of NR4A3 in addition to reduced expression and / or function of a combination of two endogenous target genes selected from combinations 1 to 600 shown in Figures 1A to 1B.

[0196] In some embodiments, the modified immune effector cells have reduced expression and / or function of several genes selected from Table 2, and several genes selected from Table 3. In some embodiments, the modified immune effector cells have reduced expression and / or function of two genes selected from Table 2, and two genes selected from Table 3. For example, in some embodiments, the modified immune effector cells have reduced expression and / or function of two genes in combinations selected from combinations 1026 to 1297 as shown in Figures 3A to 3B, and two genes in combinations selected from combinations 1 to 600 as shown in Figures 1A to 1B. In some embodiments, the modified immunoeffector cells may have reduced expression and / or function of three or more 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, or BCOR, and may also have reduced expression and / or function of three or more of BCL2L11, FLI1, CALM2, DHODH, UMPS, RBM39, SEMA7A, CHIC2, PCBP1, PBRM1, WDR6, E2F8, SERPINA3, GNAS, ZC3H12A, MAP4K1, and NR4A3. Table 2: Exemplary endogenous genes [Table 2-1] [Table 2-2] Table 3: Exemplary genes for novel regulation [Table 3-1] [Table 3-2] [Table 3-3]

[0197] III. Gene Regulation Systems In this specification, the term “gene regulatory system” refers to a protein, nucleic acid, or combination thereof that, when introduced into a cell, modulates the expression or function of an encoded gene product by altering an endogenous target DNA sequence. Many gene editing systems suitable for use in the methods of this disclosure are known in the art, and include, but are not limited to, shRNA, siRNA, zinc finger nuclease systems, TALEN systems, and CRISPR / Cas systems.

[0198] As used herein, when "regulate" is used in relation to the effects of a gene regulatory system on an endogenous target gene, "regulate" 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 that endogenous target gene.

[0199] In some embodiments, the gene regulatory system may mediate changes in the sequence of the endogenous target gene by introducing one or more mutations into the endogenous target sequence, for example, by inserting or deleting one or more nucleic acids in the endogenous target sequence. Exemplary mechanisms that can mediate changes in the endogenous target sequence include, but are not limited to, non-homologous end joining (NHEJ) (e.g., classical or alternative), microhomology-mediated end joining (MMEJ), homologous orientation repair (e.g., endogenous donor template-mediated), SDSA (synthesis-dependent single-strand annealing), single-strand annealing, or single-strand entry.

[0200] In some embodiments, the gene regulatory system may mediate changes in the epigenetic state of its endogenous target sequence. For example, in some embodiments, the gene regulatory system may mediate alterations to the covalent bonds of the DNA of its endogenous target gene (e.g., cytosine methylation and hydroxymethylation), or alterations to related histone proteins (e.g., lysine acetylation, lysine and arginine methylation, serine and threonine phosphorylation, and lysine ubiquitination and SUMOylation).

[0201] In some embodiments, the gene regulatory system may mediate changes in the expression of proteins encoded by its endogenous target genes. In such embodiments, the gene regulatory system may regulate the expression of its coding protein by modifying its endogenous target DNA sequence or by acting on the mRNA product encoded by that DNA sequence. In some embodiments, the gene regulatory system may induce the expression of a modified endogenous protein. In such embodiments, when the endogenous DNA sequence is modified by the gene regulatory system, an endogenous protein with reduced function is expressed compared to the corresponding endogenous protein in unmodified immune effector cells. In such embodiments, the expression level of the modified endogenous protein may be higher or lower than the expression level of the corresponding endogenous protein in unmodified immune cells, or it may be the same as or substantially the same as the expression level of the corresponding endogenous protein.

[0202] A. Nucleic acid-based gene regulatory systems 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 requiring an exogenous protein. In some embodiments, the nucleic acid-based gene regulatory system comprises an RNA interference molecule or antisense RNA molecule that is complementary to the target nucleic acid sequence.

[0203] An "antisense RNA molecule" refers to an RNA molecule that is complementary to an mRNA transcript, regardless of length. An antisense RNA molecule is a single-stranded RNA molecule that can be introduced into a cell, tissue, or target and reduces the expression of an endogenous target gene product through a mechanism that relies on the degradation of the target mRNA transcript mediated by RNaseH, rather than on an endogenous gene silencing pathway. In some embodiments, the antisense nucleic acid may include a modified backbone, such as a phosphorothioate, phosphorodithioate, or other backbone known in the art, or it may include unnatural nucleoside bonds. In some embodiments, the antisense nucleic acid may include locked nucleic acid (LNA).

[0204] As used herein, "RNA interference molecule" refers to an RNA polynucleotide that mediates a reduction in the expression of an endogenous target gene product by degrading the target mRNA through an endogenous gene silencing pathway (e.g., the dicer and RNA-induced silencing complex (RISC)). Examples of RNA interference agents include microRNAs (also referred to herein as "miRNAs"), short hairpin RNAs (shRNAs), small interfering RNAs (siRNAs), RNA aptamers, and morpholinos.

[0205] In some embodiments, the nucleic acid-based gene regulatory system of the present invention comprises one or more miRNAs. A miRNA is a small, naturally occurring, non-coding RNA molecule approximately 21–25 nucleotides in length. The miRNA is at least partially complementary to one or more target mRNA molecules. The miRNA can downregulate (e.g., reduce) the expression of an endogenous target gene product through translational repression, mRNA cleavage, and / or deadenylation.

[0206] In some embodiments, the nucleic acid-based gene regulatory system of the present invention comprises one or more shRNAs. shRNAs are single-stranded RNA molecules approximately 50-70 nucleotides long that form a stem-loop structure and degrade complementary mRNA sequences. shRNAs can be cloned into plasmids or non-replicating recombinant viral vectors introduced into cells so that the shRNA coding sequence is integrated into the genome. Therefore, shRNAs can reliably and steadily suppress the translation and expression of endogenous target genes.

[0207] In some embodiments, nucleic acid-based gene regulatory systems include one or more siRNAs. siRNA typically refers to a double-stranded RNA molecule approximately 21–23 nucleotides long. siRNA associates with a multi-protein complex called RNA-induced silencing complex (RISC), during which the "passenger" sense strand is enzymatically cleaved. The antisense "guide" strand contained within the activated RISC then guides the RISC to the corresponding mRNA via sequence homology, where the same nuclease cleaves the target mRNA, resulting in specific gene silencing. Optimally, siRNAs are 18, 19, 20, 21, 22, 23, or 24 nucleotides long and have a 2-base overhang at their 3' end. siRNAs can be introduced into individual cells and / or culture systems to degrade target mRNA sequences. siRNA and shRNA are further described in Fire et al., Nature, 391:19, 1998, and in U.S. Patents No. 7,732,417, No. 8,202,846, and No. 8,383,599.

[0208] In some embodiments, the nucleic acid-based gene regulatory system of the present invention comprises one or more morpholinos. When used herein, "morpholino" refers to a standard nucleic acid base in mol Morphorino refers to modified nucleic acid oligomers that are bound to a phorin ring and linked via phosphorodiamidate bonds. Like siRNA and shRNA, morpholino binds to complementary mRNA sequences. However, morpholino does not target complementary mRNA sequences to degrade them, but rather functions by sterically inhibiting mRNA translation and altering mRNA splicing.

[0209] In some embodiments, the nucleic acid-based gene regulatory system of the present invention includes 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 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 (i.e., the genes listed in Table 2) and is at least 90%, 95%, 96%, 97%, 98%, or 99% identical, or 100% identical. In some embodiments, the nucleic acid-based gene regulatory system includes 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%, or 100%, identical to an RNA sequence encoded by a set of genomic coordinates shown in Table 5A or Table 5B. Throughout this application, the reference genomic coordinates are based on genomic annotations of the GRCh38 (also known as hg38) assembly of the human genome obtained from the Genome Reference Consortium, which is available on the website of the National Center for Biotechnology Information.Tools and methods for converting genomic coordinates between one assembly and another are known in the art, and these tools and methods can be used to convert the genomic coordinates presented herein to corresponding coordinates in another assembly of the human genome (including conversion to a previous assembly prepared by the same institution or using the same algorithm (e.g., from GRCh38 to GRCh37), and conversion to assemblies prepared by different institutions or algorithms (e.g., from GRCh38 to NCBI33 prepared 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 on the National Center for Biotechnology Information website, UCSC LiftOver available on the UCSC Genome Brower website, and the Assembly Converter available on the Ensembl.org website.

[0210] In some embodiments, the nucleic acid-based gene regulatory system of the present invention includes 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%, or 100%, identical to the RNA sequence encoded by one of SEQ ID NOs. 154-498 or SEQ ID NOs. 499-813. In some embodiments, the nucleic acid-based gene regulatory system of the present invention can reduce the expression and / or function of CBLB and includes 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%, or 100%, identical to the RNA sequence encoded by one of SEQ ID NOs. 499-524. In some embodiments, the nucleic acid-based gene regulatory system of the present invention can reduce the expression and / or function of BCOR. The present invention includes 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%, or 100%, identical to the RNA sequence encoded by one of sequence numbers 708-772 or 708-764. In some embodiments, the nucleic acid-based gene regulatory system of the present invention can reduce the expression and / or function of TNFAIP3 and includes 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%, or 100%, identical to the RNA sequence encoded by one of sequence numbers 348-396 or 348-386. In some embodiments, the nucleic acid-based gene regulatory system of the present invention comprises an siRNA molecule or shRNA molecule selected from siRNA molecules or shRNA molecules known in the art, such as siRNA constructs and shRNA constructs available from commercial manufacturers such as Sigma Aldrich, Dharmacon, and ThermoFisher.

[0211] In some embodiments, the endogenous target gene is a CBLB, and its nucleic acid molecule is an shRNA encoded by a nucleic acid sequence selected from SEQ ID NOs. 41-44 (see International PCT Publication No. 2018156886) or an shRNA encoded by a nucleic acid sequence selected from SEQ ID NOs. 45-53 (see International PCT Publication No. WO2017120998). In some embodiments, the endogenous target gene is a CBLB, and its nucleic acid molecule is an siRNA containing a nucleic acid sequence selected from SEQ ID NOs. 54-63 (see International PCT Publication No. WO2018006880) or an siRNA containing a nucleic acid sequence selected from SEQ ID NOs. 64-73 (see International PCT Publication Nos. WO2018120998 and WO2018137293).

[0212] In some embodiments, the endogenous target gene is TNFAIP3, and the nucleic acid molecule is an shRNA encoded by a nucleic acid sequence selected from SEQ ID NOs. 74-95 (see U.S. Patent No. 8,324,369). In some embodiments, the endogenous target gene is TNFAIP3, and the nucleic acid molecule is an siRNA containing a nucleic acid sequence selected from SEQ ID NOs. 96-105 (see International PCT Publication No. WO2018006880).

[0213] In some embodiments, the endogenous target gene is CTLA4, and its nucleic acid molecule is an shRNA encoded by a nucleic acid sequence selected from SEQ ID NOs. 128-133 (see International PCT Publication No. WO2017120996). In some embodiments, the endogenous target gene is CTLA4, and its nucleic acid molecule is an siRNA containing a nucleic acid sequence selected from SEQ ID NOs. 134-143 (see International PCT Publication Nos. WO2017120996, WO2017120998, WO2018137295, and WO2018137293) or an siRNA containing a nucleic acid sequence selected from SEQ ID NOs. 144-153 (see International PCT Publication No. WO2018006880).

[0214] In some embodiments, the endogenous target gene is PDCD1, and its nucleic acid molecule is shRNA encoded by a nucleic acid sequence selected from SEQ ID NOs. 106-107 (see International PCT Publication No. WO2017120996). In some embodiments, the endogenous target gene is PDCD1, and its nucleic acid molecule is siRNA containing a nucleic acid sequence selected from SEQ ID NOs. 108-117 (see International PCT Publication Nos. WO2017120996, WO201712998, WO2018137295, and WO2018137293) or siRNA containing a nucleic acid sequence selected from SEQ ID NOs. 118-127 (see International PCT Publication No. WO2018006880). i) is.

[0215] In some embodiments, the nucleic acid-based gene regulatory system of the present invention includes 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 BCL2L11, FLI1, CALM2, DHODH, UMPS, RBM39, SEMA7A, CHIC2, PCBP1, PBRM1, WDR6, E2F8, SERPINA3, GNAS, ZC3H12A, MAP4K1, and NR4A3 (i.e., the genes listed in Table 3) or 100% identical. In some embodiments, the nucleic acid-based gene regulatory system of the present invention includes 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%, or 100%, identical to an RNA sequence encoded by a set of genomic coordinates shown in Tables 6A to 6H. In some embodiments, the nucleic acid-based gene regulatory system includes 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%, 99%, or 100%, identical to an RNA sequence encoded by one of sequence numbers 814 to 1566.

[0216] In some embodiments, the nucleic acid-based gene regulatory system of the present invention can reduce the expression and / or function of a target gene selected from BCL2L11, FLI1, CALM2, DHODH, UMPS, RBM39, SEMA7A, CHIC2, PCBP1, PBRM1, WDR6, E2F8, SERPINA3, and GNAS. In some embodiments, the nucleic acid-based gene regulatory system includes 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%, 99%, or 100% identical to an RNA sequence encoded by a DNA sequence defined by a set of genomic coordinates shown in one of Tables 6A or 6B. In some embodiments, the nucleic acid-based gene regulatory system includes 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%, 99%, or 100% identical to the RNA sequence encoded by one of sequence numbers 814-1064.

[0217] In some embodiments, the nucleic acid-based gene regulatory system of the present invention can reduce the expression and / or function of ZC3H12A. In some embodiments, the nucleic acid-based gene regulatory system includes 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%, 99%, or 100% identical to an RNA sequence encoded by a set of genomic coordinates shown in one of Tables 6C or 6D. In some embodiments, the nucleic acid-based gene regulatory system includes 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%, 99%, or 100% identical to an RNA sequence encoded by one of Sequence IDs 1065-1509. In some embodiments, the nucleic acid-based gene regulatory system includes 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%, 99%, or 100% identical to the RNA sequence encoded by one of sequence numbers 1065–1264.

[0218] In some embodiments, the nucleic acid-based gene regulatory system of the present invention can reduce the expression and / or function of MAP4K1. In some embodiments, the nucleic acid-based gene regulatory system includes 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%, 99%, or 100% identical to an RNA sequence encoded by a set of genomic coordinates shown in one of Tables 6E or 6F. In some embodiments, the nucleic acid-based gene regulatory system includes 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%, 99%, or 100% identical to an RNA sequence encoded by one of Sequence IDs 510-1538.

[0219] In some embodiments, the nucleic acid-based gene regulatory system of the present invention can reduce the expression and / or function of NR4A3. In some embodiments, the nucleic acid-based gene regulatory system includes 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%, 99%, or 100% identical to an RNA sequence encoded by a set of genomic coordinates shown in one of Tables 6G or 6H. In some embodiments, the nucleic acid-based gene regulatory system includes 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%, 99%, or 100% identical to an RNA sequence encoded by one of Sequence IDs 1539-1566.

[0220] In some embodiments, the nucleic acid-based gene regulatory system of the present invention includes siRNA molecules or shRNA molecules selected from siRNA molecules or shRNA molecules known in the art, such as siRNA molecules or shRNA molecules available from commercial suppliers such as Sigma Aldrich, Dharmacon, and ThermoFisher. Exemplary siRNA and shRNA constructs are listed in Tables 4A and 4B below. In some embodiments, the nucleic acid-based gene regulatory system includes two or more siRNA molecules selected from siRNA molecules known in the art, such as the siRNA constructs listed in Table 4A. In some embodiments, the nucleic acid-based gene regulatory system includes two or more shRNA molecules selected from shRNA molecules known in the art, such as the shRNA constructs listed in Table 4B. Table 4A: Exemplary siRNA construct [Table 4A-1] [Table 4A-2] [Table 4A-3] [Table 4A-4] Table 4B: Exemplary shRNA construct [Table 4B-1] [Table 4B-2] [Table 4B-3]

[0221] In some embodiments, the gene regulatory system of the present invention comprises two or more nucleic acid molecules (e.g., two or more siRNAs, two or more shRNAs, two or more RNA aptamers, or two or more morpholinos), at least one of which nucleic acid molecules is encoded by the DNA sequence of a target gene selected from IKZF1, IKZF3, GATA3, BCL3, TNIP1, TNFAIP3, NFKBIA, SMAD2, TGFBR1, TGFBR2, TANK, FOXP3, RC3H1, TRAF6, IKZF2, CBLB, PPP2R2R, NRP1, HAVCR2, LAG3, TIGIT, CTLA4, PTPN6, PDCD1, or BCOR (e.g., a gene selected from Table 2). The RNA molecule binds to a target RNA sequence that is at least 90%, 95%, 96%, 97%, 98%, or 99% identical, or 100% identical, to the target RNA sequence, and at least one of its 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 of a target gene selected from BCL2L11, FLI1, CALM2, DHODH, UMPS, RBM39, SEMA7A, CHIC2, PCBP1, PBRM1, WDR6, E2F8, SERPINA3, GNAS, ZC3H12A, MAP4K1, and NR4A3 (for example, a gene selected from Table 3).

[0222] In some embodiments, at least one of the two or more nucleic acid molecules binds to a target RNA sequence that is at least 90%, 95%, 96%, 97%, 98%, or 99%, or 100%, identical to the RNA sequence encoded by the DNA sequence defined by a series of genomic coordinates shown in Table 5A or Table 5B, and at least one of the two or more nucleic acid molecules binds to a target RNA sequence that is at least 90%, 95%, 96%, 97%, 98%, or 99%, or 100%, identical to the RNA sequence encoded by the DNA sequence defined by a series of genomic coordinates shown in Tables 6A to 6H. In some embodiments, at least one of the 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 sequence numbers 814-1566, and at least one of the 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 sequence numbers 154-498 or sequence numbers 499-813.

[0223] In some embodiments, the gene regulatory system of the present invention comprises two or more nucleic acid molecules, at least one of which is IKZF1, IKZF3, GATA3, BC The target RNA sequence is encoded by the DNA sequence of a target gene selected from L3, TNIP1, TNFAIP3, NFKBIA, SMAD2, TGFBR1, TGFBR2, TANK, FOXP3, RC3H1, TRAF6, IKZF2, CBLB, PPP2R2D, NRP1, HAVCR2, LAG3, TIGIT, CTLA4, PTPN6, PDCD1, or BCOR, and at least one of the nucleic acid molecules is encoded by the target RNA sequence of a target gene selected from BCL2L11, FLI1, CALM2, DHODH, UMPS, RBM39, SEMA7A, CHIC2, PCBP1, PBRM1, WDR6, E2F8, SERPINA3, and GNAS. In some embodiments, at least one of the two or more nucleic acid molecules binds to a target RNA sequence that is at least 90%, 95%, 96%, 97%, 98%, or 99%, or 100%, identical to the RNA sequence encoded by the DNA sequence defined by a set of genomic coordinates shown in Table 5A or Table 5B, and at least one of the two or more nucleic acid molecules binds to a target RNA sequence that is at least 90%, 95%, 96%, 97%, 98%, or 99%, or 100%, identical to the RNA sequence encoded by the DNA sequence defined by a set of genomic coordinates shown in Table 6A or Table 6B. In some embodiments, at least one of the two or more nucleic acid molecules binds to a target RNA sequence that is at least 90%, 95%, 96%, 97%, 98%, or 99%, or 100%, identical to the RNA sequence encoded by one of sequence numbers 814-1064, and at least one of the two or more nucleic acid molecules binds to a target RNA sequence that is at least 90%, 95%, 96%, 97%, 98%, or 99%, or 100%, identical to the RNA sequence encoded by one of sequence numbers 154-498 or 499-813.

[0224] In some embodiments, the gene regulatory system of the present invention comprises two or more nucleic acid molecules, at least one of which binds to a target RNA sequence encoded by the DNA sequence of CBLB, and at least one of which binds to a target RNA sequence encoded by the DNA sequence of a target gene selected from BCL2L11, FLI1, CALM2, DHODH, UMPS, RBM39, SEMA7A, CHIC2, PCBP1, PBRM1, WDR6, E2F8, SERPINA3, and GNAS. In some embodiments, at least one of the two or more nucleic acid molecules binds to a target RNA sequence that is at least 90%, 95%, 96%, 97%, 98%, or 99%, or 100%, identical to the RNA sequence encoded by one of sequence numbers 814-1064, and at least one of the two or more nucleic acid molecules binds to a target RNA sequence that is at least 90%, 95%, 96%, 97%, 98%, or 99%, or 100%, identical to the RNA sequence encoded by one of sequence numbers 499-524.

[0225] In some embodiments, the gene regulatory system of the present invention comprises two or more nucleic acid molecules, at least one of which binds to a target RNA sequence encoded by the DNA sequence of a target gene 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, or BCOR, and at least one of which binds to a target RNA sequence encoded by the DNA sequence of ZC3H12A. In some embodiments, at least one of the two or more nucleic acid molecules binds to a target RNA sequence that is at least 90%, 95%, 96%, 97%, 98%, or 99%, or 100%, identical to the RNA sequence encoded by the DNA sequence defined by a set of genomic coordinates shown in Table 5A or Table 5B, and at least one of the two or more nucleic acid molecules binds to the DNA sequence def...

Claims

[Claim 1] The invention described in the specification.

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