NR4A3-targeting polynucleotides and uses thereof

Abstract

The present disclosure provides polynucleotides capable of reducing the level of NR4A3 gene and / or NR4A3 protein in a cell (e.g., an immune cell). In some aspects, the polynucleotide includes a gRNA that specifically targets a region within the NR4A3 gene. The disclosure also provides the use of such polynucleotides for the treatment of various diseases or disorders.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This PCT application claims the benefit of priority to U.S. Provisional Application No. 63 / 365,025, filed on May 19, 2022; U.S. Provisional Application No. 63 / 382,705, filed on November 7, 2022; and U.S. Provisional Application No. 63 / 482,984, filed on February 2, 2023, each of which is incorporated herein by reference in its entirety.

[0003] Reference to a sequence listing submitted electronically

[0004] The contents of the sequence listing submitted electronically and submitted with the application (file name: 4385_105PC03_SequenceListing_ST26.XML; size: 124,447 bytes; creation date: May 19, 2023) are incorporated herein by reference in their entirety. Technical Field

[0005] The present disclosure relates to polynucleotides (e.g., guide RNAs) that can be used to reduce the level of NR4A3 genes and / or NR4A3 proteins in immune cells. The present disclosure also relates to cell-based (e.g., T cell) cancer immunotherapy that involves administering such immune cells with reduced levels of NR4A3 genes and / or NR4A3 proteins. Background Art

[0006] Cancer immunotherapy relies on enabling T cells, the immune system's primary killers of infected and diseased cells, to attack and kill tumor cells. However, immunotherapy has a significant stumbling block: T cells' killing ability can wane, a phenomenon often referred to as exhaustion. Immune checkpoint blockade, chimeric antigen receptor (CAR) T cell therapy, and T cell receptor engineered (TCR) T cell therapy are treatments that utilize functionally active T cells isolated from patients and require highly functional T cells to be effective. These T cells are engineered and expanded ex vivo to recognize specific antigens on target cancer cells.

[0007] When the immune system is forced to be active for a long time, such as with persistent viral infection or the progression of cancer, effector T cells may become exhausted. A hallmark of exhausted T cells is increased expression of immune checkpoint proteins (such as PD-1 and CTLA-4), which can cause these T cells to stop working (i.e., become non-functional). Immune checkpoint inhibitors block these checkpoint proteins and in doing so can increase the immune response against the tumor. Some studies have shown that blocking the activity of checkpoint proteins in exhausted T cells does not achieve this goal. This is important because so-called hot tumors (i.e., those that contain high levels of immune cells and therefore should be ideal candidates for responding to immunotherapy) often contain populations composed primarily of exhausted T cells. In addition, the tumor microenvironment can induce senescent and exhausted cell phenotypes. Therefore, designing strategies to reverse and / or prevent these exhausted states is critical to improving the effectiveness of immunotherapy. Summary of the Invention

[0008] Provided herein is a method for reducing the level of the NR4A3 gene and / or NR4A3 protein in an immune cell, comprising modifying the immune cell with a gene editing tool, the gene editing tool comprising a polynucleotide, the polynucleotide comprising a gRNA, wherein the gRNA is capable of specifically binding to a sequence within the NR4A3 gene and comprises, consists essentially of, or consists of a sequence set forth in any one of SEQ ID NO:94, SEQ ID NO:52, SEQ ID NO:96, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:86, SEQ ID NO:83, SEQ ID NO:55, SEQ ID NO:82, SEQ ID NO:56, SEQ ID NO:76, SEQ ID NO:57, SEQ ID NO:75, SEQ ID NO:58, SEQ ID NO:71, SEQ ID NO:61, SEQ ID NO:70, SEQ ID NO:65, SEQ ID NO:68, and SEQ ID NO:71. NO:67, and wherein after modification, the level of NR4A3 gene and / or NR4A3 protein in the immune cell is reduced compared to a reference immune cell (e.g., a corresponding immune cell not contacted with the polynucleotide). In some aspects, after modification, the level of NR4A3 gene is reduced by at least about 5%, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or about 100% compared to a reference immune cell. In some aspects, after modification, the level of NR4A3 protein is reduced by at least about 5%, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or about 100% compared to a reference immune cell.

[0009] Also provided herein is a method of reducing or preventing exhaustion of immune cells, comprising contacting an immune cell with a gene editing tool, the gene editing tool comprising a polynucleotide comprising a gRNA, wherein the gRNA is capable of specifically binding to a sequence within the NR4A3 gene and comprises, consists essentially of, or consists of a sequence set forth in any one of SEQ ID NO:94, SEQ ID NO:52, SEQ ID NO:96, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:86, SEQ ID NO:83, SEQ ID NO:55, SEQ ID NO:82, SEQ ID NO:56, SEQ ID NO:76, SEQ ID NO:57, SEQ ID NO:75, SEQ ID NO:58, SEQ ID NO:71, SEQ ID NO:61, SEQ ID NO:70, SEQ ID NO:65, SEQ ID NO:68, and SEQ ID NO:71. NO:67, and wherein after contact, compared with reference to immune cell (for example, the corresponding immune cell not contacted with polynucleotide), the depletion of immune cell reduces after continuous antigen stimulation. In some respects, compared with reference to immune cell, immune cell is more tolerant to depletion. In some respects, compared with reference to immune cell, immune cell shows persistence / survival increase when being applied to experimenter. In some respects, compared with reference to immune cell, immune cell shows the amplification / propagation of increase after continuous antigen stimulation. In some respects, compared with reference to immune cell, immune cell shows the effector function of increase to the response of continuous antigen stimulation.

[0010] Provided herein is a method for increasing cytokine production by immune cells that respond to antigenic stimulation, comprising modifying the immune cells with a gene editing tool, the gene editing tool comprising a polynucleotide comprising a gRNA, wherein the gRNA is capable of specifically binding to a sequence within the NR4A3 gene and comprises, consists essentially of, or consists of a sequence set forth in any one of SEQ ID NO:94, SEQ ID NO:52, SEQ ID NO:96, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:86, SEQ ID NO:83, SEQ ID NO:55, SEQ ID NO:82, SEQ ID NO:56, SEQ ID NO:76, SEQ ID NO:57, SEQ ID NO:75, SEQ ID NO:58, SEQ ID NO:71, SEQ ID NO:61, SEQ ID NO:70, SEQ ID NO:65, SEQ ID NO:68, and SEQ ID NO:71. NO:67, wherein after modification, compared with reference to immune cell (for example, the corresponding immune cell not modified with polynucleotides), immune cell shows the cytokine production of increase after antigen stimulation.In some aspects, cytokine includes IFN-γ, IL-2, TNF-α or their combination.In some aspects, compared with reference to immune cell, after modification, the generation of the cytokine in response to antigen stimulation increases at least about 1 times, at least about 2 times, at least about 3 times, at least about 4 times, at least about 5 times, at least about 6 times, at least about 7 times, at least about 8 times, at least about 9 times, at least about 10 times, at least about 11 times, at least about 12 times, at least about 13 times, at least about 14 times, at least about 15 times, at least about 16 times, at least about 17 times, at least about 18 times, at least about 19 times, at least about 20 times, at least about 25 times, at least about 30 times, at least about 35 times, at least about 40 times, at least about 45 times or at least about 50 times.

[0011] The present disclosure also provides a method for increasing the effector function of an immune cell that responds to sustained antigen stimulation, comprising modifying the immune cell with a gene editing tool, the gene editing tool comprising a polynucleotide, the polynucleotide comprising a gRNA, wherein the gRNA is capable of specifically binding to a sequence within the NR4A3 gene and comprises, consists essentially of, or consists of a sequence as set forth in any one of SEQ ID NO:94, SEQ ID NO:52, SEQ ID NO:96, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:86, SEQ ID NO:83, SEQ ID NO:55, SEQ ID NO:82, SEQ ID NO:56, SEQ ID NO:76, SEQ ID NO:57, SEQ ID NO:75, SEQ ID NO:58, SEQ ID NO:71, SEQ ID NO:61, SEQ ID NO:70, SEQ ID NO:65, SEQ ID NO:68, and SEQ ID NO:71. NO:67, and wherein after modification, compared with reference to immune cell (for example, the corresponding immune cell not contacted with polynucleotide), immune cell shows the effector function of increase after continuous antigen stimulation.In some respects, compared with reference to immune cell, after modification, immune cell keeps effector function in at least one round, at least two rounds or at least three rounds of other antigen stimulation assays.In some respects, effector function includes following ability: (i) killing target cell (for example, tumor cell), (ii) producing cytokine after further antigen stimulation, or (iii) (i) and (ii) both.

[0012] Some aspects of the present disclosure relate to a method of making a composition comprising immune cells having reduced levels of an NR4A3 gene and / or an NR4A3 protein, the method comprising modifying the immune cells with a gene editing tool comprising a polynucleotide comprising a gRNA, wherein the gRNA is capable of specifically binding to a sequence within the NR4A3 gene and comprises, consists essentially of, or consists of a sequence set forth in any one of SEQ ID NO:94, SEQ ID NO:52, SEQ ID NO:96, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:86, SEQ ID NO:83, SEQ ID NO:55, SEQ ID NO:82, SEQ ID NO:56, SEQ ID NO:76, SEQ ID NO:57, SEQ ID NO:75, SEQ ID NO:58, SEQ ID NO:71, SEQ ID NO:61, SEQ ID NO:70, SEQ ID NO:65, SEQ ID NO:68, and SEQ ID NO:71. NO: 67, and wherein after modification, the level of the NR4A3 gene and / or NR4A3 protein in the immune cell is reduced compared to a reference immune cell (e.g., a corresponding immune cell not contacted with the polynucleotide). In some aspects, the method further comprises combining the modified immune cell with a pharmaceutically acceptable excipient.

[0013] For any of the above methods, in some aspects, after modification, the level of NR4A3 gene is reduced by at least about 5%, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or about 100% compared to a reference immune cell. In some aspects, after modification, the level of NR4A3 protein is reduced by at least about 5%, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or about 100% compared to a reference immune cell.

[0014] Also provided herein is a method of treating a tumor in a subject in need thereof, comprising administering to the subject an immune cell that has been modified with a gene editing tool, wherein the gene editing tool comprises a polynucleotide comprising a gRNA, wherein the gRNA is capable of specifically binding to a sequence within the NR4A3 gene and comprises, consists essentially of, or consists of a sequence set forth in any one of SEQ ID NO:94, SEQ ID NO:52, SEQ ID NO:96, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:86, SEQ ID NO:83, SEQ ID NO:55, SEQ ID NO:82, SEQ ID NO:56, SEQ ID NO:76, SEQ ID NO:57, SEQ ID NO:75, SEQ ID NO:58, SEQ ID NO:71, SEQ ID NO:61, SEQ ID NO:70, SEQ ID NO:65, SEQ ID NO:68, and SEQ ID NO:67. In some aspects, the level of the NR4A3 gene in an immune cell is reduced by at least about 5%, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or about 100% compared to a reference immune cell (e.g., a corresponding immune cell that has not been contacted with the polynucleotide). In some aspects, the level of the NR4A3 protein in an immune cell is reduced by at least about 5%, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or about 100% compared to a reference immune cell.

[0015] In the above-mentioned methods for treating tumors, in some aspects, administration reduces the tumor volume in the subject compared to a reference tumor volume (e.g., the tumor volume in the subject before administration and / or the tumor volume in the subject not receiving administration). In some aspects, the tumor volume is reduced by at least about 5%, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or about 100%, compared to a reference tumor volume.

[0016] In some aspects, the tumor that can be treated using the methods provided herein is derived from a cancer comprising breast cancer, head and neck cancer, uterine cancer, brain cancer, skin cancer, kidney cancer, lung cancer, colorectal cancer, prostate cancer, liver cancer, bladder cancer, kidney cancer, pancreatic cancer, thyroid cancer, esophageal cancer, eye cancer, stomach (gastric) cancer, gastrointestinal cancer, ovarian cancer, cervical cancer, carcinoma, sarcoma, leukemia, lymphoma, myeloma, or a combination thereof.

[0017] In some aspects, the method for treating tumors provided herein also includes administering another therapeutic agent to the subject. In some aspects, another therapeutic agent includes chemotherapeutics, targeted anticancer therapy, oncolytic drugs, cytotoxic agents, immune-based therapies, cytokines, surgical procedures, radiation procedures, activators of costimulatory molecules, immune checkpoint inhibitors, vaccines, cellular immunotherapy or any combination thereof. In some aspects, another therapeutic agent is an immune checkpoint inhibitor. In some aspects, immune checkpoint inhibitors include anti-PD-1 antibodies, anti-PD-L1 antibodies, anti-LAG-3 antibodies, anti-CTLA-4 antibodies, anti-GITR antibodies, anti-TIM3 antibodies and any combination thereof. In some aspects, immune cells and another therapeutic agent are administered to the subject simultaneously. In some aspects, immune cells and another therapeutic agent are administered to the subject sequentially.

[0018] In some aspects, the immune cells are administered to the subject parenterally, intramuscularly, subcutaneously, ophthalmically, intravenously, intraperitoneally, intradermally, intraorbitally, intracerebrally, intracranially, intraspinally, intraventricularly, intrathecally, intracisternalally, intracapsularly, intratumorally, or any combination thereof.

[0019] In any of the above methods, in some aspects, the method further comprises modifying immune cells so that they have a reduced level of NR4A1 gene and / or NR4A1 protein. In some aspects, modifying immune cells so that they have a reduced level of NR4A1 gene and / or NR4A1 protein includes contacting immune cells with a gene editing tool ("NR4A1-specific gene editing tool") that can specifically target and reduce the level of NR4A1 gene and / or NR4A1 protein. In some aspects, after immune cells are contacted with NR4A1-specific gene editing tools, compared to corresponding cells that are not contacted with NR4A1-specific gene editing tools, the level of NR4A1 gene is reduced by at least about 5%, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90% or about 100%. In some aspects, after an immune cell is contacted with a NR4A1-specific gene editing tool, the level of NR4A1 protein is reduced by at least about 5%, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or about 100% compared to a corresponding cell that has not been contacted with the NR4A1-specific gene editing tool.

[0020] For any of the above methods, in some aspects, the method further comprises modifying the immune cells so that they have a reduced level of NR4A2 gene and / or NR4A2 protein. In some aspects, modifying the immune cells so that they have a reduced level of NR4A2 gene and / or NR4A2 protein includes contacting the immune cells with a gene editing tool ("NR4A1-specific gene editing tool") that can specifically target and reduce the level of NR4A2 gene and / or NR4A2 protein. In some aspects, after the immune cells are contacted with the NR4A2-specific gene editing tool, the level of the NR4A2 gene is reduced by at least about 5%, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90% or about 100% compared to the corresponding cells that are not contacted with the NR4A2-specific gene editing tool. In some aspects, after an immune cell is contacted with a NR4A2-specific gene editing tool, the level of NR4A2 protein is reduced by at least about 5%, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or about 100% compared to a corresponding cell that has not been contacted with the NR4A2-specific gene editing tool.

[0021] In some aspects, any of the methods provided above further comprises modifying an immune cell to have an elevated level of c-Jun protein. In some aspects, modifying an immune cell to have an elevated level of c-Jun protein comprises contacting the immune cell with a nucleotide sequence encoding a c-Jun protein. For example, in some aspects, the nucleotide sequence encoding the c-Jun protein comprises: (a) a nucleic acid sequence having at least 89%, at least 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity to the nucleic acid sequence set forth in SEQ ID NO:7; (b) a nucleic acid sequence having at least 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity to the nucleic acid sequence set forth in SEQ ID NO:8; (c) a nucleic acid sequence having at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity to the nucleic acid sequence set forth in SEQ ID NO:10; (d) a nucleic acid sequence having at least about 15%, at least about 16%, at least about 17%, at least about 18%, at least about 19%, at least about 20%, at least about 21%, at least about 22%, at least about 23%, at least about 24%, at least about 25%, at least about 26%, at least about 27%, at least about 28%, at least about 29%, at least about 30%, at least about 31%, at least about 32%, at least about 33%, at least about 34%, at least about 35%, at least about 36%, at least about 37%, at least about 38%, at least about 39%, at least about 40%, at least about 41%, at least about 42%, at least about 43%, at least about 44%, at least about : (i) a nucleic acid sequence having at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity to the nucleic acid sequence set forth in SEQ ID NO: 11; (ii) a nucleic acid sequence having at least 88%, at least 89%, at least 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity to the nucleic acid sequence set forth in SEQ ID NO: 12; (iii) a nucleic acid sequence having at least 88%, at least 89%, at least 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity to the nucleic acid sequence set forth in SEQ ID NO: 13; (g) a nucleic acid sequence having at least 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity to the nucleic acid sequence set forth in SEQ ID NO:14; (h) a nucleic acid sequence having at least 55%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity to the nucleic acid sequence set forth in SEQ ID NO:15;or (i) a nucleic acid sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity to the nucleic acid sequence set forth in SEQ ID NO: 16;

[0022] In some aspects, modifying an immune cell to have an elevated level of c-Jun protein comprises contacting the immune cell with a transcriptional activator capable of increasing expression of an endogenous c-Jun protein. In some aspects, the transcriptional activator is attached to a Cas protein that has been modified to lack endonuclease activity.

[0023] In some aspects, after modifying an immune cell to have an elevated level of a c-Jun protein, the level of the c-Jun protein in the immune cell is elevated by at least about 1 fold, at least about 2 fold, at least about 3 fold, at least about 4 fold, at least about 5 fold, at least about 6 fold, at least about 7 fold, at least about 8 fold, at least about 9 fold, at least about 10 fold, at least about 11 fold, at least about 12 fold, at least about 13 fold, at least about 14 fold, at least about 15 fold, at least about 16 fold, at least about 17 fold, at least about 18 fold, at least about 19 fold, at least about 20 fold, at least about 25 fold, at least about 30 fold, at least about 35 fold, at least about 40 fold, at least about 45 fold, or at least about 50 fold compared to a reference cell (e.g., a corresponding cell that has not been modified to have an elevated level of a c-Jun protein).

[0024] In some aspects, any of the methods provided above also includes modifying immune cells to express ligand-binding proteins. In some aspects, ligand-binding protein chimeric antigen receptor (CAR), T cell receptor (TCR), chimeric antibody-T cell receptor (caTCR), chimeric signaling receptor (CSR), T cell receptor mimics (TCR mimics) or a combination thereof. In some aspects, the ligand-binding protein is CAR. In some aspects, the ligand-binding protein is TCR.In some aspects, the ligand binding protein is capable of specifically binding to an antigen selected from the group consisting of CD19, TRAC, TCRβ, BCMA, CLL-1, CS1, CD38, CD19, TSHR, CD123, CD22, CD30, CD70, CD171, CD33, EGFRvIII, GD2, GD3, Tn Ag, PSMA, ROR1, ROR2, GPC1, GPC2, FLT3, FAP, TAG72, CD44v6, CEA, EPCAM, B7H3, KIT, IL-13Ra2, mesothelin, IL-11Ra, PSCA, PRSS21, VEGFR2, LewisY, CD24, PDGFR-β, SSEA-4, CD20, folate receptor α, ERBB2 (Her2 / neu), MUC1, MUC16, EGFR, NCAM, prostate enzyme, PAP, ELF2M, ephrin B2, IGF-I receptor, CAIX, LMP2, gplO O, bcr-abl, tyrosinase, EphA2, fucosyl GM1, sLe, GM3, TGS5, HMWMAA, o-acetyl-GD2, folate receptor β, TEM1 / CD248, TEM7R, CLDN6, GPRC5D, CXORF61, CD97, CD179a, ALK, polysialic acid, PLAC1, GloboH, NY-BR-1, UPK2, HAVCR1, ADRB3, PANX3, GPR20, LY6K, OR51E2, TARP, WT1, NY-ESO-1, LAGE-1a, MAGE-1a1, legumin, HPV E6, E7, MAGE Al, ETV6-AML, sperm protein 17, XAGE1, Tie 2, MAD-CT-1, MAD-CT-2, Fos-related antigen 1, p53, p53 mutant, prostein, survivin and telomerase, PCTA-1 / galectin 8, MelanA / MART1, Ras mutant, hTERT, sarcoma translocation breakpoints, ML-IAP, ERG (TMPRSS2ETS fusion gene), NA17, PAX3, androgen receptor, cyclin B1, MYCN, RhoC, TRP-2, CYP1B1, BORIS, SART3, PAX5, OY-TES1, LCK, AKAP-4, SSX2, RAGE-1, human telomerase reverse transcriptase, RU1, RU2, intestinal carboxylesterase, mut hsp70-2, CD79a, CD79b, CD72, LAIR1, FCAR, LILRA2, CD300LF, CLEC12A, BST2, EMR2, LY75, GPC3, FCRL5, IGLL1, or any combination thereof.

[0025] In some aspects, the ligand binding protein specifically binds to ROR1. In some aspects, the ligand binding protein comprises an antigen binding domain derived from R12 antibody, R11 antibody, 2A2 antibody, or any combination thereof. In some aspects, the ligand binding protein comprises a heavy chain variable domain (VH) and a light chain variable domain (VL), wherein the VH comprises the amino acid sequence shown in SEQ ID NO: 17, and wherein the VL comprises the amino acid sequence shown in SEQ ID NO: 21.

[0026] In any of the above methods, in some aspects, the gene editing tool comprises shRNA, siRNA, miRNA, antisense oligonucleotide, CRISPR, zinc finger nuclease, TALEN, meganuclease, restriction endonuclease, or any combination thereof. In some aspects, the gene editing tool is CRISPR.

[0027] Also provided herein is a composition comprising cells having reduced levels of the NR4A3 gene and / or NR4A3 protein, wherein the composition has been prepared by any of the methods provided herein.

[0028] The present disclosure also provides a composition comprising a cell expressing reduced levels of an NR4A3 gene and / or an NR4A3 protein, wherein the cell has been modified with a gRNA capable of targeting the NR4A3 gene, wherein the gRNA comprises, consists of, or consists essentially of a sequence as set forth in any one of SEQ ID NO: 94, SEQ ID NO: 52, SEQ ID NO: 96, SEQ ID NO: 53, SEQ ID NO: 54, SEQ ID NO: 86, SEQ ID NO: 83, SEQ ID NO: 55, SEQ ID NO: 82, SEQ ID NO: 56, SEQ ID NO: 76, SEQ ID NO: 57, SEQ ID NO: 75, SEQ ID NO: 58, SEQ ID NO: 71, SEQ ID NO: 61, SEQ ID NO: 70, SEQ ID NO: 65, SEQ ID NO: 68, and SEQ ID NO: 67. In some aspects, the gRNA comprises, consists of, or consists essentially of a sequence as set forth in SEQ ID NO: 94. In some aspects, the gRNA comprises, consists of, or consists essentially of the sequence set forth in SEQ ID NO: 52. In some aspects, the gRNA comprises, consists of, or consists essentially of the sequence set forth in SEQ ID NO: 96. In some aspects, the gRNA comprises, consists of, or consists essentially of the sequence set forth in SEQ ID NO: 53. In some aspects, the gRNA comprises, consists of, or consists essentially of the sequence set forth in SEQ ID NO: 54. In some aspects, the gRNA comprises, consists of, or consists essentially of the sequence set forth in SEQ ID NO: 86. In some aspects, the gRNA comprises, consists of, or consists essentially of the sequence set forth in SEQ ID NO: 83. In some aspects, the gRNA comprises, consists of, or consists essentially of the sequence set forth in SEQ ID NO: 55. In some aspects, the gRNA comprises, consists of, or consists essentially of the sequence set forth in SEQ ID NO: 82. In some aspects, the gRNA comprises, consists of, or consists essentially of the sequence set forth in SEQ ID NO: 56. In some aspects, the gRNA comprises, consists of, or consists essentially of the sequence set forth in SEQ ID NO: 76. In some aspects, the gRNA comprises, consists of, or consists essentially of the sequence set forth in SEQ ID NO: 57. In some aspects, the gRNA comprises, consists of, or consists essentially of the sequence set forth in SEQ ID NO: 75. In some aspects, the gRNA comprises, consists of, or consists essentially of the sequence set forth in SEQ ID NO: 58.In some aspects, the gRNA comprises, consists of, or consists essentially of the sequence shown in SEQ ID NO: 71. In some aspects, the gRNA comprises, consists of, or consists essentially of the sequence shown in SEQ ID NO: 61. In some aspects, the gRNA comprises, consists of, or consists essentially of the sequence shown in SEQ ID NO: 70. In some aspects, the gRNA comprises, consists of, or consists essentially of the sequence shown in SEQ ID NO: 65. In some aspects, the gRNA comprises, consists of, or consists essentially of the sequence shown in SEQ ID NO: 68. In some aspects, the gRNA comprises, consists of, or consists essentially of the sequence shown in SEQ ID NO: 67.

[0029] In some aspects, any of the above compositions further comprises a pharmaceutically acceptable excipient.

[0030] The present disclosure also provides an isolated polynucleotide comprising, consisting of, or consisting essentially of a sequence as set forth in any one of SEQ ID NO: 94, SEQ ID NO: 52, SEQ ID NO: 96, SEQ ID NO: 53, SEQ ID NO: 54, SEQ ID NO: 86, SEQ ID NO: 83, SEQ ID NO: 55, SEQ ID NO: 82, SEQ ID NO: 56, SEQ ID NO: 76, SEQ ID NO: 57, SEQ ID NO: 75, SEQ ID NO: 58, SEQ ID NO: 71, SEQ ID NO: 61, SEQ ID NO: 70, SEQ ID NO: 65, SEQ ID NO: 68, and SEQ ID NO: 67. In some aspects, the gRNA comprises, consists of, or consists essentially of a sequence as set forth in SEQ ID NO: 94. In some aspects, the gRNA comprises, consists of, or consists essentially of a sequence as set forth in SEQ ID NO: 52. In some aspects, the gRNA comprises, consists of, or consists essentially of a sequence as set forth in SEQ ID NO: 96. In some aspects, the gRNA comprises, consists of, or consists essentially of the sequence set forth in SEQ ID NO: 53. In some aspects, the gRNA comprises, consists of, or consists essentially of the sequence set forth in SEQ ID NO: 54. In some aspects, the gRNA comprises, consists of, or consists essentially of the sequence set forth in SEQ ID NO: 86. In some aspects, the gRNA comprises, consists of, or consists essentially of the sequence set forth in SEQ ID NO: 83. In some aspects, the gRNA comprises, consists of, or consists essentially of the sequence set forth in SEQ ID NO: 55. In some aspects, the gRNA comprises, consists of, or consists essentially of the sequence set forth in SEQ ID NO: 82. In some aspects, the gRNA comprises, consists of, or consists essentially of the sequence set forth in SEQ ID NO: 56. In some aspects, the gRNA comprises, consists of, or consists essentially of the sequence set forth in SEQ ID NO: 76. In some aspects, the gRNA comprises, consists of, or consists essentially of the sequence set forth in SEQ ID NO: 57. In some aspects, the gRNA comprises, consists of, or consists essentially of the sequence set forth in SEQ ID NO: 75. In some aspects, the gRNA comprises, consists of, or consists essentially of the sequence set forth in SEQ ID NO: 58. In some aspects, the gRNA comprises, consists of, or consists essentially of the sequence set forth in SEQ ID NO: 71. In some aspects, the gRNA comprises, consists of, or consists essentially of the sequence set forth in SEQ ID NO: 61.In some aspects, the gRNA comprises, consists of, or consists essentially of the sequence set forth in SEQ ID NO: 70. In some aspects, the gRNA comprises, consists of, or consists essentially of the sequence set forth in SEQ ID NO: 65. In some aspects, the gRNA comprises, consists of, or consists essentially of the sequence set forth in SEQ ID NO: 68. In some aspects, the gRNA comprises, consists of, or consists essentially of the sequence set forth in SEQ ID NO: 67.

[0031] Some aspects of the present disclosure relate to cells comprising the above-mentioned polynucleotides. In some aspects, the cell further comprises a polynucleotide encoding a ligand-binding protein. In some aspects, a ligand-binding protein chimeric antigen receptor (CAR), a T cell receptor (TCR), a chimeric antibody-T cell receptor (caTCR), a chimeric signaling receptor (CSR), a T cell receptor mimic (TCR mimic) or a combination thereof. In some aspects, the cell further comprises (i) a nucleotide sequence encoding a c-Jun protein, (ii) a transcriptional activator capable of increasing the expression of endogenous c-Jun protein, or (iii) (i) and (ii) both.

[0032] In some aspects, cell is an immune cell. In some aspects, immune cell includes lymphocyte, neutrophil, monocyte, macrophage, dendritic cell or their combination. In some aspects, lymphocyte includes T cell, tumor infiltrating lymphocyte (TIL), lymphokine activated killer cell, natural (NK) cell or their combination.

[0033] Provided herein is a kit comprising (i) a polynucleotide comprising a gRNA that specifically targets a region within the NR4A3 gene and (ii) instructions for use, wherein the polynucleotide comprises, consists essentially of, or consists of a sequence set forth in any one of SEQ ID NO:94, SEQ ID NO:52, SEQ ID NO:96, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:86, SEQ ID NO:83, SEQ ID NO:55, SEQ ID NO:82, SEQ ID NO:56, SEQ ID NO:76, SEQ ID NO:57, SEQ ID NO:75, SEQ ID NO:58, SEQ ID NO:71, SEQ ID NO:61, SEQ ID NO:70, SEQ ID NO:65, SEQ ID NO:68, and SEQ ID NO:67. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1A and Figure 1BFigure 2 shows the NR4A3-edited (“NR4A3 KO”) and control non-edited CD4 T cells generated on day 7 after 2-hour CD3 / CD28 Dynabead stimulation in five independent donors (Stim, black circles). + ( Figure 1A ) and CD8 + ( Figure 1B ) Percentage of NR4A3 expression in anti-ROR1 CAR T cells. Unstimulated cells (white circles, no Dynabeads) served as a negative control. Unpaired t-tests were used for statistical analysis between stimulation conditions. *p < 0.05, **p < 0.005, ***p < 0.001, ****p < 0.0001.

[0035] Figure 2 Figure 3 shows the expression of NR4A1-edited ("NR4A1KO"), NR4A2-edited ("NR4A2KO"), NR4A3-edited ("NR4A3KO"), and control unedited CD4 T cells from five donors on day 7 of CAR T cell generation. + (white circle) and CD8 + (Black circles) EGFR in anti-ROR1 CAR T cells + R12 + Percentage of ROR1 CAR expression.

[0036] Figure 3 Figure 2 shows continuous anti-ROR1 lysis of H1975-NLR NSCLC cells by NR4A-edited or control unedited ROR1 CAR T cells ("x" symbols) and simulated untransduced T cells (squares) in five independent donors in a sequential stimulation assay. The NR4A-edited cells shown include: NR4A1 knockout (triangle), NR4A2 knockout (star), and NR4A3 knockout (circle). The lysis of H1975-NLR target cells was quantified by measuring the total NLR intensity. The NLR intensity was normalized to the starting intensity after each round of stimulation and re-seeding. NLR-NucLight Red.

[0037] Figures 4A to 4C Shows the corresponding Figure 3 Secreted interferon-γ (IFN-γ) produced by NR4A-edited, control unedited anti-ROR1 CAR ("x" symbols), and mock untransduced T cells (squares) during sequential stimulation assay with H1975 ( Figure 4A ), interleukin-2 (IL-2) ( Figure 4B ) and tumor necrosis factor-α (TNF-α) ( Figure 4C). The NR4A-edited cells shown include: NR4A1 knockout (triangle), NR4A2 knockout (star), and NR4A3 knockout (circle). Supernatants were collected 24 hours after each reseeding, and cytokines were quantified by MSD. Each column shows data from 5 independent donors. Error bars represent the mean + / - SD of three wells. Unpaired t-test was used for statistical analysis. *p<0.05, **p<0.005, ***p<0.001, ****p<0.0001.

[0038] Figure 5A Shows the corresponding Figure 3 CD4 T cells from NR4A-edited, control unedited ROR1 CAR ("x" symbols), and mock untransduced T cells (squares) after each re-vaccination during the H1975 sequential stimulation assay. + (above) and CD8 + (Bottom) Anti-ROR1 CAR expression on T cells. NR4A-edited cells shown include: NR4A1 knockout (triangle), NR4A2 knockout (star), and NR4A3 knockout (circle). D0 shows the frequency of CAR expression at the start of the assay. Figure 5B Shows the corresponding Figure 3 Expected CD3 from NR4A-edited and control unedited anti-ROR1 CAR T cells during H1975 sequential stimulation assay + anti-ROR1 CAR + Fold change in T cell number. Calculate the expected cell number to include 25% of cells carried over to the next stimulation. Fold change is calculated as (expected number of cells from stimulation / expected number of cells from previous stimulation). Figure 5B , the bars for each of Stim1, Stim2, and Stim3 correspond to the following: (i) NR4A1 knockout (triangle; first bar); (ii) NR4A2 knockout (star; second bar); (iii) NR4A3 knockout (circle; third bar); and (iv) control unedited ROR1 CAR (x symbol; fourth bar). Each graph represents an independent donor.

[0039] Figure 6 Shows the corresponding Figure 3 H1975 stimulated the ROR1 CAR of NR4A3-edited ("NR4A3 KO") and control unedited anti-ROR1 CAR cells sequentially. + CD4 + (above) and CD8 +(Bottom) Expression of inhibitory receptors (LAG3, TIM3, CD39, and PD-1) on T cells. Paired t-test was used for statistical analysis. **p < 0.005. n = 5 independent donors.

[0040] 7A to 7C Shown are secreted interferon-γ (IFN-γ) produced by NR4A-edited and control non-edited anti-ROR1CAR T cells co-cultured with A549 (top row) or H1975 (bottom row) tumor cells in five independent donors after seven days of ROR1 antigen stimulation in the H1975 chronic stimulation assay ( Figure 7A ), interleukin-2 (IL-2) ( Figure 7B ) and tumor necrosis factor-α (TNF-α) ( Figure 7C ). Supernatant was collected 24 hours after fresh co-culture was established on the seventh day, and cytokines were quantified by MSD. Each figure represents an independent donor. In each figure, the bars correspond to the following: (i) NR4A1 knockout (triangle; first bar); (ii) NR4A2 knockout (star; second bar); (iii) NR4A3 knockout (circle; third bar); and (iv) control unedited anti-ROR1 CAR (x symbol; fourth bar). Each column shows data from 5 independent donors. Error bars represent the mean + / - SD of three wells. Unpaired t-test was used for statistical analysis. *p<0.05, **p<0.005, ***p<0.001, ****p<0.0001.

[0041] Figure 8A Figure 7 shows CD4 T cells from NR4A-edited and control unedited ROR1 CAR T cells during the H1975 chronic stimulation assay corresponding to Figure 7 + (above) and CD8 + (Bottom) ROR1 CAR expression on T cells. NR4A-edited cells are shown: NR4A1 knockout (triangles), NR4A2 knockout (stars), and NR4A3 knockout (circles). Figure 8B Figure 7 shows CD3 from NR4A-edited and control unedited ROR1 CAR T cells during a seven-day H1975 chronic stimulation assay corresponding to Figure 7 + anti-ROR1 CAR + Fold change in T cell number. Fold change was calculated as (number of cells with current E:T reset / number of cells with previous E:T reset). Each graph represents an independent donor. Figure 8B, the bars shown for each of day 2, day 4, and day 7 correspond to the following: (i) NR4A1 knockout (triangle; first bar); (ii) NR4A2 knockout (star; second bar); (iii) NR4A3 knockout (circle; third bar); and (iv) control unedited ROR1 CAR (x symbol; fourth bar). Each column shows data from 5 independent donors.

[0042] Figure 9 Anti-ROR1 CAR from NR4A3-edited ("NR4A3KO") and control unedited anti-ROR1 CAR cells on day 7 of the H1975 chronic stimulation assay corresponding to FIG. + CD4 + (above) and CD8 + (Bottom) Expression of inhibitory receptors (LAG3, TIM3, CD39, and PD-1) on T cells. n = 5 independent donors.

[0043] Figure 10A and Figure 10B Showing improved in vivo efficacy of NR4A3-edited anti-ROR1 CAR T cells. Figure 10A Shows tumor volume, and Figure 10B Shown is the survival rate of NSG mice transplanted with subcutaneous flank H1975 xenograft tumors. When the average tumor volume reached 80-120 mm 3 At 0.6×10 6 (upper panel, low dose) or 2×10 6 (lower image, high dose) CAR + T cells were treated with NR4A-edited or control unedited anti-ROR1 CAR T cells iv. Mice. n = 5 mice / group. Error bars represent mean + / - SEM. Survival curve statistics were calculated by log-rank (Manel-Cox) test. ***p < 0.001 and ***p < 0.0001. The different groups shown include: (i) NR4A1 knockout (triangle), (ii) NR4A2 knockout (star), (iii) NR4A3 knockout (circle), (iv) control unedited anti-ROR1CAR (x symbol), and mock non-transduced T cells (square).

[0044] Figure 11A and Figure 11BSerial lysis of A549-NLR and H1975-NLR cells by anti-ROR1 CAR T cells modified with the following multiple members of the NR4A family with reduced levels is shown: (1) both NR4A1 and NR4A2 ("NR4A 1+2DKO") (open circles), (2) both NR4A1 and NR4A3 ("NR4A1+3DKO") (triangles), (3) both NR4A2 and NR4A3 ("NR4A2+3DKO") (stars), and (4) NR4A1, NR4A2, and NR4A3 ("NR4ATKO") (asterisks). Anti-ROR1 CAR T cells with only NR4A3 (filled circles) with reduced levels are also shown for comparison. Simulations (untransduced T cells without ROR1 CAR or NR4A editing) (squares) are shown as controls. The lysis of H1975-NLR target cells is quantified by measuring total NLR intensity. NLR intensities were normalized to the starting intensity after each round of stimulation and reseeding. NLR-NucLight Red.

[0045] 12A to 12C A continuous stimulation assay using A549 target cells is shown (see Figure 11A ) during the period of IFN-γ production by anti-ROR1CAR T cells with reduced levels of multiple members of the NR4A family ( Figure 12A ), IL-2( Figure 12B ) and TNF-α( Figure 12C ) levels. Figure 11A and Figure 11B Supernatants were collected 24 hours after each re-seeding (ie, stim 1, stim 2, stim 3, stim 4, and stim 5), and cytokines were quantified by MSD.

[0046] 13A to 13C A continuous stimulation assay using H1975 target cells is shown (see Figure 11B ) during the period of IFN-γ production by anti-ROR1CAR T cells with reduced levels of multiple members of the NR4A family ( Figure 13A ), IL-2( Figure 13B ) and TNF-α( Figure 13C ) levels. Figure 11A and Figure 11B Supernatants were collected 24 hours after each re-seeding (ie, stim 1, stim 2, stim 3, stim 4, and stim 5), and cytokines were quantified by MSD.

[0047] Figure 14 Figure 2 shows sequential lysis of NY-ESO-1+A375-NLR melanoma cells by NR4A-edited (KO), control unedited NY-ESO-1 TCR T cells, and mock untransduced T cells in a sequential stimulation assay in three independent donors. Lysis of A375-NLR target cells was quantified by measuring total NLR counts. NLR counts were normalized to the starting counts after re-seeding after each round of stimulation. NLR–NucLight Red. Each graph shows data from three independent donors.

[0048] Figures 15A to 15C Shows the corresponding Figure 14 During the A375 sequential stimulation assay, secreted interferon-γ (IFN-γ) produced by NR4A-edited, control unedited NY-ESO-1 TCR T cells and simulated uninduced T cells ( Figure 15A ), interleukin-2 (IL-2) ( Figure 15B ) and tumor necrosis factor-α (TNF-α) ( Figure 15C ). Supernatants were collected 24 hours after each reseeding and cytokines were quantified by MSD. Each figure shows data from 3 independent donors. Figures 15A to 15C In each of the figures, for each stimulation (i.e., Stim 1, Stim 2, Stim 3, and Stim 4), the first bar (from the left) is NR4A1-edited NY-ESO-1 TCR T cells (NR4A1 KO; triangles), the second bar is NR4A2-edited NY-ESO-1 TCR T cells (NR4A2 KO; asterisks); the third bar is NR4A3-edited NY-ESO-1 TCR T cells (NR4A3 KO; filled circles); the fourth bar is unedited control NY-ESO-1 TCR T cells (control; x symbols); and the fifth bar is untransduced T cells (mock; squares).

[0049] Figure 16 Figure 2 shows the expression of NR4A3-edited and control CD19-edited CD4 T cells with c-Jun overexpression on day 7 of CAR T cell generation after 2 h PMA + ionomycin stimulation (Stim, filled circles; bar 2 for each of the sgRNA or control groups) in three independent donors. + (Left) and CD8 +(Right) Percentage of NR4A3 expression in ROR1 CAR T cells. Unstimulated cells (open circles, no PMA + ionomycin; bar 1 of each of the sgRNA or control groups) were used as negative controls. As further described in Example 9, for NR4A3 editing, the following gRNAs were used: (1) g4 (SEQ ID NO: 30), (2) g20 (SEQ ID NO: 67), (3) g29 (SEQ ID NO: 76), (4) g47 (SEQ ID NO: 94), and (5) g49 (SEQ ID NO: 96). Statistical analysis was performed using an unpaired t-test of the stimulation conditions compared to control CD19-edited ROR1 CAR T cells. **p < 0.005, ***p < 0.001, ****p < 0.0001.

[0050] Figure 17 Figure 3 shows the expression of NR4A3-edited and control CD19-edited CD4 T cells with c-Jun overexpression on day 7 of CAR T cell generation from three donors. + (open circles; bar 1 of each of sgRNA or control groups) and CD8 + (Filled circles; bar 2 for each of sgRNA or control groups) In ROR1 CAR T cells, EGFR + R12 + Percentage of ROR1 CAR expression (left) and EGFR + R12 + Geometric mean fluorescence of ROR1 CAR on T cells (right). For NR4A3 editing, the gRNA used was Figure 16 The same as described in . Unpaired t test was used for statistical analysis and the data were not significant.

[0051] Figure 18Figure 2 shows sequential anti-ROR1 lysis of H1975-NLR NSCLC cells by NR4A3-edited and control CD19-edited ROR1 CAR T cells with c-Jun overexpression in a sequential stimulation assay in three independent donors. Lysis of H1975-NLR target cells was quantified by measuring total NLR intensity. NLR intensity was normalized to the starting intensity after each round of stimulation and re-seeding. NLR–NucLight Red. Each graph shows data from one of three independent donors. For NR4A3-edited ROR1 CAR T cells, one of the following gRNAs was used: (1) g4 (SEQ ID NO: 30) (black circles), (2) g20 (SEQ ID NO: 67) (open circles), (3) g29 (SEQ ID NO: 76) (closed diamonds), (4) g47 (SEQ ID NO: 94) (open diamonds), and (5) g49 (SEQ ID NO: 96) (closed squares). H1975-NLR NSCLC cells alone (ie, no ROR1 CAR T cells) were used as a control (open squares). The "x" symbol corresponds to the control CD19-edited ROR1 CAR T cells.

[0052] 19A to 19C Shows the corresponding Figure 18 Secreted interferon-γ (IFN-γ) produced by NR4A3-edited and control CD19-edited ROR1 CAR T cells with c-Jun overexpression during the H1975 sequential stimulation assay ( Figure 19A ), interleukin-2 (IL-2) ( Figure 19B ) and tumor necrosis factor-α (TNF-α) ( Figure 19C). Supernatants were collected 24 hours after each re-inoculation and cytokines were quantified by MSD. Each figure shows data from 3 independent donors. Error bars represent the mean + / - SD of three wells. The NR4A3-edited ROR1 CAR T cells shown were edited with one of the following gRNAs: (1) g4 (SEQ ID NO: 30) (black circles; the first bar of each stimulation), (2) g20 (SEQ ID NO: 67) (open circles; the second bar of each stimulation), (3) g29 (SEQ ID NO: 76) (solid diamonds; the third bar of each stimulation), (4) g47 (SEQ ID NO: 94) (open diamonds; the fourth bar of each stimulation) and (5) g49 (SEQ ID NO: 96) (solid squares; the fifth bar of each stimulation). H1975-NLR NSCLC cells alone (i.e., no ROR1 CAR T cells) were used as controls (open squares; the last bar of each stimulation). The “x” symbols (fifth bar per stimulation) correspond to control CD19-edited ROR1 CART cells.

[0053] FIG. 20A to FIG. 20B Improved in vivo efficacy of NR4A3-edited ROR1CAR T cells with c-Jun overexpression was shown. Tumor volume ( Figure 20A ) and peripheral blood CD3 + CAR + T cell count ( Figure 20B When the average tumor volume reaches 80-120 mm 3 When using 10×10 6 mock untransduced unedited T cells or 10 × 10 6 CAR + NR4A3-edited or control CD19-edited ROR1 CAR T cells were injected intravenously into each mouse. n = 5 mice / group. Error bars represent mean + / - SEM. Mean tumor volume curves were truncated when 20% of mice / group were removed from the study due to humanitarian endpoints. Each figure shows data from one independent donor. Tukey one-way ANOVA of tumor volume and peak CD3 + CAR + Unpaired t-test was used for statistical analysis of peripheral blood fold expansion of T cell numbers. *p<0.05, **p<0.005, ****p<0.0001.

[0054] Figures 21A to 21BImproved in vivo anti-tumor activity of NR4A3 g4-edited and NR4A3 g47-edited ROR1 CART cells with c-Jun overexpression was shown. As further described in Example 11, the modified T cells were administered to NSG mice transplanted with subcutaneous flank H1975 xenograft tumors, and the tumor volume at different time points was assessed ( Figure 21A ) and the number of T cells in peripheral blood ( Figure 21B ). The modified T cells were administered at one of two doses: 0.4 × 10 6 cells / mouse (left panel, low dose) or 2×10 6 cells / mouse (right panel, high dose). Error bars represent mean + / - SEM. Mean tumor volume curves were truncated when 20% of mice / group were removed from the study due to humanitarian endpoints. DETAILED DESCRIPTION

[0055] The present disclosure generally relates to polynucleotides (e.g., isolated polynucleotides) that can reduce the level of NR4A3 genes and / or NR4A3 proteins in immune cells (e.g., T cells). As described herein, the polynucleotides of the present disclosure comprise nucleotide sequences complementary to nucleic acid sequences within the NR4A3 gene (also referred to herein as "NR4A3 targeting nucleotide sequences" or variants thereof) so that the polynucleotides described herein can interact with the NR4A3 gene, thereby reducing the level of NR4A3 genes and / or NR4A3 proteins in immune cells. Those skilled in the art will appreciate that such polynucleotides can be used in conjunction with various gene editing technologies (e.g., CRISPR / Cas systems). In addition, in some aspects, such polynucleotides can be used in combination with one or more additional nucleotide sequences described herein (e.g., encoding ligand binding proteins and / or c-Jun proteins). In some aspects, such polynucleotides can be used in combination with one or more additional nucleotide sequences that are complementary to nucleic acid sequences within other members of the NR4A family (i.e., NR4A1 and / or NR4A2). As described herein, in some aspects, by reducing the level of NR4A3 gene and / or NR4A3 protein, the polynucleotides of the present disclosure can be used to improve one or more functions of immune cells (e.g., increased persistence and / or effector activity). Reducing the level of NR4A3 gene and / or NR4A3 protein (alone or in combination with reduced levels of NR4A1 gene and / or NR4A1 protein and / or NR4A2 gene and / or NR4A2 protein) can lead to exhaustion / dysfunction-resistant cells. In addition, reducing the level of NR4A3 gene and / or NR4A3 protein (alone or in combination with reduced levels of NR4A1 gene and / or NR4A1 protein and / or NR4A2 gene and / or NR4A2 protein) can lead to maintaining anti-tumor function in the TME environment. In some aspects, the present disclosure also relates to methods for treating a variety of diseases or conditions (e.g., cancer) in a subject in need thereof, comprising administering to the subject an immune cell as described herein that has been modified to reduce the level of NR4A3 gene and / or NR4A3 protein. Additional aspects of the present disclosure are provided throughout this application.

[0056] Before describing the present disclosure in more detail, it should be understood that the present disclosure is not limited to the specific compositions or method steps described, and therefore such compositions or method steps may of course vary. It will be apparent to those skilled in the art upon reading this disclosure that each individual aspect described and illustrated herein has discrete components and features that can be easily separated or combined with the features of any other several aspects without departing from the scope or spirit of the present disclosure. Any method described can be carried out in the order of events described or in any other order that is logically possible.

[0057] The headings provided herein are not limitations of the various aspects of the disclosure, which can be defined by reference to the specification as a whole. It should also be understood that the terminology used herein is for the purpose of describing particular aspects only and is not intended to be limiting, as the scope of the disclosure will be limited only by the appended claims.

[0058] I. Terminology

[0059] To make this disclosure more easily understood, certain terms are first defined. As used in this application, unless otherwise expressly provided herein, each of the following terms shall have the meanings described below. Additional definitions are set forth throughout the application.

[0060] Throughout the disclosure, the term "a" or "an" entity refers to one or more of the entities; for example, "immune cell" is understood to refer to one or more immune cells. Therefore, the terms "a" (or "an"), "one or more" and "at least one" are used interchangeably herein.

[0061] Furthermore, when used herein, "and / or" will be considered as a specific disclosure of each of the two specified features or components in the presence or absence of the other. Thus, the term "and / or" as used in phrases herein (e.g., "A and / or B") is intended to include "A and B," "A or B," "A" (alone), and "B" (alone). Likewise, the term "and / or" as used in phrases such as "A, B, and / or C" is intended to include each of the following: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone).

[0062] It should be understood that wherever an aspect is described herein using the phrase "comprising," other similar aspects described as "consisting of" and / or "consisting essentially of" are also provided.

[0063] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure relates. For example, Concise Dictionary of Biomedicine and Molecular Biology, Juo, Pei-Show, 2nd ed., 2002, CRC Press; The Dictionary of Cell and Molecular Biology, 3rd ed., 1999, Academic Press; and Oxford Dictionary of Biochemistry and Molecular Biology, Revised, 2000, Oxford University Press provide those of skill in the art with a general dictionary of many of the terms used in this disclosure.

[0064] Units, prefixes, and symbols are expressed in the form accepted by the International System of Units (SI). Numerical ranges include the numbers defining the range. Unless otherwise indicated, amino acid sequences are written from left to right in an amino to carboxyl orientation. The headings provided herein are not limitations of the various aspects of the disclosure, which can be obtained by reference to the specification as a whole. Therefore, the terms that will be defined below are more fully defined by reference to the specification as a whole.

[0065] Abbreviations used herein are defined throughout this disclosure. Various aspects of the disclosure are further described in detail in the following subsections.

[0066] As used herein, the term "about" or "approximately" when applied to one or more values ​​of interest refers to a value similar to the reference value. In some aspects, unless otherwise specified or obvious from the context, the term "about" refers to a range of values ​​that are within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1% or less in either direction (greater than or less than) the reference value (except where such a value would exceed 100% of the possible value).

[0067] As described herein, unless otherwise indicated, any concentration range, percentage range, ratio range, or integer range should be understood to include the value of any integer within the range, and, where appropriate, fractions thereof (such as tenths and hundredths of integers).

[0068] As used herein, " administration " refers to using any of various methods well known to persons skilled in the art and delivery system by which therapeutic agents or compositions comprising therapeutic agents are physically introduced into a subject. The different routes of administration of therapeutic agents as described herein include intravenous, intraperitoneal, intramuscular, subcutaneous, spinal or other parenteral routes of administration, for example, by injection or infusion. As used herein, phrase " parenteral administration " means the mode of administration except enteral and local (topical) administration, usually by injection, and includes but is not limited to intravenous, intraperitoneal, intramuscular, intraarterial, intrathecal, intralymphatic, intralesional, intracapsular, intraorbital, intracardial, intradermal, transtracheal, intratracheal, lung, subcutaneous, subcutaneous, intraarticular, subcapsular, subarachnoid, intraventricular, intravitreal, epidural and intrasternal injection and infusion, and electroporation in vivo. Alternatively, therapeutic agents as described herein can be administered via non-parenteral route, such as local, epidermal or mucosal administration route, for example, intranasal, oral, vaginal, rectal, sublingual or topical. Administration can also be performed, for example, once, multiple times, and / or over one or more extended periods of time.

[0069] As used herein, the term "antigen" refers to any natural or synthetic immunogenic substance, such as a protein, peptide, or hapten. As used herein, the term "cognate antigen" refers to an antigen recognized by an immune cell (e.g., a T cell), thereby inducing activation of the immune cell (e.g., triggering an intracellular signal that induces effector functions, such as cytokine production and / or cell proliferation).

[0070] Nucleotide can be represented by its generally accepted single letter code.Unless otherwise indicated, nucleic acid is written from left to right with 5 ' to 3 ' orientation.Nucleotide is mentioned in this article with its well-known single letter symbol recommended by IUPAC-IUB Biochemical Nomenclature Committee.Therefore, A represents adenine, C represents cytosine, G represents guanine, T represents thymine, and U represents uracil.

[0071] It should be understood that in the disclosed sequences, T and U can be interchanged, depending on whether the sequence is DNA or RNA. For example, in the present disclosure, the gRNA spacer sequence appears in the form of DNA (A / T / C / G), while the gRNA chimeric framework appears in the form of RNA (A / U / C / G).

[0072] Amino acids may be referred to herein by either their commonly known three letter symbols or by the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission.Unless otherwise indicated, amino acid sequences are written left to right in amino to carboxyl orientation.

[0073] "Polypeptide" refers to a chain of at least two consecutively linked amino acid residues, with no upper limit to the length of the chain. One or more amino acid residues in a protein may contain modifications such as, but not limited to, glycosylation, phosphorylation, or disulfide bond formation. A "protein" may comprise one or more polypeptides. Unless otherwise indicated, the terms "protein" and "polypeptide" are used interchangeably.

[0074] As used herein, the term "nucleic acid molecule" is intended to include DNA molecules and RNA molecules. The nucleic acid molecule can be single-stranded or double-stranded and can be a cDNA.

[0075] As used herein, the term "polynucleotide" refers to a polymer of nucleotides of any length, including ribonucleotides, deoxyribonucleotides, analogs thereof, or mixtures thereof. The term refers to the primary structure of the molecule. Thus, the term includes triple-stranded, double-stranded, and single-stranded deoxyribonucleic acids ("DNA"), as well as triple-stranded, double-stranded, and single-stranded ribonucleic acids ("RNA"). It also includes modified (e.g., by alkylation and / or by capping) and unmodified polynucleotide forms. Specifically, the term "polynucleotide" includes polydeoxyribonucleotides (containing 2-deoxy-D-ribose), polyribonucleotides (containing D-ribose), including mRNA and gRNA, whether spliced ​​or unspliced, any other type of polynucleotide (which is an N- or C-glycoside of a purine or pyrimidine base), and other polymers containing an orthonucleotide backbone, such as polyamides (e.g., peptide nucleic acid "PNA") and polymorpholino polymers, and other synthetic sequence-specific nucleic acid polymers (provided that the polymer contains nucleobases in a configuration that allows base pairing and base stacking, such as those found in DNA and RNA). Unless otherwise indicated, the terms "polynucleotide," "nucleic acid," "gene," "cDNA," and "mRNA" are used interchangeably.

[0076] The term "gene" refers to a DNA segment involved in producing a polypeptide chain. It may include regions before and after the coding region (leader and trailer) and intervening sequences (introns) between individual coding segments (exons).

[0077] As used herein, the term "vector" is intended to refer to a nucleic acid molecule capable of transporting another nucleic acid connected thereto. One type of vector is a "plasmid", which refers to a circular double-stranded DNA loop to which an additional DNA segment can be connected. Another type of vector is a viral vector, in which an additional DNA segment can be connected to a viral genome. Some vectors can replicate autonomously in the host cell into which they are introduced (for example, bacterial vectors and episomal mammalian vectors with bacterial replication origins). Other vectors (for example, non-episomal mammalian vectors) can be integrated into the genome of the host cell after being introduced into the host cell, and thus replicated together with the host genome. In addition, some vectors can guide the expression of the gene to which they are operably connected. Such vectors are referred to as "recombinant expression vectors" (or simply "expression vectors") in this article. Typically, expression vectors useful in recombinant DNA technology are typically in the form of plasmids. In this specification, "plasmid" and "vector" are used interchangeably because plasmids are the most commonly used vector forms. However, other forms of expression vectors providing equivalent functions are also included, such as viral vectors (for example, replication-defective retroviruses, adenoviruses, and adeno-associated viruses).

[0078] "Cancer" refers to a wide variety of diseases characterized by the uncontrolled growth of abnormal cells in the body. Unregulated cell division and growth can form malignant tumors that invade neighboring tissues and also spread to distant parts of the body through the lymphatic system or bloodstream. As used herein, "cancer" refers to primary, metastatic, and recurrent cancers.

[0079] As used herein, the term "immune response" refers to the biological response of an exogenous agent in a vertebrate, and the response protects the organism against these agents and the disease caused thereby. Immune response is mediated by the action of cells of the immune system (e.g., T lymphocytes, B lymphocytes, natural killer (NK) cells, macrophages, eosinophils, mast cells, dendritic cells or neutrophils) and soluble macromolecules (including antibodies, cytokines and complement) produced by any of these cells or the liver, and the immune response causes selective targeting, binding, damage, destruction of invading pathogens, cells or tissues infected with pathogens, cancer cells or other abnormal cells or (in the case of autoimmunity or pathological inflammation) normal human cells or tissues and / or eliminates them from the vertebrate body. Immune response includes, for example, activation or suppression of T cells, for example effector T cells or Th cells, such as CD4 + or CD8 + cells, or T reg As used herein, the terms "T cell" and "T lymphocyte" are interchangeable and refer to any lymphocyte produced or processed by the thymus. In some aspects, the T cell is a CD4 + In some aspects, the T cells are CD8+ T cells. In some aspects, the T cells are NKT cells.

[0080] As used herein, the term "anti-tumor immune response" refers to an immune response against a tumor antigen. The ability to stimulate an immune response or the immune system may be enhanced by increased agonistic activity of T cell co-stimulatory receptors and / or increased antagonistic activity of inhibitory receptors. The increase in the ability to stimulate an immune response or the immune system may be caused by EC 50 In some aspects, the ability to stimulate an immune response or immune system activity can be enhanced by, for example, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95% or at least about 100%. In some aspects, the ability to stimulate an immune response or immune system activity can be enhanced, for example, by at least about 1.2-fold, at least about 1.4-fold, at least about 1.6-fold, at least about 1.8-fold, at least about 2-fold, at least about 3-fold, at least about 4-fold, at least about 5-fold, at least about 6-fold, at least about 7-fold, at least about 8-fold, at least about 9-fold, at least about 10-fold, or more.

[0081] "Subject" includes any human or non-human animal, and the term "non-human animal" includes, but is not limited to, vertebrates, such as non-human primates, sheep, dogs, and rodents, such as mice, rats, and guinea pigs. In some aspects, the subject is a human. The terms "subject" and "patient" are used interchangeably herein.

[0082] The term "effective amount" or "effective dose" refers to the amount of an agent (e.g., a modified immune cell disclosed herein) that provides a desired biological, therapeutic, and / or preventive result. The result can be a reduction, improvement, alleviation, reduction, delay, and / or alleviation of one or more signs, symptoms, or causes of disease, or any other desired change in a biological system. With regard to solid tumors, an effective amount comprises an amount sufficient to cause tumor shrinkage and / or sufficient to reduce tumor growth rate (e.g., inhibit tumor growth) or sufficient to prevent or delay other undesirable cell proliferation. In some aspects, an effective amount is an amount sufficient to delay tumor development. In some aspects, an effective amount is an amount sufficient to prevent or delay tumor recurrence. An effective amount can be administered in one or more administrations. An effective amount of the composition can, for example, (i) reduce the number of cancer cells; (ii) reduce the size of a tumor; (iii) inhibit, block, slow down, and prevent to some extent the infiltration of cancer cells into peripheral organs; (iv) inhibit (i.e., slow down, and prevent to some extent) tumor metastasis; (v) inhibit tumor growth; (vi) prevent or delay the occurrence and / or recurrence of a tumor; and / or (vii) alleviate to some extent one or more symptoms associated with cancer.

[0083] The ability of a therapeutic agent to promote disease regression can be assessed using a variety of methods known to skilled practitioners, such as in human subjects during clinical trials, in animal model systems predictive of efficacy in humans, or by measuring the agent's activity in in vitro assays.

[0084] As used herein, the term "standard of care" refers to a treatment that is accepted by medical experts as an appropriate treatment for a certain type of disease and is widely used by healthcare professionals. The term may be used interchangeably with any of the following terms: "best practice," "standard of medical care," and "standard therapy."

[0085] For example, an "anti-cancer agent" promotes the regression of cancer in a subject or prevents further tumor growth. In some aspects, a therapeutically effective amount of a drug promotes the regression of cancer to the extent that it eliminates the cancer.

[0086] "Promoting cancer regression" means that administration of an effective amount of a drug, alone or in combination with an anti-neoplastic agent, results in a decrease in tumor growth or size, tumor necrosis, a decrease in the severity of at least one disease symptom, an increase in the frequency and duration of disease symptom-free periods, or prevention of impairment or disability resulting from disease affliction.

[0087] The terms "effective" and "effectiveness" with respect to treatment include pharmacological efficacy and physiological safety. Pharmacological efficacy refers to the ability of a drug to promote the regression of cancer in a patient. Physiological safety refers to the level of toxicity or other adverse physiological reactions (adverse reactions) at the cellular, organ, and / or organismal level caused by drug administration.

[0088] As used herein, the term "immune checkpoint inhibitor" refers to a molecule that completely or partially reduces, inhibits, interferes with, or modulates one or more checkpoint proteins. Checkpoint proteins regulate T cell activation or function. Many checkpoint proteins are known, such as CTLA-4 and its ligands CD80 and CD86; and PD-1 and its ligands PD-L1 and PD-L2. Pardoll, DM, Nat Rev Cancer 12(4):252-64(2012). These proteins are responsible for the co-stimulatory or inhibitory interactions of T cell responses. Immune checkpoint proteins regulate and maintain self-tolerance and the duration and amplitude of physiological immune responses. Immune checkpoint inhibitors include antibodies or are derived from antibodies.

[0089] As used herein, the term "oxidative stress" refers to a condition characterized by an excess of oxidants and / or decreased levels of antioxidants. Cellular oxidants may include, but are not limited to, oxygen free radicals (superoxide anions, hydroxyl radicals, and / or peroxyl radicals); reactive non-radical oxygen species such as hydrogen peroxide and singlet oxygen; carbon free radicals; nitrogen free radicals; sulfur free radicals; and combinations thereof. In some aspects, oxidative stress conditions can result in, for example, cell damage, impaired cell performance, and / or cell death.

[0090] As used herein, the term "modified cell" refers to a cell, such as a T cell, that has undergone non-natural engineering such that the phenotype of the cell (i.e., the expression level of the NR4A3 gene and / or NR4A3 protein) is different from that of an unmodified cell (i.e., a reference cell). As can be seen from the present disclosure, the modified cells disclosed herein have been modified (e.g., modified with a gene editing tool comprising a polynucleotide described herein) to express reduced levels of NR4A3 gene and / or NR4A3 protein compared to a reference cell (e.g., a corresponding cell that has not been modified). As described herein, in some aspects, the modified cells can express normal levels (i.e., "endogenous levels") of NR4A1 gene and / or NR4A1 protein and NR4A2 gene and / or NR4A2 protein. In some aspects, the modified cells of the present disclosure can express: (i) reduced levels of NR4A3 gene and / or NR4A3 protein, and (ii) reduced levels of NR4A1 gene and / or NR4A1 protein. In some aspects, the modified cells described herein can express: (i) reduced levels of the NR4A3 gene and / or NR4A3 protein, and (ii) reduced levels of the NR4A2 gene and / or NR4A2 protein. In some aspects, the modified cells can express: (i) reduced levels of the NR4A3 gene and / or NR4A3 protein, (ii) reduced levels of the NR4A1 gene and / or NR4A1 protein, and (iii) reduced levels of the NR4A2 gene and / or NR4A2 protein. As used herein, the term "corresponding cell" refers to a cell that belongs to the same immune cell classification as the modified cell. For example, if the modified cell is a T cell, then the corresponding cell will also be a T cell. Unless otherwise specified, "modified cells having (expressing) reduced levels of the NR4A3 gene and / or NR4A3 protein" (including variants thereof) include cells (e.g., T cells) that have been modified to have reduced levels of the NR4A3 gene and / or NR4A3 protein, and: (i) endogenous levels of the NR4A1 and NR4A2 genes and NR4A1 and NR4A2 proteins; (ii) reduced levels of the NR4A1 gene and / or NR4A1 protein; (iii) reduced levels of the NR4A2 gene and / or NR4A2 protein; or (iv) reduced levels of both the NR4A1 gene and / or NR4A1 protein and reduced levels of the NR4A2 gene and / or NR4A2 protein.

[0091] As used herein, the term "endogenous expression" or "endogenous expression level" or "endogenous level" (or variants thereof) refers to gene and / or protein expression (e.g., amount, kinetics, etc.) that occurs naturally (e.g., the gene and / or protein is not directly manipulated by non-naturally occurring engineering). It can be seen from the present disclosure that in some aspects, the modified cells disclosed herein (e.g., immune cells that express a ligand binding protein and are modified with a gene editing tool comprising a polynucleotide of the present disclosure) do not express endogenous levels of the NR4A3 gene or NR4A3 protein, but because the NR4A1 and NR4A2 genes have not been modified (e.g., by CRISPR, e.g., non-naturally occurring engineering), the modified cells endogenously express the NR4A1 and NR4A2 genes and / or NR4A1 and NR4A2 proteins. As described herein, in some aspects, the modified cells that express reduced levels of the NR4A3 gene or NR4A3 protein can be further modified to also express reduced levels of the following genes or proteins: (i) the NR4A1 gene or NR4A1 protein, (ii) the NR4A2 gene or NR4A2 protein, or (iii) both (i) and (ii).

[0092] In some aspects, the modified cells are produced by introducing an exogenous or foreign nucleic acid (e.g., a polynucleotide described herein comprising a gRNA that can specifically target a region within the NR4A3 gene) into the cell. In some aspects, the exogenous or foreign nucleic acid can encode a gene editing tool disclosed herein. Nucleic acids can be introduced into cells by methods known in the art, such as, for example, electroporation (see, for example, Heiser WC Transscription Factor Protocols: Methods in Molecular Biology TM 2000;130:117-134), chemical (e.g., calcium phosphate or lipid) transfection (see, e.g., Lewis WH et al., Somatic Cell Genet. 1980 May;6(3):333-47; Chen C. et al., Mol Cell Biol. 1987 Aug;7(8):2745-2752), fusion with bacterial protoplasts containing the recombinant plasmid (see, e.g., Schaffner W. Proc Natl Acad Sci USA. 1980 Apr;77(4):2163-7), or direct microinjection of purified DNA into the cell nucleus (see, e.g., Capecchi MR Cell. 1980 Nov;22(2 Pt 2):479-88).

[0093] It will be understood that disclosures relating to "modified cells" or "cells" apply equally to a population of these cells, ie a plurality of these cells.

[0094] As used herein, the term "editing" (and grammatical variants thereof) refers to a process in which a cell (e.g., a T cell) is modified to make the cell functionally and / or structurally different from a corresponding unmodified cell. More specifically, as further described herein, provided herein are cells that have been edited so that, compared to unedited cells, the cell exhibits reduced expression of NR4A proteins and / or genes. Therefore, as used herein, "NR4A editing" refers to reduced expression of one or more members of the NR4A family (e.g., NR4A1, NR4A2, and / or NR4A3). In some aspects, NR4A-edited cells (e.g., NR4A1-edited, NR4A2-edited, and / or NR4A3-edited cells) do not exhibit expression of one or more members of the NR4A family. In some aspects, in some aspects, NR4A-edited cells exhibit some expression of members of the NR4A family, but at much lower levels than corresponding unedited cells. In some aspects, NR4A expression is reduced by at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or about 100% in NR4A-edited cells provided herein compared to corresponding unedited cells. Unless otherwise indicated, the terms "edited," "defective," and "knockout" (or variants thereof) are used interchangeably in this disclosure.

[0095] Thus, the term "NR4A3 editing" specifically refers to reduced expression of the NR4A3 gene and / or protein. In some aspects, NR4A3-edited cells do not express NR4A3 gene and / or protein. In some aspects, NR4A3-edited cells may express some expression of the NR4A3 gene and / or protein, but at a much lower level than in corresponding unedited cells. In some aspects, NR4A3 expression is reduced by at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or about 100% compared to corresponding unedited cells. Unless otherwise indicated, "NR4A3 editing," "NR4A3 deficiency," and "NR4A3 knockout" (or variants thereof) are used interchangeably herein.

[0096] As used herein, the term "elevated concentration" or "elevated level" and grammatical variations thereof refer to a higher than normal level of a substance (eg, reactive oxygen species; ROS) compared to an appropriate control (eg, healthy tissue or cells).

[0097] As used herein, the terms "reactive oxygen species" and "ROS" refer to highly reactive chemical species containing oxygen that readily react with other molecules, leading to potentially destructive changes. Reactive oxygen species include, for example, oxygen ions, free radicals, and inorganic and organic peroxides such as hydrogen peroxide, peroxides, hydroxyl radicals, lipid hydroperoxidases, and singlet oxygen. They are typically very small molecules and are highly reactive due to the presence of unpaired valence electrons. Almost all cancers are associated with elevated levels of reactive oxygen species.

[0098] As used herein, the terms "chimeric antigen receptor" and "CAR" refer to a recombinant fusion protein having an antigen-specific extracellular domain coupled to an intracellular domain that instructs the cell to perform specialized functions after the antigen binds to the extracellular domain. The terms "artificial T cell receptor," "chimeric T cell receptor," and "chimeric immune receptor" are each used interchangeably with the term "chimeric antigen receptor" herein. Chimeric antigen receptors are distinguished from other antigen binders in that they can both bind to MHC-independent antigens and transduce activation signals through their intracellular domains.

[0099] The antigen-specific extracellular domain of the chimeric antigen receptor recognizes and specifically binds to an antigen, typically an antigen expressed on the surface of a malignant tumor. When the antigen-specific extracellular domain is present, for example, with an affinity constant or interaction affinity (K) between about 0.1 pM and about 10 μM (e.g., about 0.1 pM to about 1 μM or about 0.1 pM to about 100 nM), the antigen-specific extracellular domain recognizes and specifically binds to an antigen, typically an antigen expressed on the surface of a malignant tumor. D ) when binding to an antigen, it specifically binds to the antigen. Methods for determining the affinity of an interaction are known in the art. The antigen-specific extracellular domains applicable to the CAR of the present disclosure can be any antigen-binding polypeptide, and a wide variety of such polypeptides are known in the art. In some aspects, the antigen-binding domain is a single-chain Fv (scFv). Other antibody-based recognition domains (cAb VHH (camelid antibody variable domains) and humanized forms thereof, lgNAR VH (shark antibody variable domains) and humanized forms thereof, sdAb VH (single domain antibody variable domains) and "camelized" antibody variable domains are applicable. In some aspects, recognition domains based on T cell receptors (TCRs), such as single-chain TCRs (scTv, single-chain dual-domain TCRs containing VαVβ) are also applicable.

[0100] Chimeric antigen receptor disclosed herein can also include an intracellular domain, which provides intracellular signals to cells (expressing CAR) when antigen is combined with antigen-specific extracellular domains. In some aspects, the intracellular signaling domain of CAR is responsible for activating at least one effector function of the T cells expressing chimeric receptors.

[0101] The term "intracellular domain" refers to the part of CAR that transduces effector function signals when antigen is combined with the extracellular domain and instructs T cells to perform specialized functions. Non-limiting examples of suitable intracellular domains include the ζ chain of T cell receptor or any homologue thereof (e.g., η, δ, γ or ε), MB 1 chain, 829, Fc RIII, Fc RI and a combination of signaling molecules, such as CD3.ζ. and CD28, CD27, 4-1BB, DAP-10, OX40 and combinations thereof, and other similar molecules and fragments. The intracellular signaling moieties of other members of the activation protein family, such as FcγRIII and FcεRI, can be used. Although a complete intracellular domain is generally used, it is not necessary to use a complete intracellular polypeptide in many cases. In terms of the truncated portion of the intracellular signaling domain, this truncated portion can be used to replace the complete chain, as long as it still transduces effector function signals. Therefore, the term intracellular domain means any truncated portion of the intracellular domain that is sufficient to transduce effector function signals. Typically, the antigen-specific extracellular domain is connected to the intracellular domain of the chimeric antigen receptor through a transmembrane domain. The transmembrane domain crosses the cell membrane, anchors the CAR to the T cell surface, and connects the extracellular domain to the intracellular signaling domain, thereby affecting the expression of CAR on the T cell surface. The chimeric antigen receptor can also further include one or more costimulatory domains and / or one or more spacers. The costimulatory domain is derived from the intracellular signaling domain of the costimulatory protein that enhances cytokine production, proliferation, cytotoxicity and / or in vivo persistence.

[0102] "Peptide hinge" or "spacer" connects the antigen-specific extracellular domain to the transmembrane domain. The transmembrane domain is fused to the costimulatory domain, optionally, the costimulatory domain is fused to the second costimulatory domain, and the costimulatory domain is fused to the signaling domain, not limited to CD3ζ. For example, the inclusion of a spacer domain between the antigen-specific extracellular domain and the transmembrane domain and between multiple scFvs in the case of a tandem CAR may affect the flexibility of the antigen binding domain, thereby affecting CAR function. Suitable transmembrane domains, costimulatory domains and spacers are known in the art.

[0103] As used herein, the terms "ug" and "uM" are used interchangeably with "μg" and "μΜ," respectively.

[0104] As used herein, the term "gene editing" refers to the process of changing the genetic information present in the cell genome. This gene editing can be carried out by manipulating genomic DNA, resulting in changes in genetic information. In some aspects, this gene editing may affect the expression of the edited DNA. In some aspects, this gene editing does not affect the expression of the edited DNA. In some aspects, the gene editing of the modified cells disclosed herein can be completed using the gene editing tools described herein. Non-limiting examples of gene editing tools include RNA interference molecules (e.g., shRNA, siRNA, miRNA), antisense oligonucleotides, CRISPR, zinc finger nucleases (ZFN), transcription activator-like effector nucleases (TALEN), large-range nucleases, restriction endonucleases, or any combination thereof.

[0105] As used herein, the term "nuclease" refers to an enzyme that has catalytic activity for DNA cracking. Any nuclease reagent that induces a gap or double-strand break into the desired recognition site can be used in the methods and compositions disclosed herein. Naturally occurring or natural nuclease reagents can be used, as long as the nuclease reagent induces a gap or double-strand break at the desired recognition site. Alternatively, a modified or engineered nuclease reagent can be used. "Engineering nuclease reagent" includes a nuclease engineered (modified or derived) from its native form to specifically recognize and induce a gap or double-strand break at the desired recognition site. Therefore, the engineered nuclease reagent can be derived from a natural, naturally occurring nuclease reagent, or it can be artificially produced or synthesized. The modification of the nuclease reagent can be as little as one amino acid in a protein cleavage reagent or one nucleotide in a nucleic acid cleavage reagent. In some aspects, the engineered nuclease induces a gap or double-strand break in the recognition site, wherein the recognition site is not a sequence recognized by a natural (non-engineered or non-modified) nuclease reagent. Producing a gap or double-strand break in a recognition site or other DNA can be referred to as "cutting" or "cleaving" the recognition site or other DNA in this article.

[0106] As used herein, "coding sequence" or "encoding nucleic acid" refers to a nucleic acid (RNA or DNA molecule) comprising a nucleotide sequence encoding a protein, such as a Cas9 protein, a CAR or a TCR, or a polynucleotide, such as a gRNA. The coding sequence may further include start and stop signals operably connected to regulatory elements, including promoters and polyadenylation signals, which are capable of directing expression in cells of an individual or mammal to which the nucleic acid is administered. The coding sequence may be codon-optimized.

[0107] As used herein, "complementary sequences" or "complementarity" refers to Watson-Crick (e.g., AT / U and CG) or Hoogsteen base pairing between nucleotides or nucleotide analogs of a nucleic acid molecule. "Complementarity" refers to a property shared between two nucleic acid sequences such that when the two nucleic acid sequences are aligned antiparallel to each other, the nucleotide bases at every position will be complementary.

[0108] Various aspects described herein are described in more detail in the following subsections.

[0109] II. NR4A3 Targeting Polynucleotides

[0110] Provided herein are polynucleotides (e.g., isolated polynucleotides) comprising a nucleotide sequence that can specifically bind to a target sequence within the NR4A3 gene. Without being bound by any one theory, in some aspects, the polynucleotides of the present disclosure can reduce the level of the NR4A3 gene and / or encoded protein in cells (e.g., immune cells) by binding to the target sequence within the NR4A3 gene. As further described elsewhere in this disclosure, in some aspects, reduced levels of the NR4A3 gene and / or NR4A3 protein can be associated with reduced NR4A3 activity, which can in turn improve one or more properties of the cell. Non-limiting examples of such properties are provided elsewhere in this disclosure.

[0111] The polynucleotides provided herein (e.g., including gRNAs that can specifically target regions within the NR4A3 gene) can be present in whole cells, in cell lysates, or in partially purified or substantially purified form. A polynucleotide is "isolated" or "appears to be substantially pure" when it is purified from other cellular components or other contaminants, such as other cellular nucleic acids (e.g., other chromosomal DNA, e.g., chromosomal DNA linked to isolated DNA in nature) or proteins by standard techniques (including alkaline / SDS treatment, CsCl banding, column chromatography, restriction endonucleases, agarose gel electrophoresis, and other methods well known in the art). The polynucleotides described herein can be, for example, DNA or RNA, and may or may not contain intron sequences. In some aspects, the polynucleotide is a cDNA molecule.

[0112] II.A. Binding Sequence

[0113] As described herein, the polynucleotides described herein comprise a nucleotide sequence that can specifically bind to a nucleic acid sequence within the NR4A3 gene. Such nucleotide sequences are also referred to herein as "binding sequences" or "guide sequences" or "guide RNAs" (gRNAs). Therefore, as used herein, the term "guide RNA" (gRNA) is not particularly limited, as long as it can specifically bind to a nucleic acid sequence having an NR4A3 gene and thereby reduce the level of the NR4A3 gene and / or NR4A3 protein. Non-limiting examples of such gRNAs are provided throughout this disclosure (see, for example, Table AD).

[0114] In some aspects, gRNA is DNA or RNA. In some aspects, gRNA is DNA. In some aspects, gRNA is RNA. In some aspects, DNA and / or RNA are respectively synthesized DNA and / or RNA. In some aspects, synthetic RNA or DNA include at least one non-natural core base. In some aspects, all nucleoside bases of a certain class have been replaced by non-natural nucleoside bases (for example, all uridines in the polynucleotide disclosed herein can be replaced by non-natural nucleoside bases (for example, 5-methoxyuridine or pseudouridine)). In some aspects, polynucleotides (for example, synthetic RNA or synthetic DNA) only include natural core bases, i.e., include A, C, T and U in the case of synthetic DNA, or include A, C, T and U in the case of synthetic RNA or synthetic DNA.

[0115] In some aspects, the gRNA can be between about 5 and about 100 nucleotides in length. In some aspects, the gRNA is about 5, about 6, about 7, about 8, about 9, about 10, about 15, about 20, about 25, about 30, about 35, about 40, about 45, about 50, about 60, about 70, about 80, about 90, or about 100 nucleotides in length. In some aspects, the gRNA is between about 10 and about 30 nucleotides in length (e.g., about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, about 25, about 26, about 27, about 28, about 29, or about 30 nucleotides). In some aspects, the gRNA is about 20 nucleotides in length.

[0116] In some aspects, the gRNA of the polynucleotides described herein is designed to be complementary or substantially complementary to a nucleic acid sequence within the NR4A3 gene (also referred to herein as a "target sequence"). In some aspects, the gRNA may incorporate wobble or degenerate bases to bind to multiple sequences (e.g., multiple target sequences within the NR4A3 gene; or target sequences within the NR4A3 gene and other members of the NR4A family). In some cases, the gRNA may be altered to increase stability. For example, non-natural nucleotides may be incorporated to increase resistance to degradation. In some aspects, the gRNA may be altered or designed to avoid or reduce secondary structure formation in the gRNA. In some aspects, the gRNA may be designed to optimize GC content. In some aspects, the GC content is between about 40% and about 60% (e.g., about 40%, about 45%, about 50%, about 55%, about 60%). In some aspects, the gRNA may comprise modified nucleotides, such as, but not limited to, methylated or phosphorylated nucleotides. Other methods of modifying and thereby improving one or more properties of the polynucleotides described herein are known in the art. Non-limiting examples of such modifications that can be added to the polynucleotides described herein include: a 5' cap, a 3' polyadenylation tail, a riboswitch sequence, a stability control sequence, a hairpin, a subcellular localization sequence, a detection or labeling sequence, one or more protein binding sites, non-natural nucleotides, or combinations thereof. See, for example, U.S. Publication No. 20210123046A1, the entire contents of which are incorporated herein by reference. Additional disclosures related to such modifications are provided elsewhere in this disclosure.

[0117] As described herein, in some aspects, the polynucleotides described herein include gRNAs that are fully complementary (i.e., fully complementary) to the target sequence within the NR4A3 gene. Those skilled in the art will appreciate that multiple nucleic acid sequences do not always need to be fully complementary to each other for hybridization. Therefore, in some aspects, the gRNAs of the polynucleotides described herein may contain one or more base mismatches, as long as the gRNA is able to bind to the target sequence within the NR4A3 gene of the immune cell, thereby reducing the level of the NR4A3 gene and / or NR4A3 protein in the immune cell. In some aspects, the gRNAs of the polynucleotides described herein are about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98% or about 99% complementary to the target sequence with the NR4A3 gene.

[0118] Non-limiting examples of gRNAs useful in the present disclosure are provided in Tables C and D.

[0119] In some aspects, the polynucleotides used in the present disclosure include a gRNA, wherein the gRNA comprises, consists of, or consists essentially of the nucleotide sequence set forth in SEQ ID NO: 30. For example, in some aspects, the polynucleotides include a gRNA, wherein the gRNA comprises the nucleotide sequence set forth in SEQ ID NO: 30. In some aspects, the polynucleotides include a gRNA, wherein the gRNA consists of the nucleotide sequence set forth in SEQ ID NO: 30. In some aspects, the polynucleotides include a gRNA, wherein the gRNA consists essentially of the nucleotide sequence set forth in SEQ ID NO: 30.

[0120] In some aspects, the polynucleotides used in the present disclosure include a gRNA, wherein the gRNA comprises, consists of, or consists essentially of the nucleotide sequence set forth in SEQ ID NO: 52. For example, in some aspects, the polynucleotides include a gRNA, wherein the gRNA comprises the nucleotide sequence set forth in SEQ ID NO: 52. In some aspects, the polynucleotides include a gRNA, wherein the gRNA consists of the nucleotide sequence set forth in SEQ ID NO: 52. In some aspects, the polynucleotides include a gRNA, wherein the gRNA consists essentially of the nucleotide sequence set forth in SEQ ID NO: 52.

[0121] In some aspects, the polynucleotides used in the present disclosure include a gRNA, wherein the gRNA comprises, consists of, or consists essentially of the nucleotide sequence set forth in SEQ ID NO: 53. For example, in some aspects, the polynucleotides include a gRNA, and the gRNA comprises the nucleotide sequence set forth in SEQ ID NO: 53. In some aspects, the polynucleotides include a gRNA, and the gRNA consists of the nucleotide sequence set forth in SEQ ID NO: 53. In some aspects, the polynucleotides include a gRNA, and the gRNA consists essentially of the nucleotide sequence set forth in SEQ ID NO: 53.

[0122] In some aspects, the polynucleotides used in the present disclosure include a gRNA, wherein the gRNA comprises, consists of, or consists essentially of the nucleotide sequence set forth in SEQ ID NO: 54. For example, in some aspects, the polynucleotides include a gRNA, wherein the gRNA comprises the nucleotide sequence set forth in SEQ ID NO: 54. In some aspects, the polynucleotides include a gRNA, wherein the gRNA consists of the nucleotide sequence set forth in SEQ ID NO: 54. In some aspects, the polynucleotides include a gRNA, wherein the gRNA consists essentially of the nucleotide sequence set forth in SEQ ID NO: 54.

[0123] In some aspects, the polynucleotides used in the present disclosure include a gRNA, wherein the gRNA comprises, consists of, or consists essentially of the nucleotide sequence set forth in SEQ ID NO: 55. For example, in some aspects, the polynucleotides include a gRNA, wherein the gRNA comprises the nucleotide sequence set forth in SEQ ID NO: 55. In some aspects, the polynucleotides include a gRNA, wherein the gRNA consists of the nucleotide sequence set forth in SEQ ID NO: 55. In some aspects, the polynucleotides include a gRNA, wherein the gRNA consists essentially of the nucleotide sequence set forth in SEQ ID NO: 55.

[0124] In some aspects, the polynucleotides used in the present disclosure include a gRNA, wherein the gRNA comprises, consists of, or consists essentially of the nucleotide sequence set forth in SEQ ID NO: 56. For example, in some aspects, the polynucleotides include a gRNA, and the gRNA comprises the nucleotide sequence set forth in SEQ ID NO: 56. In some aspects, the polynucleotides include a gRNA, and the gRNA consists of the nucleotide sequence set forth in SEQ ID NO: 56. In some aspects, the polynucleotides include a gRNA, and the gRNA consists essentially of the nucleotide sequence set forth in SEQ ID NO: 56.

[0125] In some aspects, the polynucleotides used in the present disclosure include a gRNA, wherein the gRNA comprises, consists of, or consists essentially of the nucleotide sequence set forth in SEQ ID NO: 57. For example, in some aspects, the polynucleotides include a gRNA, wherein the gRNA comprises the nucleotide sequence set forth in SEQ ID NO: 57. In some aspects, the polynucleotides include a gRNA, wherein the gRNA consists of the nucleotide sequence set forth in SEQ ID NO: 57. In some aspects, the polynucleotides include a gRNA, wherein the gRNA consists essentially of the nucleotide sequence set forth in SEQ ID NO: 57.

[0126] In some aspects, the polynucleotides used in the present disclosure include a gRNA, wherein the gRNA comprises, consists of, or consists essentially of the nucleotide sequence set forth in SEQ ID NO: 58. For example, in some aspects, the polynucleotides include a gRNA, and the gRNA comprises the nucleotide sequence set forth in SEQ ID NO: 58. In some aspects, the polynucleotides include a gRNA, and the gRNA consists of the nucleotide sequence set forth in SEQ ID NO: 58. In some aspects, the polynucleotides include a gRNA, and the gRNA consists essentially of the nucleotide sequence set forth in SEQ ID NO: 58.

[0127] In some aspects, the polynucleotides used in the present disclosure include a gRNA, wherein the gRNA comprises, consists of, or consists essentially of the nucleotide sequence set forth in SEQ ID NO: 59. For example, in some aspects, the polynucleotides include a gRNA, and the gRNA comprises the nucleotide sequence set forth in SEQ ID NO: 59. In some aspects, the polynucleotides include a gRNA, and the gRNA consists of the nucleotide sequence set forth in SEQ ID NO: 59. In some aspects, the polynucleotides include a gRNA, and the gRNA consists essentially of the nucleotide sequence set forth in SEQ ID NO: 59.

[0128] In some aspects, the polynucleotides used in the present disclosure include a gRNA, wherein the gRNA comprises, consists of, or consists essentially of the nucleotide sequence set forth in SEQ ID NO: 60. For example, in some aspects, the polynucleotides include a gRNA, wherein the gRNA comprises the nucleotide sequence set forth in SEQ ID NO: 60. In some aspects, the polynucleotides include a gRNA, wherein the gRNA consists of the nucleotide sequence set forth in SEQ ID NO: 60. In some aspects, the polynucleotides include a gRNA, wherein the gRNA consists essentially of the nucleotide sequence set forth in SEQ ID NO: 60.

[0129] In some aspects, the polynucleotides used in the present disclosure include a gRNA, wherein the gRNA comprises, consists of, or consists essentially of the nucleotide sequence set forth in SEQ ID NO: 61. For example, in some aspects, the polynucleotides include a gRNA, wherein the gRNA comprises the nucleotide sequence set forth in SEQ ID NO: 61. In some aspects, the polynucleotides include a gRNA, wherein the gRNA consists of the nucleotide sequence set forth in SEQ ID NO: 61. In some aspects, the polynucleotides include a gRNA, wherein the gRNA consists essentially of the nucleotide sequence set forth in SEQ ID NO: 61.

[0130] In some aspects, the polynucleotides used in the present disclosure include a gRNA, wherein the gRNA comprises, consists of, or consists essentially of the nucleotide sequence set forth in SEQ ID NO: 62. For example, in some aspects, the polynucleotides include a gRNA, wherein the gRNA comprises the nucleotide sequence set forth in SEQ ID NO: 62. In some aspects, the polynucleotides include a gRNA, wherein the gRNA consists of the nucleotide sequence set forth in SEQ ID NO: 62. In some aspects, the polynucleotides include a gRNA, wherein the gRNA consists essentially of the nucleotide sequence set forth in SEQ ID NO: 62.

[0131] In some aspects, the polynucleotides used in the present disclosure include a gRNA, wherein the gRNA comprises, consists of, or consists essentially of the nucleotide sequence set forth in SEQ ID NO: 63. For example, in some aspects, the polynucleotides include a gRNA, wherein the gRNA comprises the nucleotide sequence set forth in SEQ ID NO: 63. In some aspects, the polynucleotides include a gRNA, wherein the gRNA consists of the nucleotide sequence set forth in SEQ ID NO: 63. In some aspects, the polynucleotides include a gRNA, wherein the gRNA consists essentially of the nucleotide sequence set forth in SEQ ID NO: 63.

[0132] In some aspects, the polynucleotides used in the present disclosure include a gRNA, wherein the gRNA comprises, consists of, or consists essentially of the nucleotide sequence set forth in SEQ ID NO: 64. For example, in some aspects, the polynucleotides include a gRNA, wherein the gRNA comprises the nucleotide sequence set forth in SEQ ID NO: 64. In some aspects, the polynucleotides include a gRNA, wherein the gRNA consists of the nucleotide sequence set forth in SEQ ID NO: 64. In some aspects, the polynucleotides include a gRNA, wherein the gRNA consists essentially of the nucleotide sequence set forth in SEQ ID NO: 64.

[0133] In some aspects, the polynucleotides used in the present disclosure include a gRNA, wherein the gRNA comprises, consists of, or consists essentially of the nucleotide sequence set forth in SEQ ID NO: 65. For example, in some aspects, the polynucleotides include a gRNA, wherein the gRNA comprises the nucleotide sequence set forth in SEQ ID NO: 65. In some aspects, the polynucleotides include a gRNA, wherein the gRNA consists of the nucleotide sequence set forth in SEQ ID NO: 65. In some aspects, the polynucleotides include a gRNA, wherein the gRNA consists essentially of the nucleotide sequence set forth in SEQ ID NO: 65.

[0134] In some aspects, the polynucleotides used in the present disclosure include a gRNA, wherein the gRNA comprises, consists of, or consists essentially of the nucleotide sequence set forth in SEQ ID NO: 66. For example, in some aspects, the polynucleotides include a gRNA, wherein the gRNA comprises the nucleotide sequence set forth in SEQ ID NO: 66. In some aspects, the polynucleotides include a gRNA, wherein the gRNA consists of the nucleotide sequence set forth in SEQ ID NO: 66. In some aspects, the polynucleotides include a gRNA, wherein the gRNA consists essentially of the nucleotide sequence set forth in SEQ ID NO: 66.

[0135] In some aspects, the polynucleotides used in the present disclosure include a gRNA, wherein the gRNA comprises, consists of, or consists essentially of the nucleotide sequence set forth in SEQ ID NO: 67. For example, in some aspects, the polynucleotides include a gRNA, wherein the gRNA comprises the nucleotide sequence set forth in SEQ ID NO: 67. In some aspects, the polynucleotides include a gRNA, wherein the gRNA consists of the nucleotide sequence set forth in SEQ ID NO: 67. In some aspects, the polynucleotides include a gRNA, wherein the gRNA consists essentially of the nucleotide sequence set forth in SEQ ID NO: 67.

[0136] In some aspects, the polynucleotides used in the present disclosure include a gRNA, wherein the gRNA comprises, consists of, or consists essentially of the nucleotide sequence set forth in SEQ ID NO: 68. For example, in some aspects, the polynucleotides include a gRNA, and the gRNA comprises the nucleotide sequence set forth in SEQ ID NO: 68. In some aspects, the polynucleotides include a gRNA, and the gRNA consists of the nucleotide sequence set forth in SEQ ID NO: 68. In some aspects, the polynucleotides include a gRNA, and the gRNA consists essentially of the nucleotide sequence set forth in SEQ ID NO: 68.

[0137] In some aspects, the polynucleotides used in the present disclosure include a gRNA, wherein the gRNA comprises, consists of, or consists essentially of the nucleotide sequence set forth in SEQ ID NO: 69. For example, in some aspects, the polynucleotides include a gRNA, wherein the gRNA comprises the nucleotide sequence set forth in SEQ ID NO: 69. In some aspects, the polynucleotides include a gRNA, wherein the gRNA consists of the nucleotide sequence set forth in SEQ ID NO: 69. In some aspects, the polynucleotides include a gRNA, wherein the gRNA consists essentially of the nucleotide sequence set forth in SEQ ID NO: 69.

[0138] In some aspects, the polynucleotides used in the present disclosure include a gRNA, wherein the gRNA comprises, consists of, or consists essentially of the nucleotide sequence set forth in SEQ ID NO: 70. For example, in some aspects, the polynucleotides include a gRNA, wherein the gRNA comprises the nucleotide sequence set forth in SEQ ID NO: 70. In some aspects, the polynucleotides include a gRNA, wherein the gRNA consists of the nucleotide sequence set forth in SEQ ID NO: 70. In some aspects, the polynucleotides include a gRNA, wherein the gRNA consists essentially of the nucleotide sequence set forth in SEQ ID NO: 70.

[0139] In some aspects, the polynucleotides used in the present disclosure include a gRNA, wherein the gRNA comprises, consists of, or consists essentially of the nucleotide sequence set forth in SEQ ID NO: 71. For example, in some aspects, the polynucleotides include a gRNA, wherein the gRNA comprises the nucleotide sequence set forth in SEQ ID NO: 71. In some aspects, the polynucleotides include a gRNA, wherein the gRNA consists of the nucleotide sequence set forth in SEQ ID NO: 71. In some aspects, the polynucleotides include a gRNA, wherein the gRNA consists essentially of the nucleotide sequence set forth in SEQ ID NO: 71.

[0140] In some aspects, the polynucleotides used in the present disclosure include a gRNA, wherein the gRNA comprises, consists of, or consists essentially of the nucleotide sequence set forth in SEQ ID NO: 72. For example, in some aspects, the polynucleotides include a gRNA, wherein the gRNA comprises the nucleotide sequence set forth in SEQ ID NO: 72. In some aspects, the polynucleotides include a gRNA, wherein the gRNA consists of the nucleotide sequence set forth in SEQ ID NO: 72. In some aspects, the polynucleotides include a gRNA, wherein the gRNA consists essentially of the nucleotide sequence set forth in SEQ ID NO: 72.

[0141] In some aspects, the polynucleotides used in the present disclosure include a gRNA, wherein the gRNA comprises, consists of, or consists essentially of the nucleotide sequence set forth in SEQ ID NO: 73. For example, in some aspects, the polynucleotides include a gRNA, wherein the gRNA comprises the nucleotide sequence set forth in SEQ ID NO: 73. In some aspects, the polynucleotides include a gRNA, wherein the gRNA consists of the nucleotide sequence set forth in SEQ ID NO: 73. In some aspects, the polynucleotides include a gRNA, wherein the gRNA consists essentially of the nucleotide sequence set forth in SEQ ID NO: 73.

[0142] In some aspects, the polynucleotides used in the present disclosure include a gRNA, wherein the gRNA comprises, consists of, or consists essentially of the nucleotide sequence set forth in SEQ ID NO: 74. For example, in some aspects, the polynucleotides include a gRNA, wherein the gRNA comprises the nucleotide sequence set forth in SEQ ID NO: 74. In some aspects, the polynucleotides include a gRNA, wherein the gRNA consists of the nucleotide sequence set forth in SEQ ID NO: 74. In some aspects, the polynucleotides include a gRNA, wherein the gRNA consists essentially of the nucleotide sequence set forth in SEQ ID NO: 74.

[0143] In some aspects, the polynucleotides used in the present disclosure include a gRNA, wherein the gRNA comprises, consists of, or consists essentially of the nucleotide sequence set forth in SEQ ID NO: 75. For example, in some aspects, the polynucleotides include a gRNA, wherein the gRNA comprises the nucleotide sequence set forth in SEQ ID NO: 75. In some aspects, the polynucleotides include a gRNA, wherein the gRNA consists of the nucleotide sequence set forth in SEQ ID NO: 75. In some aspects, the polynucleotides include a gRNA, wherein the gRNA consists essentially of the nucleotide sequence set forth in SEQ ID NO: 75.

[0144] In some aspects, the polynucleotides used in the present disclosure include a gRNA, wherein the gRNA comprises, consists of, or consists essentially of the nucleotide sequence set forth in SEQ ID NO: 76. For example, in some aspects, the polynucleotides include a gRNA, wherein the gRNA comprises the nucleotide sequence set forth in SEQ ID NO: 76. In some aspects, the polynucleotides include a gRNA, wherein the gRNA consists of the nucleotide sequence set forth in SEQ ID NO: 76. In some aspects, the polynucleotides include a gRNA, wherein the gRNA consists essentially of the nucleotide sequence set forth in SEQ ID NO: 76.

[0145] In some aspects, the polynucleotides used in the present disclosure include a gRNA, wherein the gRNA comprises, consists of, or consists essentially of the nucleotide sequence set forth in SEQ ID NO: 77. For example, in some aspects, the polynucleotides include a gRNA, wherein the gRNA comprises the nucleotide sequence set forth in SEQ ID NO: 77. In some aspects, the polynucleotides include a gRNA, wherein the gRNA consists of the nucleotide sequence set forth in SEQ ID NO: 77. In some aspects, the polynucleotides include a gRNA, wherein the gRNA consists essentially of the nucleotide sequence set forth in SEQ ID NO: 77.

[0146] In some aspects, the polynucleotides used in the present disclosure include a gRNA, wherein the gRNA comprises, consists of, or consists essentially of the nucleotide sequence set forth in SEQ ID NO: 78. For example, in some aspects, the polynucleotides include a gRNA, and the gRNA comprises the nucleotide sequence set forth in SEQ ID NO: 78. In some aspects, the polynucleotides include a gRNA, and the gRNA consists of the nucleotide sequence set forth in SEQ ID NO: 78. In some aspects, the polynucleotides include a gRNA, and the gRNA consists essentially of the nucleotide sequence set forth in SEQ ID NO: 78.

[0147] In some aspects, the polynucleotides used in the present disclosure include a gRNA, wherein the gRNA comprises, consists of, or consists essentially of the nucleotide sequence set forth in SEQ ID NO: 79. For example, in some aspects, the polynucleotides include a gRNA, wherein the gRNA comprises the nucleotide sequence set forth in SEQ ID NO: 79. In some aspects, the polynucleotides include a gRNA, wherein the gRNA consists of the nucleotide sequence set forth in SEQ ID NO: 79. In some aspects, the polynucleotides include a gRNA, wherein the gRNA consists essentially of the nucleotide sequence set forth in SEQ ID NO: 79.

[0148] In some aspects, the polynucleotides used in the present disclosure include a gRNA, wherein the gRNA comprises, consists of, or consists essentially of the nucleotide sequence set forth in SEQ ID NO: 80. For example, in some aspects, the polynucleotides include a gRNA, and the gRNA comprises the nucleotide sequence set forth in SEQ ID NO: 80. In some aspects, the polynucleotides include a gRNA, and the gRNA consists of the nucleotide sequence set forth in SEQ ID NO: 80. In some aspects, the polynucleotides include a gRNA, and the gRNA consists essentially of the nucleotide sequence set forth in SEQ ID NO: 80.

[0149] In some aspects, the polynucleotides used in the present disclosure include a gRNA, wherein the gRNA comprises, consists of, or consists essentially of the nucleotide sequence set forth in SEQ ID NO: 81. For example, in some aspects, the polynucleotides include a gRNA, wherein the gRNA comprises the nucleotide sequence set forth in SEQ ID NO: 81. In some aspects, the polynucleotides include a gRNA, wherein the gRNA consists of the nucleotide sequence set forth in SEQ ID NO: 81. In some aspects, the polynucleotides include a gRNA, wherein the gRNA consists essentially of the nucleotide sequence set forth in SEQ ID NO: 81.

[0150] In some aspects, the polynucleotides used in the present disclosure include a gRNA, wherein the gRNA comprises, consists of, or consists essentially of the nucleotide sequence set forth in SEQ ID NO: 82. For example, in some aspects, the polynucleotides include a gRNA, wherein the gRNA comprises the nucleotide sequence set forth in SEQ ID NO: 82. In some aspects, the polynucleotides include a gRNA, wherein the gRNA consists of the nucleotide sequence set forth in SEQ ID NO: 82. In some aspects, the polynucleotides include a gRNA, wherein the gRNA consists essentially of the nucleotide sequence set forth in SEQ ID NO: 82.

[0151] In some aspects, the polynucleotides used in the present disclosure include a gRNA, wherein the gRNA comprises, consists of, or consists essentially of the nucleotide sequence set forth in SEQ ID NO: 83. For example, in some aspects, the polynucleotides include a gRNA, wherein the gRNA comprises the nucleotide sequence set forth in SEQ ID NO: 83. In some aspects, the polynucleotides include a gRNA, wherein the gRNA consists of the nucleotide sequence set forth in SEQ ID NO: 83. In some aspects, the polynucleotides include a gRNA, wherein the gRNA consists essentially of the nucleotide sequence set forth in SEQ ID NO: 83.

[0152] In some aspects, the polynucleotides used in the present disclosure include a gRNA, wherein the gRNA comprises, consists of, or consists essentially of the nucleotide sequence set forth in SEQ ID NO: 84. For example, in some aspects, the polynucleotides include a gRNA, wherein the gRNA comprises the nucleotide sequence set forth in SEQ ID NO: 84. In some aspects, the polynucleotides include a gRNA, wherein the gRNA consists of the nucleotide sequence set forth in SEQ ID NO: 84. In some aspects, the polynucleotides include a gRNA, wherein the gRNA consists essentially of the nucleotide sequence set forth in SEQ ID NO: 84.

[0153] In some aspects, the polynucleotides used in the present disclosure include a gRNA, wherein the gRNA comprises, consists of, or consists essentially of the nucleotide sequence set forth in SEQ ID NO: 85. For example, in some aspects, the polynucleotides include a gRNA, wherein the gRNA comprises the nucleotide sequence set forth in SEQ ID NO: 85. In some aspects, the polynucleotides include a gRNA, wherein the gRNA consists of the nucleotide sequence set forth in SEQ ID NO: 85. In some aspects, the polynucleotides include a gRNA, wherein the gRNA consists essentially of the nucleotide sequence set forth in SEQ ID NO: 85.

[0154] In some aspects, the polynucleotides used in the present disclosure include a gRNA, wherein the gRNA comprises, consists of, or consists essentially of the nucleotide sequence set forth in SEQ ID NO: 86. For example, in some aspects, the polynucleotides include a gRNA, and the gRNA comprises the nucleotide sequence set forth in SEQ ID NO: 86. In some aspects, the polynucleotides include a gRNA, and the gRNA consists of the nucleotide sequence set forth in SEQ ID NO: 86. In some aspects, the polynucleotides include a gRNA, and the gRNA consists essentially of the nucleotide sequence set forth in SEQ ID NO: 86.

[0155] In some aspects, the polynucleotides used in the present disclosure include a gRNA, wherein the gRNA comprises, consists of, or consists essentially of the nucleotide sequence set forth in SEQ ID NO: 87. For example, in some aspects, the polynucleotides include a gRNA, wherein the gRNA comprises the nucleotide sequence set forth in SEQ ID NO: 87. In some aspects, the polynucleotides include a gRNA, wherein the gRNA consists of the nucleotide sequence set forth in SEQ ID NO: 87. In some aspects, the polynucleotides include a gRNA, wherein the gRNA consists essentially of the nucleotide sequence set forth in SEQ ID NO: 87.

[0156] In some aspects, the polynucleotides used in the present disclosure include a gRNA, wherein the gRNA comprises, consists of, or consists essentially of the nucleotide sequence set forth in SEQ ID NO: 88. For example, in some aspects, the polynucleotides include a gRNA, and the gRNA comprises the nucleotide sequence set forth in SEQ ID NO: 88. In some aspects, the polynucleotides include a gRNA, and the gRNA consists of the nucleotide sequence set forth in SEQ ID NO: 88. In some aspects, the polynucleotides include a gRNA, and the gRNA consists essentially of the nucleotide sequence set forth in SEQ ID NO: 88.

[0157] In some aspects, the polynucleotides used in the present disclosure include a gRNA, wherein the gRNA comprises, consists of, or consists essentially of the nucleotide sequence set forth in SEQ ID NO: 89. For example, in some aspects, the polynucleotides include a gRNA, wherein the gRNA comprises the nucleotide sequence set forth in SEQ ID NO: 89. In some aspects, the polynucleotides include a gRNA, wherein the gRNA consists of the nucleotide sequence set forth in SEQ ID NO: 89. In some aspects, the polynucleotides include a gRNA, wherein the gRNA consists essentially of the nucleotide sequence set forth in SEQ ID NO: 89.

[0158] In some aspects, the polynucleotides used in the present disclosure include a gRNA, wherein the gRNA comprises, consists of, or consists essentially of the nucleotide sequence set forth in SEQ ID NO: 90. For example, in some aspects, the polynucleotides include a gRNA, and the gRNA comprises the nucleotide sequence set forth in SEQ ID NO: 90. In some aspects, the polynucleotides include a gRNA, and the gRNA consists of the nucleotide sequence set forth in SEQ ID NO: 90. In some aspects, the polynucleotides include a gRNA, and the gRNA consists essentially of the nucleotide sequence set forth in SEQ ID NO: 90.

[0159] In some aspects, the polynucleotides used in the present disclosure include a gRNA, wherein the gRNA comprises, consists of, or consists essentially of the nucleotide sequence set forth in SEQ ID NO: 91. For example, in some aspects, the polynucleotides include a gRNA, wherein the gRNA comprises the nucleotide sequence set forth in SEQ ID NO: 91. In some aspects, the polynucleotides include a gRNA, wherein the gRNA consists of the nucleotide sequence set forth in SEQ ID NO: 91. In some aspects, the polynucleotides include a gRNA, wherein the gRNA consists essentially of the nucleotide sequence set forth in SEQ ID NO: 91.

[0160] In some aspects, the polynucleotides used in the present disclosure include a gRNA, wherein the gRNA comprises, consists of, or consists essentially of the nucleotide sequence set forth in SEQ ID NO: 92. For example, in some aspects, the polynucleotides include a gRNA, and the gRNA comprises the nucleotide sequence set forth in SEQ ID NO: 92. In some aspects, the polynucleotides include a gRNA, and the gRNA consists of the nucleotide sequence set forth in SEQ ID NO: 92. In some aspects, the polynucleotides include a gRNA, and the gRNA consists essentially of the nucleotide sequence set forth in SEQ ID NO: 92.

[0161] In some aspects, the polynucleotides used in the present disclosure include a gRNA, wherein the gRNA comprises, consists of, or consists essentially of the nucleotide sequence set forth in SEQ ID NO: 93. For example, in some aspects, the polynucleotides include a gRNA, and the gRNA comprises the nucleotide sequence set forth in SEQ ID NO: 93. In some aspects, the polynucleotides include a gRNA, and the gRNA consists of the nucleotide sequence set forth in SEQ ID NO: 93. In some aspects, the polynucleotides include a gRNA, and the gRNA consists essentially of the nucleotide sequence set forth in SEQ ID NO: 93.

[0162] In some aspects, the polynucleotides used in the present disclosure include a gRNA, wherein the gRNA comprises, consists of, or consists essentially of the nucleotide sequence set forth in SEQ ID NO: 94. For example, in some aspects, the polynucleotides include a gRNA, and the gRNA comprises the nucleotide sequence set forth in SEQ ID NO: 94. In some aspects, the polynucleotides include a gRNA, and the gRNA consists of the nucleotide sequence set forth in SEQ ID NO: 94. In some aspects, the polynucleotides include a gRNA, and the gRNA consists essentially of the nucleotide sequence set forth in SEQ ID NO: 94.

[0163] In some aspects, the polynucleotides used in the present disclosure include a gRNA, wherein the gRNA comprises, consists of, or consists essentially of the nucleotide sequence set forth in SEQ ID NO: 95. For example, in some aspects, the polynucleotides include a gRNA, wherein the gRNA comprises the nucleotide sequence set forth in SEQ ID NO: 95. In some aspects, the polynucleotides include a gRNA, wherein the gRNA consists of the nucleotide sequence set forth in SEQ ID NO: 95. In some aspects, the polynucleotides include a gRNA, wherein the gRNA consists essentially of the nucleotide sequence set forth in SEQ ID NO: 95.

[0164] In some aspects, the polynucleotides used in the present disclosure include a gRNA, wherein the gRNA comprises, consists of, or consists essentially of the nucleotide sequence set forth in SEQ ID NO: 96. For example, in some aspects, the polynucleotides include a gRNA, wherein the gRNA comprises the nucleotide sequence set forth in SEQ ID NO: 96. In some aspects, the polynucleotides include a gRNA, wherein the gRNA consists of the nucleotide sequence set forth in SEQ ID NO: 96. In some aspects, the polynucleotides include a gRNA, wherein the gRNA consists essentially of the nucleotide sequence set forth in SEQ ID NO: 96.

[0165] In some aspects, the polynucleotides used in the present disclosure include a gRNA, wherein the gRNA comprises, consists of, or consists essentially of the nucleotide sequence set forth in SEQ ID NO: 97. For example, in some aspects, the polynucleotides include a gRNA, wherein the gRNA comprises the nucleotide sequence set forth in SEQ ID NO: 97. In some aspects, the polynucleotides include a gRNA, wherein the gRNA consists of the nucleotide sequence set forth in SEQ ID NO: 97. In some aspects, the polynucleotides include a gRNA, wherein the gRNA consists essentially of the nucleotide sequence set forth in SEQ ID NO: 97.

[0166] In some aspects, the polynucleotides used in the present disclosure include a gRNA, wherein the gRNA comprises, consists of, or consists essentially of the nucleotide sequence set forth in SEQ ID NO: 98. For example, in some aspects, the polynucleotides include a gRNA, and the gRNA comprises the nucleotide sequence set forth in SEQ ID NO: 98. In some aspects, the polynucleotides include a gRNA, and the gRNA consists of the nucleotide sequence set forth in SEQ ID NO: 98. In some aspects, the polynucleotides include a gRNA, and the gRNA consists essentially of the nucleotide sequence set forth in SEQ ID NO: 98.

[0167] In some aspects, the polynucleotides used in the present disclosure include a gRNA, wherein the gRNA comprises, consists of, or consists essentially of the nucleotide sequence set forth in SEQ ID NO: 99. For example, in some aspects, the polynucleotides include a gRNA, and the gRNA comprises the nucleotide sequence set forth in SEQ ID NO: 99. In some aspects, the polynucleotides include a gRNA, and the gRNA consists of the nucleotide sequence set forth in SEQ ID NO: 99. In some aspects, the polynucleotides include a gRNA, and the gRNA consists essentially of the nucleotide sequence set forth in SEQ ID NO: 99.

[0168] II.B. Targeting

[0169] As described herein, the polynucleotides disclosed herein are capable of specifically targeting (i.e., specifically binding to) nucleic acid sequences within the NR4A3 gene. Nuclear receptor subfamily 4A group member 3 (commonly abbreviated as NR4A3 and also referred to as MINOR, CSMF, NOR1, CHN, mitogen-induced nuclear orphan receptor, neuron-derived orphan receptor, nuclear hormone receptor NOR-1, "extraskeletal myxoid chondrosarcoma fused with EWS," and TEC) is a protein encoded by the NR4A3 gene in humans. The nuclear orphan receptor NR4A family includes NR4A1 (Nur77), NR4A2 (Nurr1), and NR4A3 (Nor-1). They function as transcription factors in a ligand-independent manner. Their function is primarily controlled by the rapid and transient induction of their expression by various extracellular signals, and therefore they are considered to be immediate early genes. NR4A is involved in various cellular functions, including apoptosis, survival, proliferation, angiogenesis, inflammation, DNA repair, and fatty acid metabolism.

[0170] The sequence of the human NR4A3 gene is located on chromosome 9 (bases 99,821,885 to 99,866,893; 45,039 bases; positive strand orientation; NCBI reference sequence: NC_000009.12). Unless otherwise indicated, the term "NR4A3 gene" as used herein refers to any nucleic acid sequence encoding an NR4A3 protein (or variant thereof).

[0171] The NR4A3 protein has three isoforms produced by alternative splicing. The sequences are shown in Table 1 below. Unless otherwise indicated and as further described herein, the polynucleotides of the present disclosure can be used to reduce the level of any known NR4A3 protein (including any isoforms and variants thereof).

[0172] Table 1. NR4A3 protein isoforms.

[0173]

[0174]

[0175] III. Modified Immune Cells

[0176] In some aspects, the present disclosure provides immune cells that have been modified with the polynucleotides described herein (i.e., including gRNAs that specifically target the NR4A3 gene). Thus, compared to corresponding immune cells ("reference cells") that have not been modified as described herein (e.g., to have reduced levels of the NR4A3 gene and / or NR4A3 protein), the modified immune cells described herein have reduced levels of the NR4A3 gene and / or NR4A3 protein. In some aspects, the reference cells include immune cells before modification with the polynucleotides described herein. In some aspects, the reference cells include corresponding immune cells that have not been modified with the polynucleotides described herein. In some aspects, the reference cells have endogenous levels of the NR4A3 gene and / or NR4A3 protein.

[0177] As used herein, the terms "reduced level," "lower level," "reduced expression level," or "lower expression level" (or variants thereof) refer to both a physical reduction (e.g., reduction in gene sequence due to editing of the genome, or reduction in protein due to reduced protein expression) and a reduction in function. For example, a reduction in NR4A3 gene levels can refer to a reduction in gene function, such as due to the introduction of a mutation that introduces a stop codon or a frameshift, to an epigenetic modification that alters transcription, or to a mutation or other change in a promoter gene or another gene that regulates NR4A3 expression. In some aspects, a reduction in NR4A3 gene levels in a modified cell refers to a reduction in the amount (e.g., concentration) of genomic DNA, pre-mRNA, and / or mRNA that can encode a functional NR4A3 protein (e.g., a wild-type NR4A3 protein) compared to a reference cell. Similarly, reduction of NR4A3 protein can refer to changes that result in the expression of functional NR4A3 protein (e.g., wild-type NR4A3 protein), including but not limited to changes (e.g., mutations or post-translational modifications) that result in loss of function (partial or complete), or to altered activity of molecules that bind to NR4A3 functional sites, such as its interactions with other cellular signaling partners.

[0178] NR4A3 gene levels (e.g., presence / absence of the entire gene or a portion thereof, or gene function) can be measured by various methods known in the art. NR4A3 protein levels (e.g., presence / absence, or quantification or protein function of NR4A3 protein or a fragment thereof) can be measured by various methods known in the art.

[0179] In some aspects, the level of the NR4A3 gene and / or NR4A3 protein in the modified immune cells described is reduced by at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or about 100% compared to a reference cell. In some aspects, the level of the NR4A3 gene and / or NR4A3 protein is completely suppressed.

[0180] In some aspects, the modified immune cells described herein have reduced levels of the NR4A3 gene compared to a reference cell. In some aspects, the level of the NR4A3 gene is reduced by at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or about 100% compared to a reference cell. In some aspects, the level of the NR4A3 gene is completely suppressed.

[0181] In some aspects, the modified immune cells described herein have reduced levels of NR4A3 protein compared to a reference cell. In some aspects, the level of NR4A3 protein is reduced by at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or about 100% compared to a reference cell. In some aspects, the level of NR4A3 protein in the modified immune cells is completely suppressed.

[0182] In some aspects, the modified immune cells described have reduced levels of both the NR4A3 gene and the NR4A3 protein compared to a reference cell. In some aspects, the levels of the NR4A3 gene and the NR4A3 protein are both reduced by at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or about 100% compared to a reference cell. In some aspects, the levels of both the NR4A3 gene and the NR4A3 protein are completely suppressed.

[0183] As can be seen from the present disclosure, any cell that can naturally express the NR4A3 gene and / or NR4A3 protein can be modified using the polynucleotides of the present disclosure. As described herein, in some aspects, cells useful in the present disclosure include immune cells. In some aspects, immune cells include lymphocytes, neutrophils, monocytes, macrophages, dendritic cells, natural killer cells, or combinations thereof. In some aspects, immune cells that can be modified to have reduced levels of the NR4A3 gene and / or NR4A3 protein include lymphocytes. In some aspects, lymphocytes are T cells (e.g., CD4+ T cells, CD8+ T cells, or both). As used herein, "modified immune cells" include daughter cells of initially modified immune cells, wherein the daughter cells also express reduced levels of the NR4A3 gene and / or NR4A3 protein.

[0184] As described herein, the modified immune cells of the present disclosure (e.g., having reduced levels of NR4A3 genes and / or NR4A3 proteins) exhibit one or more improved properties compared to corresponding cells that have not been modified to have reduced levels of NR4A3 genes and / or NR4A3 proteins ("reference cells"). Non-limiting examples of such properties include resistance to exhaustion (e.g., as indicated by reduced expression of exhaustion markers such as PD-1, CD39, TIM-3, and / or LAG-3; increased survival; and / or increased cytokine production), increased persistence / survival, increased expansion / proliferation, improved effector function (e.g., cytokine production upon antigen stimulation, lysis of cells expressing the target antigen, or both), or a combination thereof.

[0185] Assays for measuring exhaustion, cell phenotype, persistence, cytotoxicity and / or killing, proliferation, cytokine production / release, and gene expression profiles are known in the art and include, for example, flow cytometry, intracellular cytokine staining (ICS), Immune cell killing assays, Meso Scale Discovery (MSD) or similar assays, continuous antigen stimulation assays, bulk and single-cell RNAseq (see, e.g., Fron Genet. 2020; 11:220; 2019 Bioinformatics 35:i436-445; 2019 Annual Review of Biomed. Data Sci. 2:139-173), cytotoxicity / killing assays, ELISA, western blots, and other standard molecular and cell biology methods, such as those described herein or as, e.g., Current Protocols in Molecular Biology or Current Protocols in Immunology (John Wiley & Sons, Inc., 1999-2021), or elsewhere.

[0186] In some aspects, the modified cells described herein (i.e., having reduced NR4A3 genes and / or NR4A3 proteins) exhibit increased tolerance to exhaustion, e.g., increased tolerance to exhaustion following sustained antigenic stimulation, compared to corresponding cells that have not been modified to have reduced levels of NR4A3 genes and / or NR4A3 proteins ("reference cells"). In some aspects, the modified cells provided herein express, in response to sustained antigenic stimulation, (i) reduced levels of genes associated with exhaustion, (ii) increased levels of genes associated with activation, or (iii) both (i) and (ii), compared to reference cells. Non-limiting examples of such genes are described elsewhere in this disclosure.

[0187] In some aspects, the resistance to exhaustion is increased by at least about 1 fold, at least about 2 fold, at least about 3 fold, at least about 4 fold, at least about 5 fold, at least about 6 fold, at least about 7 fold, at least about 8 fold, at least about 9 fold, at least about 10 fold, at least about 11 fold, at least about 12 fold, at least about 13 fold, at least about 14 fold, at least about 15 fold, at least about 16 fold, at least about 17 fold, at least about 18 fold, at least about 19 fold, at least about 20 fold, at least about 25 fold, at least about 30 fold, at least about 35 fold, at least about 40 fold, at least about 45 fold, at least about 50 fold, at least about 75 fold, at least about 100 fold, at least about 200 fold, at least about 300 fold, at least about 400 fold, at least about 500 fold, at least about 750 fold, or at least about 1,000 fold compared to a reference cell.

[0188] In some aspects, the modified cells described herein (i.e., having reduced levels of the NR4A3 gene and / or NR4A3 protein) exhibit reduced depletion compared to a reference cell (i.e., a corresponding cell having endogenous levels of the NR4A3 gene and / or NR4A3 protein). In some aspects, the depletion is reduced by at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or about 100% compared to a reference cell.

[0189] In some aspects, the modified cells described herein (i.e., having decreased levels of the NR4A3 gene and / or NR4A3 protein) exhibit increased persistence / survival compared to a reference cell (i.e., a corresponding cell having endogenous levels of the NR4A3 gene and / or NR4A3 protein), e.g., when administered to a subject. In some aspects, the persistence / survival of the modified cells is increased by at least about 1 fold, at least about 2 fold, at least about 3 fold, at least about 4 fold, at least about 5 fold, at least about 6 fold, at least about 7 fold, at least about 8 fold, at least about 9 fold, at least about 10 fold, at least about 11 fold, at least about 12 fold, at least about 13 fold, at least about 14 fold, at least about 15 fold, at least about 16 fold, at least about 17 fold, at least about 18 fold, at least about 19 fold, at least about 20 fold, at least about 25 fold, at least about 30 fold, at least about 35 fold, at least about 40 fold, at least about 45 fold, at least about 50 fold, at least about 75 fold, at least about 100 fold, at least about 200 fold, at least about 300 fold, at least about 400 fold, at least about 500 fold, at least about 750 fold, or at least about 1,000 fold compared to a reference cell.

[0190] In some aspects, the modified cells described herein (i.e., having decreased levels of the NR4A3 gene and / or NR4A3 protein) exhibit increased expansion / proliferation compared to a reference cell (i.e., a corresponding cell having endogenous levels of the NR4A3 gene and / or NR4A3 protein), e.g., after sustained antigenic stimulation. In some aspects, the expansion / proliferation of the modified cells is increased by at least about 1 fold, at least about 2 fold, at least about 3 fold, at least about 4 fold, at least about 5 fold, at least about 6 fold, at least about 7 fold, at least about 8 fold, at least about 9 fold, at least about 10 fold, at least about 11 fold, at least about 12 fold, at least about 13 fold, at least about 14 fold, at least about 15 fold, at least about 16 fold, at least about 17 fold, at least about 18 fold, at least about 19 fold, at least about 20 fold, at least about 25 fold, at least about 30 fold, at least about 35 fold, at least about 40 fold, at least about 45 fold, at least about 50 fold, at least about 75 fold, at least about 100 fold, at least about 200 fold, at least about 300 fold, at least about 400 fold, at least about 500 fold, at least about 750 fold, or at least about 1,000 fold compared to a reference cell.

[0191] In some aspects, the modified cells described herein (i.e., cells having reduced levels of NR4A3 genes and / or NR4A3 proteins) exhibit increased effector function, e.g., increased effector function in response to sustained antigenic stimulation, compared to reference cells (i.e., cells corresponding to cells having endogenous levels of NR4A3 genes and / or NR4A3 proteins). Non-limiting examples of such effector functions include cytokine (e.g., IFN-γ, TNF-α, IL-2, or a combination thereof) production, granzyme release, cytotoxicity in response to sustained antigenic stimulation, the ability to kill / lyse antigen-expressing cells, and combinations thereof. In some aspects, the effector function of the modified cells provided herein is increased by at least about 1 fold, at least about 2 fold, at least about 3 fold, at least about 4 fold, at least about 5 fold, at least about 6 fold, at least about 7 fold, at least about 8 fold, at least about 9 fold, at least about 10 fold, at least about 11 fold, at least about 12 fold, at least about 13 fold, at least about 14 fold, at least about 15 fold, at least about 16 fold, at least about 17 fold, at least about 18 fold, at least about 19 fold, at least about 20 fold, at least about 25 fold, at least about 30 fold, at least about 35 fold, at least about 40 fold, at least about 45 fold, at least about 50 fold, at least about 75 fold, at least about 100 fold, at least about 200 fold, at least about 300 fold, at least about 400 fold, at least about 500 fold, at least about 750 fold, or at least about 1,000 fold compared to a reference cell.

[0192] In some aspects, the modified cells provided herein (i.e., having reduced levels of the NR4A3 gene and / or NR4A3 protein) have reduced expression of one or more markers associated with depletion. Non-limiting examples of such markers include TIGIT, PD-1, CD39, and combinations thereof. The expression of such markers in a large population can be measured using batch RNASeq transcriptome analysis by flow cytometry, or in some aspects, single-cell RNASeq can be used to perform single-cell transcriptome analysis. In some aspects, the expression of one or more markers associated with depletion is reduced by at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or about 100% in the modified cells of the present disclosure compared to a reference cell.

[0193] Without being bound by any one theory, in some aspects, one or more of the improved properties described above are associated with increased resistance of the modified cells to apoptosis, increased tolerance of the modified cells to immune checkpoint modulation, increased activation in response to antigenic stimulation, or a combination thereof.

[0194] As further described and demonstrated herein, in some aspects, the modified cells provided herein (i.e., having reduced levels of the NR4A3 gene and / or NR4A3 protein) are capable of maintaining anti-tumor function in a tumor microenvironment (TME) compared to a reference cell (i.e., a corresponding cell having endogenous levels of the NR4A3 gene and / or NR4A3 protein).

[0195] III.A. Other Modifications

[0196] The immune cells described herein (e.g., modified with the polynucleotides described herein to have reduced levels of the NR4A3 gene and / or NR4A3 protein) can include one or more additional modifications. In some aspects, one or more additional modifications can further improve one or more properties of the cells. Non-limiting examples of such additional modifications are further described below.

[0197] III.A.1.NR4A2

[0198] In addition to reducing the level of NR4A3 gene and / or NR4A3 protein, in some aspects, the modified cells described herein can be further modified to have reduced levels of NR4A2 gene and / or NR4A2 protein. Any suitable method known in the art can be used to reduce the level of NR4A2 gene and / or NR4A2 protein in the modified cells described herein. For example, in some aspects, any gene editing tool described herein (e.g., CRISPR / Cas system) can be used to reduce the level of NR4A2 gene and / or NR4A2 protein.

[0199] Nuclear receptor subfamily 4A group 2 member 2 (often abbreviated as NR4A2, also known as NOT, RNR1, HZF-3, NURR1, TINUR) is a protein encoded by the NR4A2 gene in humans. The NR4A2 gene is located on chromosome 2 (bases 156,324,432 to 156,332,724, NCBI reference sequence: NC_000002.12). Unless otherwise indicated, the term "NR4A2 gene" as used herein refers to any nucleic acid sequence encoding the NR4A2 protein (or its variants).

[0200] The NR4A2 protein has two isoforms produced by alternative splicing. The sequences are shown in Table 2 below. Unless otherwise indicated and as further described herein, in some aspects, the immune cells described herein have been further modified to have reduced levels of any known NR4A2 protein (including any isoforms and variants thereof). Suitable methods for reducing NR4A2 gene and / or NR4A2 protein levels are described elsewhere in this disclosure and are also known in the art.

[0201] Table 2. NR4A2 protein isoforms.

[0202]

[0203] Thus, in some aspects, the modified cells described herein have: (i) reduced levels of the NR4A3 gene and / or NR4A3 protein, and (ii) reduced levels of the NR4A2 gene and / or NR4A2 protein, as compared to a reference cell. In some aspects, the modified immune cells described herein have reduced levels of the NR4A2 gene and / or NR4A2 protein (i.e., have reduced levels of the NR4A3 gene and / or NR4A3 protein) by at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or about 100%, as compared to a reference cell. In some aspects, the levels of the NR4A2 gene and / or NR4A2 protein are completely suppressed.

[0204] In some aspects, the modified immune cells described herein (i.e., having reduced levels of NR4A3 genes and / or NR4A3 proteins) have reduced levels of NR4A2 genes compared to reference cells. In some aspects, the level of NR4A2 gene is reduced by at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95% or about 100% compared to reference cells. In some aspects, the level of NR4A2 gene is completely suppressed.

[0205] In some aspects, the modified immune cells described herein (i.e., having reduced levels of NR4A3 gene and / or NR4A3 protein) have reduced levels of NR4A2 protein compared to a reference cell. In some aspects, the level of NR4A3 protein is reduced by at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or about 100% compared to a reference cell. In some aspects, the level of NR4A2 protein in the modified immune cells is completely suppressed.

[0206] In some aspects, the modified immune cells described herein (i.e., having reduced levels of NR4A3 gene and / or NR4A3 protein) have reduced levels of both NR4A2 gene and reduced levels of NR4A2 protein compared to reference cells. In some aspects, the levels of NR4A2 gene and NR4A2 protein are reduced by at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95% or about 100% compared to reference cells. In some aspects, the levels of both NR4A2 gene and NR4A2 protein are completely suppressed.

[0207] III.A.2.NR4A1

[0208] In addition to reducing the level of NR4A3 gene and / or NR4A3 protein, in some aspects, the modified cells described herein can be further modified to have reduced levels of NR4A1 gene and / or NR4A1 protein. Any suitable method known in the art can be used to reduce the level of NR4A1 gene and / or NR4A1 protein in the modified cells described herein. For example, in some aspects, any gene editing tool described herein (e.g., CRISPR / Cas system) can be used to reduce the level of NR4A1 gene and / or NR4A1 protein.

[0209] Nuclear receptor subfamily 4A group 4A member 1 (often abbreviated as NR4A1 and also known as HMR, N10, TR3, NP10, GFRP1, NAK-1, NGFIB, and NUR77) is a protein that is encoded by the NR4A1 gene in humans. The NR4A1 gene is located on chromosome 12 (bases 52022832 to 52059507; NCBI reference sequence NC_000012.12). Unless otherwise indicated, the term "NR4A1 gene" as used herein refers to any nucleic acid sequence encoding the NR4A1 protein (or its variant).

[0210] The NR4A1 protein has three isoforms produced by alternative splicing. The sequences are shown in Table 3 below.

[0211] Table 3. NR4A1 protein isoforms.

[0212]

[0213] Thus, in some aspects, the modified cells described herein have: (i) reduced levels of the NR4A3 gene and / or NR4A3 protein, and (ii) reduced levels of the NR4A1 gene and / or NR4A1 protein, compared to a reference cell. In some aspects, the modified cells described herein have: (i) reduced levels of the NR4A3 gene and / or NR4A3 protein, (ii) reduced levels of the NR4A2 gene and / or NR4A2 protein, and (iii) reduced levels of the NR4A1 gene and / or NR4A1 protein. In some aspects, the modified immune cells described herein have reduced levels of NR4A1 gene and / or NR4A1 protein (i.e., have reduced levels of NR4A3 gene and / or NR4A3 protein) by at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or about 100% compared to a reference cell. In some aspects, the level of NR4A1 gene and / or NR4A1 protein is completely suppressed.

[0214] In some aspects, the modified immune cells described herein (i.e., having reduced levels of NR4A3 genes and / or NR4A3 proteins) have reduced levels of NR4A1 genes compared to reference cells. In some aspects, the level of NR4A1 gene is reduced by at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95% or about 100% compared to reference cells. In some aspects, the level of NR4A1 gene is completely suppressed.

[0215] In some aspects, the modified immune cells described herein (i.e., having a reduced level of NR4A3 gene and / or NR4A3 protein) have a reduced level of NR4A1 protein compared to a reference cell. In some aspects, the level of NR4A3 protein is reduced by at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95% or about 100% compared to a reference cell. In some aspects, the level of NR4A1 protein in the modified immune cells is completely suppressed.

[0216] In some aspects, the modified immune cells described herein (i.e., NR4A3 genes and / or NR4A3 proteins with reduced levels) have both reduced levels of NR4A1 genes and reduced levels of NR4A1 proteins compared to reference cells. In some aspects, the levels of NR4A1 genes and NR4A1 proteins are reduced by at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95% or about 100% compared to reference cells. In some aspects, the levels of both NR4A1 genes and NR4A1 proteins are completely suppressed.

[0217] III.A.3.c-Jun

[0218] In some aspects, the modified cells described herein (i.e., having a reduced level of NR4A3 gene and / or NR4A3 protein) are further modified to have an increased level of c-Jun protein compared to a reference cell that has not been modified to have an increased level of c-Jun protein. Any suitable method known in the art can be used to increase the level of c-Jun protein in the modified immune cells described herein. For example, in some aspects, the modified immune cells described herein have been modified to include additional nucleotide sequences encoding c-Jun protein so that the level of c-Jun protein is increased compared to the reference cells. In some aspects, the additional nucleotide sequence encoding the c-Jun protein can be part of the same polynucleotide described herein (i.e., including a gRNA that specifically targets a region within the NR4A3 gene) - i.e., a polycistronic polynucleotide. In some aspects, the additional nucleotide sequence encoding the c-Jun protein can be introduced into the immune cell as a separate polynucleotide.

[0219] In some aspects, the modified immune cells provided herein (i.e., having reduced levels of NR4A3 genes and / or NR4A3 proteins) are capable of naturally expressing c-Jun proteins (e.g., without the need to modify the cells with exogenous nucleotide sequences encoding c-Jun proteins). In some aspects, such immune cells can be further modified with a transcriptional activator (e.g., a transcriptional activator based on a CRISPR / Cas system, e.g., CRISPRa) such that expression of endogenous c-Jun proteins is increased compared to reference cells (e.g., corresponding cells not modified with a transcriptional activator).

[0220] As used herein, the term "transcriptional activator" refers to a protein that increases the transcription of a gene or a set of genes (e.g., by binding to an enhancer or promoter proximal element of a nucleic acid sequence, thereby inducing its transcription). Non-limiting examples of such transcriptional activators that can be used with the present disclosure include: transcriptional activator-like effector (TALE)-based transcriptional activators, zinc finger protein (ZFP)-based transcriptional activators, clustered regularly interspaced short palindromic repeats (CRISPR) / CRISPR-associated protein (Cas) system-based transcriptional activators, or combinations thereof. See, for example, Kabadi et al., Methods 69(2): 188-197 (September 2014), the entire contents of which are incorporated herein by reference.

[0221] In some aspects, the modified cells described herein (i.e., having reduced levels of the NR4A3 gene and / or NR4A3 protein) have been modified with a transcriptional activator based on the CRISPR / Cas system, such as CRISPR activation (CRISPRa). See, for example, Nissim et al., Molecular Cell 54: 1-13 (May 2014), the entire contents of which are incorporated herein by reference. CRISPRa is a CRISPR tool that includes the use of a modified Cas protein that lacks endonuclease activity but retains the ability to bind to its guide RNA and target DNA nucleic acid sequence. Non-limiting examples of such modified Cas proteins that can be used with the present disclosure are known in the art. See, for example, Pandelakis et al., Cell Systems 10 (1): 1-14 (January 2020), the entire contents of which are incorporated herein by reference. In some aspects, the modified Cas protein comprises a modified Cas9 protein (also referred to as "dCas9" in the art). In some aspects, the modified Cas protein comprises a modified Cas12a protein. In some aspects, the modified Cas protein useful in the present disclosure is bound to a guide polynucleotide (e.g., a small guide RNA) ("modified Cas-guide complex"), wherein the guide polynucleotide comprises a recognition sequence complementary to a region of a nucleic acid sequence encoding a protein of interest (e.g., c-Jun). In some aspects, the guide polynucleotide comprises a recognition sequence complementary to a promoter region of an endogenous nucleic acid sequence encoding a protein of interest. In some aspects, one or more transcriptional activators are attached to the modified Cas-guide complex (e.g., the N-terminus and / or C-terminus of a modified Cas protein) so that when the modified Cas-guide complex is introduced into a cell, one or more transcriptional activators can bind to a regulatory element (e.g., a promoter region) of the nucleic acid sequence to induce and / or increase expression of the encoded protein (e.g., c-Jun). In some aspects, one or more transcriptional activators can bind to a regulatory element (e.g., a promoter region) of an endogenous gene to induce and / or increase expression of the encoded protein (e.g., c-Jun). Non-limiting illustrative examples of common universal activators that can be used include the omega subunit of RNAP, VP16, VP64, and p65. See, e.g., Kabadi and Gersbach, Methods 69: 188-197 (2014), which is incorporated herein by reference in its entirety.

[0222] In some aspects, one or more transcriptional repressors (e.g., Kruppel-associated box domains (KRABs)) can be attached to a modified Cas-guide complex (e.g., the N-terminus and / or C-terminus of a modified Cas protein) such that, when introduced into a cell, the one or more transcriptional repressors can inhibit or reduce transcription of genes, e.g., genes such as those that can interfere with the expression of c-Jun (e.g., Bach2). See, e.g., US20200030379A1 and Yang et al., J Transl Med 19:459 (2021), each of which is incorporated herein by reference in its entirety. In some aspects, the modified Cas proteins useful in the present disclosure can be attached to both one or more transcriptional activators and one or more transcriptional repressors.

[0223] In some aspects, due to the above modifications (e.g., introduction of exogenously introduced c-Jun nucleotide sequences and / or transcriptional activators), the modified cells described herein (i.e., having reduced levels of NR4A3 gene and / or NR4A3 protein) overexpress, i.e., express higher levels (e.g., at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or at least about 100% higher, or at least about 1.5-fold, at least about 2-fold, at least about 3-fold, at least about 4-fold, at least about 5-fold, or at least about 10-fold higher) of c-Jun protein compared to a reference cell that has not been modified to have increased levels of c-Jun protein. The terms "expressing increased levels [or amounts]," "overexpressing," or "increased expression" (and similar forms of the phrases used herein) are used interchangeably.

[0224] c-Jun is an oncogenic transcription factor that belongs to the activator protein-1 (AP-1) family. It interacts with a variety of proteins (e.g., c-Fos) to form a dimeric complex that regulates a variety of cell signaling pathways, including cell proliferation and tumor progression. Therefore, increased c-Jun expression has been observed in certain cancers, and there is great interest in developing c-Jun antagonists to treat such cancers. See, for example, Brennan, A., et al., J Exp Clin Cancer Res 39(1):184 (September 2020).

[0225] In humans, the c-Jun protein is encoded by the JUN gene, which is located on chromosome 1 (nucleotides 58,780,791 to 58,784,047 of GenBank Accession No. NC_000001.11, minus-strand orientation). Synonyms for the JUN gene and its encoded protein are known, including "Jun proto-oncogene, AP-1 transcription factor subunit," "v-Jun avian sarcoma virus 17 oncogene homolog," "transcription factor AP-1," "Jun oncogene," "AP-1," "Jun activation domain binding protein," "p39," and "enhancer binding protein AP1." The wild-type human c-Jun protein sequence is 331 amino acids long. The amino acid and nucleic acid sequences of wild-type human c-Jun are provided in Tables 4 and 5, respectively.

[0226] Table 4. c-Jun protein sequence

[0227]

[0228] Table 5. c-Jun nucleic acid sequences

[0229]

[0230]

[0231] Unless otherwise indicated, c-Jun proteins useful in the present disclosure include wild-type human c-Jun proteins and any variants or mutants thereof. In some aspects, the c-Jun protein can be a mutant human c-Jun protein, as long as the mutant c-Jun protein does not affect the ability of the mutant to rescue dysfunctional (exhausted) T cells. In some aspects, the mutant c-Jun protein comprises at least about 70% (e.g., at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98% or at least about 99%) sequence identity to the C-terminal amino acid residues (e.g., C-terminal 50, 75, 100, 150, 200, or 250 or more residues), C-terminal portion (e.g., a quarter, a third, or a half) or C-terminal domain (e.g., ε, bZIP, and their C-terminal amino acids) of the wild-type c-Jun protein. In some aspects, the N-terminal amino acid residue (e.g., the N-terminal 50, 75, 100, or 150 or more), N-terminal portion (e.g., one quarter, one third, or one half), or N-terminal domain (e.g., delta, the transactivation domain and the amino acids N-terminal thereto) of a wild-type c-Jun protein is deleted, mutated, or inactivated.

[0232] In some aspects, the c-Jun protein comprises an inactivating mutation (e.g., a substitution, deletion, or insertion) in its transactivation domain and / or its delta domain. In some aspects, the c-Jun protein comprises one or both of the S63A and S73A mutations. In some aspects, the c-Jun protein has a deletion between residues 2 and 102 or between residues 30 and 50 compared to wild-type human c-Jun.

[0233] In some aspects, the c-Jun polypeptides useful in the modified immune cells of the present invention comprise truncated c-Jun polypeptides, as disclosed in WO2019 / 118902, which is expressly incorporated herein by reference in its entirety.

[0234] As described herein, in some aspects, the modified cells described herein (i.e., having reduced levels of the NR4A3 gene and / or NR4A3 protein) comprise a nucleotide sequence encoding a c-Jun protein, wherein the nucleotide sequence has been codon-optimized. Thus, in some aspects, the nucleotide sequence encoding the c-Jun protein described herein (also referred to herein as a "c-Jun nucleotide sequence") is different from the wild-type c-Jun nucleotide sequence (e.g., SEQ ID NO: 6).

[0235] In some aspects, the nucleotide sequence encoding the c-Jun protein has at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to any one of the nucleic acid sequences shown in SEQ ID NOs: 7 to 16. In some aspects, the nucleotide sequence encoding the c-Jun protein comprises the nucleic acid sequence shown in any one of SEQ ID NOs: 7 to 16.

[0236] In some aspects, the nucleotide sequence encoding the c-Jun protein has at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the nucleic acid sequence set forth in SEQ ID NO: 7. In some aspects, the nucleotide sequence encoding the c-Jun protein has at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the nucleic acid sequence set forth in SEQ ID NO: 7. In some aspects, the nucleotide sequence comprises the nucleic acid sequence set forth in SEQ ID NO: 7.

[0237] In some aspects, the nucleotide sequence encoding the c-Jun protein has at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the nucleic acid sequence set forth in SEQ ID NO: 8. In some aspects, the nucleotide sequence encoding the c-Jun protein has at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the nucleic acid sequence set forth in SEQ ID NO: 8. In some aspects, the nucleotide sequence comprises the nucleic acid sequence set forth in SEQ ID NO: 8.

[0238] In some aspects, the nucleotide sequence encoding the c-Jun protein has at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the nucleic acid sequence set forth in SEQ ID NO: 9. In some aspects, the nucleotide sequence encoding the c-Jun protein described herein has at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the nucleic acid sequence set forth in SEQ ID NO: 9. In some aspects, the nucleotide sequence comprises the nucleic acid sequence set forth in SEQ ID NO: 9.

[0239] In some aspects, the nucleotide sequence encoding the c-Jun protein has at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the nucleic acid sequence shown in SEQ ID NO: 10. In some aspects, the nucleotide sequence has at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the nucleic acid sequence shown in SEQ ID NO: 10. In some aspects, the nucleotide sequence comprises the nucleic acid sequence shown in SEQ ID NO: 10.

[0240] In some aspects, the nucleotide sequence encoding the c-Jun protein has at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the nucleic acid sequence shown in SEQ ID NO: 11. In some aspects, the nucleotide sequence encoding the c-Jun protein has at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the nucleic acid sequence shown in SEQ ID NO: 11. In some aspects, the nucleotide sequence comprises the nucleic acid sequence shown in SEQ ID NO: 11.

[0241] In some aspects, the nucleotide sequence encoding the c-Jun protein has at least about 80%, at least 85%, at least 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the nucleic acid sequence shown in SEQ ID NO: 12. In some aspects, the nucleotide sequence encoding the c-Jun protein has at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the nucleic acid sequence shown in SEQ ID NO: 12. In some aspects, the nucleotide sequence comprises the nucleic acid sequence shown in SEQ ID NO: 12.

[0242] In some aspects, the nucleotide sequence encoding the c-Jun protein has at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the nucleic acid sequence shown in SEQ ID NO: 13. In some aspects, the nucleotide sequence encoding the c-Jun protein has at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the nucleic acid sequence shown in SEQ ID NO: 13. In some aspects, the nucleotide sequence comprises the nucleotide sequence shown in SEQ ID NO: 13.

[0243] In some aspects, the nucleotide sequence encoding the c-Jun protein has at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the nucleic acid sequence shown in SEQ ID NO: 14. In some aspects, the nucleotide sequence encoding the c-Jun protein has at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the nucleic acid sequence shown in SEQ ID NO: 14. In some aspects, the nucleotide sequence comprises the nucleotide sequence shown in SEQ ID NO: 14.

[0244] In some aspects, the nucleotide sequence encoding the c-Jun protein has at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the nucleic acid sequence shown in SEQ ID NO: 15. In some aspects, the nucleotide sequence encoding the c-Jun protein has at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the nucleic acid sequence shown in SEQ ID NO: 15. In some aspects, the nucleotide sequence comprises the nucleotide sequence shown in SEQ ID NO: 15.

[0245] In some aspects, the nucleotide sequence encoding the c-Jun protein has at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the nucleic acid sequence shown in SEQ ID NO: 16. In some aspects, the nucleotide sequence encoding the c-Jun protein has at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the nucleic acid sequence shown in SEQ ID NO: 16. In some aspects, the nucleotide sequence comprises the nucleotide sequence shown in SEQ ID NO: 16.

[0246] In some aspects, the nucleotide sequence encoding the c-Jun protein has at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the nucleic acid sequence shown in SEQ ID NO: 16. In some aspects, the nucleotide sequence encoding the c-Jun protein has at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the nucleic acid sequence shown in SEQ ID NO: 16. In some aspects, the nucleotide sequence comprises the nucleotide sequence shown in SEQ ID NO: 16.

[0247] Table 6. Codon-optimized c-Jun nucleotide sequences

[0248]

[0249]

[0250]

[0251]

[0252] The c-Jun nucleotide sequences disclosed herein can be codon optimized using any method known in the art. For example, in some aspects, the codons of the c-Jun nucleotide sequences disclosed herein have been optimized to modify (e.g., increase or decrease) one or more of the following parameters compared to the wild-type nucleotide sequence (e.g., SEQ ID NO: 6): (i) codon adaptation index (i.e., codon usage bias); (ii) guanine-cytosine (GC) nucleotide content; (iii) mRNA secondary structure and unstable motifs; (iv) repetitive sequences (e.g., direct repeats, inverted repeats, binary repeats); (v) restriction enzyme recognition sites; or (vi) combinations thereof.

[0253] Without being bound by any one theory, in some aspects, such codon optimization can increase expression of the protein encoded by the nucleotide sequence. Thus, in some aspects, when the codon-optimized c-Jun nucleotide sequences of the present disclosure are transfected, transduced, or introduced into the modified cells described herein (i.e., having reduced levels of NR4A3 gene and / or NR4A3 protein), expression of the encoded c-Jun transcription factor is increased compared to a reference cell transfected with a wild-type nucleotide sequence (e.g., SEQ ID NO: 6). In some aspects, the codon-optimized c-Jun nucleotide sequences of the present disclosure can increase expression of the encoded c-Jun transcription factor when transfected, transduced, or introduced into the modified cells described herein (i.e., having reduced levels of NR4A3 gene and / or NR4A3 protein). NO:6) the expression of the c-Jun protein is increased by at least about 1-fold, at least about 2-fold, at least about 3-fold, at least about 4-fold, at least about 5-fold, at least about 6-fold, at least about 7-fold, at least about 8-fold, at least about 9-fold, at least about 10-fold, at least about 11-fold, at least about 12-fold, at least about 13-fold, at least about 14-fold, at least about 15-fold, at least about 16-fold, at least about 17-fold, at least about 18-fold, at least about 19-fold, at least about 20-fold, at least about 25-fold, at least about 30-fold, at least about 35-fold, at least about 40-fold, at least about 45-fold, at least about 50-fold, at least about 75-fold, at least about 100-fold, at least about 200-fold, at least about 300-fold, at least about 400-fold, at least about 500-fold, at least about 750-fold, or at least about 1,000-fold or more compared to the corresponding expression in a reference cell that is transfected, transduced, or otherwise genetically modified.

[0254] Thus, in some aspects, the modified cells described herein have: (i) reduced levels of NR4A3 gene and / or NR4A3 protein, and (ii) increased levels of c-Jun protein, compared to a reference cell that has not been modified as described herein. In some aspects, the modified cells described herein have: (i) reduced levels of NR4A3 gene and / or NR4A3 protein, (ii) reduced levels of NR4A2 gene and / or NR4A2 protein, and (iii) increased levels of c-Jun protein, compared to a reference cell that has not been modified as described herein. In some aspects, the modified cells described herein have: (i) reduced levels of NR4A3 gene and / or NR4A3 protein, (ii) reduced levels of NR4A2 gene and / or NR4A2 protein, and (iii) increased levels of c-Jun protein, compared to a reference cell that has not been modified as described herein.

[0255] As can be seen from the present disclosure, in some aspects, an increased level of c-Jun protein in the modified cells described herein (i.e., having a reduced level of NR4A3 gene and / or NR4A3 protein) can further improve and / or enhance one or more properties of the modified cells. For example, in some aspects, when an immune cell is modified to have both (1) a reduced level of NR4A3 gene and / or NR4A3 protein and (2) an increased level of c-Jun protein, one or more properties of the immune cell (e.g., described below) are improved and / or enhanced compared to a reference cell. In some aspects, the reference cell includes a corresponding immune cell that has been modified to have only a reduced level of NR4A3 gene and / or NR4A3 protein (i.e., not overexpressing c-Jun). In some aspects, the reference cell includes a corresponding immune cell that has been modified to have only an increased expression of c-Jun protein (i.e., not having a reduced level of NR4A3 gene and / or NR4A3 protein). In some aspects, the reference cell includes a corresponding immune cell that has not been modified to have both (1) a reduced level of the NR4A3 gene and / or NR4A3 protein and (2) an elevated level of the c-Jun protein. In some aspects, the reference cell includes each of: (a) a corresponding immune cell that has been modified to have only a reduced level of the NR4A3 gene and / or NR4A3 protein (i.e., without overexpressing c-Jun), (b) a corresponding immune cell that has been modified to have only increased expression of the c-Jun protein (i.e., without having a reduced level of the NR4A3 gene and / or NR4A3 protein), and (c) a corresponding immune cell that has not been modified to have both (1) a reduced level of the NR4A3 gene and / or NR4A3 protein and (2) an elevated level of the c-Jun protein.

[0256] In some aspects, the modified immune cells described herein (i.e., having decreased levels of the NR4A3 gene and / or NR4A3 protein alone or in combination with other members of the NR4A family) with elevated levels of c-Jun protein exhibit increased tolerance to depletion compared to a reference cell that does not have elevated levels of c-Jun protein. In some aspects, the resistance to depletion is increased by at least about 1 fold, at least about 2 fold, at least about 3 fold, at least about 4 fold, at least about 5 fold, at least about 6 fold, at least about 7 fold, at least about 8 fold, at least about 9 fold, at least about 10 fold, at least about 11 fold, at least about 12 fold, at least about 13 fold, at least about 14 fold, at least about 15 fold, at least about 16 fold, at least about 17 fold, at least about 18 fold, at least about 19 fold, at least about 20 fold, at least about 21 fold, at least about 22 fold, at least about 23 fold, at least about 24 fold, at least about 25 fold, at least about 26 fold, at least about 27 fold, at least about 28 fold, at least about 29 fold, at least about 30 fold, at least about 31 fold, at least about 32 fold, at least about 33 fold, at least about 34 fold, at least about 35 fold, at least about 36 fold, at least about 37 fold, at least about 38 fold, at least about 39 fold, at least about 40 fold, at least about 41 fold, at least about 42 fold, at least about 43 fold, at least about 44 fold, at least about 45 fold, at least about 46 fold, at least about 47 fold At least about 14 times, at least about 15 times, at least about 16 times, at least about 17 times, at least about 18 times, at least about 19 times, at least about 20 times, at least about 25 times, at least about 30 times, at least about 35 times, at least about 40 times, at least about 45 times, at least about 50 times, at least about 75 times, at least about 100 times, at least about 200 times, at least about 300 times, at least about 400 times, at least about 500 times, at least about 750 times, or at least about 1,000 times or more.

[0257] In some aspects, overexpression of c-Jun protein can help to further reduce the exhaustion of the modified immune cells described herein (i.e., having reduced levels of NR4A3 gene and / or NR4A3 protein alone or in combination with other members of the NR4A family). In some aspects, exhaustion is reduced by at least about 1 fold, at least about 2 fold, at least about 3 fold, at least about 4 fold, at least about 5 fold, at least about 6 fold, at least about 7 fold, at least about 8 fold, at least about 9 fold, at least about 10 fold, at least about 11 fold, at least about 12 fold, at least about 13 fold, at least about 14 fold, at least about 15 fold, at least about 16 fold, at least about 17 fold, at least about 18 fold, at least about 19 fold, at least about 20 fold, at least about 21 fold, at least about 22 fold, at least about 23 fold, at least about 24 fold, at least about 25 fold, at least about 26 fold, at least about 27 fold, at least about 28 fold, at least about 29 fold, at least about 30 fold, at least about 31 fold, at least about 32 fold, at least about 33 fold, at least about 34 fold, at least about 35 fold, at least about 36 fold, at least about 37 fold, at least about 38 fold, at least about 39 fold, at least about 40 fold, at least about 41 fold, at least about 42 fold, at least about 43 fold, at least about 44 fold, at least about 45 fold, at least about 46 fold, at least about 47 fold, at least about 48 fold, at least about 49 fold, at least At least about 14 times, at least about 15 times, at least about 16 times, at least about 17 times, at least about 18 times, at least about 19 times, at least about 20 times, at least about 25 times, at least about 30 times, at least about 35 times, at least about 40 times, at least about 45 times, at least about 50 times, at least about 75 times, at least about 100 times, at least about 200 times, at least about 300 times, at least about 400 times, at least about 500 times, at least about 750 times, or at least about 1,000 times.

[0258] In some aspects, elevated levels of c-Jun protein can help further increase the persistence / survival of the modified cells described herein (i.e., having decreased levels of the NR4A3 gene and / or NR4A3 protein alone or in combination with other members of the NR4A family), for example, when administered in vivo to a subject. In some aspects, the persistence / survival of the modified cells is increased by at least about 1 fold, at least about 2 fold, at least about 3 fold, at least about 4 fold, at least about 5 fold, at least about 6 fold, at least about 7 fold, at least about 8 fold, at least about 9 fold, at least about 10 fold, at least about 11 fold, at least about 12 fold, at least about 13 fold, at least about 15 fold, at least about 16 fold, at least about 17 fold, at least about 18 fold, at least about 19 fold, at least about 20 fold, at least about 21 fold, at least about 22 fold, at least about 23 fold, at least about 24 fold, at least about 25 fold, at least about 26 fold, at least about 27 fold, at least about 28 fold, at least about 29 fold, at least about 30 fold, at least about 31 fold, at least about 32 fold, at least about 33 fold, at least about 34 fold, at least about 35 fold, at least about 36 fold, at least about 37 fold, at least about 38 fold, at least about 39 fold, at least about 40 fold, at least about 41 fold, at least about 42 fold, at least about 43 fold, at least about 44 fold, at least about 45 fold, at least about 46 fold, at least about 47 fold, at least about 48 fold, At least about 3 times, at least about 14 times, at least about 15 times, at least about 16 times, at least about 17 times, at least about 18 times, at least about 19 times, at least about 20 times, at least about 25 times, at least about 30 times, at least about 35 times, at least about 40 times, at least about 45 times, at least about 50 times, at least about 75 times, at least about 100 times, at least about 200 times, at least about 300 times, at least about 400 times, at least about 500 times, at least about 750 times, or at least about 1,000 times or more.

[0259] In some aspects, elevated levels of c-Jun protein can help further increase the expansion / proliferation of the modified cells described herein (i.e., having decreased levels of the NR4A3 gene and / or NR4A3 protein alone or in combination with other members of the NR4A family). In some aspects, the expansion / proliferation of the modified cells is increased by at least about 1 fold, at least about 2 fold, at least about 3 fold, at least about 4 fold, at least about 5 fold, at least about 6 fold, at least about 7 fold, at least about 8 fold, at least about 9 fold, at least about 10 fold, at least about 11 fold, at least about 12 fold, at least about 13 fold, compared to a reference cell (e.g., a corresponding cell that has not been modified to have increased c-Jun expression and / or decreased expression of the NR4A gene and / or NR4A protein). times, at least about 14 times, at least about 15 times, at least about 16 times, at least about 17 times, at least about 18 times, at least about 19 times, at least about 20 times, at least about 25 times, at least about 30 times, at least about 35 times, at least about 40 times, at least about 45 times, at least about 50 times, at least about 75 times, at least about 100 times, at least about 200 times, at least about 300 times, at least about 400 times, at least about 500 times, at least about 750 times, or at least about 1,000 times or more.

[0260] In some aspects, elevated levels of c-Jun protein can contribute to further increasing the effector function of the modified cells described herein (i.e., having reduced levels of NR4A3 gene and / or NR4A3 protein alone or in combination with other members of the NR4A family). Non-limiting examples of such effector functions include cytokine (e.g., IFN-γ, TNF-α, IL-2, or a combination thereof) production, granzyme release, cytotoxicity in response to sustained antigen stimulation, the ability to kill / lyse antigen-expressing cells, and combinations thereof. In some aspects, the effector function of the modified cells described herein is increased by at least about 1 fold, at least about 2 fold, at least about 3 fold, at least about 4 fold, at least about 5 fold, at least about 6 fold, at least about 7 fold, at least about 8 fold, at least about 9 fold, at least about 10 fold, at least about 11 fold, at least about 12 fold, at least about 13 fold, at least about 14 fold, at least about 15 fold, at least about 16 fold, at least about 17 fold, at least about 18 fold, at least about 19 fold, at least about 20 fold, at least about 21 fold, at least about 22 fold, at least about 23 fold, at least about 24 fold, at least about 25 fold, at least about 26 fold, at least about 27 fold, at least about 28 fold, at least about 29 fold, at least about 30 fold, at least about 31 fold, at least about 32 fold, at least about 33 fold, at least about 34 fold, at least about 35 fold, at least about 36 fold, at least about 37 fold, at least about 38 fold, at least about 39 fold, at least about 4 At least about 13 times, at least about 14 times, at least about 15 times, at least about 16 times, at least about 17 times, at least about 18 times, at least about 19 times, at least about 20 times, at least about 25 times, at least about 30 times, at least about 35 times, at least about 40 times, at least about 45 times, at least about 50 times, at least about 75 times, at least about 100 times, at least about 200 times, at least about 300 times, at least about 400 times, at least about 500 times, at least about 750 times, or at least about 1,000 times or more.

[0261] In some aspects, the modified immune cells described herein that have been further modified to have elevated levels of c-Jun protein (e.g., with reduced levels of NR4A3 gene and / or NR4A3 protein) have reduced expression of one or more exhaustion markers, including but not limited to TIGIT, PD-1, and CD39. The expression of exhaustion markers in a large population can be measured using bulk RNASeq transcriptome analysis by flow cytometry, or in some aspects, single-cell RNASeq can be used to perform single-cell transcriptome analysis. In some aspects, expression of one or more depletion markers is reduced by at least about 1.5-fold, at least about 2-fold, at least about 2.5-fold, at least about 3.0-fold, at least about 3.5-fold, at least about 4-fold, at least about 4.5-fold, at least about 5-fold, at least about 10-fold, at least about 15-fold, at least about 20-fold, at least about 25-fold, at least about 30-fold, at least about 35-fold, at least about 40-fold, at least about 45-fold, at least about 50-fold, at least about 55-fold, at least about 60-fold, at least about 65-fold, at least about 70-fold, at least about 75-fold, at least about 80-fold, at least about 85-fold, at least about 90-fold, at least about 95-fold, or at least about 100-fold or more compared to a reference cell (e.g., a corresponding cell that has not been modified to have increased c-Jun expression and / or decreased expression of the NR4A3 gene and / or NR4A3 protein).

[0262] Thus, it can be seen from at least the above disclosure that, in some aspects, immune cells that have been modified to express (1) reduced levels of the NR4A3 gene and / or NR4A3 protein, and (2) increased levels of the c-Jun protein exhibit enhanced in vivo anti-tumor activity compared to reference cells. In some aspects, immune cells that have been modified to express (1) reduced levels of the NR4A3 gene and / or NR4A3 protein, and (2) increased levels of the c-Jun protein exhibit greater survival and / or persistence in vivo compared to reference cells. As further described elsewhere in this disclosure, in some aspects, the reference cell includes one or more of the following: (a) a corresponding immune cell that has been modified to have only a reduced level of the NR4A3 gene and / or NR4A3 protein (i.e., without overexpressing c-Jun), (b) a corresponding immune cell that has been modified to have only increased expression of the c-Jun protein (i.e., without having a reduced level of the NR4A3 gene and / or NR4A3 protein), and (c) a corresponding immune cell that has not been modified to have both (1) a reduced level of the NR4A3 gene and / or NR4A3 protein and (2) an increased level of the c-Jun protein.

[0263] III.A.4. Ligand-binding proteins

[0264] In some aspects, the modified cells described herein (e.g., having reduced levels of NR4A3 genes and / or NR4A3 proteins) are further modified to express ligand binding proteins. As used herein, the term "ligand binding protein" refers to any protein capable of binding to a molecule of interest (i.e., a ligand) (e.g., an antigen or peptide / MHC complex expressed on a tumor cell). In some aspects, the ligand binding protein is a chimeric binding protein. As used herein, the term "chimeric binding protein" refers to a protein that is capable of binding to one or more ligands (e.g., an antigen (e.g., comprising an antigen binding portion)) and is produced by connecting two or more polynucleotide sequences that originally encoded separate proteins. Unless otherwise indicated, the terms are used interchangeably in this disclosure.

[0265] Any suitable method known in the art can be used to express the ligand binding protein. For example, in some aspects, the modified cells described herein have been further modified to include additional nucleotide sequences encoding the ligand binding protein. In some aspects, the additional nucleotide sequences encoding the ligand binding protein can be part of the same polynucleotide described herein (i.e., including a gRNA that specifically targets a region within the NR4A3 gene) - i.e., a multicistronic polynucleotide. In some aspects, the additional nucleotide sequences encoding the ligand binding protein can be introduced into the cell as separate polynucleotides.

[0266] Thus, in some aspects, the modified cells described herein (i) express a ligand-binding protein and have (ii) a reduced level of NR4A3 gene and / or NR4A3 protein compared to a reference cell. In some aspects, the modified cells described herein (i) express a ligand-binding protein and have (ii) a reduced level of NR4A3 gene and / or NR4A3 protein and (iii) an increased level of c-Jun protein compared to a reference cell. In some aspects, the modified cells described herein (i) express a ligand-binding protein and (ii) have a reduced level of NR4A3 gene and / or NR4A3 protein, and (iii) have a reduced level of NR4A2 gene and / or NR4A2 protein compared to a reference cell. In some aspects, the modified cells described herein (i) express a ligand-binding protein and have (ii) a reduced level of NR4A3 gene and / or NR4A3 protein, (iii) a reduced level of NR4A2 gene and / or NR4A2 protein, and (iv) an increased level of c-Jun protein compared to a reference cell. In some aspects, the modified cells described herein (i) express the ligand binding protein and have (ii) reduced levels of the NR4A3 gene and / or NR4A3 protein, (iii) reduced levels of the NR4A2 gene and / or NR4A2 protein, and (iv) reduced levels of the NR4A1 gene and / or NR4A1 protein, compared to a reference cell. In some aspects, the modified cells described herein (i) express the ligand binding protein and have (ii) reduced levels of the NR4A3 gene and / or NR4A3 protein, (iii) reduced levels of the NR4A2 gene and / or NR4A2 protein, (iv) reduced levels of the NR4A1 gene and / or NR4A1 protein, and (v) increased levels of c-Jun protein, compared to a reference cell.

[0267] Non-limiting examples of ligand binding proteins (e.g., chimeric binding proteins) that can be used in the present disclosure include chimeric antigen receptors (CARs), T cell receptors (TCRs) (e.g., engineered TCRs), chimeric antibody-T cell receptors (caTCRs), chimeric signaling receptors (CSRs), T cell receptor mimics (TCR mimics), and combinations thereof.

[0268] In some aspects, the chimeric binding protein comprises a CAR.

[0269] In some aspects, CAR is designed as standard CAR. In " standard CAR ", different components (for example, extracellular targeting domain, transmembrane domain and intracellular signal transduction / activation domain) are linearly constructed as a single fusion protein. In some aspects, CAR is designed as first generation CAR. "First generation" CAR consists of extracellular binding domain, hinge region, transmembrane domain and one or more intracellular signal transduction domains. All first generation CARs include CD3 ζ chain domain as intracellular signal transduction domain. In some aspects, CAR is designed as second generation CAR. "Second generation" CAR additionally contains costimulatory domain (for example, CD28 or 4-1BB). In some aspects, CAR is designed as third generation CAR. "Third generation" CAR is similar to second generation CAR, except that they include multiple costimulatory domains (for example, CD28-4-1BB or CD28-OX40). In some aspects, CAR is designed as fourth generation CAR. "Fourth generation" CAR (also referred to as TRUCK or armored CAR) additionally contains additional factors that can further improve function. For example, in some aspects, the fourth generation CAR contains in addition CAR cytokines that can be released when signal transduction in targeted tumor tissue.In some aspects, the fourth generation CAR includes one or more additional elements, such as homing genes and suicide genes, which can help further regulate the activity of CAR.In some aspects, CAR is designed as split CAR.In " split CAR " system, one or more components of CAR (for example, extracellular targeting domain, transmembrane domain and intracellular signal transduction / activation domain) are divided into two or more parts so that it depends on multiple inputs for promoting complete functional receptor assembly.In some aspects, CAR is designed as switchable CAR.Utilizing " switchable CAR ", CAR can be switched (for example, instantaneously) to open (open switch CAR) or close (close switch CAR) in the presence of stimulation.The other examples of CAR that can be used together with the present disclosure are described in, for example, US2020 / 0172879 A1 and US 2019 / 0183932A1, each of which is incorporated herein by reference in its entirety.

[0270] In some aspects, the construct encoding engineered T cell receptor (TCR) herein is also referred to as " transgenic TCR " in the art. TCR is a molecule present on the surface of T cells that is responsible for identifying antigen fragments as peptides combined with major histocompatibility complex (MHC) molecules. TCR is a heterodimer consisting of two different protein chains. In some aspects, TCR is composed of α (α) chain and β (β) chain (encoded by TRA and TRB, respectively). In some aspects, TCR is composed of γ and δ (γ / δ) chain (encoded by TRG and TRD, respectively). When TCR is combined with the antigen peptide (peptide / MHC) presented by MHC molecules, T lymphocytes are activated by signal transduction. In some aspects, TCR is engineered (transgenic) TCR. As used herein, the term "engineered TCR" or "engineered T cell receptor" refers to a T cell receptor (TCR) that has been isolated or engineered to specifically bind to a major histocompatibility complex (MHC) / peptide target antigen with a desired affinity and introduced into a population of immune cells (e.g., T cells, NK cells and / or TILs).

[0271] In some aspects, the chimeric binding protein comprises a chimeric antibody T cell receptor (caTCR). As used herein, "chimeric antibody-T cell receptor" or "caTCR" comprises an antibody portion that specifically binds to an antigen of interest and (ii) a T cell receptor module that can raise at least one TCR-related signaling molecule. In some aspects, the antibody portion and the T cell receptor module are fused together. In some aspects, the chimeric binding protein comprises a chimeric signaling receptor (CSR). "Chimeric signaling receptor" or "CSR" comprises a ligand binding domain that specifically binds to a target ligand and a costimulatory signaling domain that can provide a stimulation signal to immune cells expressing CSR. Non-limiting examples of caTCR and CSR are further described in US10,822,413B2, which is incorporated herein by reference in its entirety.

[0272] In some aspects, the chimeric binding protein comprises a T cell receptor mimic (TCR mimic). As used herein, the term "T cell receptor mimic" or "TCR mimic" refers to an antibody (or fragment thereof) that has been engineered to recognize a tumor antigen, wherein the tumor antigen is displayed in the context of an HLA molecule. As will be apparent to those skilled in the art, these antibodies can simulate the specificity of TCR. Non-limiting examples of TCR mimics are provided, for example, in US2009 / 0226474A1 and US2019 / 0092876 A1, each of which is incorporated herein by reference in its entirety.

[0273] In some aspects, chimeric binding protein can be associated with gene editing tools (for example, CRISPR-Cas system), wherein the activation of chimeric binding protein can induce the activation of gene editing tools so that the expression and / or activity of one or more genes are regulated in cells. For example, in some aspects, cells as described herein (for example, T cells) are modified to include chimeric binding protein (for example, CAR) connected to protease and a single guide RNA of the regulatory region (for example, promoter) of the targeting gene of interest. In some aspects, the cell is modified to also include a joint for activating T cells (LAT), for example, by a joint and gene editing tool compound. The activation of chimeric binding protein (for example, by antigen stimulation) allows the release of gene editing tools for nuclear localization and gene expression regulation. Other aspects of such chimeric binding protein are provided in other places of the present disclosure. See also Pietrobon et al., Int J Mol Sci 22 (19): 10828 (October 2021), the entire contents of which are incorporated herein by reference.

[0274] As described herein, chimeric binding proteins useful in the present disclosure comprise an antigen binding domain, a transmembrane domain, a co-stimulatory domain, an intracellular signaling domain, or a combination thereof. In some aspects, the antigen binding domain recognizes and specifically binds to an antigen. Non-limiting examples of antigens include: AFP (alpha-fetoprotein), αvβ6 or another integrin, BCMA, Braf, B7-H3, B7-H6, CA9 (carbonic anhydrase 9), CCL-1 (CC motif chemokine ligand 1), CD5, CD19, CD20, CD21, CD22, CD23, CD24, CD30, CD33, CD38, CD40, CD44, CD44v6, CD44v7 / 8, CD45, CD47, CD56, CD66e, CD70, CD 74, CD79a, CD79b, CD98, CD123, CD138, CD171, CD352, CEA (carcinoembryonic antigen), claudin 18.2, claudin 6, c-MET, DLL3 (delta-like protein 3), DLL4, ENPP3 (ectonucleotide pyrophosphatase / phosphodiesterase family member 3), EpCAM, EPG-2 (epithelial glycoprotein 2), EPG-40, ephrin B2, EPHa2 (ephrine receptor A2), ERBB dimer, estrogen receptor, ETB R (endothelin B receptor), FAP-α (fibroblast activation protein α), fetal AchR (fetal acetylcholine receptor), FBP (folate binding protein), FCRL5, FR-α (folate receptor α), GCC (guanylate cyclase C), GD2, GD3, GPC2 (glypican 2), GPC3, gp100 (glycoprotein 100), GPNMB (glycoprotein NMB), GPRC5D (G protein-coupled receptor 5D), HER2, HER3, HER4, hepatitis B surface antigen, HL A-A1 (human leukocyte antigen Al), HLA-A2 (human leukocyte antigen A2), HMW-MAA (human high molecular weight melanoma-associated antigen), IGF1R (insulin-like growth factor 1 receptor), Igκ, Igλ, IL-22Ra (IL-22 receptor alpha), IL-13Ra2 (IL-13 receptor alpha 2), KDR (kinase insert domain receptor), LI cell adhesion molecule (LI-CAM), Liv-1, LRRC8A (leucine-rich repeat-containing protein 8 family member A), Lewis Y, melanoma-associated antigen (MAGE)-A1, MAGE-A3, MAGE-A6, MART-1 (melan A), murine cytomegalovirus (MCMV), MCSP (melanoma-associated chondroitin sulfate proteoglycan), mesothelin, mucin 1 (MUC1), MUC16, MHC / peptide complexes (e.g., with those derived from AFP, KRAS, HPV (e.g.,HPV E6 or E7), NY-ESO, MAGE-A and WT1 peptide complexed HLA-A), NCAM (neural cell adhesion molecule), nectin-4, NKG2D (natural killer cell family 2 member D) ligand, NY-ESO, carcinoembryonic antigen, PD-1, PD-L1, PRAME (melanoma preferentially expressed antigen), progesterone receptor, PSA (prostate specific antigen), PSCA (prostate stem cell antigen), PSMA (prostate specific membrane antigen), ROR1, ROR2, SIRPα (signal regulatory protein α), SLIT, SLITRK6 (NTRK-like protein 6), STEAP1 (prostate six segment transmembrane epithelial antigen 1), survivin, TAG72 (tumor-associated glycoprotein 72), TPBG (trophoblast glycoprotein), TRAC, TCRβ, Trop-2, VEGFR1 (vascular endothelial growth factor receptor 1), VEGFR2, and antigens from HIV, HBV, HCV, HPV and other pathogens or their combinations. In some aspects, the antigen binding domain of the chimeric binding protein described herein specifically binds ROR1. In some aspects, the antigen binding domain of the chimeric binding protein specifically binds GPC2. In some aspects, the antigen binding domain of the chimeric binding protein specifically binds a tumor antigen,The tumor antigen is derived from: alpha-fetoprotein (AFP), CD19, BCMA, CLL-1, CS1, CD38, CD19, TSHR, CD123, CD22, CD30, CD171, CD33, EGFRvIII, GD2, GD3, TnAg, PSMA, ROR1, ROR2, GPC1, GPC2, FLT3, FAP, TAG72, CD44v6, CEA, EPCAM, B7H3, KIT, IL-13Ra2, mesothelin, IL-1 lRa, PSCA, PRSS21, VEGFR2, LewisY, CD24, PDGFR-β, SSEA-4, CD20, folate receptor alpha, ERBB2 (Her2 / neu), MUC1, MUC16, EGFR, NCAM, prostate enzyme, PAP, ELF2M, ephrin B2, IGF-I receptor, CAIX, LMP2, gplOO, bcr-abl, tyrosinase, EphA2, fucosyl GM1, sLe, GM3, TGS5, HMW MAA, o-acetyl-GD2, folate receptor beta, TEM1 / CD248, TEM7R, CLDN6, GPRC5D, CXORF61, CD97, CD179a, ALK, polysialic acid, PLAC1, GloboH, NY-BR-1, UPK2, HAVCR1, ADRB3, PANX3, GPR20, LY6K, OR51E2, TARP, WT1, NY-ESO-1, LAGE-1a, MAGE-A1, legumain, HPV E6, E7, MAGE A1, ETV6-AML, sperm protein 17, XAGE1, Tie 2, MAD-CT-1, MAD-CT-2, Fos-related antigen 1, p53, p53 mutant, prostein, surviving, telomerase, PCTA-1 / galectin 8, MelanA / MART1, Ras mutants (e.g.,HRAS, KRAS, NRAS), hTERT, sarcoma translocation breakpoints, ML-IAP, ERG (TMPRSS2 ETS fusion gene), NA17, PAX3, androgen receptor, cyclin Bl, MYCN, RhoC, TRP-2, CYP1B1, BORIS, SART3, PAX5, OY-TES1, LCK, AKAP-4, SSX2, RAGE-1, human telomerase reverse transcriptase, RU1, RU2, intestinal carboxylesterase, mut hsp70-2, CD79a, CD79b, CD72, LAIR1, FCAR, LILRA2, CD300LF, CLEC12A, BST2, EMR2, LY75, GPC3, FCRL5, IGLL1, CD2, CD3ε, CD4, CD5, CD7, the extracellular portion of the APRIL protein, neoantigens, or any combination thereof.

[0275] As further described elsewhere in the present disclosure, the antigen binding domain of the chimeric binding protein can be any polypeptide capable of binding to one or more antigens. In some aspects, the antigen binding domain comprises or is derived from Ig NAR, Fab fragment, Fab' fragment, F(ab)'2 fragment, F(ab)'3 fragment, Fv, single chain variable fragment (scFv), double-scFv, (scFv)2, miniantibodies, double antibodies, three antibodies, four antibodies, intrabody, disulfide-stabilized Fv protein (dsFv), one-body antibodies, nanobodies, and antigen binding regions derived from antibodies, which can specifically bind to any protein of interest, ligand, receptor, receptor fragment, peptide aptamer, or a combination thereof. In some aspects, the antigen binding domain is a single chain Fv (scFv).

[0276] In some aspects, the chimeric binding proteins described herein include an intracellular signaling domain that transduces effector function signals after the antigen is bound to the extracellular domain and instructs cells (e.g., T cells) expressing the chimeric binding proteins to perform specialized functions. Non-limiting examples of intracellular signaling domains include intracellular signaling domain regions derived from CD3ζ, FcRγ, common FcRγ (FCER1G), FcγRIIa, FcRβ (FcεRib), CD3γ, CD3δ, CD3ε, CD22, CD79a, CD79b, CD278 ("ICOS"), FcεRI, CD66d, CD32, DAP10, DAP12, or any combination thereof. In some aspects, the intracellular signaling domain includes a CD3ζ intracellular signaling domain. In some aspects, the chimeric binding proteins include the entire intracellular domain of a protein disclosed herein. In some aspects, the intracellular domain is truncated. A truncated portion of the intracellular domain may be used in place of the complete chain as long as it still transduces the effector function signal. Thus, the term intracellular domain is intended to include any truncated portion of the intracellular domain that is sufficient to transduce the effector function signal.

[0277] In some aspects, the chimeric binding protein expressed in the modified immune cells described herein (for example, NR4A3 genes and / or NR4A3 proteins with reduced levels) further comprises a membrane spaning domain. In some aspects, the antigen binding domain is connected to the intracellular domain of the chimeric binding protein through a membrane spaning domain. In some aspects, the antigen binding domain is connected to the membrane spaning domain of the chimeric binding protein (for example, CAR) through a joint. In some aspects, comprising a joint between the antigen binding domain and the membrane spaning domain can affect the flexibility of the antigen binding domain, thereby improving the chimeric binding protein function.

[0278] Any membrane-spanning domain known in the art can be used for chimeric binding proteins (e.g., CAR) as described herein. In some aspects, the membrane-spanning domain is artificial (e.g., engineered membrane-spanning domain). In some aspects, the membrane-spanning domain is derived from a naturally occurring polypeptide. In some aspects, the membrane-spanning domain includes a membrane-spanning domain from a naturally occurring polypeptide. Non-limiting examples of transmembrane domains include KIRDS2, OX40, CD2, CD27, LFA-1 (CD11a, CD18), ICOS (CD278), 4-1BB (CD137), GITR, CD40, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD160, CD19, IL2Rβ, IL2Rγ, IL7Rα, ITGA1, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11d, ITGAE, CD103, ITGAL, CD11a, LFA-1, ITGAM, CD 11b, ITGAX, CD11c, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, TNFR2, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (tactile), CEACAM1, CRTAM, Ly9 (CD229), CD160 (BY55), PSGL1, CD100 (SEMA4D), SLAMF6 (NTB-A, Ly108), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, ​​PAG / Cbp, NKG2D, NKG2C, CD19, or any combination thereof. In some aspects, the transmembrane domain comprises a CD28 transmembrane domain.

[0279] As described herein, in some aspects, chimeric binding protein (e.g., CAR) includes one or more costimulatory domains (e.g., second and third generation CAR). Without being bound by any one theory, these costimulatory domains can further improve the amplification, activation, memory, persistence and / or effector function of modified immune cells as described herein (e.g., NR4A3 genes and / or NR4A3 proteins with reduced levels, and engineered to express ligand-binding proteins). In some aspects, the transmembrane domain is fused to the costimulatory domain, optionally the costimulatory domain is fused to the second costimulatory domain, and the costimulatory domain is fused to the signaling domain, not limited to CD3 ζ. The non-limiting examples of costimulatory domain include interleukin-2 receptor (IL-2R), interleukin-12 receptor (IL-12R), IL-7, IL-21, IL-23, IL-15, CD2, CD3, CD4, CD7, CD8, CD27, CD28, CD30, CD40, 4-1BB / CD137, ICOS, lymphocyte function-associated antigen-1 (LFA-1), LIGHT, NKG2C, OX40, DAP10 or any combination thereof. In some aspects, costimulatory domain includes 4-1BB / CD137 costimulatory domain.

[0280] Also disclosed herein is a cell population comprising one or more of the modified cells described above (e.g., having a reduced level of NR4A3 gene and / or NR4A3 protein, and a ligand binding protein). In some aspects, the immune cell population is a pure population. In some aspects, the pure population comprises at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least 99% of cells belonging to the same immune cell type (e.g., 99% of immune cells are lymphocytes). In some aspects, the immune cell population comprises one, two, three, four, or five different cell types, for example, an immune cell population comprising two cell types may comprise lymphocytes and dendritic cells.

[0281] In some aspects, the cell colony disclosed herein comprises, is composed of, or is essentially composed of lymphocytes. In some aspects, the modified immune cell colony disclosed herein comprises lymphocytes, wherein lymphocytes are selected from the group consisting of: T cells, tumor infiltrating lymphocytes (TILs), lymphokine-activated killer cells, natural killer (NK) T cells, and any combination thereof. In some specific aspects, lymphocytes are T cells. In some specific aspects, lymphocytes are NK cells.

[0282] As will be apparent to those skilled in the art, in some aspects, the cells described herein have been modified using a combination of methods. For example, in some aspects, the cells have been modified to have reduced levels of the NR4A3 gene and / or NR4A3 protein and to comprise (i) an exogenous nucleotide sequence encoding one or more proteins (e.g., a ligand binding protein, e.g., a CAR or TCR) and (ii) an exogenous transcriptional activator (e.g., CRISPRa) that increases expression of an endogenous protein (e.g., c-Jun). In some aspects, the cells have been modified to have reduced levels of the NR4A3 gene and / or NR4A3 protein and to comprise (i) an exogenous nucleotide sequence encoding a first protein (e.g., a ligand binding protein, e.g., a CAR or TCR) and (ii) an exogenous nucleotide sequence encoding a second protein (e.g., a c-Jun protein). In some aspects, the cell has been modified to have a reduced level of the NR4A3 gene and / or NR4A3 protein and comprises (i) an exogenous nucleotide sequence encoding one or more proteins (e.g., a ligand binding protein, e.g., a CAR or TCR), (ii) an exogenous transcriptional activator (e.g., CRISPRa) that increases expression of an endogenous protein (e.g., c-Jun), and (iii) an exogenous nucleotide sequence encoding a second protein (e.g., a c-Jun protein). As described herein, in some aspects, the exogenous nucleotide sequence encoding the first protein and the second protein can be part of a single polycistronic vector.

[0283] In some aspects, the modified immune cells disclosed herein are T cells. In some aspects, the T cells include CAR. In some aspects, the modified T cells (CAR T cells) that can be prepared for expressing CAR are, for example, CD8 + T cells or CD4 + T cells. In some aspects, the CAR expressing cells disclosed herein are CAR T cells, such as single CAR T cells, genome-edited CAR T cells, double CAR T cells or series CAR T cells. In some aspects, the modified cells disclosed herein are NK cells. In some aspects, NK cells include CAR. In some aspects, CAR NK cells are single CAR NK cells, double CAR NK cells or series CAR NKT cells. In some aspects, the modified cells of the present disclosure include T cells and NK cells. In some aspects, T cells and NK cells both include CAR. Examples of such CAR T cells and CAR NK cells are provided in International Application No. PCT / US2019 / 044195 (disclosed as WO2020028400A1), which are incorporated herein by reference in their entirety.

[0284] In some aspects, the modified immune cells can be any immune cell type. In some aspects, the cells are modified immune cells for any adoptive cell transfer (ACT) therapy (also referred to as adoptive cell therapy). ACT therapy can be autologous therapy or allogeneic therapy. In some aspects, ACT therapy includes but is not limited to CAR T therapy, tumor infiltrating lymphocytes (TIL) therapy, NK cell therapy or any combination thereof.

[0285] In some aspects, the modified immune cells are TILs for TIL therapy. The use of TILs as adoptive cell transfer therapy to treat cancer has been studied for more than two decades, and TIL adoptive cell therapy is used to treat melanoma. Rosenberg SA et al., (July 2011). Clinical Cancer Research 17(13):4550–7 (July 2011). In adoptive T cell transfer therapy, TILs are expanded in vitro from surgically removed tumors that have been cut into small fragments or from single cell suspensions separated from tumor fragments. Multiple separate cultures are established, grown separately, and specific tumor recognition is determined. TILs are expanded over the course of several weeks. The selected TIL lines that exhibit the best tumor reactivity are then further expanded in a "rapid expansion protocol" (REP), which uses anti-CD3 activation and typically lasts for two weeks. TILs grown in culture can be modified at any time during the ex vivo process to reduce the expression of the NR4A3 gene and / or NR4A3 protein (alone or in combination with other members of the NR4A family, such as NR4A1 and / or NR4A2). After the last REP, the TILs are returned to the patient. The method may also include a preliminary chemotherapy regimen to reduce endogenous lymphocytes, thereby providing sufficient pathways for the adoptively transferred TILs to surround the tumor site.

[0286] In some aspects, the modified immune cells disclosed herein (e.g., T cells) include a T cell receptor (TCR), such as an engineered T cell receptor. In some aspects, the modified immune cells disclosed herein (e.g., T cells) may include a chimeric antigen receptor (CAR) that specifically binds to a tumor antigen. In some aspects, the modified immune cells (e.g., lymphocytes) are T cells with a T cell receptor, such as an engineered TCR. As used herein, the term "engineered TCR" or "engineered T cell receptor" refers to an engineered T cell receptor (TCR) that specifically binds to a major histocompatibility complex (MHC) / peptide target antigen with a desired affinity, and the antigen is selected, cloned, and / or subsequently introduced into a T cell colony.

[0287] In some aspects, the CAR or TCR that may be expressed on the modified cells disclosed herein specifically binds (i.e., targets) one or more antigens expressed on tumor cells, such as malignant B cells, malignant T cells, or malignant plasma cells.

[0288] In some aspects, the modified cells of the present disclosure can express a T cell receptor (TCR) that targets an antigen. The T cell receptor is a heterodimer composed of two different transmembrane polypeptide chains: an α chain and a β chain, each composed of a constant region and a variable region, the constant region anchors the chain in the T cell surface membrane, and the variable region recognizes and binds to the antigen presented by MHC. The TCR complex associates with six polypeptides that form two heterodimers, CD3γε and CD3δε, and one homodimer, CD3ζ, which together form the CD3 complex. T cell receptor engineered T cell therapy utilizes modifications of T cells that retain these complexes to specifically target antigens expressed by specific tumor cells.

[0289] In some aspects, modified TCR-engineered cells can target two main types: shared tumor-associated antigens (shared TAAs) and unique tumor-associated antigens (unique TAAs), or tumor-specific antigens. The former can include but are not limited to testicular cancer (CT) antigens, overexpressed antigens, and differentiation antigens, while the latter can include but are not limited to new antigens and tumor virus antigens. Human papillomavirus (HPV) E6 protein and HPV E7 protein fall into the category of tumor virus antigens.

[0290] In some aspects, modified TCR engineering cells can target CT antigens, such as melanoma-associated antigens (MAGE), including but not limited to MAGE-A1, MAGE-A2, MAGE-A3, MAGE-A4, MAGE-A6, MAGE-A8, MAGE-A9.23, MAGE-A10, and MAGE-A12. In some aspects, modified TCR engineering cells can target glycoprotein (gp100), melanoma antigens recognized by T cells (MART-1), and / or tyrosinase, which are mainly present in melanoma and normal melanocytes. In some aspects, modified TCR engineering cells can target Wilms' tumor 1 (WT1), an overexpressed antigen that is highly expressed in most acute myeloid leukemias (AML), acute lymphocytic leukemias, almost every type of solid tumor, and several key tissues (such as heart tissue). In some aspects, the modified TCR-engineered cells can target mesothelin, another overexpressed antigen that is highly expressed in mesothelioma but also present on mesothelial cells in several tissues, including the trachea.

[0291] IV. Methods of Producing Modified Cells

[0292] The present disclosure also provides methods for producing or preparing modified cells as described herein (i.e., having reduced levels of NR4A3 genes and / or NR4A3 proteins). In some aspects, such methods include contacting cells (e.g., immune cells) with gene editing tools, wherein the gene editing tools (e.g., including polynucleotides of the present disclosure, including gRNAs that can specifically target sequences within the NR4A3 gene) are capable of reducing the expression of the NR4A3 gene and / or NR4A3 protein. In some aspects, after contact, the level of the NR4A3 gene and / or NR4A3 protein in the cell is reduced by at least about 5%, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90% or about 100%. In some aspects, after contact, the cell does not express any level of NR4A3 gene and / or NR4A3 protein. In some aspects, the modified cells can be further modified to also have: (i) reduced levels of the NR4A1 gene and / or NR4A1 protein, (ii) reduced levels of the NR4A2 gene and / or NR4A2 protein, or (iii) both (i) and (ii).

[0293] Thus, in some aspects, the modified cells described herein have reduced levels of the NR4A3 gene and / or NR4A3 protein, but have endogenous levels of both the NR4A1 gene and / or NR4A1 protein and the NR4A2 gene and / or NR4A2 protein. In some aspects, the modified cells described herein have: (i) reduced levels of the NR4A3 gene and / or NR4A3 protein, (ii) reduced levels of the NR4A1 gene and / or NR4A1 protein, and (iii) endogenous levels of the NR4A2 gene and / or NR4A2 protein. In some aspects, (i) the level of the NR4A3 gene and / or NR4A3 protein is reduced by at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 71%, at least about 72%, at least about 73%, at least about 74%, at least about 75%, at least about 76%, at least about 77%, at least about 78%, at least about 79%, at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, at least about 100%, at least about 101%, at least about 102%, at least about 103%, at least about 104%, at least about 105%, at least about 106%, at least about 107%, at least about 108%, at least about 109%, at least about 110%, at least about 111%, at least about 112%, at least about 113%, at least about 114%, at least about 115%, at least about 116%, at least about In some aspects, the modified cells produced using the above methods express neither the NR4A3 gene and / or NR4A3 protein nor the NR4A1 gene and / or NR4A1 protein.

[0294] In some aspects, the modified cells described herein have: (i) reduced levels of the NR4A3 gene and / or NR4A3 protein, (ii) reduced levels of the NR4A2 gene and / or NR4A2 protein, and (iii) endogenous levels of the NR4A1 gene and / or NR4A1 protein. In some aspects, the level of (i) the NR4A3 gene and / or NR4A3 protein is reduced by at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 100%, at least about 101%, at least about 102%, at least about 103%, at least about 104%, at least about 105%, at least about 106%, at least about 107%, at least about 108%, at least about 109%, at least about 110%, at least about 111%, at least about 112%, at least about 113%, at least about 114%, at least about 115%, at least about 116%, at least about 117%, at least about 118%, at least about 119%, at least about 120%, at In some aspects, the modified cells produced using the above methods express neither the NR4A3 gene and / or NR4A3 protein nor the NR4A2 gene and / or NR4A2 protein.

[0295] In some aspects, the modified cells described herein have: (i) reduced levels of the NR4A3 gene and / or NR4A3 protein, (ii) reduced levels of the NR4A1 gene and / or NR4A1 protein, and (iii) reduced levels of the NR4A2 gene and / or NR4A2 protein. In some aspects, (i) the level of the NR4A3 gene and / or NR4A3 protein is reduced by at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or about 100%, and (ii) the level of the NR4A1 gene and / or NR4A1 protein is reduced by at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or about 100%, compared to a reference cell (e.g., a corresponding cell that has not been modified to, for example, have endogenous levels of all members of the NR4A family). or about 100%, and (iii) the level of the NR4A2 gene and / or NR4A2 protein is reduced by at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or about 100%.

[0296] When the levels of multiple members of the NR4A family are reduced, in some aspects, these levels can be reduced using the same gene editing tool (e.g., the levels of different NR4A family members are reduced using a CRISPR / Cas system). When the levels of multiple members of the NR4A family are reduced, in some aspects, these levels can be reduced using different gene editing tools (e.g., the levels of the NR4A3 gene and / or NR4A3 protein are reduced using CRISPR / Cas, and the levels of the NR4A2 gene and / or NR4A2 protein are reduced using antisense oligonucleotides).

[0297] Gene editing (e.g., base editing) can be performed using any editing tool known in the art. For example, in some aspects, cells (e.g., immune cells) can be modified using techniques such as CRISPR / Cas, TALEN, zinc finger nucleases (ZFNs), large-range nucleases, restriction endonucleases, interfering RNA (RNAi), antisense oligonucleotides, or a combination thereof. In some aspects, shRNA, siRNA, or miRNA can also be used to modify the NR4A3 gene and / or expression. All of these exemplary techniques will be discussed in more detail below. In some aspects, the method for reducing the expression of the NR4A3 gene and / or NR4A3 protein includes the use of one or more gene editing tools (e.g., two, three, or more tools). In some aspects, the method for reducing the expression of the NR4A3 gene and / or NR4A3 protein includes at least one method acting on NR4A3 DNA (e.g., CRISPR) or RNA (e.g., antisense oligonucleotides) and at least one method acting on the NR4A3 protein (e.g., inhibiting binding to a cell signaling partner or post-translational modification).

[0298] In some aspects, the modified cells produced using the above methods can be further modified to express ligand-binding proteins (e.g., CAR or transgenic TCR). For example, in some aspects, the method for producing modified cells provided herein includes contacting cells (e.g., immune cells) with gene editing tools, which can reduce the level of NR4A3 genes and / or NR4A3 proteins (e.g., including polynucleotides of the present disclosure) and nucleotide sequences encoding ligand-binding proteins. In some aspects, gene editing tools and nucleotide sequences encoding ligand-binding proteins are contacted with cells simultaneously. For example, in some aspects, cells are contacted with a single polynucleotide comprising both gene editing tools and nucleotide sequences encoding ligand-binding proteins. In some aspects, cells are contacted with a first polynucleotide comprising gene editing tools and a second polynucleotide comprising a nucleotide sequence encoding ligand-binding proteins simultaneously. In some aspects, gene editing tools and nucleotide sequences encoding ligand-binding proteins are contacted with cells in sequence.

[0299] In some aspects, the above-mentioned method for producing modified cells may further include contacting the cell with a nucleotide sequence encoding a c-Jun protein. As described herein, contacting the cell with a nucleotide sequence encoding a c-Jun protein can increase the c-Jun protein expression of the cell. When the cell is capable of naturally expressing c-Jun protein, in some aspects, the above-mentioned method may include contacting the cell with a transcriptional activator (e.g., a transcriptional activator based on a CRISPR / Cas system, e.g., CRISPRa) to increase the expression of endogenous c-Jun protein. As described herein, in some aspects, the cell may be contacted with a nucleotide sequence encoding a c-Jun protein and a transcriptional activator (e.g., a transcriptional activator based on a CRISPR / Cas system, e.g., CRISPRa) that can increase the expression of endogenous c-Jun protein. In some aspects, the nucleotide sequence encoding the c-Jun protein and / or the transcriptional activator are contacted with the cell simultaneously with a gene editing tool. For example, in some aspects, the cell is contacted with a single polynucleotide comprising (i) a nucleotide sequence encoding a c-Jun protein and / or a transcriptional activator and (ii) both a gene editing tool. In some aspects, the cell is contacted simultaneously with a first polynucleotide comprising a nucleotide sequence encoding a c-Jun protein and / or a transcriptional activator and a second polynucleotide comprising a gene editing tool. In some aspects, the gene editing tool and the nucleotide sequence encoding a c-Jun protein and / or a transcriptional activator are contacted with the cell sequentially.

[0300] Thus, in some aspects, provided herein is a method for producing a modified cell of the present disclosure, comprising contacting a cell with (i) a gene editing tool (e.g., a polynucleotide described herein) capable of reducing the level of the NR4A3 gene and / or NR4A3 protein, (ii) a nucleotide sequence encoding a ligand-binding protein, and (iii) a nucleotide sequence encoding a c-Jun protein and / or a transcriptional activator as described herein. In some aspects, the gene editing tool, the nucleotide sequence encoding the ligand-binding protein, and the nucleotide sequence encoding the c-Jun protein and / or a transcriptional activator are contacted with the cell simultaneously. For example, in some aspects, the cell is contacted with a single polynucleotide comprising (i) a gene editing tool, (ii) a nucleotide sequence encoding a ligand-binding protein, and (iii) a nucleotide sequence encoding a c-Jun protein and / or a transcriptional activator. In some aspects, the cell is contacted simultaneously with: (i) a first nucleotide comprising a gene editing tool, (ii) a second nucleotide sequence encoding a ligand-binding protein, and (iii) a third nucleotide sequence encoding a c-Jun protein and / or a transcriptional activator. In some aspects, at least two of the following are contacted sequentially with a cell: (i) a gene editing tool, (ii) a nucleotide sequence encoding a ligand binding protein, and (iii) a nucleotide sequence encoding a c-Jun protein and / or a transcriptional activator.

[0301] As described herein, in some aspects, the modified cells that can be produced using the methods provided herein include immune cells. In some aspects, the immune cells include lymphocytes, neutrophils, monocytes, macrophages, dendritic cells, or a combination thereof. In some aspects, the lymphocytes include T cells, tumor infiltrating lymphocytes (TIL), lymphokine-activated killer cells, natural (NK) cells, or a combination thereof. In some aspects, the lymphocytes are T cells, such as CD4 T cells. + T cells or CD8 + T cells. In some aspects, lymphocytes express chimeric antigen receptors (e.g., CD8+T cells expressing CAR and / or CD4+T cells expressing CAR). In some aspects, lymphocytes express engineered TCRs (e.g., engineered CD8+T cells expressing TCR and / or engineered CD4+T cells expressing TCR). In some aspects, lymphocytes are tumor infiltrating lymphocytes (TIL). In some aspects, TILs are CD8 + In some aspects, TIL is CD4 + TIL.

[0302] For any of the methods provided above or described elsewhere in the present disclosure that include contacting a cell (e.g., an immune cell) with any of the following, contact can occur in vivo, ex vivo, or in vitro: (i) a gene editing tool that can specifically target one or more members of the NR4A family (e.g., a polynucleotide described herein, comprising a gRNA that specifically targets the NR4A3 gene and / or NR4A3 protein), (ii) a nucleotide sequence encoding a c-Jun protein and / or a transcriptional activator described herein, (iii) a nucleotide sequence encoding a ligand-binding protein, or (iv) any combination thereof. In some aspects, contact occurs in vivo (e.g., gene therapy). In some aspects, contact occurs in vitro. In some aspects, contact occurs ex vivo. In some aspects, the cell is an autologous cell. In some aspects, the cell is a heterologous cell.

[0303] As can be seen from the present disclosure, in order for the gene editing tools described herein to exert their intended effects (e.g., reducing the level of the NR4A3 gene and / or NR4A3 protein), the gene editing tools must be able to enter the cell and bind to the gene of interest. In some aspects, any delivery vehicle known in the art for delivering a molecule of interest to a cell can be used. See, for example, U.S. Patent No. 10,047,355B2, which is incorporated herein by reference. Other disclosures related to usable vectors are provided elsewhere in this disclosure.

[0304] In some aspects, the gene editing tools that can be used in the present disclosure can remove the entire gene encoding the target protein (e.g., NR4A3 protein). In some aspects, the gene editing tool removes a portion (e.g., one or more exons) of the genome encoding the target protein (e.g., NR4A3 protein). In some aspects, the gene editing tool (e.g., base editor) modifies a specific nucleotide base without producing an insertion or deletion (indel). As used herein, the term "indel" refers to the insertion or deletion of a nucleotide base in a nucleic acid, which can result in a frameshift mutation in a gene coding region. Non-limiting examples of base editors are disclosed in U.S. Publication No. 2017 / 0121693, published on May 4, 2017, which is incorporated herein by reference in its entirety.

[0305] IV.A. Gene Editing Tools

[0306] Provided below are exemplary gene editing tools that can be used with the present disclosure.

[0307] IV.A.1. CRISPR / Cas systems

[0308] In some aspects, the gene editing tools that can be used in the present disclosure include CRISPR / Cas systems. This system can use, for example, Cas9 nuclease or nucleic acid molecules encoding Cas9 nuclease, and in some cases, the nucleic acid molecules are codon-optimized (e.g., T cells, such as T cells expressing CAR) for the desired cell type to be expressed therein. As further described herein, in some aspects, this system can include Cas9 nuclease protein.

[0309] The CRISPR / Cas system uses Cas nuclease, such as Cas9 nuclease, which targets genomic sites by compounding with guide RNA (e.g., synthetic guide RNA) (gRNA), which hybridizes to the target DNA sequence immediately before the NGG motif identified by the Cas nuclease (e.g., Cas9). This results in a double-strand break of three nucleotides upstream of the NGG motif. The unique ability of the CRISPR / Cas9 system is the ability to simultaneously target multiple different genomic loci by coexpressing a single Cas9 protein with two or more gRNAs (e.g., at least one, two, three, four, five, six, seven, eight, nine or ten gRNAs). This system can also use a guide RNA comprising two separate molecules. In some aspects, bimolecular gRNA includes crRNA samples ("CRISPR RNA" or "targeting sub-RNA" or "crRNA" or "crRNA repeats") molecules and corresponding tracrRNA samples ("trans-acting CRISPR RNA" or "activating sub-RNA" or "tracrRNA" or "scaffold") molecules.

[0310] The crRNA comprises a DNA targeting segment (single strand) of the gRNA and a segment of nucleotides that form half of the double-stranded RNA (dsRNA) duplex of the protein-binding segment of the gRNA. The corresponding tracrRNA (activator RNA) comprises a segment of nucleotides that forms the other half of the dsRNA duplex of the protein-binding segment of the gRNA. Therefore, a segment of nucleotides of the crRNA complements and hybridizes with a segment of nucleotides of the tracrRNA to form a dsRNA duplex of the protein-binding domain of the gRNA. Therefore, it can be said that each crRNA has a corresponding tracrRNA. The crRNA also provides a single-stranded DNA targeting segment. Therefore, the gRNA comprises a sequence that hybridizes with the target sequence (e.g., NR4A3 mRNA) and the tracrRNA. Therefore, the crRNA and tracrRNA (as a corresponding pair) hybridize to form the gRNA. If used for intracellular modification, the exact sequence and / or length of a given crRNA or tracrRNA molecule can be designed to be specific to the species (e.g., humans) in which the RNA molecule will be used.

[0311] The natural genes encoding three elements (Cas9, tracrRNA and crRNA) are usually organized in an operon. Naturally occurring CRISPR RNA is different according to the Cas9 system and organism, but generally comprises a targeting segment with a length of 21 to 72 nucleotides, flanked by two directional repeats (DR) of 21 to 46 nucleotides in length (see, for example, WO2014 / 131833). In the case of Streptococcus pyogenes (S.pyogenes), DR is 36 nucleotides long and the targeting segment is 30 nucleotides long. DR located at 3' is complementary and hybridized with the corresponding tracrRNA, and tracrRNA is bound to the Cas9 protein.

[0312] Alternatively, the CRISPR system used herein can further use a fused crRNA-tracrRNA construct (i.e., a single transcript), which works together with codon-optimized Cas9. This single RNA is commonly referred to as guide RNA or gRNA. In gRNA, the crRNA portion is identified as the "target sequence" of a given recognition site, and tracrRNA is commonly referred to as a "scaffold." In short, a short DNA fragment containing a target sequence is inserted into a guide RNA expression plasmid. The gRNA expression plasmid includes a target sequence (about 20 nucleotides in some aspects), which is a form of tracrRNA sequence (scaffold) and an appropriate promoter active in the cell and the elements necessary for correct processing in eukaryotic cells. Many systems rely on customized complementary oligomers, which anneal to form double-stranded DNA and are then cloned into the gRNA expression plasmid.

[0313] The gRNA expression cassette and the Cas9 expression cassette are then introduced into the cells. See, e.g., Mali P et al. (2013) Science 2013 Feb 15; 339(6121): 823-6; Jinek M et al. Science 2012 Aug 17; 337(6096): 816-21; Hwang WY et al. Nat Biotechnol 2013 Mar; 31(3): 227-9; Jiang W et al. Nat Biotechnol 2013 Mar; 31(3): 233-9; and Cong L et al. Science 2013 Feb 15; 339(6121): 819-23, each of which is incorporated herein by reference in its entirety. See, for example, WO / 2013 / 176772A1, WO / 2014 / 065596A1, WO / 2014 / 089290A1, WO / 2014 / 093622A2, WO / 2014 / 099750A2, and WO / 2013142578A1, each of which is herein incorporated by reference in its entirety.

[0314] In some aspects, Cas9 nuclease can be provided in the form of protein. For example, in some aspects, cells for the present disclosure (for example, immune cells expressing CAR or TCR) can be modified by introducing Cas9 nuclease protein and nucleic acid molecules comprising gRNA (for example, with NR4A genes and / or NR4A proteins with reduced levels). In some aspects, Cas9 nuclease protein and nucleic acid molecules comprising gRNA can be sequentially introduced into cells. In some aspects, Cas9 nuclease protein and nucleic acid molecules comprising gRNA can be introduced into cells simultaneously. For example, in some aspects, simultaneous administration includes but is not limited to introducing Cas9 nuclease protein and nucleic acid molecules comprising gRNA as a separate composition. In some aspects, Cas9 protein can be provided in the form of a complex with nucleic acid molecules comprising gRNA (that is, as a single composition).

[0315] In some aspects, Cas9 nuclease can be provided in the form of nucleic acid encoding protein. Therefore, in some aspects, the cell (for example, expressing CAR or TCR immune cell) for the present disclosure can be modified by introducing the first nucleic acid molecule encoding Cas9 nuclease protein and the second nucleic acid molecule comprising gRNA (for example, with the NR4A gene and / or NR4A protein with reduced levels). In some aspects, the first nucleic acid molecule and the second nucleic acid molecule can be introduced into the cell in sequence. In some aspects, the first nucleic acid molecule and the second nucleic acid molecule can be introduced into the cell simultaneously. For example, in some aspects, the first nucleic acid molecule and the second nucleic acid molecule can be introduced into the cell simultaneously but as a separate composition. In some aspects, the first nucleic acid molecule and the second nucleic acid molecule can be a part of a single polynucleotide, and the cell is modified to include a single polynucleotide. In some aspects, the nucleic acid molecule comprising gene editing tool also includes guide RNA (for example, synthetic guide RNA disclosed herein) and encoding Cas nuclease (for example, Cas9 nuclease) nucleic acid.

[0316] The nucleic acid encoding the Cas9 nuclease can be RNA (e.g., messenger RNA (mRNA)) or DNA. In some aspects, the gRNA can be provided in the form of RNA. In some aspects, the gRNA can be provided in the form of a DNA encoding RNA. In some aspects, the gRNA can be provided in the form of a separate crRNA and tracrRNA molecule or a separate DNA molecule encoding crRNA and tracrRNA, respectively.

[0317] In some aspects, gRNA comprises a third nucleic acid sequence that encodes clustered regularly spaced short palindromic repeats (CRISPR) RNA (crRNA) and trans-activated CRISPR RNA (tracrRNA). In some aspects, Cas protein is a type I Cas protein. In some aspects, Cas protein is a type II Cas protein. In some aspects, type II Cas protein is Cas9. In some aspects, type II Cas (e.g., Cas9) is a human codon-optimized Cas.

[0318] In some aspects, Cas proteins are "nickases" that can produce single-strand breaks (i.e., "nicks") in a target nucleic acid sequence without cutting the two chains of double-stranded DNA (dsDNA). For example, Cas9 comprises two nuclease domains: a RuvC-like nuclease domain and an HNH-like nuclease domain, which are responsible for cleaving the relative DNA chains. Mutations in any one of these two domains can produce nickases. Examples of mutations that produce nickases can be found in, for example, WO / 2013 / 176772A1 and WO / 2013 / 142578A1, each of which is incorporated herein by reference.

[0319] In some aspects, two separate Cas proteins (e.g., nickases) that are specific for the target site on each chain of dsDNA can produce a protruding sequence complementary to the protruding sequence on another nucleic acid, or a separate region on the same nucleic acid. The protruding ends produced by contacting the nucleic acid with two specific nickases for the target site on both chains of dsDNA can be 5' or 3' protruding ends. For example, a first nickase can produce a single-strand break on the first chain of dsDNA, and a second nickase can produce a single-strand break on the second chain of dsDNA, so as to produce a protruding sequence. The target site of each nickase that produces a single-strand break can be selected so that the protruding end sequence produced is complementary to the protruding end sequence on different nucleic acid molecules. The complementary protruding ends of two different nucleic acid molecules can be annealed by methods disclosed herein. In some aspects, the target site of the nickase on the first chain is different from the target site of the nickase on the second chain.

[0320] In some aspects, the expression of the NR4A3 gene and the NR4A3 protein encoded therein is reduced by contacting the cell with, for example, a CRISPR (e.g., CRISPR-Cas9 system) that is specific for the NR4A3 gene. As further described elsewhere in the present disclosure, in some aspects, the cells described herein (e.g., immune cells expressing CAR or TCR and / or c-Jun protein with elevated levels) are further modified to reduce (i) NR4A1 gene and / or protein, (ii) NR4A2 gene and / or protein or (iii) (i) and (ii) both levels. Therefore, in some aspects, CRISPR is specific for the NR4A1 gene. Therefore, in some aspects, after contact with CRISPR, the cell (e.g., immune cell expressing CAR or TCR) has: (i) reduced levels of NR4A1 gene and / or protein, (ii) endogenous levels of NR4A2 gene and / or protein, and (iii) endogenous levels of NR4A3 gene and / or protein. In some aspects, CRISPR is specific for the NR4A2 gene. Thus, in some aspects, upon contact with CRISPR, cells (e.g., immune cells expressing CAR or TCR) have: (i) endogenous levels of NR4A1 gene and / or protein, (ii) reduced levels of NR4A2 gene and / or protein, and (iii) endogenous levels of NR4A3 gene and / or protein. In some aspects, CRISPR is specific to the NR4A3 gene. Thus, in some aspects, upon contact with CRISPR, cells (e.g., immune cells expressing CAR or TCR) have: (i) endogenous levels of NR4A1 gene and / or protein, (ii) endogenous levels of NR4A2 gene and / or protein, and (iii) reduced levels of NR4A3 gene and / or protein.

[0321] In some aspects, CRISPR targets multiple NR4A genes. For example, in some aspects, CRISPR can target NR4A1 genes and NR4A2 genes. Therefore, in some aspects, after contacting with CRISPR, cells (e.g., immune cells expressing CAR or TCR) have: (i) reduced levels of NR4A1 genes and / or proteins, (ii) reduced levels of NR4A2 genes and / or proteins, and (iii) endogenous levels of NR4A3 genes and / or proteins. In some aspects, CRISPR can target NR4A1 genes and NR4A3 genes. Therefore, in some aspects, after contacting with CRISPR, cells (e.g., immune cells expressing CAR or TCR) have: (i) reduced levels of NR4A1 genes and / or proteins, (ii) endogenous expression of NR4A2 genes and / or proteins, and (iii) reduced levels of NR4A3 genes and / or proteins. In some aspects, CRISPR can target NR4A2 genes and / or NR4A3 genes. In some aspects, after contact with CRISPR, cells (e.g., immune cells expressing CAR or TCR) have: (i) endogenous levels of NR4A1 gene and / or protein, (ii) reduced levels of NR4A2 gene and / or protein, and (iii) reduced levels of NR4A3 gene and / or protein. In some aspects, CRISPR can target NR4A1 gene, NR4A2 gene, and NR4A3 gene. Therefore, in some aspects, after contact with CRISPR, cells (e.g., immune cells expressing CAR or TCR) have: (i) reduced levels of NR4A1 gene and / or protein, (ii) reduced levels of NR4A2 gene and / or protein, and (iii) reduced levels of NR4A3 gene and / or protein.

[0322] In some aspects, gene editing using CRISPR reduces NR4A3 gene levels by at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or about 100% relative to the NR4A3 gene levels observed in a reference cell (e.g., a corresponding cell that has not undergone gene editing using CRISPR). In some aspects, NR4A3 gene levels can be measured using any technique known in the art, such as by digital droplet PCR.

[0323] In some aspects, the nucleic acid encoding gRNA and / or Cas9 disclosed herein is RNA or DNA. In some aspects, the RNA or DNA encoding gRNA and / or Cas9 disclosed herein is synthetic RNA or synthetic DNA respectively. In some aspects, synthetic RNA or DNA include at least one non-natural core base. In some aspects, all nucleoside bases of a certain class have been replaced by non-natural nucleoside bases (for example, all uridines in the polynucleotide disclosed herein can be replaced by non-natural nucleoside bases (for example, 5-methoxyuridine or pseudouridine)). In some aspects, polynucleotides (for example, synthetic RNA or synthetic DNA) only include natural core bases, i.e., include A, C, T and U in the case of synthetic DNA, or include A, C, T and U in the case of synthetic RNA or synthetic DNA.

[0324] In general, the CRISPR gene editing methods disclosed herein comprise contacting a cell (e.g., an immune cell) in vivo, in vitro, or ex vivo with:

[0325] (i) Cas9 or a nucleic acid encoding Cas9; and

[0326] (ii) at least one NR4A3 gene guide RNA (gRNA) or a nucleic acid encoding a gRNA,

[0327] wherein the gRNA targets a sequence in the NR4A3 gene (e.g., an intronic and / or exonic sequence), wherein contacting the cell with Cas9 and at least one gRNA results in decreased expression of the NR4A3 gene and / or NR4A3 protein.

[0328] In some aspects, the gRNA that can be used to reduce the level of the NR4A3 gene in a cell (e.g., an immune cell) comprises any one or more gRNAs provided in Tables C and D. For example, in some aspects, the gRNA that can be used to target the NR4A3 gene comprises, consists of, or essentially consists of any one or more of the sequences shown in SEQ ID NOs: 30, 52-57, 58, 61, 65, 67, 68, 70, 71, 75, 76, 82, 83, 86, 94, and 96. In some aspects, the gRNA that can be used to target the NR4A3 gene comprises, consists of, or essentially consists of the sequence shown in SEQ ID NO: 30. In some aspects, the gRNA that can be used to target the NR4A3 gene comprises the sequence shown in SEQ ID NO: 30. In some aspects, the gRNA that can be used to target the NR4A3 gene consists of the sequence shown in SEQ ID NO: 30. In some aspects, the gRNA that can be used to target the NR4A3 gene consists essentially of the sequence shown in SEQ ID NO: 30. In some aspects, the gRNA useful for targeting the NR4A3 gene comprises, consists of, or consists essentially of the sequence set forth in SEQ ID NO: 52. In some aspects, the gRNA useful for targeting the NR4A3 gene comprises, consists of, or consists essentially of the sequence set forth in SEQ ID NO: 52. In some aspects, the gRNA useful for targeting the NR4A3 gene consists of, consists of, or consists essentially of the sequence set forth in SEQ ID NO: 52. In some aspects, the gRNA useful for targeting the NR4A3 gene comprises, consists of, or consists essentially of the sequence set forth in SEQ ID NO: 53. In some aspects, the gRNA useful for targeting the NR4A3 gene comprises, consists of, or consists essentially of the sequence set forth in SEQ ID NO: 53. In some aspects, the gRNA useful for targeting the NR4A3 gene consists of, consists of, or consists essentially of the sequence set forth in SEQ ID NO: 53. In some aspects, the gRNA that can be used to target the NR4A3 gene comprises, consists of, or consists essentially of the sequence set forth in SEQ ID NO: 54. In some aspects, the gRNA that can be used to target the NR4A3 gene comprises, consists of, or consists essentially of the sequence set forth in SEQ ID NO: 54. In some aspects, the gRNA that can be used to target the NR4A3 gene consists of, consists of, or consists essentially of the sequence set forth in SEQ ID NO: 54. In some aspects, the gRNA that can be used to target the NR4A3 gene comprises, consists of, or consists essentially of the sequence set forth in SEQ ID NO: 55.In some aspects, the gRNA useful for targeting the NR4A3 gene comprises the sequence set forth in SEQ ID NO: 55. In some aspects, the gRNA useful for targeting the NR4A3 gene consists of the sequence set forth in SEQ ID NO: 55. In some aspects, the gRNA useful for targeting the NR4A3 gene consists essentially of the sequence set forth in SEQ ID NO: 55. In some aspects, the gRNA useful for targeting the NR4A3 gene comprises, consists of, or consists essentially of the sequence set forth in SEQ ID NO: 56. In some aspects, the gRNA useful for targeting the NR4A3 gene comprises, consists of, or consists essentially of the sequence set forth in SEQ ID NO: 56. In some aspects, the gRNA useful for targeting the NR4A3 gene comprises, consists of, or consists essentially of the sequence set forth in SEQ ID NO: 57. In some aspects, the gRNA useful for targeting the NR4A3 gene comprises the sequence set forth in SEQ ID NO: 57. In some aspects, the gRNA useful for targeting the NR4A3 gene consists of the sequence set forth in SEQ ID NO: 57. In some aspects, the gRNA useful for targeting the NR4A3 gene consists essentially of the sequence set forth in SEQ ID NO: 57. In some aspects, the gRNA useful for targeting the NR4A3 gene comprises, consists of, or consists essentially of the sequence set forth in SEQ ID NO: 58. In some aspects, the gRNA useful for targeting the NR4A3 gene comprises, consists of, or consists essentially of the sequence set forth in SEQ ID NO: 58. In some aspects, the gRNA useful for targeting the NR4A3 gene consists essentially of the sequence set forth in SEQ ID NO: 58. In some aspects, the gRNA useful for targeting the NR4A3 gene comprises, consists of, or consists essentially of the sequence set forth in SEQ ID NO: 59. In some aspects, the gRNA useful for targeting the NR4A3 gene comprises, consists of, or consists essentially of the sequence set forth in SEQ ID NO: 59. In some aspects, the gRNA useful for targeting the NR4A3 gene consists of the sequence set forth in SEQ ID NO: 59. In some aspects, the gRNA that can be used to target the NR4A3 gene consists essentially of the sequence shown in SEQ ID NO: 59. In some aspects, the gRNA that can be used to target the NR4A3 gene comprises, consists of, or consists essentially of the sequence shown in SEQ ID NO: 60. In some aspects, the gRNA that can be used to target the NR4A3 gene comprises the sequence shown in SEQ ID NO: 60.In some aspects, the gRNA useful for targeting the NR4A3 gene consists of the sequence set forth in SEQ ID NO: 60. In some aspects, the gRNA useful for targeting the NR4A3 gene consists essentially of the sequence set forth in SEQ ID NO: 60. In some aspects, the gRNA useful for targeting the NR4A3 gene comprises, consists of, or consists essentially of the sequence set forth in SEQ ID NO: 61. In some aspects, the gRNA useful for targeting the NR4A3 gene comprises, consists of, or consists essentially of the sequence set forth in SEQ ID NO: 61. In some aspects, the gRNA useful for targeting the NR4A3 gene consists essentially of the sequence set forth in SEQ ID NO: 61. In some aspects, the gRNA useful for targeting the NR4A3 gene comprises, consists of, or consists essentially of the sequence set forth in SEQ ID NO: 62. In some aspects, the gRNA useful for targeting the NR4A3 gene comprises, consists of, or consists essentially of the sequence set forth in SEQ ID NO: 62. In some aspects, the gRNA useful for targeting the NR4A3 gene consists of the sequence set forth in SEQ ID NO: 62. In some aspects, the gRNA useful for targeting the NR4A3 gene consists essentially of the sequence set forth in SEQ ID NO: 62. In some aspects, the gRNA useful for targeting the NR4A3 gene comprises, consists of, or consists essentially of the sequence set forth in SEQ ID NO: 63. In some aspects, the gRNA useful for targeting the NR4A3 gene comprises, consists of, or consists essentially of the sequence set forth in SEQ ID NO: 63. In some aspects, the gRNA useful for targeting the NR4A3 gene consists essentially of the sequence set forth in SEQ ID NO: 63. In some aspects, the gRNA useful for targeting the NR4A3 gene comprises, consists of, or consists essentially of the sequence set forth in SEQ ID NO: 64. In some aspects, the gRNA useful for targeting the NR4A3 gene comprises, consists of, or consists essentially of the sequence set forth in SEQ ID NO: 64. In some aspects, the gRNA useful for targeting the NR4A3 gene comprises, consists of, or consists essentially of the sequence set forth in SEQ ID NO: 64. In some aspects, the gRNA useful for targeting the NR4A3 gene consists essentially of the sequence set forth in SEQ ID NO: 64. In some aspects, the gRNA that can be used to target the NR4A3 gene comprises, consists of, or consists essentially of the sequence set forth in SEQ ID NO: 65. In some aspects, the gRNA that can be used to target the NR4A3 gene comprises, consists of, or consists essentially of the sequence set forth in SEQ ID NO: 65. In some aspects, the gRNA that can be used to target the NR4A3 gene comprises, consists of, or consists essentially of the sequence set forth in SEQ ID NO: 65.In some aspects, the gRNA useful for targeting the NR4A3 gene consists essentially of the sequence set forth in SEQ ID NO: 65. In some aspects, the gRNA useful for targeting the NR4A3 gene comprises, consists of, or consists essentially of the sequence set forth in SEQ ID NO: 66. In some aspects, the gRNA useful for targeting the NR4A3 gene comprises, consists of, or consists essentially of the sequence set forth in SEQ ID NO: 66. In some aspects, the gRNA useful for targeting the NR4A3 gene consists essentially of the sequence set forth in SEQ ID NO: 66. In some aspects, the gRNA useful for targeting the NR4A3 gene comprises, consists of, or consists essentially of the sequence set forth in SEQ ID NO: 67. In some aspects, the gRNA useful for targeting the NR4A3 gene comprises, consists of, or consists essentially of the sequence set forth in SEQ ID NO: 67. In some aspects, the gRNA useful for targeting the NR4A3 gene comprises, consists of, or consists essentially of the sequence set forth in SEQ ID NO: 67. In some aspects, the gRNA useful for targeting the NR4A3 gene consists essentially of the sequence set forth in SEQ ID NO: 67. In some aspects, the gRNA useful for targeting the NR4A3 gene comprises, consists of, or consists essentially of the sequence set forth in SEQ ID NO: 68. In some aspects, the gRNA useful for targeting the NR4A3 gene comprises, consists of, or consists essentially of the sequence set forth in SEQ ID NO: 68. In some aspects, the gRNA useful for targeting the NR4A3 gene consists of, consists essentially of the sequence set forth in SEQ ID NO: 68. In some aspects, the gRNA useful for targeting the NR4A3 gene comprises, consists of, or consists essentially of the sequence set forth in SEQ ID NO: 69. In some aspects, the gRNA useful for targeting the NR4A3 gene comprises, consists of, or consists essentially of the sequence set forth in SEQ ID NO: 69. In some aspects, the gRNA useful for targeting the NR4A3 gene comprises, consists of, or consists essentially of the sequence set forth in SEQ ID NO: 69. In some aspects, the gRNA useful for targeting the NR4A3 gene consists of, consists essentially of the sequence set forth in SEQ ID NO: 69. In some aspects, the gRNA that can be used to target the NR4A3 gene comprises, consists of, or consists essentially of the sequence set forth in SEQ ID NO: 70. In some aspects, the gRNA that can be used to target the NR4A3 gene comprises, consists of, or consists essentially of the sequence set forth in SEQ ID NO: 70. In some aspects, the gRNA that can be used to target the NR4A3 gene consists of the sequence set forth in SEQ ID NO: 70. In some aspects, the gRNA that can be used to target the NR4A3 gene consists essentially of the sequence set forth in SEQ ID NO: 70.In some aspects, the gRNA useful for targeting the NR4A3 gene comprises, consists of, or consists essentially of the sequence set forth in SEQ ID NO: 71. In some aspects, the gRNA useful for targeting the NR4A3 gene comprises, consists of, or consists essentially of the sequence set forth in SEQ ID NO: 71. In some aspects, the gRNA useful for targeting the NR4A3 gene consists of, consists of, or consists essentially of the sequence set forth in SEQ ID NO: 71. In some aspects, the gRNA useful for targeting the NR4A3 gene comprises, consists of, or consists essentially of the sequence set forth in SEQ ID NO: 72. In some aspects, the gRNA useful for targeting the NR4A3 gene comprises, consists of, or consists essentially of the sequence set forth in SEQ ID NO: 72. In some aspects, the gRNA useful for targeting the NR4A3 gene consists of, consists of, or consists essentially of the sequence set forth in SEQ ID NO: 72. In some aspects, the gRNA useful for targeting the NR4A3 gene comprises, consists of, or consists essentially of the sequence set forth in SEQ ID NO: 73. In some aspects, the gRNA useful for targeting the NR4A3 gene comprises, consists of, or consists essentially of the sequence set forth in SEQ ID NO: 73. In some aspects, the gRNA useful for targeting the NR4A3 gene consists of, consists of, or consists essentially of the sequence set forth in SEQ ID NO: 73. In some aspects, the gRNA useful for targeting the NR4A3 gene comprises, consists of, or consists essentially of the sequence set forth in SEQ ID NO: 74. In some aspects, the gRNA useful for targeting the NR4A3 gene comprises, consists of, or consists essentially of the sequence set forth in SEQ ID NO: 74. In some aspects, the gRNA useful for targeting the NR4A3 gene consists of, consists of, or consists essentially of the sequence set forth in SEQ ID NO: 74. In some aspects, the gRNA that can be used to target the NR4A3 gene comprises, consists of, or consists essentially of the sequence set forth in SEQ ID NO: 75. In some aspects, the gRNA that can be used to target the NR4A3 gene comprises, consists of, or consists essentially of the sequence set forth in SEQ ID NO: 75. In some aspects, the gRNA that can be used to target the NR4A3 gene consists of the sequence set forth in SEQ ID NO: 75. In some aspects, the gRNA that can be used to target the NR4A3 gene consists essentially of the sequence set forth in SEQ ID NO: 75.In some aspects, the gRNA useful for targeting the NR4A3 gene comprises, consists of, or consists essentially of the sequence set forth in SEQ ID NO: 76. In some aspects, the gRNA useful for targeting the NR4A3 gene comprises, consists of, or consists essentially of the sequence set forth in SEQ ID NO: 76. In some aspects, the gRNA useful for targeting the NR4A3 gene consists of, consists of, or consists essentially of the sequence set forth in SEQ ID NO: 76. In some aspects, the gRNA useful for targeting the NR4A3 gene comprises, consists of, or consists essentially of the sequence set forth in SEQ ID NO: 77. In some aspects, the gRNA useful for targeting the NR4A3 gene comprises, consists of, or consists essentially of the sequence set forth in SEQ ID NO: 77. In some aspects, the gRNA useful for targeting the NR4A3 gene consists of, consists of, or consists essentially of the sequence set forth in SEQ ID NO: 77. In some aspects, the gRNA useful for targeting the NR4A3 gene comprises, consists of, or consists essentially of the sequence set forth in SEQ ID NO: 78. In some aspects, the gRNA useful for targeting the NR4A3 gene comprises, consists of, or consists essentially of the sequence set forth in SEQ ID NO: 78. In some aspects, the gRNA useful for targeting the NR4A3 gene consists of, consists of, or consists essentially of the sequence set forth in SEQ ID NO: 78. In some aspects, the gRNA useful for targeting the NR4A3 gene comprises, consists of, or consists essentially of the sequence set forth in SEQ ID NO: 79. In some aspects, the gRNA useful for targeting the NR4A3 gene comprises, consists of, or consists essentially of the sequence set forth in SEQ ID NO: 79. In some aspects, the gRNA useful for targeting the NR4A3 gene consists of, consists of, or consists essentially of the sequence set forth in SEQ ID NO: 79. In some aspects, the gRNA that can be used to target the NR4A3 gene comprises, consists of, or consists essentially of the sequence set forth in SEQ ID NO: 80. In some aspects, the gRNA that can be used to target the NR4A3 gene comprises, consists of, or consists essentially of the sequence set forth in SEQ ID NO: 80. In some aspects, the gRNA that can be used to target the NR4A3 gene consists of the sequence set forth in SEQ ID NO: 80. In some aspects, the gRNA that can be used to target the NR4A3 gene consists essentially of the sequence set forth in SEQ ID NO: 80.In some aspects, the gRNA useful for targeting the NR4A3 gene comprises, consists of, or consists essentially of the sequence set forth in SEQ ID NO: 81. In some aspects, the gRNA useful for targeting the NR4A3 gene comprises, consists of, or consists essentially of the sequence set forth in SEQ ID NO: 81. In some aspects, the gRNA useful for targeting the NR4A3 gene consists of, consists essentially of the sequence set forth in SEQ ID NO: 81. In some aspects, the gRNA useful for targeting the NR4A3 gene comprises, consists of, or consists essentially of the sequence set forth in SEQ ID NO: 82. In some aspects, the gRNA useful for targeting the NR4A3 gene comprises, consists of, or consists essentially of the sequence set forth in SEQ ID NO: 82. In some aspects, the gRNA useful for targeting the NR4A3 gene consists of, consists essentially of the sequence set forth in SEQ ID NO: 82. In some aspects, the gRNA useful for targeting the NR4A3 gene comprises, consists of, or consists essentially of the sequence set forth in SEQ ID NO: 83. In some aspects, the gRNA useful for targeting the NR4A3 gene comprises, consists of, or consists essentially of the sequence set forth in SEQ ID NO: 83. In some aspects, the gRNA useful for targeting the NR4A3 gene consists of, consists essentially of the sequence set forth in SEQ ID NO: 83. In some aspects, the gRNA useful for targeting the NR4A3 gene comprises, consists of, or consists essentially of the sequence set forth in SEQ ID NO: 84. In some aspects, the gRNA useful for targeting the NR4A3 gene comprises, consists of, or consists essentially of the sequence set forth in SEQ ID NO: 84. In some aspects, the gRNA useful for targeting the NR4A3 gene consists of, consists essentially of the sequence set forth in SEQ ID NO: 84. In some aspects, the gRNA that can be used to target the NR4A3 gene comprises, consists of, or consists essentially of the sequence set forth in SEQ ID NO: 85. In some aspects, the gRNA that can be used to target the NR4A3 gene comprises, consists of, or consists essentially of the sequence set forth in SEQ ID NO: 85. In some aspects, the gRNA that can be used to target the NR4A3 gene consists of the sequence set forth in SEQ ID NO: 85. In some aspects, the gRNA that can be used to target the NR4A3 gene consists essentially of the sequence set forth in SEQ ID NO: 85.In some aspects, the gRNA useful for targeting the NR4A3 gene comprises, consists of, or consists essentially of the sequence set forth in SEQ ID NO: 86. In some aspects, the gRNA useful for targeting the NR4A3 gene comprises, consists of, or consists essentially of the sequence set forth in SEQ ID NO: 86. In some aspects, the gRNA useful for targeting the NR4A3 gene consists of, consists essentially of the sequence set forth in SEQ ID NO: 86. In some aspects, the gRNA useful for targeting the NR4A3 gene comprises, consists of, or consists essentially of the sequence set forth in SEQ ID NO: 87. In some aspects, the gRNA useful for targeting the NR4A3 gene comprises, consists of, or consists essentially of the sequence set forth in SEQ ID NO: 87. In some aspects, the gRNA useful for targeting the NR4A3 gene consists of, consists essentially of the sequence set forth in SEQ ID NO: 87. In some aspects, the gRNA useful for targeting the NR4A3 gene comprises, consists of, or consists essentially of the sequence set forth in SEQ ID NO: 88. In some aspects, the gRNA useful for targeting the NR4A3 gene comprises, consists of, or consists essentially of the sequence set forth in SEQ ID NO: 88. In some aspects, the gRNA useful for targeting the NR4A3 gene consists of, consists essentially of the sequence set forth in SEQ ID NO: 88. In some aspects, the gRNA useful for targeting the NR4A3 gene comprises, consists of, or consists essentially of the sequence set forth in SEQ ID NO: 89. In some aspects, the gRNA useful for targeting the NR4A3 gene comprises, consists of, or consists essentially of the sequence set forth in SEQ ID NO: 89. In some aspects, the gRNA useful for targeting the NR4A3 gene consists of, consists essentially of the sequence set forth in SEQ ID NO: 89. In some aspects, the gRNA that can be used to target the NR4A3 gene comprises, consists of, or consists essentially of the sequence set forth in SEQ ID NO: 90. In some aspects, the gRNA that can be used to target the NR4A3 gene comprises, consists of, or consists essentially of the sequence set forth in SEQ ID NO: 90. In some aspects, the gRNA that can be used to target the NR4A3 gene consists of the sequence set forth in SEQ ID NO: 90. In some aspects, the gRNA that can be used to target the NR4A3 gene consists essentially of the sequence set forth in SEQ ID NO: 90.In some aspects, the gRNA useful for targeting the NR4A3 gene comprises, consists of, or consists essentially of the sequence set forth in SEQ ID NO: 91. In some aspects, the gRNA useful for targeting the NR4A3 gene comprises, consists of, or consists essentially of the sequence set forth in SEQ ID NO: 91. In some aspects, the gRNA useful for targeting the NR4A3 gene consists of, consists of, or consists essentially of the sequence set forth in SEQ ID NO: 91. In some aspects, the gRNA useful for targeting the NR4A3 gene comprises, consists of, or consists essentially of the sequence set forth in SEQ ID NO: 92. In some aspects, the gRNA useful for targeting the NR4A3 gene comprises, consists of, or consists essentially of the sequence set forth in SEQ ID NO: 92. In some aspects, the gRNA useful for targeting the NR4A3 gene consists of, consists of, or consists essentially of the sequence set forth in SEQ ID NO: 92. In some aspects, the gRNA useful for targeting the NR4A3 gene comprises, consists of, or consists essentially of the sequence set forth in SEQ ID NO: 93. In some aspects, the gRNA useful for targeting the NR4A3 gene comprises, consists of, or consists essentially of the sequence set forth in SEQ ID NO: 93. In some aspects, the gRNA useful for targeting the NR4A3 gene consists of, consists essentially of the sequence set forth in SEQ ID NO: 93. In some aspects, the gRNA useful for targeting the NR4A3 gene comprises, consists of, or consists essentially of the sequence set forth in SEQ ID NO: 94. In some aspects, the gRNA useful for targeting the NR4A3 gene comprises, consists of, or consists essentially of the sequence set forth in SEQ ID NO: 94. In some aspects, the gRNA useful for targeting the NR4A3 gene consists of, consists essentially of the sequence set forth in SEQ ID NO: 94. In some aspects, the gRNA that can be used to target the NR4A3 gene comprises, consists of, or consists essentially of the sequence set forth in SEQ ID NO: 95. In some aspects, the gRNA that can be used to target the NR4A3 gene comprises, consists of, or consists essentially of the sequence set forth in SEQ ID NO: 95. In some aspects, the gRNA that can be used to target the NR4A3 gene consists of the sequence set forth in SEQ ID NO: 95. In some aspects, the gRNA that can be used to target the NR4A3 gene consists essentially of the sequence set forth in SEQ ID NO: 95.In some aspects, the gRNA useful for targeting the NR4A3 gene comprises, consists of, or consists essentially of the sequence set forth in SEQ ID NO: 96. In some aspects, the gRNA useful for targeting the NR4A3 gene comprises, consists of, or consists essentially of the sequence set forth in SEQ ID NO: 96. In some aspects, the gRNA useful for targeting the NR4A3 gene consists of, consists essentially of the sequence set forth in SEQ ID NO: 96. In some aspects, the gRNA useful for targeting the NR4A3 gene comprises, consists of, or consists essentially of the sequence set forth in SEQ ID NO: 97. In some aspects, the gRNA useful for targeting the NR4A3 gene comprises, consists of, or consists essentially of the sequence set forth in SEQ ID NO: 97. In some aspects, the gRNA useful for targeting the NR4A3 gene consists of, consists essentially of the sequence set forth in SEQ ID NO: 97. In some aspects, the gRNA useful for targeting the NR4A3 gene comprises, consists of, or consists essentially of the sequence set forth in SEQ ID NO: 98. In some aspects, the gRNA useful for targeting the NR4A3 gene comprises, consists of, or consists essentially of the sequence set forth in SEQ ID NO: 98. In some aspects, the gRNA useful for targeting the NR4A3 gene consists of, consists essentially of the sequence set forth in SEQ ID NO: 98. In some aspects, the gRNA useful for targeting the NR4A3 gene comprises, consists of, or consists essentially of the sequence set forth in SEQ ID NO: 99. In some aspects, the gRNA useful for targeting the NR4A3 gene comprises, consists of, or consists essentially of the sequence set forth in SEQ ID NO: 99. In some aspects, the gRNA useful for targeting the NR4A3 gene consists of, consists essentially of the sequence set forth in SEQ ID NO: 99.

[0329] As described herein, in some aspects, the gene editing method may further include reducing the levels of: (i) NR4A1 gene and / or NR4A1 protein, (ii) NR4A2 gene and / or NR4A2 protein, or (iii) (i) and (ii) both. In some aspects, the gRNA that can be used to target the NR4A1 gene comprises, consists of, or is essentially composed of the sequence shown in SEQ ID NO: 25. In some aspects, the gRNA that can be used to target the NR4A1 gene comprises, consists of, or is essentially composed of the sequence shown in SEQ ID NO: 25. In some aspects, the gRNA that can be used to target the NR4A1 gene consists of, consists of, or is essentially composed of the sequence shown in SEQ ID NO: 25. In some aspects, the gRNA that can be used to target the NR4A1 gene comprises, consists of, or is essentially composed of the sequence shown in SEQ ID NO: 26. In some aspects, the gRNA that can be used to target the NR4A1 gene comprises, consists of, or is essentially composed of the sequence shown in SEQ ID NO: 26. In some aspects, the gRNA that can be used to target the NR4A1 gene consists of the sequence set forth in SEQ ID NO: 26. In some aspects, the gRNA that can be used to target the NR4A1 gene consists essentially of the sequence set forth in SEQ ID NO: 26. In some aspects, the gRNA that can be used to target the NR4A2 gene comprises, consists of, or consists essentially of the sequence set forth in SEQ ID NO: 27. In some aspects, the gRNA that can be used to target the NR4A2 gene comprises the sequence set forth in SEQ ID NO: 27. In some aspects, the gRNA that can be used to target the NR4A1 gene consists of the sequence set forth in SEQ ID NO: 27. In some aspects, the gRNA that can be used to target the NR4A1 gene consists essentially of the sequence set forth in SEQ ID NO: 27. In some aspects, the gRNA that can be used to target the NR4A2 gene comprises, consists of, or consists essentially of the sequence set forth in SEQ ID NO: 28. In some aspects, the gRNA that can be used to target the NR4A2 gene comprises the sequence set forth in SEQ ID NO: 28. In some aspects, the gRNA that can be used to target the NR4A1 gene consists of the sequence set forth in SEQ ID NO: 28. In some aspects, the gRNA that can be used to target the NR4A1 gene consists essentially of the sequence set forth in SEQ ID NO: 28. In some aspects, the gRNA that can be used to target the NR4A2 gene comprises, consists of, or consists essentially of the sequence set forth in SEQ ID NO: 29. In some aspects, the gRNA that can be used to target the NR4A2 gene comprises the sequence set forth in SEQ ID NO: 29. In some aspects, the gRNA that can be used to target the NR4A1 gene consists of the sequence set forth in SEQ ID NO: 29.In some aspects, the gRNA that can be used to target the NR4A1 gene consists essentially of the sequence shown in SEQ ID NO:29.

[0330] As used herein, the term "contacting" (e.g., contacting a cell (e.g., an immune cell) with at least one gRNA and at least one Cas9) is intended to include incubating at least one gRNA and at least one Cas protein (e.g., Cas9) in a cell in vitro (e.g., adding gRNA and / or Cas protein, or nucleic acid encoding gRNA and / or Cas9 protein to cultured cells) or contacting the cell in vivo or ex vivo.

[0331] The step of contacting the NR4A3 gene target sequence with at least one gRNA and at least one Cas protein (e.g., Cas9) as disclosed herein (or at least one nucleic acid encoding them) can be performed in any suitable manner. For example, cells (e.g., immune cells) can be treated under cell culture conditions. It should be understood that cells contacted with at least one gRNA and at least one Cas protein (e.g., Cas9 protein) (or at least one nucleic acid encoding them) disclosed herein can also be contacted simultaneously or subsequently with another reagent (e.g., a vector comprising at least one nucleic acid sequence encoding CAR or TCR). In some aspects, after contacting the cells in vitro or ex vivo, the method further comprises introducing the cells into the subject, thereby treating or improving the symptoms of the disease or illness (e.g., cancer).

[0332] For ex vivo methods, the cells may include autologous cells, i.e., one or more immune cells obtained from a subject in which the target polynucleotide sequence (e.g., NR4A3 gene) is to be altered in one or more cells (i.e., the donor and recipient are the same individual). The advantage of autologous cells is that any immune-based cell rejection is avoided. Alternatively, the cells may be heterologous, such as obtained from a donor. Typically, when cells are obtained from a donor, they will be obtained from a donor that is sufficiently immunologically compatible with the recipient, i.e., will not be subject to transplant rejection, to reduce or eliminate the need for immunosuppression. In some aspects, the cells are obtained from a xenogeneic source, i.e., a non-human mammal that has been genetically engineered to be immunologically compatible with the recipient or recipient species. Methods for determining immunocompatibility are known in the art and include tissue typing to assess donor-recipient compatibility of HLA and ABO determinants. See, for example, Transplantation Immunology, Bach and Auchincloss, eds. (Wiley, John & Sons, incorporated in 1994).

[0333] In some aspects, the present disclosure provides a method for producing a modified immune cell, comprising ex vivo altering the NR4A3 gene sequence in a cell (e.g., an immune cell (e.g., a T cell)) by contacting the NR4A3 gene sequence in the cell with a Cas9 protein (or a nucleic acid encoding such a Cas9 protein) and a gRNA targeting a motif in the NR4A3 gene (e.g., a motif, wherein the gRNA directs the Cas9 protein to the target gene and hybridizes with the target motif, wherein the NR4A3 gene is partially or completely cut, and wherein the cleavage efficiency is about 10% to about 100%). Non-limiting examples of such gRNAs are provided herein (see, e.g., Table A, Table C, and Table D). As described herein, in some aspects, the method for producing a modified immune cell described herein comprises altering the NR4A gene sequence by contacting the cell with a first nucleic acid molecule encoding the Cas9 protein and a second nucleic acid molecule comprising a gRNA targeting one or more members of the NR4A gene family. In some aspects, the first and nucleic acid molecules are contacted with the cell sequentially. In some aspects, the first and nucleic acid molecules are contacted with the cell simultaneously. For example, in some aspects, a cell is contacted with a single polynucleotide comprising a first nucleic acid molecule encoding a Cas9 protein and a second nucleic acid molecule comprising a gRNA.

[0334] In some aspects, the cells have been modified (e.g., transfected) with a nucleic acid (e.g., a vector) encoding a CAR or TCR before, after, or concurrently with the above-described altering step.

[0335] In some aspects, the cleavage efficiency is at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or about 100%.

[0336] The CRISPR / Cas system of the present disclosure can use gRNA spacer sequences of different lengths, depending on the Cas used, such as Cas9. Cas9 from different species must be paired with their corresponding gRNA to form a functional ribonucleoprotein (RNP) complex. In other words, due to the differences in the spacer sequence and the chimeric framework sequence, the chimeric gRNA frameworks engineered from different bacterial species can have different lengths.

[0337] In some aspects, the gRNA spacer sequence can be at least 18 nucleotides (e.g., 18, 19, 20, 21 or 22 nucleotides) long. For example, the length of the Streptococcus pyogenes gRNA spacer sequence combined with Streptococcus pyogenes Cas9 in the gRNA is 20 nucleotides, while the length of the Staphylococcus aureus gRNA spacer sequence combined with Staphylococcus aureus (S.aureus) Cas9 in the gRNA is 21 nucleotides. In some aspects, the gRNA spacer sequence can include 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34 or 35 nucleotides. In specific aspects, the gRNA comprises a spacer sequence consisting of 18 to 22 consecutive nucleotides (e.g., 20) corresponding to a subsequence of exon 3 of the NR4A3 gene. In some aspects, the gRNA comprises a spacer sequence consisting of 18 to 22 consecutive nucleotides (e.g., 20) corresponding to a subsequence of exon 4 of the NR4A3 gene. In some aspects, the gRNA comprises a spacer sequence consisting of 18 to 22 consecutive nucleotides (e.g., 20) corresponding to a subsequence of exon 3 or exon 4 of the NR4A3 gene.

[0338] Although a perfect match between the gRNA spacer sequence and the DNA strand it binds to on the NR4A3 gene is preferred, mismatches between the gRNA spacer sequence and the NR4A3 target sequence are also permitted, as long as they still result in a reduction in NR4A3 gene levels or a reduction in NR4A3 gene function. A "seed" sequence of 8-12 consecutive nucleotides on the gRNA that is fully complementary to the target NR4A3 sequence is preferred for correct recognition of the target sequence on the NR4A3 gene. The remainder of the gRNA spacer sequence may contain one or more mismatches.

[0339] In general, gRNA activity is negatively correlated with the number of mismatches. Preferably, the gRNA spacer sequence of the present disclosure contains less than 7 mismatches. In some aspects, the gRNA spacer sequence contains 7 mismatches, 6 mismatches, 5 mismatches, 4 mismatches, 3 mismatches with the corresponding NR4A3 gene target sequence, more preferably 2 mismatches or less, and even more preferably no mismatches. The fewer the number of nucleotides in the gRNA, the fewer the number of mismatches allowed. Binding affinity is believed to depend on the sum of the matched gRNA-DNA combinations.

[0340] The gRNA spacer of the present disclosure can be selected to minimize the off-target effects of the CRISPR / Cas editing system. Therefore, in some aspects, the gRNA spacer is selected so that when compared with all other genomic nucleotide sequences in the cell, it contains at least two mispairings. In some aspects, the gRNA spacer is selected so that when compared with all other genomic nucleotide sequences in the cell, it contains at least one mispairing. It will be understood by those skilled in the art that a variety of techniques can be used to select suitable gRNA spacer to minimize off-target effects (e.g., bioinformatics analysis).

[0341] In some aspects, the gRNA spacer sequence comprises, consists of, or consists essentially of a spacer sequence of SEQ ID NOs: 31-42.

[0342] In some aspects, the gRNA spacer sequence comprises, consists of, or consists essentially of a spacer sequence comprising at least one, two, three, four, or five nucleotide mismatches compared to the DNA sequence of any one of SEQ ID NOs: 31-42.

[0343] In some aspects, editing efficiency can be increased by targeting multiple positions. Therefore, in some aspects, the methods disclosed herein include using a gRNA targeting an upstream position of exon 1 of the NR4A3 gene. In some aspects, the methods disclosed herein include using 2, 3, 4, 5, 6, 7, 8, 9 or 10 gRNAs targeting an upstream position of exon 1 of the NR4A3 gene. In addition, in some aspects, the methods disclosed herein include using a gRNA targeting an downstream position of exon 4 of the NR4A3 gene. In some aspects, the methods disclosed herein include using 2, 3, 4, 5, 6, 7, 8, 9 or 10 gRNAs targeting an downstream position of exon 4 of the NR4A3 gene.

[0344] In some aspects, the two gRNAs are complementary and / or hybridize to sequences on the same strand of the NR4A3 gene. In some aspects, the two gRNAs are complementary and / or hybridize to sequences on opposite strands of the NR4A3 gene. In some aspects, the two gRNAs are not complementary and / or hybridize to sequences on opposite strands of the NR4A3 gene. In some aspects, the two gRNAs are complementary and / or hybridize to overlapping target motifs of the NR4A3 gene. In some aspects, the two gRNAs are complementary and / or hybridize to offset target motifs of the NR4A3 gene.

[0345] In general, the gRNA of the present disclosure may comprise any variant of its sequence or chemical modification, as long as it allows the corresponding Cas protein (e.g., Cas9 protein) to bind to the target sequence and subsequently excise (all or part of) the NR4A3 gene.

[0346] The Cas protein (e.g., Cas9) used in the methods disclosed herein is an endonuclease that cleaves nucleic acids and is encoded by the CRISPR locus of many bacterial genomes and participates in the type II CR ISPR system. Cas9 protein is produced by numerous bacterial species, including Streptococcus pyogenes, Staphylococcus aureus, Streptococcus thermophilus, Neisseria meningitidis, etc. Therefore, the Cas9 protein that can be used in the present disclosure can be derived from any suitable bacteria known in the art. The limiting examples of such bacteria include Streptococcus pyogenes, Streptococcus mutans, Streptococcus pneumonia, Streptococcus aureus, Streptococcus thermophilus, Campylobacter jejuni, Neisseria meningitidis, Pasteurella multocida, Listeria innocua, and Francisella novicida. Methods disclosed herein can be implemented with any Cas9 known in the art. In some aspects, Cas9 is wild-type Cas9. In some aspects, Cas9 is a mutant Cas9 with enhanced enzymatic activity or a fusion protein comprising a Cas9 portion. In some aspects, the Cas9 nuclease protein is a Streptococcus pyogenes Cas9 protein.

[0347] Because Cas9 nuclease proteins are typically expressed in bacteria, when designing and preparing Cas9 recombinant proteins, it may be advantageous to modify their nucleic acid sequences for optimal expression in eukaryotic cells (e.g., mammalian cells). Therefore, in some aspects, the nucleic acids encoding Cas9 used in the methods disclosed herein have been codon-optimized for expression in eukaryotic cells, for example, for expression in cells of human subjects in need thereof.

[0348] In some aspects, the Cas9 protein used in the methods disclosed herein includes one or more amino acid substitutions or modifications. In some aspects, one or more amino acid substitutions include conservative amino acid substitutions. In some cases, substitution and / or modification can prevent or reduce proteolytic degradation and / or extend the half-life of the polypeptide in the cell. In some aspects, the Cas9 protein can include peptide bond replacements (e.g., urea, thiourea, carbamate, sulfonylurea, etc.). In some aspects, the Cas9 protein can include naturally occurring amino acids. In some aspects, the Cas9 protein can include alternative amino acids (e.g., D-amino acids, β-amino acids, homocysteine, phosphoserine, etc.). In some aspects, the Cas9 protein can include modifications to include heterologous moieties (e.g., pegylation, glycosylation, lipidation, acetylation, capping, etc.).

[0349] Although the methods disclosed herein are generally implemented using Cas9 proteins, it is foreseeable that in some aspects, the Cas protein can be Cas1, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7 or Cas8. In some aspects, the Cas protein is a Cas9 protein from any bacterial species or a functional portion thereof. In some specific aspects, the Cas9 protein used in the methods disclosed herein is a Streptococcus pyogenes or Staphylococcus aureus Cas9 protein or a functional portion thereof, or a nucleic acid encoding such a Cas9 or a functional portion thereof. Non-limiting examples of other usable Cas nucleases are known in the art and are described in, for example, US 9,970,001 B2, US 10,221,398 B2 and US2020 / 0190487 A1, each of which is incorporated herein by reference in its entirety. In some aspects, the Cas nucleases useful in the present disclosure include type I Cas proteins. Non-limiting examples of type I Cas proteins include Cas3, Cas5, Cas6, Cas7, Cas8a, Cas8b, Cas8c, Cas10d, Cse1, Cse2, Csy1, Csy2, Csy3, and variants thereof. In some aspects, the Cas nucleases useful in the present disclosure include type II Cas proteins. Non-limiting examples of type II Cas proteins include Cas9, Csn2, Cas4, and variants thereof. In some aspects, the Cas nucleases useful in the present disclosure include type III Cas proteins. Non-limiting examples include Cas10, Csm2, Cmr5, Csx10, Csx11, and variants thereof. In some aspects, the Cas nucleases useful in the present disclosure include type IV Cas proteins. Non-limiting examples of such Cas proteins include Csf1. In some aspects, the Cas nucleases useful in the present disclosure include type V Cas proteins. Non-limiting examples include Cas12, Cas12a (Cpf1), Cas12b (C2c1), Cas12c (C2c3), Cas12d (CasY), Cas12e (Cas X), Cas12f (Cas14, C2c10), Cas12g, Cas12h, Cas12i, Cas12k (C2c5), C2c4, C2c8, C2c9 and variants thereof. In some aspects, the Cas nucleases useful in the present disclosure comprise type VI Cas proteins. Non-limiting examples of type VI Cas proteins include Cas13, Cas13a (C2c2), Cas13b, Cas13c, Cas13d and variants thereof.

[0350] In some cases, the Cas proteins useful in the present disclosure comprise orthologs or homologs of the above-mentioned Cas proteins. The terms "orthologue" (also referred to herein as "ortholog") and "homologue" (also referred to herein as "homolog") are well known in the art. By way of further guidance, a "homologue" of a protein as used herein is a protein of the same species that has the same or similar function as a protein to which it is homologous. Homologous proteins may, but need not, be structurally related, or only partially structurally related. As used herein, "orthologs" of a protein are proteins of different species that perform the same or similar function as a protein to which it is an ortholog. Orthologous proteins may, but need not, be structurally related, or only partially structurally related.

[0351] As used herein, "functional portion" refers to a portion of a peptide, such as Cas9, which retains the ability to compound with at least one gRNA and cleave a target sequence, resulting in reduced expression of the NR4A3 gene and / or NR4A3 protein. In some aspects, the functional portion comprises a combination of operably connected Cas9 protein functional domains, the functional domains being selected from the group consisting of: a DNA binding domain, at least one RNA binding domain, a helicase domain, and an endonuclease domain. In some aspects, the functional domains form a non-covalent complex. In some aspects, the functional domains form a fusion complex (e.g., a fusion protein). In some aspects, the functional domains are chemically connected (e.g., through one or more spacers or joints). In some aspects, the functional domains are conjugated.

[0352] It should be understood that the present disclosure contemplates various ways of contacting the NR4A3 gene with at least one gRNA and at least one Cas protein (e.g., Cas9). In some aspects, exogenous Cas proteins (e.g., Cas9) can be introduced into cells in the form of polypeptides. In some aspects, Cas proteins (e.g., Cas9) can be conjugated or fused to cell-penetrating polypeptides or cell-penetrating peptides. As used herein, "cell-penetrating polypeptides" and "cell-penetrating peptides" refer to polypeptides or peptides that promote the uptake of molecules into cells, respectively. The cell-penetrating polypeptide may contain a detectable label.

[0353] In some aspects, the Cas protein (e.g., Cas9) can be conjugated or fused to a charged protein (e.g., a protein carrying a positive charge, a negative charge, or all neutral charges). This connection can be covalent. In some aspects, the Cas protein (e.g., Cas9) can be fused to a peptide with a superimposed charge to significantly increase the ability of the Cas protein (e.g., Cas9) to penetrate cells. See Cronican et al. ACS Chem.Biol.5(8):747-52(2010). In some aspects, the Cas protein (e.g., Cas9) can be fused to a protein transduction domain (PTD) to facilitate its entry into cells. Exemplary PTDs include, but are not limited to, Tat, oligoarginine, and penetratin. Therefore, in some specific aspects, the methods disclosed herein can be implemented using a Cas protein (e.g., Cas9 protein), comprising a Cas protein fused to a cell-penetrating peptide, a Cas protein fused to a PTD, a Cas protein fused to a tat domain, a Cas protein fused to an oligoarginine domain, a Cas protein fused to a penetratin domain, or a combination thereof.

[0354] In some aspects, Cas protein (e.g., Cas9) can be introduced into cells in the form of nucleic acids encoding Cas protein (e.g., Cas9), such as immune cells disclosed herein, for example, immune cells expressing CAR or TCR and / or having elevated levels of c-Jun protein, which contain target polynucleotide sequences, i.e., NR4A3 genes. The process of introducing nucleic acid into cells can be achieved by any suitable technique. Suitable techniques include calcium phosphate or lipid-mediated transfection, electroporation, and transduction or infection using viral vectors. In some aspects, nucleic acid comprises DNA. In some aspects, nucleic acid comprises modified DNA as described herein. In some aspects, nucleic acid comprises mRNA. In some aspects, nucleic acid comprises modified mRNA as described herein (e.g., synthetic modified mRNA).

[0355] The gRNA sequences and / or nucleic acid sequences encoding Cas9 used in the methods disclosed herein can be chemically modified to enhance, for example, their stability (e.g., to increase their plasma half-life after administration to a subject in need thereof). Possible chemical modifications of the gRNA and / or nucleic acid sequences encoding, for example, Cas9 disclosed herein will be discussed in detail below in this specification.

[0356] In some aspects, the entire gRNA is chemically modified. In some aspects, only the gRNA spacer is chemically modified. In some aspects, the gRNA spacer and gRNA framework sequences are chemically modified. Non-limiting examples of specific chemical modifications are disclosed in detail below.

[0357] Therefore, in some aspects of the methods disclosed herein, by expression from one or more delivery vectors encoding thereof, Cas protein (for example, Cas9) and one or more gRNA are provided to target cells. In some aspects, the above-mentioned vectors or the vector for introducing gRNA or gRNA and Cas9 in target cells are viral vectors. In some aspects, the above-mentioned vectors or the vector for introducing gRNA or gRNA and Cas9 in target cells are non-viral vectors. In some aspects, the viral vector is adeno-associated vector (AAV), lentiviral vector (LV), retroviral vector, adenoviral vector, herpes virus vector or a combination thereof. AAV vectors can be based on one or more of several capsid types, including AAVI, AAV2, AAV5, AAV6, AAV8 and AAV9. In some aspects, AAV vectors are AAVDJ-8, AAV2DJ9 or a combination thereof.

[0358] In addition to the methods disclosed above, the present disclosure also provides compositions for implementing the disclosed methods. Accordingly, the present disclosure provides nucleic acids encoding at least one of the above-mentioned gRNAs.

[0359] Also provided is a composition and / or at least one Cas9. In some aspects, the nucleic acid encoding Cas9 encodes (i) Cas9 from Staphylococcus aureus, (ii) Cas9 from Streptococcus pyogenes, (iii) a mutant Cas9 derived from Cas9 from Staphylococcus aureus or Cas9 from Streptococcus pyogenes, wherein the mutant protein retains Cas9 activity, (iv) a fusion protein comprising a Cas9 portion, or (v) a combination thereof.

[0360] In some aspects, one or more gene editing tools (e.g., the tools disclosed herein) can be used to modify the cells of the disclosure.

[0361] IV.A.2. TALEN

[0362] In some aspects, gene editing tools that can be used to edit (e.g., reduce or inhibit) the expression of the NR4A3 gene and / or NR4A3 protein are nuclease reagents, such as transcription activator-like effector nucleases (TALENs). TAL effector nucleases are a class of sequence-specific nucleases that can be used to generate double-strand breaks at specific target sequences in the genomes of prokaryotes or eukaryotes. TAL effector nucleases are produced by fusing a natural or engineered transcription activator-like (TAL) effector or a functional portion thereof to a catalytic domain of a nuclease (such as, for example, FokI).

[0363] The unique modular TAL effector DNA binding domain allows the design of proteins with potential for any given DNA recognition specificity. Thus, the DNA binding domain of the TAL effector nuclease can be engineered to recognize specific DNA target sites, thereby being used to generate double-strand breaks on the desired target sequence. See WO 2010 / 079430; Morbitzer et al., (2010) PNAS 10.1073 / pnas.1013133107; Scholze & Boch (2010) Virulence 1:428-432; Christian et al., Genetics (2010) 186:757-761; Li et al., (2010) Nuc. Acids Res. (2010) doi: 10.1093 / nar / gkq704; and Miller et al., (2011) Nature Biotechnology 29:143-148; all are incorporated herein by reference in their entirety.

[0364] Non-limiting examples of suitable TAL nucleases and methods for making suitable TAL nucleases are disclosed, for example, in U.S. Patent Application Nos. 2011 / 0...

Claims

1. A method for reducing the level of NR4A3 gene and / or NR4A3 protein in immune cells, comprising modifying the immune cells with a gene editing tool, wherein the gene editing tool comprises a polynucleotide, the polynucleotide comprises a gRNA, wherein the gRNA is capable of specifically binding to a sequence within the NR4A3 gene, and comprises, consists essentially of, or consists of a sequence as shown in any one of the following: SEQ ID NO:94, SEQ ID NO:52, SEQ ID NO:96, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:86, SEQ ID NO:83, SEQ ID NO:55, SEQ ID NO:82, SEQ ID NO:56, SEQ ID NO:76, SEQ ID NO:57, SEQ ID NO:75, SEQ ID NO:58, SEQ ID NO:71, SEQ ID NO:61, SEQ ID NO:70, SEQ ID NO:65, SEQ ID NO:68 and SEQ ID NO:67, and wherein after the modification, the level of the NR4A3 gene and / or the NR4A3 protein in the immune cell is reduced compared to a reference immune cell (e.g., a corresponding immune cell that has not been contacted with the polynucleotide).

2. The method of claim 1, wherein after the modification, the level of the NR4A3 gene is reduced by at least about 5%, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90% or about 100% compared to the reference immune cell.

3. The method of claim 1 or claim 2, wherein after the modification, the level of the NR4A3 protein is reduced by at least about 5%, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90% or about 100% compared to the reference immune cell.

4. A method of reducing or preventing the exhaustion of immune cells, comprising contacting the immune cells with a gene editing tool, the gene editing tool comprising a polynucleotide, the polynucleotide comprising a gRNA, wherein the gRNA is capable of specifically binding to a sequence within the NR4A3 gene, and comprises, consists essentially of, or consists of a sequence as shown in any one of the following: SEQ ID NO:94, SEQ ID NO:52, SEQ ID NO:96, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:86, SEQ ID NO:83, SEQ ID NO:55, SEQ ID NO:82, SEQ ID NO:56, SEQ ID NO:76, SEQ ID NO:57, SEQ ID NO:75, SEQ ID NO:58, SEQ ID NO:71, SEQ ID NO:61, SEQ ID NO:70, SEQ ID NO:65, SEQ ID NO:68 and SEQ ID NO:67, and wherein after the contacting, the exhaustion of the immune cell is reduced after sustained antigen stimulation compared to a reference immune cell (e.g., a corresponding immune cell that has not been contacted with the polynucleotide).

5. The method of claim 8, wherein the immune cell is more resistant to depletion than the reference immune cell.

6. The method of claim 8 or claim 9, wherein the immune cell exhibits increased persistence / survival when administered to a subject compared to the reference immune cell.

7. The method of any one of claims 8 to 10, wherein the immune cell exhibits increased expansion / proliferation upon sustained antigen stimulation compared to the reference immune cell.

8. The method of any one of claims 8 to 11, wherein the immune cell exhibits increased effector function in response to sustained antigenic stimulation compared to the reference immune cell.

9. A method for increasing cytokine production by immune cells that respond to antigenic stimulation, comprising modifying the immune cells with a gene editing tool, the gene editing tool comprising a polynucleotide, the polynucleotide comprising a gRNA, wherein the gRNA is capable of specifically binding to a sequence within the NR4A3 gene and comprises, consists essentially of, or consists of a sequence as shown in any one of the following: SEQ ID NO:94, SEQ ID NO:52, SEQ ID NO:96, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:86, SEQ ID NO:83, SEQ ID NO:55, SEQ ID NO:82, SEQ ID NO:56, SEQ ID NO:76, SEQ ID NO:57, SEQ ID NO:75, SEQ ID NO:58, SEQ ID NO:71, SEQ ID NO:61, SEQ ID NO:70, SEQ ID NO:65, SEQ ID NO:68 and SEQ ID NO:67, wherein after the modification, the immune cell exhibits increased cytokine production after antigen stimulation compared to a reference immune cell (e.g., a corresponding immune cell not modified with the polynucleotide).

10. The method of claim 11, wherein the cytokine comprises IFN-γ, IL-2, TNF-α, or a combination thereof.

11. The method of claim 11 or 12, wherein after the modification, the production of the cytokine in response to the antigen stimulation is increased by at least about 1 fold, at least about 2 fold, at least about 3 fold, at least about 4 fold, at least about 5 fold, at least about 6 fold, at least about 7 fold, at least about 8 fold, at least about 9 fold, at least about 10 fold, at least about 11 fold, at least about 12 fold, at least about 13 fold, at least about 14 fold, at least about 15 fold, at least about 16 fold, at least about 17 fold, at least about 18 fold, at least about 19 fold, at least about 20 fold, at least about 25 fold, at least about 30 fold, at least about 35 fold, at least about 40 fold, at least about 45 fold, or at least about 50 fold compared to the reference immune cell.

12. A method for increasing the effector function of an immune cell that responds to sustained antigenic stimulation, comprising modifying the immune cell with a gene editing tool, the gene editing tool comprising a polynucleotide, the polynucleotide comprising a gRNA, wherein the gRNA is capable of specifically binding to a sequence within the NR4A3 gene, and comprises, consists essentially of, or consists of a sequence as shown in any one of the following: SEQ ID NO:94, SEQ ID NO:52, SEQ ID NO:96, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:86, SEQ ID NO:83, SEQ ID NO:55, SEQ ID NO:82, SEQID NO:56, SEQ ID NO:76, SEQ ID NO:57, SEQ ID NO:75, SEQ ID NO:58, SEQ ID NO:71, SEQ ID NO:61, SEQ ID NO:70, SEQ ID NO:65, SEQ ID NO:68 and SEQ ID NO:67, and wherein after the modification, the immune cell exhibits increased effector function after sustained antigen stimulation compared to a reference immune cell (e.g., a corresponding immune cell that has not been contacted with the polynucleotide).

13. The method of claim 14, wherein after said modification, said immune cell retains effector function in at least one, at least two, or at least three additional antigen stimulation assays compared to a reference immune cell.

14. The method of claim 14 or 15, wherein the effector function comprises the ability to: (i) kill target cells (e.g., tumor cells), (ii) produce cytokines upon further antigenic stimulation, or (iii) both (i) and (ii).

15. A method for preparing a composition comprising immune cells having reduced levels of NR4A3 gene and / or NR4A3 protein, the method comprising modifying the immune cells with a gene editing tool, the gene editing tool comprising a polynucleotide, the polynucleotide comprising a gRNA, wherein the gRNA is capable of specifically binding to a sequence within the NR4A3 gene, and comprises, consists essentially of, or consists of a sequence as shown in any one of the following: SEQ ID NO:94, SEQ ID NO:52, SEQ ID NO:96, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:86, SEQ ID NO:83, SEQ ID NO:55, SEQ ID NO:82, SEQ ID NO:56, SEQ ID NO:76, SEQ ID NO:57, SEQ ID NO:75, SEQ ID NO:58, SEQ ID NO:71, SEQ ID NO:61, SEQ ID NO:70, SEQ ID NO:65, SEQ ID NO:68 and SEQ ID NO:67, and wherein after the modification, the level of the NR4A3 gene and / or the NR4A3 protein in the immune cell is reduced compared to a reference immune cell (e.g., a corresponding immune cell that has not been contacted with the polynucleotide).

16. The method of claim 15, further comprising combining the modified immune cells with a pharmaceutically acceptable excipient.

17. The method of any one of claims 1 to 16, wherein after the modification, the level of the NR4A3 gene is reduced by at least about 5%, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or about 100% compared to the reference immune cell.

18. The method of any one of claims 1 to 17, wherein after said modification, the level of said NR4A3 protein is reduced by at least about 5%, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or about 100% compared to said reference immune cell.

19. A method of treating a tumor in a subject in need thereof, comprising administering to the subject an immune cell that has been modified with a gene editing tool, the gene editing tool comprising a polynucleotide, the polynucleotide comprising a gRNA, wherein the gRNA is capable of specifically binding to a sequence within the NR4A3 gene, and comprises, consists essentially of, or consists of a sequence as shown in any one of the following: SEQ ID NO:94, SEQ ID NO:52, SEQ ID NO:96, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:86, SEQ ID NO:83, SEQ ID NO:55, SEQ ID NO:82, SEQ ID NO:56, SEQ ID NO:76, SEQ ID NO:57, SEQ ID NO:75, SEQ ID NO:58, SEQ ID NO:71, SEQ ID NO:61, SEQ ID NO:70, SEQ ID NO:65, SEQ ID NO:68 and SEQ ID NO:

67.

20. The method of claim 19, wherein the level of the NR4A3 gene in the immune cell is reduced by at least about 5%, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or about 100% compared to a reference immune cell (e.g., a corresponding immune cell that has not been contacted with the polynucleotide).

21. The method of claim 20, wherein the level of the NR4A3 protein in the immune cell is reduced by at least about 5%, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or about 100% compared to the reference immune cell.

22. The method of any one of claims 19 to 21, wherein the administration reduces the tumor volume in the subject as compared to a reference tumor volume (e.g., the tumor volume in the subject before the administration and / or the tumor volume in a subject not receiving the administration).

23. The method of claim 22, wherein the tumor volume is reduced by at least about 5%, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or about 100% compared to the reference tumor volume.

24. The method of any one of claims 19 to 23, wherein the tumor is derived from a cancer selected from the group consisting of breast cancer, head and neck cancer, uterine cancer, brain cancer, skin cancer, kidney cancer, lung cancer, colorectal cancer, prostate cancer, liver cancer, bladder cancer, kidney cancer, pancreatic cancer, thyroid cancer, esophageal cancer, eye cancer, stomach (gastric) cancer, gastrointestinal cancer, ovarian cancer, cervical cancer, carcinoma, sarcoma, leukemia, lymphoma, myeloma, or a combination thereof.

25. The method of any one of claims 19 to 24, comprising administering to the subject an additional therapeutic agent.

26. The method of claim 25, wherein the additional therapeutic agent comprises a chemotherapeutic drug, a targeted anti-cancer therapy, an oncolytic drug, a cytotoxic agent, an immune-based therapy, a cytokine, a surgical procedure, a radiation procedure, an activator of a co-stimulatory molecule, an immune checkpoint inhibitor, a vaccine, a cellular immunotherapy, or any combination thereof.

27. The method of claim 26, wherein the additional therapeutic agent is an immune checkpoint inhibitor.

28. The method of claim 26 or 27, wherein the immune checkpoint inhibitor comprises an anti-PD-1 antibody, an anti-PD-L1 antibody, an anti-LAG-3 antibody, an anti-CTLA-4 antibody, an anti-GITR antibody, an anti-TIM3 antibody, and any combination thereof.

29. The method of any one of claims 25 to 28, wherein the immune cells and the additional therapeutic agent are administered to the subject simultaneously.

30. The method of any one of claims 25 to 28, wherein the immune cell and the additional therapeutic agent are administered sequentially to the subject.

31. The method of any one of claims 25 to 30, wherein the immune cell is administered to the subject parenterally, intramuscularly, subcutaneously, ocularly, intravenously, intraperitoneally, intradermally, intraorbitally, intracerebrally, intracranially, intraspinally, intraventricularly, intrathecally, intracisternalally, intracapsules, intratumorally, or any combination thereof.

32. The method of any one of claims 1 to 31, further comprising modifying the immune cell to have a reduced level of the NR4A1 gene and / or NR4A1 protein.

33. The method of claim 32, wherein modifying the immune cell to have a reduced level of the NR4A1 gene and / or the NR4A1 protein comprises contacting the immune cell with a gene editing tool that can specifically target and reduce the level of the NR4A1 gene and / or the NR4A1 protein ("NR4A1-specific gene editing tool").

34. A method as claimed in claim 33, wherein after the immune cells are contacted with the NR4A1-specific gene editing tool, the level of the NR4A1 gene is reduced by at least about 5%, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90% or about 100% compared to corresponding cells that have not been contacted with the NR4A1-specific gene editing tool.

35. A method as described in claim 33 or 34, wherein after the immune cells are contacted with the NR4A1-specific gene editing tool, the level of the NR4A1 protein is reduced by at least about 5%, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90% or about 100% compared to corresponding cells that have not been contacted with the NR4A1-specific gene editing tool.

36. The method of any one of claims 1 to 35, further comprising modifying the immune cell to have a reduced level of the NR4A2 gene and / or NR4A2 protein.

37. The method of claim 36, wherein modifying the immune cell to have a reduced level of the NR4A2 gene and / or the NR4A2 protein comprises contacting the immune cell with a gene editing tool that can specifically target and reduce the level of the NR4A2 gene and / or the NR4A2 protein ("NR4A1-specific gene editing tool").

38. A method as claimed in claim 37, wherein after the immune cells are contacted with the NR4A2-specific gene editing tool, the level of the NR4A2 gene is reduced by at least about 5%, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90% or about 100% compared to corresponding cells that have not been contacted with the NR4A2-specific gene editing tool.

39. A method as described in claim 37 or 38, wherein after the immune cells are contacted with the NR4A2-specific gene editing tool, the level of the NR4A2 protein is reduced by at least about 5%, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90% or about 100% compared to corresponding cells that have not been contacted with the NR4A2-specific gene editing tool.

40. The method of any one of claims 1 to 39, further comprising modifying the immune cell to have elevated levels of c-Jun protein.

41. The method of claim 40, wherein modifying the immune cell to have an elevated level of c-Jun protein comprises contacting the immune cell with a nucleotide sequence encoding a c-Jun protein.

42. The method of claim 41, wherein the nucleotide sequence encoding the c-Jun protein comprises: (a) a nucleic acid sequence having at least 89%, at least 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity to the nucleic acid sequence set forth in SEQ ID NO:7; (b) a nucleic acid sequence having at least 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity to the nucleic acid sequence set forth in SEQ ID NO:8; (c) a nucleic acid sequence having at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity to the nucleic acid sequence set forth in SEQ ID NO: 10; (d) a nucleic acid sequence having at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity to the nucleic acid sequence set forth in SEQ ID NO: 11; (e) a nucleic acid sequence having at least 88%, at least 89%, at least 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity to the nucleic acid sequence set forth in SEQ ID NO: 12; (f) a nucleic acid sequence having at least 82%, at least 83%, at least 84%, at least 85%, at least 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity to the nucleic acid sequence set forth in SEQ ID NO: 13; (g) a nucleic acid sequence having at least 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity to the nucleic acid sequence set forth in SEQ ID NO: 14; (h) a nucleic acid sequence having at least 55%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity to the nucleic acid sequence set forth in SEQ ID NO: 15; or (i) a nucleic acid sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity to the nucleic acid sequence set forth in SEQ ID NO:

16.

43. The method of any one of claims 40 to 42, wherein modifying the immune cell to have an elevated level of c-Jun protein comprises contacting the immune cell with a transcriptional activator capable of increasing expression of endogenous c-Jun protein.

44. The method of claim 43, wherein the transcriptional activator is attached to a Cas protein that has been modified to lack endonuclease activity.

45. methods as described in any one of claims 40 to 44, wherein after modifying the immune cell so that it has an elevated level of c-Jun protein, the level of the c-Jun protein in the immune cell is increased by at least about 1 fold, at least about 2 fold, at least about 3 fold, at least about 4 fold, at least about 5 fold, at least about 6 fold, at least about 7 fold, at least about 8 fold, at least about 9 fold, at least about 10 fold, at least about 11 fold, at least about 12 fold, at least about 13 fold, at least about 14 fold, at least about 15 fold, at least about 16 fold, at least about 17 fold, at least about 18 fold, at least about 19 fold, at least about 20 fold, at least about 25 fold, at least about 30 fold, at least about 35 fold, at least about 40 fold, at least about 45 fold, or at least about 50 fold compared to a reference cell (e.g., a corresponding cell that has not been modified to have an elevated level of the c-Jun protein).

46. ​​The method of any one of claims 1 to 45, further comprising modifying the immune cell so that it expresses a ligand binding protein.

47. A method as claimed in claim 46, wherein the ligand binding protein is a chimeric antigen receptor (CAR), a T cell receptor (TCR), a chimeric antibody-T cell receptor (caTCR), a chimeric signaling receptor (CSR), a T cell receptor mimetic (TCR mimetic) or a combination thereof.

48. The method of claim 47, wherein the ligand binding protein is a CAR.

49. The method of claim 47, wherein the ligand binding protein is a TCR.

50. The method of any one of claims 47 to 49, wherein the ligand-binding protein is capable of specifically binding to an antigen selected from the group consisting of CD19, TRAC, TCRβ, BCMA, CLL-1, CS1, CD38, CD19, TSHR, CD123, CD22, CD30, CD70, CD171, CD33, EGFRvIII, GD2, GD3, Tn Ag, PSMA, ROR1, ROR2, GPC1, GPC2, FLT3, FAP, TAG72, CD44v6, CEA, EPCAM, B7H3, KIT, IL-13Ra2, mesothelin, IL-llRa, PSCA, PRSS21, VEGFR2, LewisY, CD24, PDGFR-β, SSEA-4, CD20, folate receptor α, ERBB2 (Her2 / neu), MUC1, MUC16, EGFR, NCAM, prostate enzyme, PAP, ELF2M, ephrin B2, IGF-I receptor, CAIX, LMP2, gplO O, bcr-abl, tyrosinase, EphA2, fucosyl GM1, sLe, GM3, TGS5, HMWMAA, o-acetyl-GD2, folate receptor β, TEM1 / CD248, TEM7R, CLDN6, GPRC5D, CXORF61, CD97, CD179a, ALK, polysialic acid, PLAC1, GloboH, NY-BR-1, UPK2, HAVCR1, ADRB3, PANX3, GPR20, LY6K, OR51E2, TARP, WTl, NY-ESO-1, LAGE-la, MAGE-Al, legumin, HPV E6, E7, MAGE Al, ETV6-AML, sperm protein 17, XAGE1, Tie 2, MAD-CT-1, MAD-CT-2, Fos-related antigen 1, p53, p53 mutant, prostein, survivin and telomerase, PCTA-1 / galectin 8, MelanA / MARTl, Ras mutant, hTERT, sarcoma translocation breakpoints, ML-IAP, ERG (TMPRSS2 ETS fusion gene), NA17, PAX3, androgen receptor, cyclin B1, MYCN, RhoC, TRP-2, CYP1B1, BORIS, SART3, PAX5, OY-TES1, LCK, AKAP-4, SSX2, RAGE-1, human telomerase reverse transcriptase, RU1, RU2, intestinal carboxylesterase, mut hsp70-2, CD79a, CD79b, CD72, LAIR1, FCAR, LILRA2, CD300LF, CLEC12A, BST2, EMR2, LY75, GPC3, FCRL5, IGLL1, or any combination thereof.

51. The method of claim 50, wherein the ligand binding protein specifically binds to ROR1.

52. The method of claim 51, wherein the ligand binding protein comprises an antigen binding domain derived from the R12 antibody, the R11 antibody, the 2A2 antibody, or any combination thereof.

53. A method as described in claim 51 or claim 52, wherein the ligand binding protein comprises a heavy chain variable domain (VH) and a light chain variable domain (VL), wherein the VH comprises the amino acid sequence shown in SEQ ID NO: 17, and wherein the VL comprises the amino acid sequence shown in SEQ ID NO:

21.

54. The method of any one of claims 1 to 53, wherein the gene editing tool comprises shRNA, siRNA, miRNA, antisense oligonucleotides, CRISPR, zinc finger nucleases, TALENs, meganucleases, restriction endonucleases, or any combination thereof.

55. The method of claim 54, wherein the gene editing tool is CRISPR.

56. A composition comprising cells having reduced levels of NR4A3 gene and / or NR4A3 protein, wherein the composition has been prepared by the method of any one of claims 15 to 18.

57. A composition comprising a cell that expresses a reduced level of a NR4A3 gene and / or a NR4A3 protein, wherein the cell has been modified with a gRNA capable of targeting the NR4A3 gene, wherein the gRNA comprises, consists of, or consists essentially of a sequence as shown in any one of the following: SEQ ID NO:94, SEQ ID NO:52, SEQ ID NO:96, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:86, SEQ ID NO:83, SEQ ID NO:55, SEQ ID NO:82, SEQ ID NO:56, SEQ ID NO:76, SEQ ID NO:57, SEQ ID NO:75, SEQ ID NO:58, SEQ ID NO:71, SEQ ID NO:61, SEQ ID NO:70, SEQ ID NO:65, SEQ ID NO:68 and SEQ ID NO:

67.

58. A composition as described in claim 57, wherein the gRNA comprises, consists of, or essentially consists of the sequence shown in SEQ ID NO:

94.

59. A composition as described in claim 57, wherein the gRNA comprises, consists of, or consists essentially of the sequence shown in SEQ ID NO:

52.

60. A composition as described in claim 57, wherein the gRNA comprises, consists of, or consists essentially of the sequence shown in SEQ ID NO:

96.

61. A composition as described in claim 57, wherein the gRNA comprises, consists of, or consists essentially of the sequence shown in SEQ ID NO:

53.

62. A composition as described in claim 57, wherein the gRNA comprises, consists of, or consists essentially of the sequence shown in SEQ ID NO:

54.

63. A composition as described in claim 57, wherein the gRNA comprises, consists of, or consists essentially of the sequence shown in SEQ ID NO:

86.

64. A composition as described in claim 57, wherein the gRNA comprises, consists of, or consists essentially of the sequence shown in SEQ ID NO:

83.

65. A composition as described in claim 57, wherein the gRNA comprises, consists of, or consists essentially of the sequence shown in SEQ ID NO:

55.

66. A composition as described in claim 57, wherein the gRNA comprises, consists of, or consists essentially of the sequence shown in SEQ ID NO:

82.

67. A composition as described in claim 57, wherein the gRNA comprises, consists of, or consists essentially of the sequence shown in SEQ ID NO:

56.

68. A composition as described in claim 57, wherein the gRNA comprises, consists of, or consists essentially of the sequence shown in SEQ ID NO:

76.

69. A composition as described in claim 57, wherein the gRNA comprises, consists of, or consists essentially of the sequence shown in SEQ ID NO:

57.

70. A composition as described in claim 57, wherein the gRNA comprises, consists of, or consists essentially of the sequence shown in SEQ ID NO:

75.

71. A composition as described in claim 57, wherein the gRNA comprises, consists of, or consists essentially of the sequence shown in SEQ ID NO:

58.

72. A composition as described in claim 57, wherein the gRNA comprises, consists of, or consists essentially of the sequence shown in SEQ ID NO:

71.

73. A composition as described in claim 57, wherein the gRNA comprises, consists of, or consists essentially of the sequence shown in SEQ ID NO:

61.

74. A composition as described in claim 57, wherein the gRNA comprises, consists of, or consists essentially of the sequence shown in SEQ ID NO:

70.

75. A composition as described in claim 57, wherein the gRNA comprises, consists of, or consists essentially of the sequence shown in SEQ ID NO:

65.

76. A composition as described in claim 57, wherein the gRNA comprises, consists of, or consists essentially of the sequence shown in SEQ ID NO:

68.

77. A composition as described in claim 57, wherein the gRNA comprises, consists of, or consists essentially of the sequence shown in SEQ ID NO:

67.

78. The composition of any one of claims 56 to 77, further comprising a pharmaceutically acceptable excipient.

79. An isolated polynucleotide comprising, consisting of, or consisting essentially of a sequence as set forth in any one of the following: SEQ ID NO:94, SEQ ID NO:52, SEQ ID NO:96, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:86, SEQ ID NO:83, SEQ ID NO:55, SEQ ID NO:82, SEQ ID NO:56, SEQ ID NO:76, SEQ ID NO:57, SEQ ID NO:75, SEQ ID NO:58, SEQ ID NO:71, SEQ ID NO:61, SEQ ID NO:70, SEQ ID NO:65, SEQ ID NO:68 and SEQ ID NO:

67.

80. A composition as described in claim 79, which comprises, consists of or essentially consists of the sequence shown in SEQ ID NO:

94.

81. A composition as described in claim 79, which comprises, consists of or essentially consists of the sequence shown in SEQ ID NO:

52.

82. A composition as described in claim 79, which comprises, consists of or essentially consists of the sequence shown in SEQ ID NO:

96.

83. A composition as described in claim 79, which comprises, consists of or essentially consists of the sequence shown in SEQ ID NO:

53.

84. A composition as described in claim 79, which comprises, consists of or essentially consists of the sequence shown in SEQ ID NO:

54.

85. A composition as described in claim 79, which comprises, consists of or essentially consists of the sequence shown in SEQ ID NO:

86.

86. A composition as described in claim 79, which comprises, consists of or essentially consists of the sequence shown in SEQ ID NO:

83.

87. A composition as described in claim 79, which comprises, consists of or essentially consists of the sequence shown in SEQ ID NO:

55.

88. A composition as described in claim 79, which comprises, consists of or essentially consists of the sequence shown in SEQ ID NO:

82.

89. A composition as described in claim 79, which comprises, consists of or essentially consists of the sequence shown in SEQ ID NO:

56.

90. A composition as described in claim 79, which comprises, consists of or essentially consists of the sequence shown in SEQ ID NO:

76.

91. A composition as described in claim 79, which comprises, consists of or essentially consists of the sequence shown in SEQ ID NO:

57.

92. A composition as described in claim 79, which comprises, consists of or essentially consists of the sequence shown in SEQ ID NO:

75.

93. A composition as described in claim 79, which comprises, consists of or essentially consists of the sequence shown in SEQ ID NO:

58.

94. A composition as described in claim 79, which comprises, consists of or essentially consists of the sequence shown in SEQ ID NO:

71.

95. A composition as described in claim 79, which comprises, consists of or essentially consists of the sequence shown in SEQ ID NO:

61.

96. A composition as described in claim 79, which comprises, consists of or essentially consists of the sequence shown in SEQ ID NO:

70.

97. A composition as described in claim 79, which comprises, consists of or essentially consists of the sequence shown in SEQ ID NO:

65.

98. A composition as described in claim 79, which comprises, consists of or essentially consists of the sequence shown in SEQ ID NO:

68.

99. A composition as described in claim 79, which comprises, consists of or essentially consists of the sequence shown in SEQ ID NO:

67.

100. A cell comprising the polynucleotide of any one of claims 79 to 99.

101. The cell of claim 100, further comprising a polynucleotide encoding a ligand binding protein.

102. The cell of claim 101, wherein the ligand binding protein is a chimeric antigen receptor (CAR), a T cell receptor (TCR), a chimeric antibody-T cell receptor (caTCR), a chimeric signaling receptor (CSR), a T cell receptor mimetic (TCR mimetic), or a combination thereof.

103. The cell of any one of claims 100 to 102, further comprising (i) a nucleotide sequence encoding a c-Jun protein, (ii) a transcriptional activator capable of increasing the expression of an endogenous c-Jun protein, or (iii) both (i) and (ii).

104. The cell of any one of claims 100 to 103, which is an immune cell.

105. The cell of claim 104, wherein the immune cell comprises a lymphocyte, a neutrophil, a monocyte, a macrophage, a dendritic cell, or a combination thereof.

106. The cell of claim 105, wherein the lymphocytes comprise T cells, tumor infiltrating lymphocytes (TILs), lymphokine-activated killer cells, natural (NK) cells, or a combination thereof.

107. A kit comprising (i) a polynucleotide comprising a gRNA that specifically targets a region within the NR4A3 gene and (ii) instructions for use, wherein the polynucleotide comprises, consists essentially of, or consists of a sequence as set forth in any one of the following: SEQ ID NO:94, SEQ ID NO:52, SEQ ID NO:96, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:86, SEQ ID NO:83, SEQ ID NO:55, SEQ ID NO:82, SEQ ID NO:56, SEQ ID NO:76, SEQ ID NO:57, SEQ ID NO:75, SEQ ID NO:58, SEQ ID NO:71, SEQ ID NO:61, SEQ ID NO:70, SEQ ID NO:65, SEQ ID NO:68 and SEQ ID NO:67.

Images