Polynucleotides Targeting NR4A3 and Their Use

Abstract

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

Description

Technical Field

[0001] Cross - Reference to Related Applications This PCT application claims the benefit of priority of U.S. Provisional Application No. 63 / 365,025, filed May 19, 2022; U.S. Provisional Application No. 63 / 382,705, filed November 7, 2022; and U.S. Provisional Application No. 63 / 482,984, filed February 2, 2023, each of which is hereby incorporated by reference in its entirety.

[0002] Reference to Electronically Submitted Sequence Listing The content of the sequence listing was submitted electronically (Name: 4385_105PC03_SequenceListing_ST26.XML; Size: 124,447 bytes; and Creation Date: May 19, 2023), filed together with this application, and is hereby incorporated by reference in its entirety.

[0003] The present disclosure relates to polynucleotides (e.g., guide RNAs) that can be used to reduce the level of the NR4A3 gene and / or NR4A3 protein in immune cells. The present disclosure also relates to cell - based (e.g., T - cell) cancer immunotherapy involving the administration of such immune cells having a reduced level of the NR4A3 gene and / or NR4A3 protein.

Background Art

[0004] Cancer immunotherapy relies on T cells, which are the main killers of the immune system for infected and diseased cells, to attack and kill tumor cells. However, there is an important obstacle to immunotherapy: the ability of T cells to kill can decline (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 use functionally active T cells isolated from patients and require highly functional T cells to be effective. These T cells are engineered to recognize specific antigens on target cancer cells and are expanded ex vivo.

[0005] When the immune system is forced to be active for a long time, for example, by persistent viral infection or the progressive development of cancer, effector T cells can become exhausted. One characteristic of exhausted T cells is the increased expression of immune checkpoint proteins such as PD-1 and CTLA-4, which can cause the detachment of these T cells (i.e., become non-functional). Immune checkpoint inhibitors can block these checkpoint proteins, thereby increasing the immune response against tumors. Some studies have suggested that blocking the activity of checkpoint proteins in exhausted T cells does not achieve that purpose. This is important because so-called inflammatory tumors, i.e., tumors that contain high levels of immune cells and should thus be ideal candidates for responding to immunotherapy, often possess populations composed almost entirely of exhausted T cells. Moreover, the tumor microenvironment can induce an aged and exhausted cell phenotype. Therefore, devising strategies to reverse and / or prevent these exhausted states is extremely important for improving the effectiveness of immunotherapy.

Summary of the Invention

[0006] A method for reducing the level of the NR4A3 gene and / or NR4A3 protein in immune cells, 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 shown 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, and after modification, the level of the NR4A3 gene and / or NR4A3 protein in the immune cells is reduced when compared to a reference immune cell (e.g., the corresponding immune cell not contacted with the polynucleotide). In some embodiments, after 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% when compared to the reference immune cell. In some embodiments, after 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% when compared to the reference immune cell.

[0007] Also provided herein is a method of reducing or preventing exhaustion in immune cells, the method comprising contacting 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 the gRNA 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, and wherein after the contacting, the immune cells exhibit less exhaustion after persistent antigen stimulation as compared to a reference immune cell (e.g., a corresponding immune cell not contacted with the polynucleotide). In some embodiments, the immune cells are more resistant to exhaustion as compared to a reference immune cell. In some embodiments, the immune cells exhibit increased persistence / survival when administered to a subject as compared to a reference immune cell. In some embodiments, the immune cells exhibit increased expansion / proliferation in response to persistent antigen stimulation as compared to a reference immune cell. In some embodiments, the immune cells exhibit increased effector function in response to persistent antigen stimulation as compared to a reference immune cell.

[0008] A method for increasing the production of cytokines by immune cells that respond to antigenic stimulation, 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 shown 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, and after modification, the immune cells exhibit increased cytokine production upon antigenic stimulation as compared to reference immune cells (e.g., corresponding immune cells not modified with the polynucleotide). In some embodiments, the cytokines include IFN-γ, IL-2, TNF-α, or a combination thereof. In some embodiments, after modification, the production of cytokines that respond to antigenic 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 as compared to reference immune cells.

[0009] The present disclosure also provides a method of increasing the effector function of immune cells that respond to persistent antigen stimulation, 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: 67, and wherein after modification, the immune cells exhibit an increased effector function upon persistent antigen stimulation as compared to a reference immune cell (e.g., the corresponding immune cell not contacted with the polynucleotide). In some embodiments, after modification, the immune cells retain effector function for at least 1, at least 2, or at least 3 additional rounds of antigen stimulation assays as compared to a reference immune cell. In some embodiments, the effector function comprises (i) the ability to kill target cells (e.g., tumor cells), (ii) the ability to produce cytokines upon further antigen stimulation, or (iii) both (i) and (ii).

[0010] Some aspects of the present disclosure are methods of preparing a composition comprising immune cells having reduced levels of the NR4A3 gene and / or the NR4A3 protein, the methods comprising modifying the immune cells with a gene editing tool comprising a polynucleotide comprising a gRNA, the gRNA being capable of specifically binding to a sequence within the NR4A3 gene, the sequence comprising, consisting essentially of, or consisting of any one of the sequences set forth in 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 modification, the level of the NR4A3 gene and / or the NR4A3 protein in the immune cells is reduced when compared to a reference immune cell (e.g., the corresponding immune cell not contacted with the polynucleotide). In some aspects, the method further comprises combining the modified immune cells with a pharmaceutically acceptable excipient.

[0011] For any of the above methods, in some aspects, after 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% when compared to a reference immune cell. In some aspects, after 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% when compared to a reference immune cell.

[0012] Also provided herein is a method of treating a tumor in a subject in need of treating a tumor, the method comprising administering to the subject immune cells modified 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: 67. In some embodiments, the level of the NR4A3 gene in the immune cells 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% as compared to a reference immune cell (e.g., a corresponding immune cell not contacted with the polynucleotide). In some embodiments, the level of the NR4A3 protein in the immune cells 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% as compared to a reference immune cell.

[0013] In some embodiments of the above method of treating a tumor, administering reduces the tumor volume in the subject as compared to a reference tumor volume (e.g., the tumor volume in the subject before administration and / or the tumor volume in a subject not receiving the administration). In some embodiments, 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 at least about 100% as compared to the reference tumor volume.

[0014] In some embodiments, the tumors that can be treated by the methods provided herein are derived from cancers including 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 combinations thereof.

[0015] In some embodiments, the methods of treating the tumors provided herein further comprise administering an additional therapeutic agent to the subject. In some embodiments, the additional therapeutic agent comprises a chemotherapeutic agent, a targeted anti-cancer therapy, an oncolytic agent, a cytotoxic agent, an immune-based therapy, a cytokine, a surgical procedure, a radiation treatment, an activator of a co-stimulatory molecule, an immune checkpoint inhibitor, a vaccine, a cellular immunotherapy, or any combination thereof. In some embodiments, the additional therapeutic agent is an immune checkpoint inhibitor. In some embodiments, 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. In some embodiments, the immune cells and the additional therapeutic agent are administered to the subject simultaneously. In some embodiments, the immune cells and the additional therapeutic agent are administered to the subject sequentially.

[0016] In some embodiments, the immune cells are administered to the subject by injection, intramuscularly, subcutaneously, intraocularly, intravenously, intraperitoneally, intradermally, intraorbitally, intracranially, intraspinally, intraventricularly, intrathecally, intracapsularly, intraarticularly, intratumorally, or any combination thereof.

[0017] In any of the above methods, in some embodiments, the method further comprises modifying the immune cells to have a reduced level of the NR4A1 gene and / or the NR4A1 protein. In some embodiments, modifying the immune cells to have a reduced level of the NR4A1 gene and / or the NR4A1 protein comprises contacting the immune cells with a gene editing tool (an "NR4A1-specific gene editing tool") capable of specifically targeting the NR4A1 gene and / or the NR4A1 protein and reducing its level. In some embodiments, after contacting the immune cells with the NR4A1-specific gene editing tool, the level of the NR4A1 gene is 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% reduced compared to the corresponding cells not contacted with the NR4A1-specific gene editing tool. In some embodiments, after contacting the immune cells with the NR4A1-specific gene editing tool, the level of the NR4A1 protein is 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% reduced compared to the corresponding cells not contacted with the NR4A1-specific gene editing tool.

[0018] For any of the above methods, in some embodiments, the method further comprises modifying immune cells to have a reduced level of the NR4A2 gene and / or the NR4A2 protein. In some embodiments, modifying immune cells to have a reduced level of the NR4A2 gene and / or the NR4A2 protein comprises contacting the immune cells with a gene editing tool (an "NR4A1-specific gene editing tool") that can specifically target the NR4A2 gene and / or the NR4A2 protein and reduce its level. In some embodiments, after contacting the immune cells with the NR4A2-specific gene editing tool, the level of the NR4A2 gene is 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% reduced compared to the corresponding cells that were not contacted with the NR4A2-specific gene editing tool. In some embodiments, after contacting the immune cells with the NR4A2-specific gene editing tool, the level of the NR4A2 protein is 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% reduced compared to the corresponding cells that were not contacted with the NR4A2-specific gene editing tool.

[0019] In some embodiments, any of the methods provided above further comprises modifying immune cells to have increased levels of c-Jun protein. In some embodiments, modifying immune cells to have increased levels of c-Jun protein comprises contacting the immune cells with a nucleotide sequence encoding c-Jun protein. In some embodiments, the nucleotide sequence encoding c-Jun protein is: (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 with the nucleic acid sequence shown 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 with the nucleic acid sequence shown 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 with the nucleic acid sequence shown 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 with the nucleic acid sequence shown 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 with the nucleic acid sequence shown 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 with 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 with 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 with 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 with the nucleic acid sequence set forth in SEQ ID NO: 16;

[0020] In some embodiments, modifying an immune cell to have an increased level of c-Jun protein involves contacting the immune cell with a transcriptional activator capable of increasing the expression of endogenous c-Jun protein. In some embodiments, the transcriptional activator is bound to a Cas protein modified to lack endonuclease activity.

[0021] In some embodiments, after modifying immune cells to have increased levels of c-Jun protein, the level of c-Jun protein in the immune cells is 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 increased compared to a reference cell (e.g., a corresponding cell not modified to have increased levels of c-Jun protein).

[0022] In some embodiments, any of the methods provided above further comprises modifying immune cells to express a ligand-binding protein. In some embodiments, 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. In some embodiments, the ligand-binding protein is a CAR. In some embodiments, the ligand-binding protein is a TCR. In some embodiments, the ligand-binding protein is 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, Lewis Y, CD24, PDGFR-beta, SSEA-4, CD20, folate receptor alpha, ERBB2 (Her2 / neu), MUC1, MUC16, EGFR, NCAM, prostase, PAP, ELF2M, ephrin B2, IGF-I receptor, CAIX, LMP2, gp100, bcr-abl, tyrosinase, EphA2, fucosyl GM1, sLe, GM3, TGS5, HMWMAA, 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,It is capable of specifically binding to an antigen selected from E7, MAGE A1, ETV6-AML, sperm protein 17, XAGE1, Tie2, MAD-CT-1, MAD-CT-2, Fos-related antigen 1, p53, p53 variants, prostain, survivin and telomerase, PCTA-1 / galectin 8, Melan-A / MART1, Ras variants, hTERT, sarcoma translocation breakpoint, 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.,

[0023] In some embodiments, the ligand-binding protein specifically binds to ROR1. In some embodiments, the ligand-binding protein comprises an antigen-binding domain derived from an R12 antibody, an R11 antibody, a 2A2 antibody, or any combination thereof. In some embodiments, the ligand-binding protein comprises a heavy chain variable domain (VH) and a light chain variable domain (VL), wherein VH comprises the amino acid sequence shown in SEQ ID NO: 17 and VL comprises the amino acid sequence shown in SEQ ID NO: 21.,

[0024] In any of the above methods, in some embodiments, the gene editing tool comprises shRNA, siRNA, miRNA, antisense oligonucleotide, CRISPR, zinc finger nuclease, TALEN, meganuclease, restriction endonuclease, or any combination thereof. In some embodiments, the gene editing tool is CRISPR.,

[0025] Also provided herein are compositions comprising cells having reduced levels of the NR4A3 gene and / or the NR4A3 protein, wherein the compositions are prepared by any of the methods provided herein.

[0026] The present disclosure also provides a composition comprising cells that express reduced levels of the NR4A3 gene and / or the NR4A3 protein, wherein the cells are modified with a gRNA that can target the NR4A3 gene, and the gRNA comprises, consists of, or consists essentially of a sequence shown 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 embodiments, the gRNA comprises, consists of, or consists essentially of the sequence shown in SEQ ID NO: 94. In some embodiments, the gRNA comprises, consists of, or consists essentially of the sequence shown in SEQ ID NO: 52. In some embodiments, the gRNA comprises, consists of, or consists essentially of the sequence shown in SEQ ID NO: 96. In some embodiments, the gRNA comprises, consists of, or consists essentially of the sequence shown in SEQ ID NO: 53. In some embodiments, the gRNA comprises, consists of, or consists essentially of the sequence shown in SEQ ID NO: 54. In some embodiments, the gRNA comprises, consists of, or consists essentially of the sequence shown in SEQ ID NO: 86. In some embodiments, the gRNA comprises, consists of, or consists essentially of the sequence shown in SEQ ID NO: 83. In some embodiments, the gRNA comprises, consists of, or consists essentially of the sequence shown in SEQ ID NO: 55. In some embodiments, the gRNA comprises, consists of, or consists essentially of the sequence shown in SEQ ID NO: 82. In some embodiments, the gRNA comprises, consists of, or consists essentially of the sequence shown in SEQ ID NO: 56. In some embodiments, the gRNA comprises, consists of, or consists essentially of the sequence shown in SEQ ID NO: 76. In some embodiments, the gRNA comprises, consists of, or consists essentially of the sequence shown in SEQ ID NO: 57. In some embodiments, the gRNA comprises, consists of, or consists essentially of the sequence shown in SEQ ID NO: 75.In some embodiments, the gRNA comprises, consists of, or consists essentially of the sequence set forth in SEQ ID NO: 58. In some embodiments, the gRNA comprises, consists of, or consists essentially of the sequence set forth in SEQ ID NO: 71. In some embodiments, the gRNA comprises, consists of, or consists essentially of the sequence set forth in SEQ ID NO: 61. In some embodiments, the gRNA comprises, consists of, or consists essentially of the sequence set forth in SEQ ID NO: 70. In some embodiments, the gRNA comprises, consists of, or consists essentially of the sequence set forth in SEQ ID NO: 65. In some embodiments, the gRNA comprises, consists of, or consists essentially of the sequence set forth in SEQ ID NO: 68. In some embodiments, the gRNA comprises, consists of, or consists essentially of the sequence set forth in SEQ ID NO: 67.

[0027] In some embodiments, any of the above compositions further comprises a pharmaceutically acceptable excipient.

[0028] In addition, the present disclosure provides an isolated polynucleotide comprising, consisting of, or consisting essentially of the sequence shown 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 embodiments, the gRNA comprises, consists of, or consists essentially of the sequence shown in SEQ ID NO: 94. In some embodiments, the gRNA comprises, consists of, or consists essentially of the sequence shown in SEQ ID NO: 52. In some embodiments, the gRNA comprises, consists of, or consists essentially of the sequence shown in SEQ ID NO: 96. In some embodiments, the gRNA comprises, consists of, or consists essentially of the sequence shown in SEQ ID NO: 53. In some embodiments, the gRNA comprises, consists of, or consists essentially of the sequence shown in SEQ ID NO: 54. In some embodiments, the gRNA comprises, consists of, or consists essentially of the sequence shown in SEQ ID NO: 86. In some embodiments, the gRNA comprises, consists of, or consists essentially of the sequence shown in SEQ ID NO: 83. In some embodiments, the gRNA comprises, consists of, or consists essentially of the sequence shown in SEQ ID NO: 55. In some embodiments, the gRNA comprises, consists of, or consists essentially of the sequence shown in SEQ ID NO: 82. In some embodiments, the gRNA comprises, consists of, or consists essentially of the sequence shown in SEQ ID NO: 56. In some embodiments, the gRNA comprises, consists of, or consists essentially of the sequence shown in SEQ ID NO: 76. In some embodiments, the gRNA comprises, consists of, or consists essentially of the sequence shown in SEQ ID NO: 57. In some embodiments, the gRNA comprises, consists of, or consists essentially of the sequence shown in SEQ ID NO: 75. In some embodiments, the gRNA comprises, consists of, or consists essentially of the sequence shown in SEQ ID NO: 58.In some embodiments, the gRNA comprises, consists of, or consists essentially of the sequence set forth in SEQ ID NO: 71. In some embodiments, the gRNA comprises, consists of, or consists essentially of the sequence set forth in SEQ ID NO: 61. In some embodiments, the gRNA comprises, consists of, or consists essentially of the sequence set forth in SEQ ID NO: 70. In some embodiments, the gRNA comprises, consists of, or consists essentially of the sequence set forth in SEQ ID NO: 65. In some embodiments, the gRNA comprises, consists of, or consists essentially of the sequence set forth in SEQ ID NO: 68. In some embodiments, the gRNA comprises, consists of, or consists essentially of the sequence set forth in SEQ ID NO: 67.

[0029] Some embodiments of the present disclosure relate to cells comprising the polynucleotides described above. In some embodiments, the cell further comprises a polynucleotide encoding a ligand-binding protein. In some embodiments, 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. In some embodiments, the cell further comprises (i) a nucleotide sequence encoding the c-Jun protein, (ii) a transcriptional activator capable of increasing the expression of the endogenous c-Jun protein, or (iii) both (i) and (ii).

[0030] In some embodiments, the cell is an immune cell. In some embodiments, the immune cells include lymphocytes, neutrophils, monocytes, macrophages, dendritic cells, or combinations thereof. In some embodiments, the lymphocytes include T cells, tumor-infiltrating lymphocytes (TILs), lymphokine-activated killer cells, natural (NK) cells, or combinations thereof.

[0031] This specification provides 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 the sequence shown 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

[0032]

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Mode for Carrying Out the Invention

[0033] The present disclosure generally relates to polynucleotides (e.g., isolated polynucleotides) capable of reducing the level of the NR4A3 gene and / or NR4A3 protein in immune cells (e.g., T cells). As described herein, the polynucleotides of the present disclosure are such that the polynucleotides described herein can interact with the NR4A3 gene, thereby reducing the level of the NR4A3 gene and / or NR4A3 protein in immune cells, and include a nucleotide sequence complementary to a nucleic acid sequence within the NR4A3 gene (also referred to herein as the "NR4A3 targeting nucleotide sequence" or a variant thereof). As will be apparent to those skilled in the art, such polynucleotides can be used with various gene editing techniques (e.g., the CRISPR / Cas system). Also, in some embodiments, such polynucleotides can be used in combination with one or more additional nucleotide sequences described herein (e.g., encoding a ligand binding protein and / or a c-Jun protein). In some embodiments, 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 embodiments, by reducing the level of the NR4A3 gene and / or NR4A3 protein, the polynucleotides of the present disclosure are useful in improving one or more functions of immune cells (e.g., increased persistence and / or effector activity). Reducing the level of the NR4A3 gene and / or NR4A3 protein (either alone or in combination with reduced levels of the NR4A1 gene and / or NR4A1 protein and / or NR4A2 gene and / or NR4A2 protein) can result in exhausted / dysfunction-resistant cells. Further, reducing the level of the NR4A3 gene and / or NR4A3 protein (either alone or in combination with reduced levels of the NR4A1 gene and / or NR4A1 protein and / or NR4A2 gene and / or NR4A2 protein) can result in the maintenance of anti-tumor function in the TME environment.In some aspects, the disclosure also provides a method of treating a wide range of diseases or disorders (e.g., cancer) in a subject in need of treatment for a wide range of diseases or disorders (e.g., cancer), the method comprising administering to the subject an immune cell described herein that has been modified to have a reduced level of the NR4A3 gene and / or the NR4A3 protein. Additional aspects of the disclosure are provided throughout this application.

[0034] Before describing the disclosure in more detail, it is to be understood that the disclosure is not limited to the specific compositions or process steps described, as such may, of course, vary. As will be apparent to those skilled in the art upon reading this disclosure, each of the individual aspects described and illustrated herein can be readily separated from or combined with the features of any of the other several aspects without departing from the scope or spirit of the disclosure. Any described method can be performed in the order of events described or in any other logically possible order.

[0035] The headings provided herein are not limitations of the various aspects of the disclosure, which can be defined in their entirety by reference to the specification as a whole. It is also to 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 is defined solely by the appended claims.

[0036] I. Terms To make the disclosure more readily understandable, certain terms are first defined. As used in this application, unless otherwise expressly provided herein, each of the following terms shall have the meaning set forth below. Additional definitions are provided throughout this application.

[0037] Throughout this disclosure, the term "a" or "an" entity refers to one or more of that entity; for example, "an immune cell" is understood to represent one or more immune cells. Thus, the terms "a" (or "an"), "one or more", and "at least one" may be used interchangeably herein.

[0038] Furthermore, as used herein, "and / or" is to be construed as a specific disclosure of each of the two specified features or components, with or without the other. Thus, the term "and / or" as used in phrases such as "A and / or B" herein is intended to include "A and B", "A or B", "A" (alone), and "B" (alone). Similarly, the term "and / or" as used in phrases such as "A, B, and / or C" is intended to include each of the following aspects: 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).

[0039] It is understood that when an aspect is described herein using the word "comprising", similar aspects are also provided with respect to "consisting of" and / or "consisting essentially of" where separately described.

[0040] 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 pertains. For example, the 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 the Oxford Dictionary of Biochemistry and Molecular Biology, Revised, 2000, Oxford University Press provide many common dictionaries of terms used in the present disclosure to one of ordinary skill in the art.

[0041] Units, prefixes, and symbols are shown in their approved forms of the International System of Units (SI). Numerical ranges are inclusive of the numbers defining the range. Unless otherwise indicated, amino acid sequences are written left to right in the amino to carboxy direction. The headings provided herein are not limitations of the various aspects of the disclosure, which may be provided by reference to the specification as a whole. Accordingly, the terms defined immediately below are more fully defined by reference to the specification as a whole thereof.

[0042] The abbreviations used herein are defined throughout the present disclosure. The various aspects of the present disclosure are described in further detail in the following subsections.

[0043] As used herein, the term "about" or "approximately" when applied to one or more values of interest, refers to a value similar to the recited reference value. In some embodiments, the term "approximately" refers to a range of values that fall within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less in either (greater than or less than) direction of the recited reference value, unless otherwise stated or otherwise apparent from the context, for values that would otherwise fall outside the possible range of 100% of the possible values.

[0044] As described herein, any concentration range, percentage range, ratio range or integer range is to be understood to include any integer value within the recited range and, where appropriate, fractions thereof (e.g., one tenth and one hundredth of an integer), unless otherwise indicated.

[0045] As used herein, "administering" refers to the physical introduction of a therapeutic agent or a composition comprising a therapeutic agent to a subject using any of a variety of methods and delivery systems known to those of skill in the art. The various routes of administration for the therapeutic agents described herein include intravenous, intraperitoneal, intramuscular, subcutaneous, spinal or other parenteral routes of administration, e.g., by injection or infusion. The phrase "parenteral administration" as used herein means a mode of administration other than enteral and topical administration, usually by injection, and includes, but is not limited to, intravenous, intraperitoneal, intramuscular, intraarterial, intrathecal, intralymphatic, intralesional, intraarticular, intraorbital, intracardiac, intradermal, transtracheal, intratracheal, pulmonary, subcutaneous, subepidermal, intraarticular, subcapsular, subarachnoid, intraventricular, intravitreal, epidural, and intrasternal injection and infusion, as well as in vivo electroporation. Alternatively, the therapeutic agents described herein may be administered via routes other than parenteral routes, e.g., topical, epidermal, or mucosal routes of administration, e.g., intranasally, orally, vaginally, rectally, sublingually, or topically. Administering may also be carried out, for example, once, a plurality of times, and / or over one or more extended periods of time.

[0046] 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 "alloantigen" refers to an antigen that is recognized by immune cells (e.g., T cells), thereby inducing activation of the immune cells (e.g., inducing effector functions such as cytokine production and / or triggering intracellular signals for cell proliferation).

[0047] Nucleotides are referred to by their generally recognized one-letter codes. Unless otherwise indicated, nucleic acids are written left to right in the 5' to 3' direction. Nucleotides are referred to herein by their generally known one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission. Thus, A represents adenine, C represents cytosine, G represents guanine, T represents thymine, and U represents uracil.

[0048] It should be understood that T and U are interchangeable in the disclosed sequences depending on whether the sequence is DNA or RNA. For example, the gRNA spacer sequence is presented as DNA (A / T / C / G) in the present disclosure, while the gRNA chimeric frame is presented as RNA (A / U / C / G).

[0049] Amino acids are referred to herein by their generally 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 the amino to carboxy direction.

[0050] "Polypeptide" refers to a chain containing at least two contiguous amino acid residues, and there is 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. "Protein" may contain one or more polypeptides. Unless otherwise specified, the terms "protein" and "polypeptide" may be used interchangeably.

[0051] 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 cDNA.

[0052] As used herein, the term "polynucleotide" refers to a polymer of nucleotides of any length that includes ribonucleotides, deoxyribonucleotides, analogs thereof, or mixtures thereof. This term refers to the primary structure of a molecule. Thus, the term includes triple-stranded, double-stranded, and single-stranded deoxyribonucleic acid ("DNA"), as well as triple-stranded, double-stranded, and single-stranded ribonucleic acid ("RNA"). It also includes polynucleotides in modified and unmodified forms, for example, modified by alkylation and / or capping. More 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 that is an N- or C-glycoside of a purine or pyrimidine base, and other polymers containing non-nucleotide backbones, such as polyamides (e.g., peptide nucleic acid "PNA") and polymorpholino polymers, and other sequence-specific synthetic nucleic acid polymers provided that the polymer contains nucleobases in a configuration that allows base pairing and base stacking as seen in DNA and RNA. Unless otherwise indicated, the terms "polynucleotide," "nucleic acid," "gene," "cDNA," and "mRNA" may be used interchangeably.

[0053] The term "gene" means a segment of DNA involved in producing a polypeptide chain. It may include regions preceding and following the coding region (leader and trailer) as well as intervening sequences (introns) between individual coding segments (exons).

[0054] The term "vector", as used herein, is intended to refer to a nucleic acid molecule capable of transporting another nucleic acid to which it is linked. One type of vector is a "plasmid", which refers to a circular double-stranded DNA loop into which additional DNA segments can be ligated. Another type of vector is a viral vector, into which additional DNA segments can be ligated to the viral genome. A given vector is capable of self-replication in the host cell into which it is introduced (e.g., a bacterial vector having a bacterial origin of replication and an episomal mammalian vector). Other vectors (e.g., non-episomal mammalian vectors) can be integrated into the genome of the host cell upon introduction into the host cell and are thereby replicated along with the host genome. Moreover, a given vector is capable of directing the expression of a functionally linked gene. Such vectors are referred to herein as "recombinant expression vectors" (or simply, "expression vectors"). Usually, the expression vectors utilized in recombinant DNA technology are often in the form of plasmids. Since plasmids are the most commonly used form of vectors, the terms "plasmid" and "vector" may be used interchangeably herein. However, other forms of expression vectors, such as viral vectors (e.g., replication-defective retroviruses, adenoviruses, and adeno-associated viruses), are also included and perform equivalent functions.

[0055] "Cancer" means a broad group of various diseases characterized by the uncontrolled growth of abnormal cells in the body. Uncontrolled cell division and growth result in the formation of malignant tumors that invade adjacent tissues and can also metastasize to distant parts of the body through the lymphatic system or bloodstream. "Cancer", as used herein, refers to primary, metastatic, and recurrent cancers.

[0056] As used herein, the term "immune response" refers to a biological response within a vertebrate to a foreign substance, which response protects the organism against these substances and the diseases they cause. An immune response results in the selective targeting, binding, damaging, destruction, and / or elimination from the vertebrate body of invading pathogens, pathogen-infected cells or tissues, cancerous or other abnormal cells, or, in the case of autoimmunity or pathological inflammation, normal human cells or tissues, 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. The immune response includes, for example, activation or inhibition of T cells, such as effector T cells or Th cells, such as CD4 + or CD8 + or inhibition of Treg cells. As used herein, the terms "T cell" and "T lymphocyte" are interchangeable and refer to any lymphocyte produced and processed by the thymus. In some embodiments, the T cell is a CD4 + T cell. In some embodiments, the T cell is a CD8 + T cell. In some embodiments, the T cell is an NKT cell.

[0057] As used herein, the term "anti-tumor immune response" refers to an immune response against a tumor antigen. The increased ability to stimulate an immune response or the immune system can be due to improved agonistic activity of T cell co-stimulatory receptors and / or improved antagonistic activity of inhibitory receptors. The increased ability to stimulate an immune response or the immune system can be determined in assays that measure immune responses, such as cytokine or chemokine release, cytolytic activity (determined directly on target cells or indirectly through detection of CD107a or granzyme), and assays that measure changes in proliferation, EC 50Or it can be reflected in the doubling rate of the maximum activity level. In some embodiments, the ability to stimulate an immune response or immune system activity can be improved, for example, 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 at least about 100%. In some embodiments, the ability to stimulate an immune response or immune system activity can be improved, 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.

[0058] The "subject" includes any human or non-human animal. 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 embodiments, the subject is a human. The terms "subject" and "patient" are used interchangeably herein.

[0059] The term "effective amount" or "effective dosage" refers to the amount of an agent (e.g., a modified immune cell disclosed herein) that provides a desired biological, therapeutic, and / or prophylactic result. The result can be a reduction, improvement, alleviation, attenuation, delay, and / or mitigation of one or more of the signs, symptoms, or causes of a disease, or any other desired modification of a biological system. With respect to solid tumors, an effective amount includes an amount sufficient to shrink the tumor and / or to reduce the rate of tumor growth (e.g., to inhibit tumor growth) or to prevent or delay other unwanted cell proliferation. In some embodiments, an effective amount is an amount sufficient to delay tumor development. In some embodiments, an effective amount is an amount sufficient to prevent or delay tumor recurrence. An effective amount can be administered in one or more administrations. The effective amount of a composition can, for example, (i) reduce the number of cancer cells, (ii) reduce tumor size, (iii) inhibit, delay, slow to some extent, or stop cancer cell infiltration into peripheral organs, (iv) inhibit tumor metastasis (i.e., slow to some extent or stop), (v) inhibit tumor growth, (vi) prevent or delay the development and / or recurrence of tumors, and / or (vii) reduce to some extent one or more of the symptoms associated with cancer.

[0060] The ability of a therapeutic agent to promote disease regression can be evaluated using a variety of methods known to skilled practitioners, e.g., in human subjects during clinical trials, in animal model systems that predict efficacy in humans, or by assaying the activity of the agent in in vitro assays.

[0061] As used herein, the term "standard of care" refers to a treatment that is accepted by medical experts as an appropriate treatment for a given type of disease and is widely used by healthcare professionals. The term can be used interchangeably with any of the following terms: "best practice", "standard medical care", and "standard therapy".

[0062] By way of example, an “anticancer agent” promotes cancer regression in a subject or prevents further tumor growth. In some embodiments, a therapeutically effective amount of the drug promotes cancer regression to the point of eliminating the cancer.

[0063] “Promoting cancer regression” means that administering an effective amount of the drug alone or in combination with an antineoplastic agent results in a reduction 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 periods without disease symptoms, or prevention of dysfunction or impairment due to disease pain.

[0064] As used herein, the terms “effective” and “efficacy” with respect to a treatment include both pharmacological efficacy and physiological safety. Pharmacological efficacy refers to the ability of the drug to promote cancer regression in a patient. Physiological safety refers to the level of toxicity at the cellular, organ, and / or organism level resulting from administration of the drug, or the level of other adverse physiological effects (adverse reactions).

[0065] As used herein, the term “immune checkpoint inhibitor” refers to a molecule that wholly or partially reduces, inhibits, interferes with, or modulates one or more checkpoint proteins. Checkpoint proteins control T cell activation or function. A number of 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, D.M., Nat Rev Cancer 12(4):252-64 (2012). These proteins are responsible for co-stimulatory or inhibitory interactions of the T cell response. Immune checkpoint proteins control and maintain self-tolerance as well as the duration and extent of the physiological immune response. Immune checkpoint inhibitors include or are derived from antibodies.

[0066] As used herein, the term "oxidative stress" refers to a condition characterized by an excess of oxidizing substances and / or a decrease in antioxidant levels. Cellular oxidizing substances can include, but are not limited to, oxygen radicals (superoxide anion, hydroxyl radical, and / or peroxyl radical); reactive non-radical oxygen species, such as hydrogen peroxide and singlet oxygen; carbon radicals; nitrogen radicals; sulfur radicals; and combinations thereof. In some embodiments, the state of oxidative stress can result in, for example, cell damage, cell dysfunction, and / or cell death.

[0067] As used herein, the term "modified cell" refers to a cell that has been subjected to a non-natural manipulation 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), for example, a T cell. As will become apparent from the present disclosure, the modified cells disclosed herein are modified to express a reduced level of the NR4A3 gene and / or NR4A3 protein as compared to a reference cell (e.g., a corresponding unmodified cell) (e.g., with a gene editing tool comprising a polynucleotide described herein). As described herein, in some embodiments, the modified cell may express normal levels (i.e., "endogenous levels") of the NR4A1 gene and / or NR4A1 protein and the NR4A2 gene and / or NR4A2 protein. In some embodiments, the modified cells of the present disclosure may express (i) a reduced level of the NR4A3 gene and / or NR4A3 protein and (ii) a reduced level of the NR4A1 gene and / or NR4A1 protein. In some embodiments, the modified cells described herein may express (i) a reduced level of the NR4A3 gene and / or NR4A3 protein and (ii) a reduced level of the NR4A2 gene and / or NR4A2 protein. In some embodiments, the modified cell may express (i) a reduced level of the NR4A3 gene and / or NR4A3 protein, (ii) a reduced level of the NR4A1 gene and / or NR4A1 protein, and (iii) a reduced level 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, the corresponding cell will also be a T cell.Unless otherwise indicated, "modified cells having (expressing) reduced levels of the NR4A3 gene and / or the NR4A3 protein" (including such types thereof) include cells (e.g., T cells) modified to have reduced levels of the NR4A3 gene and / or the NR4A3 protein and: (i) endogenous levels of the NR4A1 and NR4A2 genes and the NR4A1 and NR4A2 proteins; (ii) reduced levels of the NR4A1 gene and / or the NR4A1 protein; (iii) reduced levels of the NR4A2 gene and / or the NR4A2 protein; or (iv) reduced levels of both the NR4A1 gene and / or the NR4A1 protein and the NR4A2 gene and / or the NR4A2 protein.

[0068] As used herein, the terms "endogenous expression" or "endogenous expression level" or "endogenous level" (or such types thereof) refer to the expression of naturally occurring genes and / or proteins (e.g., amount, kinetics, etc.) (e.g., genes and / or proteins not directly manipulated by non-naturally occurring manipulations). As will be apparent from the present disclosure, in some embodiments, the modified cells disclosed herein (e.g., immune cells expressing a ligand-binding protein and modified with a gene editing tool comprising a polynucleotide of the present disclosure) do not express the endogenous level of the NR4A3 gene or the NR4A3 protein, but since the NR4A1 and NR4A2 genes are not modified (e.g., by CRISPR, e.g., by non-naturally occurring manipulations), the modified cells endogenously express the NR4A1 and NR4A2 genes and / or the NR4A1 and NR4A2 proteins. As described herein, in some embodiments, modified cells expressing reduced levels of the NR4A3 gene or the NR4A3 protein can be further modified to also express reduced levels of (i) the NR4A1 gene or the NR4A1 protein, (ii) the NR4A2 gene or the NR4A2 protein, or (iii) both (i) and (ii).

[0069] In some embodiments, the modified cells are generated by introducing into the cells a foreign or exogenous nucleic acid (e.g., a polynucleotide described herein that can specifically target a region within the NR4A3 gene, such as a gRNA). In some embodiments, the foreign or exogenous nucleic acid can encode a gene editing tool disclosed herein. The nucleic acid can be introduced into the cells by methods known in the art, such as, for example, electroporation (see, e.g., Heiser W.C. Transcription Factor Protocols: Methods in Molecular Biology™ 2000;130:117-134), chemical (e.g., calcium phosphate or lipid) transfection (see, e.g., Lewis W.H., et al., Somatic Cell Genet. 1980 May;6(3):333-47; Chen C., et al., Mol Cell Biol. 1987 August;7(8):2745-2752), fusion with bacterial protoplasts containing a recombinant plasmid (see, e.g., Schaffner W. Proc Natl Acad Sci USA. 1980 April;77(4):2163-7), or direct microinjection of purified DNA into the nucleus of the cell (see, e.g., Capecchi M.R. Cell. 1980 November;22(2 Pt 2):479-88).

[0070] It should be understood that the disclosure referring to "modified cells" or "cells" is equally applicable to populations of those cells, i.e., multiple of those cells.

[0071] As used herein, the term "edited" (and its grammatical variants) refers to a process by which a cell (e.g., a T cell) is modified such that it is functionally and / or structurally different from the corresponding unmodified cell. More specifically, as further described herein, the cells provided herein are edited such that the cell exhibits reduced expression of an NR4A protein and / or gene when compared to an unedited cell. Thus, the term "NR4A-edited" as used herein refers to reduced expression of one or more members of the NR4A family (e.g., NR4A1, NR4A2, and / or NR4A3). In some embodiments, an NR4A-edited cell (e.g., an NR4A1-edited, NR4A2-edited, and / or NR4A3-edited cell) does not exhibit expression of one or more members of the NR4A family. In some embodiments, in some embodiments, an NR4A-edited cell exhibits some expression of a member of the NR4A family but at a level that is significantly reduced compared to the corresponding unedited cell. In some embodiments, NR4A expression in the NR4A-edited cells provided herein 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 the corresponding unedited cell. Unless otherwise indicated, the terms "edited," "deficient," and "knocked out" (or their variants) are used interchangeably in this disclosure.

[0072] Accordingly, the term "NR4A3-edited" refers specifically to a reduced expression of the NR4A3 gene and / or protein. In some embodiments, NR4A3-edited cells do not exhibit expression of the NR4A3 gene and / or protein. In some embodiments, NR4A3-edited cells may exhibit some expression of the NR4A3 gene and / or protein, but at levels that are substantially reduced compared to corresponding non-edited cells. In some embodiments, compared to corresponding non-edited cells, 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%. Unless otherwise indicated, "NR4A3-edited", "NR4A3-deficient", and "NR4A3-knocked out" (or the like) are used interchangeably herein.

[0073] As used herein, the terms "elevated concentration" or "elevated level" and their grammatical variants refer to a substance (e.g., reactive oxygen species; ROS) that exceeds normal levels compared to a suitable control (e.g., healthy tissue or cells).

[0074] As used herein, the terms "reactive oxygen species" and "ROS" refer to highly reactive oxygen-containing chemicals that can readily react with other molecules and cause potentially damaging modifications. Reactive oxygen species include, for example, oxygen ions, both inorganic and organic free radicals and peroxides, such as hydrogen peroxide, superoxide, hydroxyl radical, lipid hydroperoxidase, and singlet oxygen. They are typically very small molecules and are highly reactive due to the presence of unpaired valence shell electrons. Almost all cancers are associated with elevated concentrations of reactive oxygen species.

[0075] 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 directs a cell to perform a special function upon binding of an antigen to the extracellular domain. The terms "artificial T cell receptor", "chimeric T cell receptor", and "chimeric immunoreceptor" may each be used interchangeably herein with the term "chimeric antigen receptor". Chimeric antigen receptors are distinguished from other antigen-binding agents by both their ability to bind MHC-independent antigens and to transmit activation signals through their intracellular domains.

[0076] The antigen-specific extracellular domain of a chimeric antigen receptor recognizes and specifically binds an antigen, typically a surface-expressed antigen of a malignancy. The antigen-specific extracellular domain binds the antigen with an affinity constant or affinity of interaction (K D ) of, for example, from about 0.1 pM to about 10 μM, such as from about 0.1 pM to about 1 μM or from about 0.1 pM to about 100 nM, thereby specifically binding the antigen. Methods for determining the affinity of interaction are known in the art. Suitable antigen-specific extracellular domains for use in the CARs of the present disclosure can be any antigen-binding polypeptide, and a variety are known in the art. In some embodiments, the antigen-binding domain is a single-chain Fv (scFv). Other antibody-based recognition domains (cAb VHH (camelid antibody variable domain) and humanized versions thereof, lgNAR VH (shark antibody variable domain) and humanized versions thereof, sdAb VH (single-domain antibody variable domain) and "camelized" antibody variable domains) are suitable for use. In some embodiments, T cell receptor (TCR)-based recognition domains, such as single-chain TCRs (scTvs, single-chain two-domain TCRs containing V alpha V beta), are also suitable for use.

[0077] The chimeric antigen receptors disclosed herein may also include an intracellular domain that provides an intracellular signal to the cell (expressing the CAR) upon antigen binding to the antigen-specific extracellular domain. In some embodiments, the intracellular signaling domain of the CAR is responsible for activating at least one of the effector functions of the T cells expressing the chimeric receptor.

[0078] The term "intracellular domain" refers to the portion of the CAR that transmits an effector function signal upon antigen binding to the extracellular domain and directs the T cell to perform a specific function. Non-limiting examples of suitable intracellular domains include either the zeta chain of the T cell receptor or a homolog thereof (e.g., eta, delta, gamma, or epsilon), the MB1 chain, 829, Fc RIII, Fc RI, and combinations of signaling molecules, such as CD3 zeta, and CD28, CD27, 4-1BB, DAP-10, OX40, and combinations thereof, as well as similar molecules and fragments. The intracellular signaling portions of other members of the family of activating proteins, such as FcγRIII and FcεRI, can be used. Usually, the entire intracellular domain is used, whereas in many cases, it may not be necessary to use the entire intracellular polypeptide. To the extent that truncated portions of the intracellular signaling domain can be utilized, such truncated portions can be used in place of the intact chain as long as they still introduce an effector function signal. Thus, the term "intracellular domain" is meant to include any truncated portion of the intracellular domain that is sufficient to introduce an effector function signal. Typically, the antigen-specific extracellular domain is linked to the intracellular domain of the chimeric antigen receptor by a transmembrane domain. The transmembrane domain traverses the cell membrane, anchors the CAR to the T cell surface, connects the extracellular domain to the intracellular signaling domain, and thus affects the expression of the CAR on the T cell surface. The chimeric antigen receptor may also further include one or more co-stimulatory domains and / or one or more spacers. The co-stimulatory domain is derived from the intracellular signaling domain of a co-stimulatory protein that improves cytokine production, proliferation, cytotoxicity, and / or persistence in vivo.

[0079] A "peptide hinge" or "spacer" connects an antigen-specific extracellular domain to a transmembrane domain. The transmembrane domain is fused to a co-stimulatory domain, and optionally the co-stimulatory domain is fused to a second co-stimulatory domain, and the co-stimulatory domain is fused to a 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 in the case of tandem CARs between multiple scFvs, can affect the flexibility of the antigen-binding domain(s) and thereby the CAR function. Suitable transmembrane domains, co-stimulatory domains, and spacers are known in the art.

[0080] As used herein, the terms "ug" and "uM" are used interchangeably with "μg" and "μΜ", respectively.

[0081] As used herein, the term "gene editing" refers to the process of changing the genetic information present in a cell's genome. This gene editing can be carried out by manipulating genomic DNA that results in modification of the genetic information. In some embodiments, such gene editing can affect the expression of the edited DNA. In some embodiments, such gene editing does not affect the expression of the edited DNA. In some embodiments, the gene editing of the modified cells disclosed herein can be performed 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), meganucleases, restriction endonucleases, or any combination thereof.

[0082] As used herein, the term "nuclease" refers to an enzyme having catalytic activity for DNA cleavage. Any nuclease agent that induces a nick or double-strand break at a desired recognition site can be used in the methods and compositions disclosed herein. Native or native nuclease agents can be used as long as the nuclease agent induces a nick or double-strand break at the desired recognition site. Alternatively, modified or engineered nuclease agents can be used. An "engineered nuclease agent" includes a nuclease that has been engineered (modified or derived) from its native form to specifically recognize a desired recognition site and induce a nick or double-strand break therein. Thus, an engineered nuclease agent can be derived from a native, naturally occurring nuclease agent, or it can be artificially created or synthesized. Modifications of nuclease agents can be as small as one amino acid in a protein cleavage agent or one nucleotide in a nucleic acid cleavage agent. In some embodiments, the engineered nuclease induces a nick or double-strand break at a recognition site that was not the sequence that would have been recognized by the native (unengineered or unmodified) nuclease agent. Creating a nick or double-strand break in a recognition site or other DNA can be referred to herein as "cutting" or "cleaving" the recognition site or other DNA.

[0083] As used herein, "coding sequence" or "coding nucleic acid" means a nucleic acid (RNA or DNA molecule) that includes 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 can further include start and stop signals operably linked to regulatory elements including a promoter and a polyadenylation signal capable of directing expression in the cells of the individual or mammal to which the nucleic acid is administered. The coding sequence can be codon optimized.

[0084] "Complementary" or "complementarity", as used herein, refers to Watson-Crick (e.g., A-T / U and C-G) or Hoogsteen base pairing between nucleotides or nucleotide analogs of nucleic acid molecules. "Complementarity" refers to the property shared between two nucleic acid sequences such that when the two sequences are aligned in an antiparallel orientation to each other, the nucleotide bases at each position are complementary.

[0085] The various aspects described herein are described in more detail in the following subsections.

[0086] II. NR4A3-Targeting Polynucleotides Provided herein are polynucleotides (e.g., isolated polynucleotides) that include nucleotide sequences capable of specifically binding to target sequences within the NR4A3 gene. Without being bound by any one theory, in some aspects, by binding to a target sequence within the NR4A3 gene, the polynucleotides of the present disclosure can reduce the level of the NR4A3 gene and / or the encoded protein in a cell (e.g., an immune cell). As further described elsewhere in the present disclosure, in some aspects, the reduced levels of the NR4A3 gene and / or the NR4A3 protein can be associated with reduced NR4A3 activity, which in turn can improve one or more properties of the cell. Non-limiting examples of such properties are provided elsewhere in the present disclosure.

[0087] The polynucleotides provided herein (e.g., including gRNAs that can specifically target target regions within the NR4A3 gene) can exist in whole cells, in cell lysates, or in a partially purified or substantially pure form. The polynucleotides are purified from other cellular components or other contaminating substances, such as other cellular nucleic acids (e.g., other chromosomal DNAs, e.g., chromosomal DNAs that are substantially linked to the DNA to be isolated) or proteins, by standard techniques including alkaline / SDS treatment, CsCl banding, column chromatography, restriction enzymes, agarose gel electrophoresis, and others well known in the art, and are then “isolated” or “substantially purified”. The polynucleotides described herein can be, for example, DNA or RNA, and can or cannot contain intron sequences. In some embodiments, the polynucleotide is a cDNA molecule.

[0088] II.A. Binding sequence As described herein, the polynucleotides described herein include nucleotide sequences that can specifically bind to nucleic acid sequences within the NR4A3 gene. Such nucleotide sequences are also referred to herein as “binding sequences” or “guide sequences” or “guide RNAs” (gRNAs). Thus, the term “guide RNA” (gRNA), as used herein, is not particularly limited as long as it can specifically bind to a nucleic acid sequence having the NR4A3 gene, thereby reducing the level of the NR4A3 gene and / or the NR4A3 protein. Non-limiting examples of such gRNAs are provided throughout the present disclosure (see, for example, Tables A - D).

[0089] In some embodiments, the gRNA is DNA or RNA. In some embodiments, the gRNA is DNA. In some embodiments, the gRNA is RNA. In some embodiments, the DNA and / or RNA are each synthetic DNA and / or RNA. In some embodiments, the synthetic RNA or DNA contains at least one unnatural nucleobase. In some embodiments, all nucleobases of a given class are replaced with unnatural nucleobases (e.g., all uridines in the polynucleotides disclosed herein can be replaced with unnatural nucleobases such as 5-methoxyuridine or pseudouridine). In some embodiments, the polynucleotide (e.g., synthetic RNA or synthetic DNA) contains only natural nucleobases, i.e., A, C, T, and U in the case of synthetic DNA, or A, C, T, and U in the case of synthetic RNA or synthetic DNA.

[0090] In some embodiments, the gRNA can be about 5 to about 100 nucleotides in length. In some embodiments, 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 embodiments, the gRNA is about 10 to about 30 nucleotides (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 length. In some embodiments, the gRNA is about 20 nucleotides in length.

[0091] In some embodiments, the gRNA of the polynucleotide described herein is designed to be complementary or substantially complementary to a nucleic acid sequence within the NR4A3 gene (also referred to herein as the "target sequence"). In some embodiments, the gRNA can incorporate wobbles or degenerate bases to bind to multiple sequences (e.g., multiple target sequences within the NR4A3 gene; or a target sequence within the NR4A3 gene and target sequences within other members of the NR4A family). In some cases, the gRNA can be modified to increase stability. For example, non-natural nucleotides can be incorporated to increase resistance to degradation. In some embodiments, the gRNA can be modified or designed to avoid or reduce secondary structure formation in the gRNA. In some embodiments, the gRNA can be designed to optimize the G-C content. In some embodiments, the G-C content is from about 40% to about 60% (e.g., about 40%, about 45%, about 50%, about 55%, about 60%). In some embodiments, the gRNA can contain modified nucleotides, such as, but not limited to, methylated or phosphorylated nucleotides. Additional methods for 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, riboswitch sequences, stability control sequences, hairpins, intracellular localization sequences, detection or labeling sequences, binding sites for one or more proteins, non-natural nucleotides, or combinations thereof. See, for example, U.S. Publication No. 20210123046A1, which is incorporated herein by reference in its entirety. Additional disclosure regarding such modifications is provided elsewhere in this disclosure.

[0092] As described herein, in some embodiments, the polynucleotides described herein comprise a gRNA that is sufficiently complementary (i.e., perfectly complementary) to a target sequence within the NR4A3 gene. As will be apparent to those skilled in the art, perfect complementarity is not always required for multiple nucleic acid sequences to hybridize to each other. Thus, in some embodiments, as long as the gRNA is capable of binding to a target sequence within the NR4A3 gene of an immune cell, thereby reducing the level of the NR4A3 gene and / or NR4A3 protein in the immune cell, the gRNA of the polynucleotides described herein may contain one or more base mismatches. In some embodiments, the gRNA of the polynucleotides described herein is 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 a target sequence having the NR4A3 gene.

[0093] Non-limiting examples of gRNAs useful for the present disclosure are provided in Tables C and D.

[0094] In some embodiments, the polynucleotides useful for the present disclosure comprise a gRNA, and the gRNA comprises, consists of, or consists essentially of the nucleotide sequence set forth in SEQ ID NO: 30. For example, in some embodiments, the polynucleotide comprises a gRNA comprising the nucleotide sequence set forth in SEQ ID NO: 30. In some embodiments, the polynucleotide comprises a gRNA consisting of the nucleotide sequence set forth in SEQ ID NO: 30. In some embodiments, the polynucleotide comprises a gRNA consisting essentially of the nucleotide sequence set forth in SEQ ID NO: 30.

[0095] In some embodiments, the polynucleotide useful for the present disclosure comprises a gRNA, and the gRNA comprises, consists of, or consists essentially of the nucleotide sequence set forth in SEQ ID NO: 52. For example, in some embodiments, the polynucleotide comprises a gRNA comprising the nucleotide sequence set forth in SEQ ID NO: 52. In some embodiments, the polynucleotide comprises a gRNA consisting of the nucleotide sequence set forth in SEQ ID NO: 52. In some embodiments, the polynucleotide comprises a gRNA consisting essentially of the nucleotide sequence set forth in SEQ ID NO: 52.

[0096] In some embodiments, the polynucleotide useful for the present disclosure comprises a gRNA, and the gRNA comprises, consists of, or consists essentially of the nucleotide sequence set forth in SEQ ID NO: 53. For example, in some embodiments, the polynucleotide comprises a gRNA comprising the nucleotide sequence set forth in SEQ ID NO: 53. In some embodiments, the polynucleotide comprises a gRNA consisting of the nucleotide sequence set forth in SEQ ID NO: 53. In some embodiments, the polynucleotide comprises a gRNA consisting essentially of the nucleotide sequence set forth in SEQ ID NO: 53.

[0097] In some embodiments, the polynucleotide useful for the present disclosure comprises a gRNA, and the gRNA comprises, consists of, or consists essentially of the nucleotide sequence set forth in SEQ ID NO: 54. For example, in some embodiments, the polynucleotide comprises a gRNA comprising the nucleotide sequence set forth in SEQ ID NO: 54. In some embodiments, the polynucleotide comprises a gRNA consisting of the nucleotide sequence set forth in SEQ ID NO: 54. In some embodiments, the polynucleotide comprises a gRNA consisting essentially of the nucleotide sequence set forth in SEQ ID NO: 54.

[0098] In some embodiments, the polynucleotide useful for the present disclosure comprises a gRNA, and the gRNA comprises, consists of, or consists essentially of the nucleotide sequence set forth in SEQ ID NO: 55. For example, in some embodiments, the polynucleotide comprises a gRNA comprising the nucleotide sequence set forth in SEQ ID NO: 55. In some embodiments, the polynucleotide comprises a gRNA consisting of the nucleotide sequence set forth in SEQ ID NO: 55. In some embodiments, the polynucleotide comprises a gRNA consisting essentially of the nucleotide sequence set forth in SEQ ID NO: 55.

[0099] In some embodiments, the polynucleotide useful for the present disclosure comprises a gRNA, and the gRNA comprises, consists of, or consists essentially of the nucleotide sequence set forth in SEQ ID NO: 56. For example, in some embodiments, the polynucleotide comprises a gRNA comprising the nucleotide sequence set forth in SEQ ID NO: 56. In some embodiments, the polynucleotide comprises a gRNA consisting of the nucleotide sequence set forth in SEQ ID NO: 56. In some embodiments, the polynucleotide comprises a gRNA consisting essentially of the nucleotide sequence set forth in SEQ ID NO: 56.

[0100] In some embodiments, the polynucleotide useful for the present disclosure comprises a gRNA, and the gRNA comprises, consists of, or consists essentially of the nucleotide sequence set forth in SEQ ID NO: 57. For example, in some embodiments, the polynucleotide comprises a gRNA comprising the nucleotide sequence set forth in SEQ ID NO: 57. In some embodiments, the polynucleotide comprises a gRNA consisting of the nucleotide sequence set forth in SEQ ID NO: 57. In some embodiments, the polynucleotide comprises a gRNA consisting essentially of the nucleotide sequence set forth in SEQ ID NO: 57.

[0101] In some embodiments, the polynucleotide useful for the present disclosure comprises a gRNA, and the gRNA comprises, consists of, or consists essentially of the nucleotide sequence set forth in SEQ ID NO: 58. For example, in some embodiments, the polynucleotide comprises a gRNA comprising the nucleotide sequence set forth in SEQ ID NO: 58. In some embodiments, the polynucleotide comprises a gRNA consisting of the nucleotide sequence set forth in SEQ ID NO: 58. In some embodiments, the polynucleotide comprises a gRNA consisting essentially of the nucleotide sequence set forth in SEQ ID NO: 58.

[0102] In some embodiments, the polynucleotide useful for the present disclosure comprises a gRNA, and the gRNA comprises, consists of, or consists essentially of the nucleotide sequence set forth in SEQ ID NO: 59. For example, in some embodiments, the polynucleotide comprises a gRNA comprising the nucleotide sequence set forth in SEQ ID NO: 59. In some embodiments, the polynucleotide comprises a gRNA consisting of the nucleotide sequence set forth in SEQ ID NO: 59. In some embodiments, the polynucleotide comprises a gRNA consisting essentially of the nucleotide sequence set forth in SEQ ID NO: 59.

[0103] In some embodiments, the polynucleotide useful for the present disclosure comprises a gRNA, and the gRNA comprises, consists of, or consists essentially of the nucleotide sequence set forth in SEQ ID NO: 60. For example, in some embodiments, the polynucleotide comprises a gRNA comprising the nucleotide sequence set forth in SEQ ID NO: 60. In some embodiments, the polynucleotide comprises a gRNA consisting of the nucleotide sequence set forth in SEQ ID NO: 60. In some embodiments, the polynucleotide comprises a gRNA consisting essentially of the nucleotide sequence set forth in SEQ ID NO: 60.

[0104] In some embodiments, the polynucleotide useful for the present disclosure comprises a gRNA, and the gRNA comprises, consists of, or consists essentially of the nucleotide sequence shown in SEQ ID NO: 61. For example, in some embodiments, the polynucleotide comprises a gRNA comprising the nucleotide sequence shown in SEQ ID NO: 61. In some embodiments, the polynucleotide comprises a gRNA consisting of the nucleotide sequence shown in SEQ ID NO: 61. In some embodiments, the polynucleotide comprises a gRNA consisting essentially of the nucleotide sequence shown in SEQ ID NO: 61.

[0105] In some embodiments, the polynucleotide useful for the present disclosure comprises a gRNA, and the gRNA comprises, consists of, or consists essentially of the nucleotide sequence shown in SEQ ID NO: 62. For example, in some embodiments, the polynucleotide comprises a gRNA comprising the nucleotide sequence shown in SEQ ID NO: 62. In some embodiments, the polynucleotide comprises a gRNA consisting of the nucleotide sequence shown in SEQ ID NO: 62. In some embodiments, the polynucleotide comprises a gRNA consisting essentially of the nucleotide sequence shown in SEQ ID NO: 62.

[0106] In some embodiments, the polynucleotide useful for the present disclosure comprises a gRNA, and the gRNA comprises, consists of, or consists essentially of the nucleotide sequence shown in SEQ ID NO: 63. For example, in some embodiments, the polynucleotide comprises a gRNA comprising the nucleotide sequence shown in SEQ ID NO: 63. In some embodiments, the polynucleotide comprises a gRNA consisting of the nucleotide sequence shown in SEQ ID NO: 63. In some embodiments, the polynucleotide comprises a gRNA consisting essentially of the nucleotide sequence shown in SEQ ID NO: 63.

[0107] In some embodiments, the polynucleotide useful for the present disclosure comprises a gRNA, and the gRNA comprises, consists of, or consists essentially of the nucleotide sequence shown in SEQ ID NO: 64. For example, in some embodiments, the polynucleotide comprises a gRNA comprising the nucleotide sequence shown in SEQ ID NO: 64. In some embodiments, the polynucleotide comprises a gRNA consisting of the nucleotide sequence shown in SEQ ID NO: 64. In some embodiments, the polynucleotide comprises a gRNA consisting essentially of the nucleotide sequence shown in SEQ ID NO: 64.

[0108] In some embodiments, the polynucleotide useful for the present disclosure comprises a gRNA, and the gRNA comprises, consists of, or consists essentially of the nucleotide sequence shown in SEQ ID NO: 65. For example, in some embodiments, the polynucleotide comprises a gRNA comprising the nucleotide sequence shown in SEQ ID NO: 65. In some embodiments, the polynucleotide comprises a gRNA consisting of the nucleotide sequence shown in SEQ ID NO: 65. In some embodiments, the polynucleotide comprises a gRNA consisting essentially of the nucleotide sequence shown in SEQ ID NO: 65.

[0109] In some embodiments, the polynucleotide useful for the present disclosure comprises a gRNA, and the gRNA comprises, consists of, or consists essentially of the nucleotide sequence shown in SEQ ID NO: 66. For example, in some embodiments, the polynucleotide comprises a gRNA comprising the nucleotide sequence shown in SEQ ID NO: 66. In some embodiments, the polynucleotide comprises a gRNA consisting of the nucleotide sequence shown in SEQ ID NO: 66. In some embodiments, the polynucleotide comprises a gRNA consisting essentially of the nucleotide sequence shown in SEQ ID NO: 66.

[0110] In some embodiments, the polynucleotide useful for the present disclosure comprises a gRNA, and the gRNA comprises, consists of, or consists essentially of the nucleotide sequence set forth in SEQ ID NO: 67. For example, in some embodiments, the polynucleotide comprises a gRNA comprising the nucleotide sequence set forth in SEQ ID NO: 67. In some embodiments, the polynucleotide comprises a gRNA consisting of the nucleotide sequence set forth in SEQ ID NO: 67. In some embodiments, the polynucleotide comprises a gRNA consisting essentially of the nucleotide sequence set forth in SEQ ID NO: 67.

[0111] In some embodiments, the polynucleotide useful for the present disclosure comprises a gRNA, and the gRNA comprises, consists of, or consists essentially of the nucleotide sequence set forth in SEQ ID NO: 68. For example, in some embodiments, the polynucleotide comprises a gRNA comprising the nucleotide sequence set forth in SEQ ID NO: 68. In some embodiments, the polynucleotide comprises a gRNA consisting of the nucleotide sequence set forth in SEQ ID NO: 68. In some embodiments, the polynucleotide comprises a gRNA consisting essentially of the nucleotide sequence set forth in SEQ ID NO: 68.

[0112] In some embodiments, the polynucleotide useful for the present disclosure comprises a gRNA, and the gRNA comprises, consists of, or consists essentially of the nucleotide sequence set forth in SEQ ID NO: 69. For example, in some embodiments, the polynucleotide comprises a gRNA comprising the nucleotide sequence set forth in SEQ ID NO: 69. In some embodiments, the polynucleotide comprises a gRNA consisting of the nucleotide sequence set forth in SEQ ID NO: 69. In some embodiments, the polynucleotide comprises a gRNA consisting essentially of the nucleotide sequence set forth in SEQ ID NO: 69.

[0113] In some embodiments, the polynucleotide useful for the present disclosure comprises a gRNA, and the gRNA comprises, consists of, or consists essentially of the nucleotide sequence shown in SEQ ID NO: 70. For example, in some embodiments, the polynucleotide comprises a gRNA comprising the nucleotide sequence shown in SEQ ID NO: 70. In some embodiments, the polynucleotide comprises a gRNA consisting of the nucleotide sequence shown in SEQ ID NO: 70. In some embodiments, the polynucleotide comprises a gRNA consisting essentially of the nucleotide sequence shown in SEQ ID NO: 70.

[0114] In some embodiments, the polynucleotide useful for the present disclosure comprises a gRNA, and the gRNA comprises, consists of, or consists essentially of the nucleotide sequence shown in SEQ ID NO: 71. For example, in some embodiments, the polynucleotide comprises a gRNA comprising the nucleotide sequence shown in SEQ ID NO: 71. In some embodiments, the polynucleotide comprises a gRNA consisting of the nucleotide sequence shown in SEQ ID NO: 71. In some embodiments, the polynucleotide comprises a gRNA consisting essentially of the nucleotide sequence shown in SEQ ID NO: 71.

[0115] In some embodiments, the polynucleotide useful for the present disclosure comprises a gRNA, and the gRNA comprises, consists of, or consists essentially of the nucleotide sequence shown in SEQ ID NO: 72. For example, in some embodiments, the polynucleotide comprises a gRNA comprising the nucleotide sequence shown in SEQ ID NO: 72. In some embodiments, the polynucleotide comprises a gRNA consisting of the nucleotide sequence shown in SEQ ID NO: 72. In some embodiments, the polynucleotide comprises a gRNA consisting essentially of the nucleotide sequence shown in SEQ ID NO: 72.

[0116] In some embodiments, the polynucleotide useful for the present disclosure comprises a gRNA, and the gRNA comprises, consists of, or consists essentially of the nucleotide sequence set forth in SEQ ID NO: 73. For example, in some embodiments, the polynucleotide comprises a gRNA comprising the nucleotide sequence set forth in SEQ ID NO: 73. In some embodiments, the polynucleotide comprises a gRNA consisting of the nucleotide sequence set forth in SEQ ID NO: 73. In some embodiments, the polynucleotide comprises a gRNA consisting essentially of the nucleotide sequence set forth in SEQ ID NO: 73.

[0117] In some embodiments, the polynucleotide useful for the present disclosure comprises a gRNA, and the gRNA comprises, consists of, or consists essentially of the nucleotide sequence set forth in SEQ ID NO: 74. For example, in some embodiments, the polynucleotide comprises a gRNA comprising the nucleotide sequence set forth in SEQ ID NO: 74. In some embodiments, the polynucleotide comprises a gRNA consisting of the nucleotide sequence set forth in SEQ ID NO: 74. In some embodiments, the polynucleotide comprises a gRNA consisting essentially of the nucleotide sequence set forth in SEQ ID NO: 74.

[0118] In some embodiments, the polynucleotide useful for the present disclosure comprises a gRNA, and the gRNA comprises, consists of, or consists essentially of the nucleotide sequence set forth in SEQ ID NO: 75. For example, in some embodiments, the polynucleotide comprises a gRNA comprising the nucleotide sequence set forth in SEQ ID NO: 75. In some embodiments, the polynucleotide comprises a gRNA consisting of the nucleotide sequence set forth in SEQ ID NO: 75. In some embodiments, the polynucleotide comprises a gRNA consisting essentially of the nucleotide sequence set forth in SEQ ID NO: 75.

[0119] In some embodiments, the polynucleotide useful for the present disclosure comprises a gRNA, and the gRNA comprises, consists of, or consists essentially of the nucleotide sequence set forth in SEQ ID NO: 76. For example, in some embodiments, the polynucleotide comprises a gRNA comprising the nucleotide sequence set forth in SEQ ID NO: 76. In some embodiments, the polynucleotide comprises a gRNA consisting of the nucleotide sequence set forth in SEQ ID NO: 76. In some embodiments, the polynucleotide comprises a gRNA consisting essentially of the nucleotide sequence set forth in SEQ ID NO: 76.

[0120] In some embodiments, the polynucleotide useful for the present disclosure comprises a gRNA, and the gRNA comprises, consists of, or consists essentially of the nucleotide sequence set forth in SEQ ID NO: 77. For example, in some embodiments, the polynucleotide comprises a gRNA comprising the nucleotide sequence set forth in SEQ ID NO: 77. In some embodiments, the polynucleotide comprises a gRNA consisting of the nucleotide sequence set forth in SEQ ID NO: 77. In some embodiments, the polynucleotide comprises a gRNA consisting essentially of the nucleotide sequence set forth in SEQ ID NO: 77.

[0121] In some embodiments, the polynucleotide useful for the present disclosure comprises a gRNA, and the gRNA comprises, consists of, or consists essentially of the nucleotide sequence set forth in SEQ ID NO: 78. For example, in some embodiments, the polynucleotide comprises a gRNA comprising the nucleotide sequence set forth in SEQ ID NO: 78. In some embodiments, the polynucleotide comprises a gRNA consisting of the nucleotide sequence set forth in SEQ ID NO: 78. In some embodiments, the polynucleotide comprises a gRNA consisting essentially of the nucleotide sequence set forth in SEQ ID NO: 78.

[0122] In some embodiments, the polynucleotide useful for the present disclosure comprises a gRNA, and the gRNA comprises, consists of, or consists essentially of the nucleotide sequence shown in SEQ ID NO: 79. For example, in some embodiments, the polynucleotide comprises a gRNA comprising the nucleotide sequence shown in SEQ ID NO: 79. In some embodiments, the polynucleotide comprises a gRNA consisting of the nucleotide sequence shown in SEQ ID NO: 79. In some embodiments, the polynucleotide comprises a gRNA consisting essentially of the nucleotide sequence shown in SEQ ID NO: 79.

[0123] In some embodiments, the polynucleotide useful for the present disclosure comprises a gRNA, and the gRNA comprises, consists of, or consists essentially of the nucleotide sequence shown in SEQ ID NO: 80. For example, in some embodiments, the polynucleotide comprises a gRNA comprising the nucleotide sequence shown in SEQ ID NO: 80. In some embodiments, the polynucleotide comprises a gRNA consisting of the nucleotide sequence shown in SEQ ID NO: 80. In some embodiments, the polynucleotide comprises a gRNA consisting essentially of the nucleotide sequence shown in SEQ ID NO: 80.

[0124] In some embodiments, the polynucleotide useful for the present disclosure comprises a gRNA, and the gRNA comprises, consists of, or consists essentially of the nucleotide sequence shown in SEQ ID NO: 81. For example, in some embodiments, the polynucleotide comprises a gRNA comprising the nucleotide sequence shown in SEQ ID NO: 81. In some embodiments, the polynucleotide comprises a gRNA consisting of the nucleotide sequence shown in SEQ ID NO: 81. In some embodiments, the polynucleotide comprises a gRNA consisting essentially of the nucleotide sequence shown in SEQ ID NO: 81.

[0125] In some embodiments, the polynucleotide useful for the present disclosure comprises a gRNA, and the gRNA comprises, consists of, or consists essentially of the nucleotide sequence shown in SEQ ID NO: 82. For example, in some embodiments, the polynucleotide comprises a gRNA comprising the nucleotide sequence shown in SEQ ID NO: 82. In some embodiments, the polynucleotide comprises a gRNA consisting of the nucleotide sequence shown in SEQ ID NO: 82. In some embodiments, the polynucleotide comprises a gRNA consisting essentially of the nucleotide sequence shown in SEQ ID NO: 82.

[0126] In some embodiments, the polynucleotide useful for the present disclosure comprises a gRNA, and the gRNA comprises, consists of, or consists essentially of the nucleotide sequence shown in SEQ ID NO: 83. For example, in some embodiments, the polynucleotide comprises a gRNA comprising the nucleotide sequence shown in SEQ ID NO: 83. In some embodiments, the polynucleotide comprises a gRNA consisting of the nucleotide sequence shown in SEQ ID NO: 83. In some embodiments, the polynucleotide comprises a gRNA consisting essentially of the nucleotide sequence shown in SEQ ID NO: 83.

[0127] In some embodiments, the polynucleotide useful for the present disclosure comprises a gRNA, and the gRNA comprises, consists of, or consists essentially of the nucleotide sequence shown in SEQ ID NO: 84. For example, in some embodiments, the polynucleotide comprises a gRNA comprising the nucleotide sequence shown in SEQ ID NO: 84. In some embodiments, the polynucleotide comprises a gRNA consisting of the nucleotide sequence shown in SEQ ID NO: 84. In some embodiments, the polynucleotide comprises a gRNA consisting essentially of the nucleotide sequence shown in SEQ ID NO: 84.

[0128] In some embodiments, the polynucleotide useful for the present disclosure comprises a gRNA, and the gRNA comprises, consists of, or consists essentially of the nucleotide sequence set forth in SEQ ID NO: 85. For example, in some embodiments, the polynucleotide comprises a gRNA comprising the nucleotide sequence set forth in SEQ ID NO: 85. In some embodiments, the polynucleotide comprises a gRNA consisting of the nucleotide sequence set forth in SEQ ID NO: 85. In some embodiments, the polynucleotide comprises a gRNA consisting essentially of the nucleotide sequence set forth in SEQ ID NO: 85.

[0129] In some embodiments, the polynucleotide useful for the present disclosure comprises a gRNA, and the gRNA comprises, consists of, or consists essentially of the nucleotide sequence set forth in SEQ ID NO: 86. For example, in some embodiments, the polynucleotide comprises a gRNA comprising the nucleotide sequence set forth in SEQ ID NO: 86. In some embodiments, the polynucleotide comprises a gRNA consisting of the nucleotide sequence set forth in SEQ ID NO: 86. In some embodiments, the polynucleotide comprises a gRNA consisting essentially of the nucleotide sequence set forth in SEQ ID NO: 86.

[0130] In some embodiments, the polynucleotide useful for the present disclosure comprises a gRNA, and the gRNA comprises, consists of, or consists essentially of the nucleotide sequence set forth in SEQ ID NO: 87. For example, in some embodiments, the polynucleotide comprises a gRNA comprising the nucleotide sequence set forth in SEQ ID NO: 87. In some embodiments, the polynucleotide comprises a gRNA consisting of the nucleotide sequence set forth in SEQ ID NO: 87. In some embodiments, the polynucleotide comprises a gRNA consisting essentially of the nucleotide sequence set forth in SEQ ID NO: 87.

[0131] In some embodiments, the polynucleotide useful for the present disclosure comprises a gRNA, and the gRNA comprises, consists of, or consists essentially of the nucleotide sequence set forth in SEQ ID NO: 88. For example, in some embodiments, the polynucleotide comprises a gRNA comprising the nucleotide sequence set forth in SEQ ID NO: 88. In some embodiments, the polynucleotide comprises a gRNA consisting of the nucleotide sequence set forth in SEQ ID NO: 88. In some embodiments, the polynucleotide comprises a gRNA consisting essentially of the nucleotide sequence set forth in SEQ ID NO: 88.

[0132] In some embodiments, the polynucleotide useful for the present disclosure comprises a gRNA, and the gRNA comprises, consists of, or consists essentially of the nucleotide sequence set forth in SEQ ID NO: 89. For example, in some embodiments, the polynucleotide comprises a gRNA comprising the nucleotide sequence set forth in SEQ ID NO: 89. In some embodiments, the polynucleotide comprises a gRNA consisting of the nucleotide sequence set forth in SEQ ID NO: 89. In some embodiments, the polynucleotide comprises a gRNA consisting essentially of the nucleotide sequence set forth in SEQ ID NO: 89.

[0133] In some embodiments, the polynucleotide useful for the present disclosure comprises a gRNA, and the gRNA comprises, consists of, or consists essentially of the nucleotide sequence set forth in SEQ ID NO: 90. For example, in some embodiments, the polynucleotide comprises a gRNA comprising the nucleotide sequence set forth in SEQ ID NO: 90. In some embodiments, the polynucleotide comprises a gRNA consisting of the nucleotide sequence set forth in SEQ ID NO: 90. In some embodiments, the polynucleotide comprises a gRNA consisting essentially of the nucleotide sequence set forth in SEQ ID NO: 90.

[0134] In some embodiments, the polynucleotide useful for the present disclosure comprises a gRNA, and the gRNA comprises, consists of, or consists essentially of the nucleotide sequence set forth in SEQ ID NO: 91. For example, in some embodiments, the polynucleotide comprises a gRNA comprising the nucleotide sequence set forth in SEQ ID NO: 91. In some embodiments, the polynucleotide comprises a gRNA consisting of the nucleotide sequence set forth in SEQ ID NO: 91. In some embodiments, the polynucleotide comprises a gRNA consisting essentially of the nucleotide sequence set forth in SEQ ID NO: 91.

[0135] In some embodiments, the polynucleotide useful for the present disclosure comprises a gRNA, and the gRNA comprises, consists of, or consists essentially of the nucleotide sequence set forth in SEQ ID NO: 92. For example, in some embodiments, the polynucleotide comprises a gRNA comprising the nucleotide sequence set forth in SEQ ID NO: 92. In some embodiments, the polynucleotide comprises a gRNA consisting of the nucleotide sequence set forth in SEQ ID NO: 92. In some embodiments, the polynucleotide comprises a gRNA consisting essentially of the nucleotide sequence set forth in SEQ ID NO: 92.

[0136] In some embodiments, the polynucleotide useful for the present disclosure comprises a gRNA, and the gRNA comprises, consists of, or consists essentially of the nucleotide sequence set forth in SEQ ID NO: 93. For example, in some embodiments, the polynucleotide comprises a gRNA comprising the nucleotide sequence set forth in SEQ ID NO: 93. In some embodiments, the polynucleotide comprises a gRNA consisting of the nucleotide sequence set forth in SEQ ID NO: 93. In some embodiments, the polynucleotide comprises a gRNA consisting essentially of the nucleotide sequence set forth in SEQ ID NO: 93.

[0137] In some embodiments, the polynucleotide useful for the present disclosure comprises a gRNA, and the gRNA comprises, consists of, or consists essentially of the nucleotide sequence set forth in SEQ ID NO: 94. For example, in some embodiments, the polynucleotide comprises a gRNA comprising the nucleotide sequence set forth in SEQ ID NO: 94. In some embodiments, the polynucleotide comprises a gRNA consisting of the nucleotide sequence set forth in SEQ ID NO: 94. In some embodiments, the polynucleotide comprises a gRNA consisting essentially of the nucleotide sequence set forth in SEQ ID NO: 94.

[0138] In some embodiments, the polynucleotide useful for the present disclosure comprises a gRNA, and the gRNA comprises, consists of, or consists essentially of the nucleotide sequence set forth in SEQ ID NO: 95. For example, in some embodiments, the polynucleotide comprises a gRNA comprising the nucleotide sequence set forth in SEQ ID NO: 95. In some embodiments, the polynucleotide comprises a gRNA consisting of the nucleotide sequence set forth in SEQ ID NO: 95. In some embodiments, the polynucleotide comprises a gRNA consisting essentially of the nucleotide sequence set forth in SEQ ID NO: 95.

[0139] In some embodiments, the polynucleotide useful for the present disclosure comprises a gRNA, and the gRNA comprises, consists of, or consists essentially of the nucleotide sequence set forth in SEQ ID NO: 96. For example, in some embodiments, the polynucleotide comprises a gRNA comprising the nucleotide sequence set forth in SEQ ID NO: 96. In some embodiments, the polynucleotide comprises a gRNA consisting of the nucleotide sequence set forth in SEQ ID NO: 96. In some embodiments, the polynucleotide comprises a gRNA consisting essentially of the nucleotide sequence set forth in SEQ ID NO: 96.

[0140] In some embodiments, the polynucleotide useful for the present disclosure comprises a gRNA, and the gRNA comprises, consists of, or consists essentially of the nucleotide sequence set forth in SEQ ID NO: 97. For example, in some embodiments, the polynucleotide comprises a gRNA comprising the nucleotide sequence set forth in SEQ ID NO: 97. In some embodiments, the polynucleotide comprises a gRNA consisting of the nucleotide sequence set forth in SEQ ID NO: 97. In some embodiments, the polynucleotide comprises a gRNA consisting essentially of the nucleotide sequence set forth in SEQ ID NO: 97.

[0141] In some embodiments, the polynucleotide useful for the present disclosure comprises a gRNA, and the gRNA comprises, consists of, or consists essentially of the nucleotide sequence set forth in SEQ ID NO: 98. For example, in some embodiments, the polynucleotide comprises a gRNA comprising the nucleotide sequence set forth in SEQ ID NO: 98. In some embodiments, the polynucleotide comprises a gRNA consisting of the nucleotide sequence set forth in SEQ ID NO: 98. In some embodiments, the polynucleotide comprises a gRNA consisting essentially of the nucleotide sequence set forth in SEQ ID NO: 98.

[0142] In some embodiments, the polynucleotide useful for the present disclosure comprises a gRNA, and the gRNA comprises, consists of, or consists essentially of the nucleotide sequence set forth in SEQ ID NO: 99. For example, in some embodiments, the polynucleotide comprises a gRNA comprising the nucleotide sequence set forth in SEQ ID NO: 99. In some embodiments, the polynucleotide comprises a gRNA consisting of the nucleotide sequence set forth in SEQ ID NO: 99. In some embodiments, the polynucleotide comprises a gRNA consisting essentially of the nucleotide sequence set forth in SEQ ID NO: 99.

[0143] II.B. Target As described herein, the polynucleotides of the present disclosure are capable of specifically targeting (i.e., specifically binding to) nucleic acid sequences within the NR4A3 gene. Commonly abbreviated as NR4A3, MINOR, CSMF, NOR1, CHN, mitogen-inducible nuclear orphan receptor, neuron-derived orphan receptor, nuclear hormone receptor NOR-1, "Chondrosarcoma, Extraskeletal Myxoid, Fused to EWS", and TEC, is a protein encoded by the NR4A3 gene in humans. The NR4A family of orphan nuclear receptors includes NR4A1 (Nur77), NR4A2 (Nurr1), and NR4A3 (Nor-1). They act ligand-independently as transcription factors. Their functions are mainly controlled by the rapid and transient induction of their expression by various extracellular signals and are thus considered immediate-early genes. NR4A is involved in various cellular functions including apoptosis, survival, proliferation, angiogenesis, inflammation, DNA repair, and fatty acid metabolism.

[0144] The sequence for the human NR4A3 gene is on chromosome 9 (base 99,821,885 - 99,866,893; 45,039 bases; plus 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).

[0145] The NR4A3 protein has three isoforms generated 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 levels of any known NR4A3 protein (including any isoform and variant thereof).

Table 1-1

Table 1-2

[0146] III. Modified Immune Cells In some embodiments, the disclosure provides immune cells modified with the polynucleotides described herein (i.e., comprising gRNAs that specifically target the NR4A3 gene). Thus, the modified immune cells described herein have reduced levels of the NR4A3 gene and / or the NR4A3 protein when compared to corresponding immune cells (the "reference cells") that are not modified as described herein (e.g., having reduced levels of the NR4A3 gene and / or the NR4A3 protein). In some embodiments, the reference cells include immune cells prior to modification with the polynucleotides described herein. In some embodiments, the reference cells include corresponding immune cells that were not modified with the polynucleotides described herein. In some embodiments, the reference cells have endogenous levels of the NR4A3 gene and / or the NR4A3 protein.

[0147] As used herein, the terms "reduced level", "lower level", "reduced expression level", or "lower expression level" (or the like) refer to both a reduction in the physical level (e.g., less gene sequence from the genome, or less protein due to decreased protein expression) and a reduction in function. For example, a reduction in the level of the NR4A3 gene may refer to a decrease in gene function due to, for example, the introduction of a mutation that introduces a stop codon or frameshift, an epigenetic modification that would alter transcription, or a mutation or other change in a promoter gene or another gene that controls NR4A3 expression. In some embodiments, a reduction in the level of the NR4A3 gene in a modified cell refers to a decrease in the amount (e.g., concentration) of genomic DNA, pre-mRNA, and / or mRNA capable of encoding a functional NR4A3 protein, e.g., a wild-type NR4A3 protein, as compared to a reference cell. Similarly, a reduction in the NR4A3 protein may refer to a change (e.g., a mutation or post-translational modification) that causes a (partial or complete) loss of function, including but not limited to a change that results in the expression of a functional NR4A3 protein, e.g., a wild-type NR4A3 protein, or the activity of a molecule that binds to a functional site of NR4A3 that modifies its interaction with other cell signaling partners.

[0148] The NR4A3 gene level (e.g., the presence / absence of the whole gene or a part thereof, or gene function) can be measured by various methods known in the art. The NR4A3 protein level (e.g., the presence / absence of the NR4A3 protein or a fragment thereof, or quantification or protein function) can be measured by various methods known in the art.

[0149] In some embodiments, 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% when compared to reference cells. In some embodiments, the level of the NR4A3 gene and / or NR4A3 protein is completely inhibited.

[0150] In some embodiments, the modified immune cells described herein have a reduced level of the NR4A3 gene when compared to reference cells. In some embodiments, 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% when compared to reference cells. In some embodiments, the level of the NR4A3 gene is completely inhibited.

[0151] In some embodiments, the modified immune cells described herein have a reduced level of NR4A3 protein when compared to a reference cell. In some embodiments, 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% when compared to a reference cell. In some embodiments, the level of NR4A3 protein in the modified immune cells is completely inhibited.

[0152] In some embodiments, the modified immune cells described have both a reduced level of the NR4A3 gene and a reduced level of the NR4A3 protein when compared to a reference cell. In some embodiments, both the level of the NR4A3 gene and the level of the NR4A3 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% when compared to a reference cell. In some embodiments, both the levels of the NR4A3 gene and the NR4A3 protein are completely inhibited.

[0153] As is apparent from the present disclosure, any cell capable of naturally expressing the NR4A3 gene and / or the NR4A3 protein can be modified using the polynucleotides of the present disclosure. As described herein, in some embodiments, cells useful for the present disclosure include immune cells. In some embodiments, immune cells include lymphocytes, neutrophils, monocytes, macrophages, dendritic cells, natural killer cells, or combinations thereof. In some embodiments, immune cells that can be modified to have reduced levels of the NR4A3 gene and / or the NR4A3 protein include lymphocytes. In some embodiments, the lymphocytes are T cells (e.g., CD4+ T cells, CD8+ T cells, or both). As used herein, "modified immune cells" includes progeny cells of the initially modified immune cells, and the progeny cells also express reduced levels of the NR4A3 gene and / or the NR4A3 protein.

[0154] As described herein, the modified immune cells of the present disclosure (e.g., having reduced levels of the NR4A3 gene and / or the NR4A3 protein) exhibit one or more improved properties when compared to corresponding cells that have not been modified to have reduced levels of the NR4A3 gene and / or the NR4A3 protein ("reference cells"). Non-limiting examples of such properties include resistance to exhaustion (e.g., 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 combinations thereof.

[0155] Assays useful 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), INCUCYTE® immune cell killing assay, Meso Scale Discovery (MSD) or similar assays, persistent 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 blot and other standard molecular and cell biological methods, such as those described herein or, for example, in Current Protocols in Molecular Biology or Current Protocols in Immunology (John Wiley & Sons, Inc., 1999-2021) or as described elsewhere.

[0156] In some embodiments, the modified cells described herein (i.e., having reduced levels of the NR4A3 gene and / or NR4A3 protein) exhibit increased resistance to exhaustion, for example, after persistent antigen stimulation, compared to corresponding cells that have not been modified to have reduced levels of the NR4A3 gene and / or NR4A3 protein ("reference cells"). In some embodiments, in response to persistent antigen stimulation, the modified cells provided herein express (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.

[0157] In some embodiments, the resistance to fatigue 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 as compared to reference cells.

[0158] In some embodiments, the modified cells described herein (i.e., having reduced levels of the NR4A3 gene and / or NR4A3 protein) exhibit reduced fatigue as compared to reference cells (i.e., corresponding cells having endogenous levels of the NR4A3 gene and / or NR4A3 protein). In some embodiments, the fatigue 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% as compared to reference cells.

[0159] In some embodiments, the modified cells described herein (i.e., having reduced levels of the NR4A3 gene and / or NR4A3 protein) exhibit increased persistence / survival when compared to reference cells (i.e., corresponding cells having endogenous levels of the NR4A3 gene and / or NR4A3 protein), for example, when administered to a subject. In some embodiments, the persistence / survival of the modified cells is 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 increased compared to the reference cells.

[0160] In some embodiments, the modified cells described herein (i.e., having reduced levels of the NR4A3 gene and / or NR4A3 protein) exhibit increased growth / proliferation when compared to reference cells (i.e., corresponding cells having endogenous levels of the NR4A3 gene and / or NR4A3 protein), for example, by persistent antigen stimulation. In some embodiments, the growth / proliferation of the modified cells is 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 increased when compared to reference cells.

[0161] In some embodiments, the modified cells described herein (i.e., having reduced levels of the NR4A3 gene and / or NR4A3 protein) exhibit increased effector function, for example, in response to persistent antigen stimulation, when compared to reference cells (i.e., corresponding cells having endogenous levels of the NR4A3 gene and / or NR4A3 protein). Non-limiting examples of such effector functions include cytokine production (e.g., IFN-γ, TNF-α, IL-2, or combinations thereof), granzyme release, cytotoxicity in response to persistent antigen stimulation, the ability to kill / lyse antigen-expressing cells, and combinations thereof. In some embodiments, the effector function of the modified cells provided herein is 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 increased when compared to reference cells.

[0162] In some embodiments, 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 exhaustion. Non-limiting examples of such markers include TIGIT, PD-1, CD39, and combinations thereof. The expression of such markers can be measured in bulk populations by flow cytometry using bulk RNAseq transcriptome analysis or, in some embodiments, single cell transcriptome analysis can be performed using single cell RNAseq. In some embodiments, the expression of one or more markers associated with exhaustion is 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% reduced in the modified cells of the present disclosure when compared to reference cells.

[0163] Without being bound by any one theory, in some embodiments, one or more of the improved properties described above are related to increased resistance of the modified cells to apoptosis, increased resistance of the modified cells to immune checkpoint control, increased activation in response to antigen stimulation, or combinations thereof.

[0164] As further described and demonstrated herein, in some embodiments, 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 the tumor microenvironment (TME) when compared to reference cells (i.e., corresponding cells having endogenous levels of the NR4A3 gene and / or NR4A3 protein).

[0165] III.A. Other Modifications 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) may contain one or more additional modifications. In some embodiments, one or more additional modifications may further improve one or more properties of the cells. Non-limiting examples of such additional modifications are further described below.

[0166] III.A.1.NR4A2 In addition to reduced levels of the NR4A3 gene and / or NR4A3 protein, in some embodiments, the modified cells described herein may be further modified to have reduced levels of the NR4A2 gene and / or NR4A2 protein. Any suitable method known in the art may be used to reduce the levels of the NR4A2 gene and / or NR4A2 protein in the modified cells described herein. For example, in some embodiments, the levels of the NR4A2 gene and / or NR4A2 protein may be reduced using any of the gene editing tools described herein (e.g., the CRISPR / Cas system).

[0167] Nuclear receptor subfamily 4 group A member 2, commonly abbreviated as NR4A2 and 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 (base pairs 156,324,432 - 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 a variant thereof).

[0168] The NR4A2 protein has two isoforms generated by alternative splicing. The sequences are shown in Table 2 below. Unless otherwise indicated and as further described herein, in some embodiments, the immune cells described herein are further modified to have reduced levels of any known NR4A2 protein (including any isoforms and variants thereof). Suitable methods for reducing the level of the NR4A2 gene and / or the NR4A2 protein are described elsewhere in this disclosure and are also known in the art.

Table 2

[0169] Thus, in some embodiments, the modified cells described herein have (i) reduced levels of the NR4A3 gene and / or the NR4A3 protein and (ii) reduced levels of the NR4A2 gene and / or the NR4A2 protein when compared to a reference cell. In some embodiments, the level of the NR4A2 gene and / or the NR4A2 protein in the modified immune cells described herein (i.e., having reduced levels of the NR4A3 gene and / or the NR4A3 protein) is 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% reduced when compared to a reference cell. In some embodiments, the level of the NR4A2 gene and / or the NR4A2 protein is completely inhibited.

[0170] In some embodiments, the modified immune cells described herein (i.e., having reduced levels of the NR4A3 gene and / or NR4A3 protein) have reduced levels of the NR4A2 gene when compared to reference cells. In some embodiments, the level of the 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% when compared to reference cells. In some embodiments, the level of the NR4A2 gene is completely inhibited.

[0171] In some embodiments, the modified immune cells described herein (i.e., having reduced levels of the NR4A3 gene and / or NR4A3 protein) have reduced levels of the NR4A2 protein when compared to reference cells. In some embodiments, the level of the 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% when compared to reference cells. In some embodiments, the level of the NR4A2 protein in the modified immune cells is completely inhibited.

[0172] In some embodiments, the modified immune cells described herein (i.e., having reduced levels of the NR4A3 gene and / or NR4A3 protein) have reduced levels of the NR4A2 gene and reduced levels of the NR4A2 protein when compared to reference cells. In some embodiments, both the level of the NR4A2 gene and the level of the 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% when compared to reference cells. In some embodiments, the levels of both the NR4A2 gene and the NR4A2 protein are completely inhibited.

[0173] III.A.2.NR4A1 In addition to reduced levels of the NR4A3 gene and / or NR4A3 protein, in some embodiments, the modified cells described herein can be further modified to have reduced levels of the NR4A1 gene and / or NR4A1 protein. Any suitable method known in the art can be used to reduce the levels of the NR4A1 gene and / or NR4A1 protein in the modified cells described herein. For example, in some embodiments, the levels of the NR4A1 gene and / or NR4A1 protein can be reduced using any of the gene editing tools described herein (e.g., the CRISPR / Cas system).

[0174] Nuclear receptor subfamily 4 group A member 1, commonly abbreviated as NR4A1 and also known as HMR, N10, TR3, NP10, GFRP1, NAK-1, NGFIB, and NUR77, is a protein encoded by the NR4A1 gene in humans. The NR4A1 gene is located on chromosome 12 (base pairs 52022832 - 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 variants).

[0175] The NR4A1 protein has three isoforms generated by alternative splicing. The sequences are shown in Table 3 below.

Table 3-1

Table 3-2

[0176] Thus, in some embodiments, 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 when compared to a reference cell. In some embodiments, 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 embodiments, the level of the NR4A1 gene and / or NR4A1 protein in the modified immune cells described herein (i.e., having reduced levels 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% when compared to a reference cell. In some embodiments, the level of the NR4A1 gene and / or NR4A1 protein is completely inhibited.

[0177] In some embodiments, the modified immune cells described herein (i.e., having reduced levels of the NR4A3 gene and / or NR4A3 protein) have reduced levels of the NR4A1 gene when compared to reference cells. In some embodiments, the level of the 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% when compared to reference cells. In some embodiments, the level of the NR4A1 gene is completely inhibited.

[0178] In some embodiments, the modified immune cells described herein (i.e., having reduced levels of the NR4A3 gene and / or NR4A3 protein) have reduced levels of the NR4A1 protein when compared to reference cells. In some embodiments, the level of the 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% when compared to reference cells. In some embodiments, the level of the NR4A1 protein in the modified immune cells is completely inhibited.

[0179] In some embodiments, the modified immune cells described herein (i.e., having reduced levels of the NR4A3 gene and / or the NR4A3 protein) have reduced levels of the NR4A1 gene and reduced levels of the NR4A1 protein when compared to reference cells. In some embodiments, both the level of the NR4A1 gene and the level of the NR4A1 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% when compared to reference cells. In some embodiments, the levels of both the NR4A1 gene and the NR4A1 protein are completely inhibited.

[0180] III.A.3.c-Jun In some embodiments, the modified cells described herein (i.e., having reduced levels of the NR4A3 gene and / or NR4A3 protein) are further modified to have increased levels of c-Jun protein when compared to reference cells that are not modified to have increased levels 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 embodiments, the modified immune cells described herein are modified to contain an additional nucleotide sequence encoding c-Jun protein such that the level of c-Jun protein is increased as compared to reference cells. In some embodiments, the additional nucleotide sequence encoding c-Jun protein can be part of the same polynucleotide described herein (i.e., containing a gRNA that specifically targets a region within the NR4A3 gene)-i.e., a part of a polycistronic polynucleotide. In some embodiments, the additional nucleotide sequence encoding c-Jun protein can be introduced into the immune cells as a separate polynucleotide.

[0181] In some embodiments, the modified immune cells provided herein (i.e., having reduced levels of the NR4A3 gene and / or NR4A3 protein) are capable of naturally expressing c-Jun protein (e.g., without modifying the cells with an exogenous nucleotide sequence encoding c-Jun protein). In some embodiments, such immune cells can be further modified with a transcriptional activator (e.g., a CRISPR / Cas system-based transcriptional activator, e.g., CRISPRa) such that the expression of endogenous c-Jun protein is increased as compared to reference cells (e.g., corresponding cells not modified with a transcriptional activator).

[0182] As used herein, the term "transcription activator" refers to a protein that increases the transcription of a gene or set of genes (e.g., by binding to an enhancer or promoter-proximal element of a nucleic acid sequence and thereby inducing its transcription). Non-limiting examples of such transcription activators that may be used in the present disclosure include transcription activator-like effector (TALE)-based transcription activators, zinc finger protein (ZFP)-based transcription activators, clustered regularly interspaced short palindromic repeats (CRISPR) / CRISPR-associated protein (Cas) system-based transcription activators, or combinations thereof. See, for example, Kabadi et al., Methods 69(2):188-197 (Sep. 2014), which is incorporated herein by reference in its entirety.

[0183] In some embodiments, the modified cells described herein (i.e., having reduced levels of the NR4A3 gene and / or NR4A3 protein) are modified with a CRISPR / Cas system-based transcriptional activator, such as CRISPR activation (CRISPRa). See, for example, Bissim et al., Molecular Cell 54:1-13 (May 2014), which is incorporated herein by reference in its entirety. CRISPRa is a type of CRISPR tool that involves 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 in the present disclosure are known in the art. See, for example, Pandelakis et al., Cell Systems 10(1):1-14 (Jan. 2020), which is incorporated herein by reference in its entirety. In some embodiments, the modified Cas protein includes a modified Cas9 protein (also referred to in the art as "dCas9"). In some embodiments, the modified Cas protein includes a modified Cas12a protein. In some embodiments, the modified Cas protein useful for the present disclosure is bound to a guide polynucleotide (e.g., a short guide RNA) ("modified Cas-guide complex"), and the guide polynucleotide includes a recognition sequence that is complementary to a region of the nucleic acid sequence encoding the target protein (e.g., c-Jun). In some embodiments, the guide polynucleotide includes a recognition sequence that is complementary to the promoter region of an endogenous nucleic acid sequence encoding the target protein. In some embodiments, one or more transcriptional activators are bound to the modified Cas-guide complex (e.g., the N and / or C termini of the modified Cas protein), such that when the modified Cas-guide complex is introduced into a cell, the one or more transcriptional activators can bind to regulatory elements of the nucleic acid sequence (e.g., the promoter region), thereby inducing and / or increasing the expression of the encoded protein (e.g., c-Jun).In some embodiments, one or more transcriptional activators can bind to regulatory elements (e.g., promoter regions) of endogenous genes, thereby inducing and / or increasing the expression of the encoded protein (e.g., c-Jun). Non-limiting, exemplary examples of common, general activators that can be used include the omega subunit of RNAP, VP16, VP64, and p65. See, for example, Kabadi and Gersbach, Methods 69:188-197 (2014), which is incorporated herein by reference in its entirety.

[0184] In some embodiments, one or more transcriptional repressors (e.g., Kruppel-associated box domain (KRAB)) can be bound to a modified Cas-guide complex (e.g., the N and / or C terminus of a modified Cas protein), such that when introduced into a cell, the one or more transcriptional repressors can inhibit the expression of a gene, e.g., transcription of something that can interfere with the expression of c-Jun (e.g., Bach2), etc., or reduce it. See, for example, US20200030379A1 and Yang et al., J Transl Med 19:459 (2021), each of which is incorporated herein by reference in its entirety. In some embodiments, modified Cas proteins useful for the present disclosure can be bound to both one or more transcriptional activators and one or more transcriptional repressors.

[0185] In some embodiments, due to the modifications described above (e.g., the introduction of an exogenously introduced c-Jun nucleotide sequence and / or a transcriptional activator), the modified cells described herein (i.e., having a reduced level of the NR4A3 gene and / or NR4A3 protein) express the c-Jun protein at a higher level (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% or more, or at least about 1.5, at least about 2, at least about 3, at least about 4, at least about 5, or at least about 10-fold higher) than reference cells that are not modified to overexpress the c-Jun protein, i.e., have an increased level of the c-Jun protein. The terms "express at an increased level [or amount]", "overexpress", or "have increased expression of" (and similar forms of the phrases used herein) are used interchangeably.

[0186] c-Jun is an oncogenic transcription factor belonging to the activator protein-1 (AP-1) family. It interacts with various proteins (e.g., c-Fos) to form dimeric complexes that regulate a diverse range of cell signaling pathways including cell proliferation and tumor progression. Thus, increased c-Jun expression has been observed in certain cancers and has been of great interest in developing c-Jun antagonists for treating such cancers. See, for example, Brennan, A., et al., J Exp Clin Cancer Res 39(1):184 (Sep. 2020).

[0187] In humans, the c-Jun protein is encoded by the JUN gene located on chromosome 1 (nucleotides 58,780,791 to 58,784,047, minus strand direction of GenBank accession number NC_000001.11). Synonyms for the JUN gene and the protein it encodes are known and include "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 in length. The amino acid and nucleic acid sequences of wild-type human c-Jun are provided in Tables 4 and 5, respectively. [Table 4] [Table 5-1] [Table 5-2]

[0188] Unless otherwise indicated, c-Jun proteins useful for the present disclosure include both wild-type human c-Jun protein and any variants or mutants thereof. In some embodiments, 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 variant to rescue dysfunctional (exhausted) T cells. In some embodiments, the mutant c-Jun protein has 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., the C-terminal 50, 75, 100, 150, 200, or 250 or more residues), C-terminal portion (e.g., one-fourth, one-third, or one-half), or C-terminal domain (e.g., epsilon, bZIP, and its C-terminal amino acids) of the wild-type c-Jun protein. In some embodiments, the N-terminal amino acid residues (e.g., the N-terminal 50, 75, 100, or 150 or more), N-terminal portion (e.g., one-fourth, one-third, or one-half), or N-terminal domain (e.g., delta, transactivation domain, and its N-terminal amino acids) of the wild-type c-Jun protein are deleted, mutated, or otherwise inactivated.

[0189] In some embodiments, the c-Jun protein includes activating mutations (e.g., substitutions, deletions, or insertions) in its transactivation domain and / or its delta domain. In some embodiments, the c-Jun protein includes one or both of the S63A and S73A mutations. In some embodiments, the c-Jun protein has a deletion at residues 2-102 or residues 30-50 when compared to wild-type human c-Jun.

[0190] In some embodiments, the c-Jun polypeptide useful for the modified immune cells includes a truncated c-Jun polypeptide as disclosed in WO2019 / 118902, which is hereby expressly incorporated by reference in its entirety.

[0191] As described herein, in some embodiments, the modified cells described herein (i.e., having reduced levels of the NR4A3 gene and / or NR4A3 protein) contain a nucleotide sequence encoding the c-Jun protein, and the nucleotide sequence is codon-optimized. Thus, in some embodiments, the nucleotide sequence encoding the c-Jun protein described herein (also referred to herein as the "c-Jun nucleotide sequence") is different from that of the wild-type c-Jun nucleotide sequence (e.g., SEQ ID NO: 6).

[0192] In some embodiments, 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 with any one of the nucleic acid sequences shown in SEQ ID NOs: 7-16. In some embodiments, the nucleotide sequence encoding the c-Jun protein comprises the nucleic acid sequence shown in any one of SEQ ID NOs: 7-16.

[0193] In some embodiments, 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 with the nucleic acid sequence shown in SEQ ID NO: 7. In some embodiments, 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 with the nucleic acid sequence shown in SEQ ID NO: 7. In some embodiments, the nucleotide sequence comprises the nucleic acid sequence shown in SEQ ID NO: 7.

[0194] In some embodiments, 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 with the nucleic acid sequence shown in SEQ ID NO: 8. In some embodiments, 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 with the nucleic acid sequence shown in SEQ ID NO: 8. In some embodiments, the nucleotide sequence comprises the nucleic acid sequence shown in SEQ ID NO: 8.

[0195] In some embodiments, 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 with the nucleic acid sequence shown in SEQ ID NO: 9. In some embodiments, 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 with the nucleic acid sequence shown in SEQ ID NO: 9. In some embodiments, the nucleotide sequence comprises the nucleic acid sequence shown in SEQ ID NO: 9.

[0196] In some embodiments, 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 with the nucleic acid sequence shown in SEQ ID NO: 10. In some embodiments, the nucleotide sequence has at least 96%, at least 97%, at least 98%, or at least 99% with the nucleic acid sequence shown in SEQ ID NO: 10. In some embodiments, the nucleotide sequence comprises the nucleic acid sequence shown in SEQ ID NO: 10.

[0197] In some embodiments, 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 with the nucleic acid sequence shown in SEQ ID NO: 11. In some embodiments, 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 with the nucleic acid sequence shown in SEQ ID NO: 11. In some embodiments, the nucleotide sequence comprises the nucleic acid sequence shown in SEQ ID NO: 11.

[0198] In some embodiments, 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 with the nucleic acid sequence shown in SEQ ID NO: 12. In some embodiments, 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 with the nucleic acid sequence shown in SEQ ID NO: 12. In some embodiments, the nucleotide sequence comprises the nucleic acid sequence shown in SEQ ID NO: 12.

[0199] In some embodiments, 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 with the nucleic acid sequence shown in SEQ ID NO: 13. In some embodiments, 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 with the nucleic acid sequence shown in SEQ ID NO: 13. In some embodiments, the nucleotide sequence comprises the nucleotide sequence shown in SEQ ID NO: 13.

[0200] In some embodiments, 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 with the nucleic acid sequence shown in SEQ ID NO: 14. In some embodiments, 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 with the nucleic acid sequence shown in SEQ ID NO: 14. In some embodiments, the nucleotide sequence comprises the nucleotide sequence shown in SEQ ID NO: 14.

[0201] In some embodiments, 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 with the nucleic acid sequence shown in SEQ ID NO: 15. In some embodiments, 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 with the nucleic acid sequence shown in SEQ ID NO: 15. In some embodiments, the nucleotide sequence comprises the nucleotide sequence shown in SEQ ID NO: 15.

[0202] In some embodiments, 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 with the nucleic acid sequence shown in SEQ ID NO: 16. In some embodiments, 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 with the nucleic acid sequence shown in SEQ ID NO: 16. In some embodiments, the nucleotide sequence comprises the nucleotide sequence shown in SEQ ID NO: 16.

[0203] In some embodiments, 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 with the nucleic acid sequence shown in SEQ ID NO: 16. In some embodiments, 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 with the nucleic acid sequence shown in SEQ ID NO: 16. In some embodiments, the nucleotide sequence comprises the nucleotide sequence shown in SEQ ID NO: 16.

Table 6-1

Table 6-2

Table 6-3

Table 6-4

[0204] The c-Jun nucleotide sequences disclosed in this specification can be codon-optimized using any method known in the art. For example, in some embodiments, the codons of the c-Jun nucleotide sequences disclosed herein are 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 frequency bias); (ii) guanine-cytosine (GC) nucleotide content; (iii) mRNA secondary structure and destabilizing motifs; (iv) repeat sequences (e.g., direct repeats, inverted repeats, dyad repeats); (v) restriction enzyme recognition sites; or (vi) combinations thereof.

[0205] Without being bound by any one theory, in some embodiments, such codon optimization can increase the expression of the protein encoded by the nucleotide sequence. Thus, in some embodiments, the codon-optimized c-Jun nucleotide sequences of the present disclosure, when transfected into the modified cells described herein (i.e., having reduced levels of the NR4A3 gene and / or NR4A3 protein) as compared to reference cells transfected with the wild-type nucleotide sequence (e.g., SEQ ID NO: 6), transduced, or otherwise introduced, can increase the expression of the encoded c-Jun transcription factor. In some embodiments, the expression of the c-Jun protein is 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 increased as compared to the corresponding expression in reference cells when transfected, transduced, or otherwise genetically modified with the wild-type nucleotide sequence (e.g., SEQ ID NO: 6).

[0206] Thus, in some embodiments, the modified cells described herein have (i) a reduced level of the NR4A3 gene and / or NR4A3 protein, and (ii) an increased level of the c-Jun protein when compared to reference cells that are not modified as described herein. In some embodiments, the modified cells described herein have (i) a reduced level of the NR4A3 gene and / or NR4A3 protein, (ii) a reduced level of the NR4A2 gene and / or NR4A2 protein, and (iii) an increased level of the c-Jun protein when compared to reference cells that are not modified as described herein. In some embodiments, the modified cells described herein have (i) a reduced level of the NR4A3 gene and / or NR4A3 protein, (ii) a reduced level of the NR4A2 gene and / or NR4A2 protein, (iii) a reduced level of the NR4A1 gene and / or NR4A1 protein, and (iv) an increased level of the c-Jun protein when compared to reference cells that are not modified as described herein.

[0207] As is apparent from the present disclosure, in some embodiments, increasing the level of c-Jun protein in the modified cells described herein (i.e., having reduced levels of the NR4A3 gene and / or NR4A3 protein) can further improve and / or enhance one or more properties of the modified cells. For example, in some embodiments where immune cells are modified to have both (1) reduced levels of the NR4A3 gene and / or NR4A3 protein and (2) increased levels of c-Jun protein, one or more properties of the immune cells (e.g., as described below) are improved and / or enhanced when compared to reference cells. In some embodiments, the reference cells include corresponding immune cells modified to have only reduced levels of the NR4A3 gene and / or NR4A3 protein (i.e., not overexpressing c-Jun). In some embodiments, the reference cells include corresponding immune cells modified to have only increased expression of c-Jun protein (i.e., not having reduced levels of the NR4A3 gene and / or NR4A3 protein). In some embodiments, the reference cells include corresponding immune cells not modified to have both (1) reduced levels of the NR4A3 gene and / or NR4A3 protein and (2) increased levels of c-Jun protein. In some embodiments, the reference cells include each of the following: (a) corresponding immune cells modified to have only reduced levels of the NR4A3 gene and / or NR4A3 protein (i.e., not overexpressing c-Jun), (b) corresponding immune cells modified to have only increased expression of c-Jun protein (i.e., not having reduced levels of the NR4A3 gene and / or NR4A3 protein), and (c) corresponding immune cells not modified to have both (1) reduced levels of the NR4A3 gene and / or NR4A3 protein and (2) increased levels of c-Jun protein.

[0208] In some embodiments, the modified immune cells described herein having increased levels of c-Jun protein (i.e., having reduced levels of the NR4A3 gene and / or NR4A3 protein alone or in combination with other members of the NR4A family) exhibit increased resistance to exhaustion when compared to reference cells that do not have increased levels of c-Jun protein. In some embodiments, the resistance to exhaustion is 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 reduced as compared to reference cells (e.g., corresponding cells not modified to have increased c-Jun expression and / or reduced expression of the NR4A gene and / or NR4A protein).

[0209] In some embodiments, overexpression of the c-Jun protein can assist in further reducing exhaustion in the modified immune cells described herein (i.e., having reduced levels of the NR4A3 gene and / or the NR4A3 protein alone or in combination with other members of the NR4A family). In some embodiments, exhaustion is 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 reduced when compared to reference cells (e.g., corresponding cells that were not modified to have increased c-Jun expression and / or reduced expression of the NR4A gene and / or the NR4A protein).

[0210] In some embodiments, increased levels of c-Jun protein may assist in further increasing the persistence / survival of the modified cells described herein (i.e., having reduced levels of the NR4A3 gene and / or NR4A3 protein, alone or in combination with other members of the NR4A family) when administered to a subject, e.g., in vivo. In some embodiments, the persistence / survival of the modified cells is 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 reduced when compared to a reference cell (e.g., a corresponding cell that has not been modified to have increased c-Jun expression and / or reduced expression of the NR4A gene and / or NR4A protein).

[0211] In some embodiments, the increased levels of c-Jun protein may assist in further increasing the growth / proliferation of the modified cells described herein (i.e., having reduced levels of the NR4A3 gene and / or NR4A3 protein, either alone or in combination with other members of the NR4A family). In some embodiments, the growth / proliferation of the modified cells is 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 reduced when compared to reference cells (e.g., corresponding cells that have not been modified to have increased c-Jun expression and / or reduced expression of the NR4A gene and / or NR4A protein).

[0212] In some embodiments, increased levels of c-Jun protein may assist in further increasing the effector function of the modified cells described herein (i.e., having reduced levels of the 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 production (e.g., IFN-γ, TNF-α, IL-2, or combinations thereof), granzyme release, cytotoxicity in response to persistent antigen stimulation, the ability to kill / lyse antigen-expressing cells, and combinations thereof. In some embodiments, the effector function of the modified cells described herein is 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 reduced compared to a reference cell (e.g., a corresponding cell not modified to have increased c-Jun expression and / or reduced expression of the NR4A gene and / or NR4A protein).

[0213] In some embodiments, the modified immune cells described herein further modified to have increased levels of c-Jun protein (e.g., having reduced levels of the 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. Expression of the exhaustion markers can be measured in the bulk population by flow cytometry using bulk RNA Seq transcriptome analysis or in some embodiments, single cell transcriptome analysis can be performed using single cell RNA Seq. In some embodiments, expression of one or more exhaustion markers is 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 reduced when compared to a reference cell (e.g., a corresponding cell not modified to have increased c-Jun expression and / or reduced expression of the NR4A3 gene and / or NR4A3 protein).

[0214] Thus, at least as apparent from the foregoing disclosure, in some embodiments, immune cells engineered to exhibit (1) reduced levels of the NR4A3 gene and / or NR4A3 protein and (2) increased levels of the c-Jun protein exhibit improved in vivo anti-tumor activity when compared to reference cells. In some embodiments, immune cells engineered to exhibit (1) reduced levels of the NR4A3 gene and / or NR4A3 protein and (2) increased levels of the c-Jun protein exhibit higher survival and / or persistence in vivo when compared to reference cells. As further described elsewhere, in some embodiments, reference cells include one or more of the following: (a) corresponding immune cells engineered to have only reduced levels of the NR4A3 gene and / or NR4A3 protein (i.e., not overexpressing c-Jun), (b) corresponding immune cells engineered to have only increased expression of the c-Jun protein (i.e., not having reduced levels of the NR4A3 gene and / or NR4A3 protein), and (c) corresponding immune cells not engineered to have both (1) reduced levels of the NR4A3 gene and / or NR4A3 protein and (2) increased levels of the c-Jun protein.

[0215] III.A.4. Ligand-Binding Protein In some embodiments, the modified cells described herein (e.g., having reduced levels of the NR4A3 gene and / or NR4A3 protein) are further modified to express a ligand-binding protein. 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 embodiments, 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., including an antigen-binding site)) and that is created by the joining of two or more polynucleotide sequences that originally encode different proteins. Unless otherwise indicated, the term may be used interchangeably in this disclosure.

[0216] Any suitable method known in the art can be used to express the ligand-binding protein. For example, in some embodiments, the modified cells described herein are further modified to include an additional nucleotide sequence encoding the ligand-binding protein. In some embodiments, the additional nucleotide sequence encoding the ligand-binding protein can be a part of the same polynucleotide described herein (i.e., including the gRNA that specifically targets a region within the NR4A3 gene)-i.e., a part of a polycistronic polynucleotide. In some embodiments, the additional nucleotide sequence encoding the ligand-binding protein can be introduced into the cell as a separate polynucleotide.

[0217] Thus, in some embodiments, the modified cells described herein, when compared to a reference cell, (i) express a ligand-binding protein, and (ii) have a reduced level of the NR4A3 gene and / or NR4A3 protein. In some embodiments, the modified cells, when compared to a reference cell, (i) express a ligand-binding protein, (ii) have a reduced level of the NR4A3 gene and / or NR4A3 protein and (iii) have an increased level of c-Jun protein. In some embodiments, the modified cells described herein, when compared to a reference cell, (i) express a ligand-binding protein, (ii) have a reduced level of the NR4A3 gene and / or NR4A3 protein, and (iii) have a reduced level of the NR4A2 gene and / or NR4A2 protein. In some embodiments, the modified cells described herein, when compared to a reference cell, (i) express a ligand-binding protein, (ii) have a reduced level of the NR4A3 gene and / or NR4A3 protein, (iii) have a reduced level of the NR4A2 gene and / or NR4A2 protein, and (iv) have an increased level of c-Jun protein. In some embodiments, the modified cells described herein, when compared to a reference cell, (i) express a ligand-binding protein, (ii) have a reduced level of the NR4A3 gene and / or NR4A3 protein, (iii) have a reduced level of the NR4A2 gene and / or NR4A2 protein, and (iv) have a reduced level of the NR4A1 gene and / or NR4A1 protein. In some embodiments, the modified cells described herein, when compared to a reference cell, (i) express a ligand-binding protein, (ii) have a reduced level of the NR4A3 gene and / or NR4A3 protein, (iii) have a reduced level of the NR4A2 gene and / or NR4A2 protein, (iv) have a reduced level of the NR4A1 gene and / or NR4A1 protein, and (v) have an increased level of c-Jun protein.

[0218] Non-limiting examples of ligand-binding proteins (e.g., chimeric binding proteins) useful for 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), TCR mimics, and combinations thereof.

[0219] In some embodiments, the chimeric binding protein includes a CAR.

[0220] In some embodiments, the CAR is designed as a standard CAR. In a "standard CAR", different components (e.g., an extracellular targeting domain, a transmembrane domain, and an intracellular signaling / activation domain) are linearly constructed as a single fusion protein. In some embodiments, the CAR is designed as a first-generation CAR. A "first-generation" CAR is composed of an extracellular binding domain, a hinge region, a transmembrane domain, and one or more intracellular signaling domains. All first-generation CARs contain the CD3ζ chain domain as the intracellular signaling domain. In some embodiments, the CAR is designed as a second-generation CAR. A "second-generation" CAR additionally contains a co-stimulatory domain (e.g., CD28 or 4-1BB). In some embodiments, the CAR is designed as a third-generation CAR. A "third-generation" CAR is similar to a second-generation CAR except that they contain multiple co-stimulatory domains (e.g., CD28-4-1BB or CD28-OX40). In some embodiments, the CAR is designed as a fourth-generation CAR. A "fourth-generation" CAR (also known as a TRUCK or an enhanced CAR) additionally contains additional factors that can further improve its function. For example, in some embodiments, the fourth-generation CAR additionally contains a cytokine that can be released by CAR signaling in the targeted tumor tissue. In some embodiments, the fourth-generation CAR includes one or more additional elements such as homing and suicide genes that can assist in further controlling the activity of the CAR. In some embodiments, the CAR is designed as a split CAR. In a "split CAR" system, one or more components of the CAR (e.g., an extracellular target domain, a transmembrane domain, and an intracellular signaling / activation domain) are divided into two or more parts such that they depend on multiple inputs that promote the assembly of an intact functional receptor. In some embodiments, the CAR is designed as a switchable CAR. Using a "switchable CAR", the CAR can be switched (e.g., temporarily) on (on-switch CAR) or off (off-switch CAR) in the presence of a stimulant.Additional examples of CARs that can be used in the present disclosure are described, for example, in US2020 / 0172879A1 and US2019 / 0183932A1, each of which is incorporated herein by reference in its entirety.

[0221] In some embodiments, the constructs herein encode an engineered T cell receptor (TCR), also referred to in the art as a “transgenic TCR.” A TCR is a molecule found on the surface of T cells that is responsible for recognizing fragments of antigens as peptides bound to major histocompatibility complex (MHC) molecules. A TCR is a heterodimer composed of two different protein chains. In some embodiments, the TCR consists of an alpha (α) chain and a beta (β) chain, encoded by TRA and TRB, respectively. In some embodiments, the TCR consists of gamma and delta (γ / δ) chains, encoded by TRG and TRD, respectively. When a TCR engages an antigenic peptide presented by an MHC molecule (peptide / MHC), the T lymphocyte is activated via signal transduction. In some embodiments, the TCR is an engineered (transgenic) TCR. As used herein, the term “engineered TCR” or “engineered T cell receptor” refers to an isolated or engineered T cell receptor (TCR) that specifically binds to a major histocompatibility gene complex (MHC) / peptide target antigen with a desired affinity and has been introduced into a population of immune cells, such as T cells, NK cells, and / or TILs.

[0222] In some embodiments, the chimeric binding protein comprises a chimeric antibody-T cell receptor (caTCR). As used herein, "chimeric antibody-T cell receptor" or "caTCR" comprises (i) an antibody site that specifically binds to a target antigen and (ii) a T cell receptor module capable of mobilizing at least one TCR-related signaling molecule. In some embodiments, the antibody site and the T cell receptor module are fused together. In some embodiments, 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 co-stimulatory signaling domain capable of providing a stimulating signal to an immune cell expressing the CSR. Non-limiting examples of caTCR and CSR are further described in US 10,822,413 B2, which is incorporated herein by reference in its entirety.

[0223] In some embodiments, 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) engineered to recognize a tumor antigen, which is presented in association with an HLA molecule. As will be apparent to those skilled in the art, these antibodies can mimic the specificity of a TCR. Non-limiting examples of TCR mimics are provided, for example, in US 2009 / 0226474 A1 and US 2019 / 0092876 A1, each of which is incorporated herein by reference in its entirety.

[0224] In some embodiments, the chimeric binding protein can be associated with a gene editing tool (e.g., the CRISPR-Cas system), and activation of the chimeric binding protein can induce activation of the gene editing tool such that the expression and / or activity of one or more genes is regulated in a cell. For example, in some embodiments, the cells described herein (e.g., T cells) are modified to include a chimeric binding protein (e.g., a CAR) linked to a protease and a single guide RNA that targets a regulatory region (e.g., a promoter) of a gene of interest. In some embodiments, the cells are further modified to include, for example, via a linker, a linker for T cell activation (LAT) that is complexed with the gene editing tool. Activation of the chimeric binding protein (e.g., via antigen stimulation) enables release of the gene editing tool for nuclear localization and regulation of gene expression. Additional embodiments of such chimeric binding proteins are provided elsewhere in this disclosure. See also Pietrobon et al., Int J Mol Sci 22(19):10828 (Oct. 2021) (incorporated herein by reference in its entirety).

[0225] As described herein, chimeric binding proteins useful for the present disclosure include an antigen-binding domain, a transmembrane domain, a co-stimulatory domain, an intracellular signaling domain, or combinations thereof. In some embodiments, the antigen-binding domain recognizes and specifically binds 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 (C-C motif chemokine ligand 1), CD5, CD19, CD20, CD21, CD22, CD23, CD24, CD30, CD33, CD38, CD40, CD44, CD44v6, CD44v7 / 8, CD45, CD47, CD56, CD66e, CD70, CD74, 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 (Ephrin receptor A2), ERBB dimer, estrogen receptor, ETBR (endothelin B receptor), FAP-α (fibroblast activation protein α), fetal AchR (fetal acetylcholine receptor), FBP (folate-binding protein), FCRL5, FR-α (folate receptor alpha), 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, HLA-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), Ig kappa, Ig lambda, 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 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 complex (e.g., HLA-A complexed with peptides derived from AFP, KRAS, HPV (e.g., HPV E6 or E7), NY-ESO, MAGE-A, and WT1), NCAM (neural cell adhesion molecule), nectin-4, NKG2D (natural killer group 2 member D) ligand, NY-ESO, fetal cancer antigen, PD-1, PD-L1, PRAME (antigen preferentially expressed in melanoma), progesterone receptor, PSA (prostate-specific antigen), PSCA (prostate stem cell antigen), PSMA (prostate-specific membrane antigen), ROR1, ROR2, SIRPα (signal regulatory protein alpha), SLIT, SLITRK6 (NTRK-like protein 6), STEAP1 (six-transmembrane epithelial antigen of prostate 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 combinations thereof are included. In some embodiments, the antigen-binding domain of the chimeric binding protein described herein specifically binds to ROR1. In some embodiments, the antigen-binding domain of the chimeric binding protein specifically binds to GPC2. In some embodiments, the antigen-binding domain of the chimeric binding protein specifically binds to a tumor antigen, and the tumor antigen is alpha-fetoprotein (AFP), CD19, BCMA, CLL-1, CS1, CD38, CD19, TSHR, CD123, CD22, CD30, 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, Lewis Y, CD24, PDGFR-beta, SSEA-4, CD20, folate receptor alpha,Derived from ERBB2 (Her2 / neu), MUC1, MUC16, EGFR, NCAM, prostate specific antigen, PAP, ELF2M, ephrin B2, IGF-I receptor, CAIX, LMP2, gp100, bcr-abl, tyrosinase, EphA2, fucosyl GM1, sLe, GM3, TGS5, HMWMAA, 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 variants, prostain, survivin, telomerase, PCTA-1 / galectin 8, Melan-A / MART1, Ras variants (e.g., HRAS, KRAS, NRAS), hTERT, sarcoma translocation breakpoint, 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, CD2, CD3ε, CD4, CD5, CD7, the extracellular portion of the APRIL protein, neoantigen, or any combination thereof.,

[0226] As further described elsewhere in this disclosure, the antigen-binding domain of a chimeric binding protein can be any polypeptide capable of binding one or more antigens. In some embodiments, the antigen-binding domain comprises, or is derived from, an Ig NAR, Fab fragment, Fab′ fragment, F(ab)′2 fragment, F(ab)′3 fragment, Fv, single-chain variable fragment (scFv), bis-scFv, (scFv)2, minibody, diabody, triabody, tetrabody, intrabody, disulfide-stabilized Fv protein (dsFv), uniobody, nanobody, and an antigen-binding region, ligand, receptor, receptor fragment, peptide aptamer, or combination thereof that can specifically bind to any of the proteins of interest. In some embodiments, the antigen-binding domain is a single-chain Fv (scFv).

[0227] In some embodiments, the chimeric binding proteins described herein include an intracellular signaling domain that transmits an effector function signal upon binding of an antigen to the extracellular domain and directs a cell (e.g., a T cell) expressing the chimeric binding protein to perform a special function. Non-limiting examples of intracellular signaling domains include intracellular signaling domain regions derived from CD3 zeta, FcR gamma, common FcR gamma (FCER1G), Fc gamma RIIa, FcR beta (Fc epsilon Rib), CD3 gamma, CD3 delta, CD3 epsilon, CD22, CD79a, CD79b, CD278 (“ICOS”), FcεRI, CD66d, CD32, DAP10, DAP12, or any combination thereof. In some embodiments, the intracellular signaling domain comprises the CD3 zeta intracellular signaling domain. In some embodiments, the chimeric binding protein includes the entire intracellular domain of the proteins disclosed herein. In some embodiments, the intracellular domain is a truncated form. The truncated portion of the intracellular domain can be used in place of the intact chain as long as it still transmits the effector function signal. Thus, the term intracellular domain is meant to include any truncated portion of the intracellular domain sufficient to transmit the effector function signal.

[0228] In some embodiments, the chimeric binding protein that can be expressed in the modified immune cells described herein (e.g., having a reduced level of the NR4A3 gene and / or NR4A3 protein) further comprises a transmembrane domain. In some embodiments, the antigen-binding domain can be linked to the intracellular domain of the chimeric binding protein by the transmembrane domain. In some embodiments, the antigen-binding domain is connected to the transmembrane domain of the chimeric binding protein (e.g., CAR) by a linker. In some embodiments, the inclusion of a linker between the antigen-binding domain and the transmembrane domain can affect the flexibility of the antigen-binding domain, thereby improving chimeric binding protein function.

[0229] Any transmembrane domain known in the art can be used in the chimeric binding proteins (e.g., CARs) described herein. In some embodiments, the transmembrane domain is artificial (e.g., engineered transmembrane domain). In some embodiments, the transmembrane domain is derived from a naturally occurring polypeptide. In some embodiments, the transmembrane domain comprises a transmembrane 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, IL2R beta, IL2R gamma, IL7Rα, ITGA1, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11d, ITGAE, CD103, ITGAL, CD11a, LFA-1, ITGAM, CD11b, 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, the transmembrane domain region of CD19, or any combination thereof. In some embodiments, the transmembrane domain comprises the CD28 transmembrane domain.

[0230] As described herein, in some embodiments, a chimeric binding protein (e.g., a CAR) includes one or more co-stimulatory domains (e.g., second and third generation CARs). Without being bound by any one theory, these co-stimulatory domains may further improve the expansion, activation, memory, persistence, and / or effector function of the modified immune cells described herein (e.g., having reduced levels of the NR4A3 gene and / or NR4A3 protein and engineered to express a ligand-binding protein). In some embodiments, the transmembrane domain is fused to a co-stimulatory domain, optionally the co-stimulatory domain is fused to a second co-stimulatory domain, and the co-stimulatory domain is fused to a signaling domain not limited to CD3ζ. Non-limiting examples of co-stimulatory domains 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 embodiments, the co-stimulatory domain includes the 4-1BB / CD137 co-stimulatory domain.

[0231] Also disclosed herein is a population of cells comprising one or more of the modified cells described above (e.g., having reduced levels of the NR4A3 gene and / or NR4A3 protein and a ligand-binding protein). In some embodiments, the population of immune cells is a pure population. In some embodiments, a 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% cells belonging to the same immune cell type (e.g., 99% of the immune cells are lymphocytes). In some embodiments, the population of immune cells comprises 1, 2, 3, 4, or 5 different cell types, e.g., a population of immune cells comprising 2 cell types may comprise lymphocytes and dendritic cells.

[0232] In some embodiments, the population of cells disclosed herein comprises, consists of, or consists essentially of lymphocytes. In some embodiments, the population of modified immune cells disclosed herein comprises lymphocytes, which 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 embodiments, the lymphocytes are T cells. In some specific embodiments, the lymphocytes are NK cells.

[0233] As will be apparent to those skilled in the art, in some embodiments, the cells described herein are modified using a combination of multiple approaches. For example, in some embodiments, the cells are modified to have reduced levels of the NR4A3 gene and / or NR4A3 protein and (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 the expression of an endogenous protein (e.g., c-Jun). In some embodiments, the cells are modified to have reduced levels of the NR4A3 gene and / or NR4A3 protein and (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., including the c-Jun protein). In some embodiments, the cells are modified to have reduced levels of the NR4A3 gene and / or NR4A3 protein and (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 the expression of an endogenous protein (e.g., c-Jun), and (iii) an exogenous nucleotide sequence encoding a second protein (the c-Jun protein). As described herein, in some embodiments, the exogenous nucleotide sequences encoding the first and second proteins can be part of a single polycistronic vector.

[0234] In some embodiments, the modified immune cells disclosed herein are T cells. In some embodiments, the T cells include a CAR. In some embodiments, modified T cells (CAR T cells) that can be prepared to express a CAR are, for example, CD8 + T cells or CD4 +They are T cells. In some embodiments, the CAR-expressing cells disclosed herein are CAR T cells, e.g., mono CAR T cells, genome-edited CAR T cells, dual CAR T cells, or tandem CAR T cells. In some embodiments, the modified cells disclosed herein are NK cells. In some embodiments, the NK cells comprise a CAR. In some embodiments, the CAR NK cells are mono CAR NK cells, dual CAR NK cells, or tandem CAR NK T cells. In some embodiments, the modified cells of the disclosure comprise both T cells and NK cells. In some embodiments, both the T cells and NK cells comprise a CAR. Examples of such CAR T cells and CAR NK cells are provided in International Application No. PCT / US2019 / 044195 (published as WO2020028400A1), which is incorporated herein by reference in its entirety.

[0235] In some embodiments, the modified immune cells can be any immune cell type. In some embodiments, the cells are modified immune cells for any adoptive cell transfer (ACT) therapy (also known as adoptive cell therapy). The ACT therapy can be autologous or allogeneic. In some embodiments, the ACT therapy includes CAR T therapy, tumor-infiltrating lymphocyte (TIL) therapy, NK cell therapy, or any combination thereof.

[0236] In some embodiments, the modified immune cells are TILs for TIL therapy. The use of TILs as adoptive cell transfer therapy for treating cancer has been studied for over 20 years using TIL adoptive cell therapy for 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 ex vivo from surgically resected tumors cut into small pieces or from single cell suspensions isolated from tumor fragments. Multiple individual cultures are established, grown separately, and assayed for specific tumor recognition. TILs are expanded over the course of several weeks. The selected TIL lines that exhibit the best tumor reactivity are then further expanded using a "rapid expansion protocol" (REP) that uses anti-CD3 activation for a typical period of two weeks. TILs grown in culture can be modified at any time during the ex vivo process such that the expression of the NR4A3 gene and / or the NR4A3 protein is reduced (either alone or in combination with other members of the NR4A family, such as NR4A1 and / or NR4A2). The final TILs after REP are injected back into the patient. The process may also involve a pre-chemotherapy regimen to deplete endogenous lymphocytes to provide sufficient access for the adoptively transferred TILs to surround the tumor site.

[0237] In some embodiments, the modified immune cells disclosed herein, such as T cells, comprise a T cell receptor (TCR), such as an engineered T cell receptor. In some embodiments, the modified immune cells disclosed herein, such as T cells, may comprise a chimeric antigen receptor (CAR) that specifically binds to a tumor antigen. In some embodiments, the modified immune cells, such as lymphocytes, are T cells having a T cell receptor, such as an engineered TCR. As used herein, the term "engineered TCR" or "engineered T cell receptor" refers to a T cell receptor (TCR) that has been selected, cloned, and / or later introduced into a population of T cells and engineered to specifically bind with a desired affinity to a major histocompatibility complex (MHC) / peptide target antigen.

[0238] In some embodiments, the CAR or TCR that can 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.

[0239] In some embodiments, the modified cells of the 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 consisting of a constant region that anchors the chain within the T cell surface membrane and a variable region that recognizes and binds to an antigen presented by MHC. The TCR complex is associated with six polypeptides that form two heterodimers (CD3γε and CD3δε) and one homodimer (CD3ζ), which together form the CD3 complex. T cell therapies engineered with T cell receptors utilize the modification of T cells that retain these complexes to specifically target antigens expressed by specific tumor cells.

[0240] In some embodiments, the engineered TCR-engineered cells can target major 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, cancer-testis (CT) antigens, overexpressed antigens, and differentiation antigens, whereas the latter can include, but are not limited to, neoantigens and oncoviral antigens. Human papillomavirus (HPV) E6 protein and HPV E7 protein belong to the category of oncoviral antigens.

[0241] In some embodiments, the engineered TCR-engineered cells can target melanoma-associated antigens (MAGE), including but not limited to, CT antigens such as MAGE-A1, MAGE-A2, MAGE-A3, MAGE-A4, MAGE-A6, MAGE-A8, MAGE-A9.23, MAGE-A10, and MAGE-A12. In some embodiments, the engineered TCR-engineered cells can target glycoprotein (gp100), melanoma antigen recognized by T cells (MART-1), and / or tyrosinase (which are mainly found in melanoma and normal melanocytes). In some embodiments, the engineered TCR-engineered cells can target Wilms tumor 1 (WT1), an overexpressed antigen that is highly expressed in most acute myeloid leukemia (AML), acute lymphoblastic leukemia, almost all types of solid tumors, and some important tissues such as heart tissue. In some embodiments, the engineered TCR-engineered cells can target mesothelin, another type of overexpressed antigen that is highly expressed in mesothelioma but also present on mesothelial cells of some tissues including the trachea.

[0242] IV. Methods for generating engineered cells The present disclosure also provides methods for generating or preparing the modified cells described herein (i.e., having reduced levels of the NR4A3 gene and / or NR4A3 protein). In some embodiments, such methods include contacting a cell (e.g., an immune cell) with a gene editing tool, wherein the gene editing tool (e.g., comprising a polynucleotide of the present disclosure that can specifically target a sequence within the NR4A3 gene and comprises a gRNA) is capable of reducing the expression of the NR4A3 gene and / or NR4A3 protein. In some embodiments, after contacting, the level of the NR4A3 gene and / or 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% in the cell. In some embodiments, after contacting, the cell does not express any level of the NR4A3 gene and / or NR4A3 protein. In some embodiments, the modified cell can be further modified to 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).

[0243] Thus, in some embodiments, the modified cells described herein have reduced levels of the NR4A3 gene and / or protein, but have endogenous levels of both the NR4A1 gene and / or protein and the NR4A2 gene and / or protein. In some embodiments, the modified cells described herein have (i) reduced levels of the NR4A3 gene and / or protein, (ii) reduced levels of the NR4A1 gene and / or protein, and (iii) endogenous levels of the NR4A2 gene and / or protein. In some embodiments, compared to a reference cell (e.g., a corresponding cell that is not modified and has endogenous levels of the NR4A3 gene and / or protein and the endogenous levels of the NR4A1 gene and / or protein), the level of (i) the NR4A3 gene and / or 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 the level of (ii) the NR4A1 gene and / or 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%. In some embodiments, the modified cells generated using the methods described above do not express either the NR4A3 gene and / or protein or the NR4A1 gene and / or protein.

[0244] In some embodiments, 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 embodiments, compared to a reference cell (e.g., a corresponding cell having endogenous levels of the NR4A3 gene and / or NR4A3 protein and the NR4A2 gene and / or NR4A2 protein that is not modified), 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 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or about 100%, and the level of (ii) 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%. In some embodiments, the modified cells generated using the methods described above do not express either the NR4A3 gene and / or NR4A3 protein or the NR4A2 gene and / or NR4A2 protein.

[0245] In some embodiments, the modified cells described herein have (i) reduced levels of the NR4A3 gene and / or the NR4A3 protein, (ii) reduced levels of the NR4A1 gene and / or the NR4A1 protein, and (iii) reduced levels of the NR4A2 gene and / or the NR4A2 protein. In some embodiments, compared to a reference cell (e.g., a corresponding cell that is not modified, e.g., has endogenous levels of all members of the NR4A family), the level of (i) the NR4A3 gene and / or the 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%; (ii) the level of the NR4A1 gene and / or the 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%; and (iii) the level of the NR4A2 gene and / or the 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%.

[0246] When the levels of multiple members of the NR4A family are reduced, in some embodiments, the levels can be reduced with the same gene editing tool (e.g., the levels of different NR4A family members are reduced by the CRISPR / Cas system). When the levels of multiple members of the NR4A family are reduced, in some embodiments, the levels can be reduced with different gene editing tools (e.g., the levels of the NR4A3 gene and / or protein are reduced using CRISPR / Cas, and the levels of the NR4A2 gene and / or protein are reduced using antisense oligonucleotides).

[0247] Gene editing, e.g., base editing, can be performed using any editing tool known in the art. For example, in some embodiments, cells (e.g., immune cells) can be modified using techniques such as CRISPR / Cas, TALEN, zinc finger nucleases (ZFNs), meganucleases, restriction endonucleases, interfering RNA (RNAi), antisense oligonucleotides, or combinations thereof. In some embodiments, the NR4A3 gene and / or expression can also be modified using shRNA, siRNA, or miRNA. All of these exemplary techniques are discussed in more detail below. In some embodiments, the methods used to reduce the expression of the NR4A3 gene and / or protein include using one or more gene editing tools (e.g., two, three, or more tools). In some embodiments, the methods used to reduce the expression of the NR4A3 gene and / or protein include at least one method that acts on NR4A3 DNA (e.g., CRISPR) or at least one method that acts on NR4A3 RNA (e.g., antisense oligonucleotides) and at least one method that acts on the NR4A3 protein (e.g., inhibition of binding to a cellular signaling partner or post-translational modification).

[0248] In some embodiments, the modified cells generated using the above method can be further modified to express a ligand-binding protein (e.g., a CAR or a transgenic TCR). For example, in some embodiments, the method for generating the modified cells provided herein comprises contacting a cell (e.g., an immune cell) with a gene editing tool (e.g., comprising a polynucleotide of the present disclosure) capable of reducing the level of the NR4A3 gene and / or the NR4A3 protein and a nucleotide sequence encoding a ligand-binding protein. In some embodiments, the gene editing tool and the nucleotide sequence encoding the ligand-binding protein are contacted with the cell simultaneously. For example, in some embodiments, the cell is contacted with a single polynucleotide comprising both the gene editing tool and the nucleotide sequence encoding the ligand-binding protein. In some embodiments, the cell is contacted simultaneously with a first polynucleotide comprising the gene editing tool and a second polynucleotide comprising the nucleotide sequence encoding the ligand-binding protein. In some embodiments, the gene editing tool and the nucleotide sequence encoding the ligand-binding protein are contacted with the cell sequentially.

[0249] In some embodiments, the above-described method of generating modified cells may further include contacting the cells with a nucleotide sequence encoding the c-Jun protein. As described herein, contacting the cells with a nucleotide sequence encoding the c-Jun protein can increase the expression of the c-Jun protein by the cells. If the cells are capable of naturally expressing the c-Jun protein, in some embodiments, the method may include contacting the cells with a transcriptional activator (e.g., a CRISPR / Cas system-based transcriptional activator, e.g., CRISPRa) such that the expression of the endogenous c-Jun protein is increased. As described herein, in some embodiments, the cells may be contacted with both a nucleotide sequence encoding the c-Jun protein and a transcriptional activator (e.g., a CRISPR / Cas system-based transcriptional activator, e.g., CRISPRa) capable of increasing endogenous c-Jun protein expression. In some embodiments, the nucleotide sequence encoding the c-Jun protein and / or the transcriptional activator are contacted with the cells simultaneously with the gene editing tool. For example, in some embodiments, the cells are contacted with a single polynucleotide comprising both (i) a nucleotide sequence encoding the c-Jun protein and / or the transcriptional activator and (ii) the gene editing tool. In some embodiments, the cells are contacted simultaneously with a first polynucleotide comprising a nucleotide sequence encoding the c-Jun protein and / or the transcriptional activator and a second polynucleotide comprising the gene editing tool. In some embodiments, the gene editing tool and the nucleotide sequence encoding the c-Jun protein and / or the transcriptional activator are contacted with the cells sequentially.

[0250] Accordingly, in some aspects, provided herein is a method of generating a modified cell of the present disclosure, the method comprising contacting a cell with (i) a gene editing tool capable of reducing the level of the NR4A3 gene and / or NR4A3 protein (e.g., the polynucleotide described herein), (ii) a nucleotide sequence encoding a ligand-binding protein, and (iii) a nucleotide encoding c-Jun protein and / or a transcriptional activator described herein. In some aspects, the gene editing tool, the nucleotide sequence encoding the ligand-binding protein, and the nucleotide encoding c-Jun protein and / or the transcriptional activator are contacted with the cell simultaneously. For example, in some aspects, the cell is contacted with a single polynucleotide comprising (i) the gene editing tool, (ii) the nucleotide sequence encoding the ligand-binding protein, and (iii) the nucleotide encoding c-Jun protein and / or the transcriptional activator. In some aspects, the cell is contacted simultaneously with (i) a first nucleotide comprising the gene editing tool, (ii) a second nucleotide sequence encoding the ligand-binding protein, and (iii) a third nucleotide sequence encoding c-Jun protein and / or the transcriptional activator. In some aspects, at least two of the following are contacted with the cell sequentially: (i) the gene editing tool, (ii) the nucleotide sequence encoding the ligand-binding protein, and (iii) the nucleotide sequence encoding c-Jun protein and / or the transcriptional activator.

[0251] As described herein, in some aspects, modified cells that can be generated using the methods provided herein include immune cells. In some aspects, the immune cells include lymphocytes, neutrophils, monocytes, macrophages, dendritic cells, or combinations thereof. In some aspects, the lymphocytes include T cells, tumor-infiltrating lymphocytes (TILs), lymphokine-activated killer cells, natural (NK) cells, or combinations thereof. In some aspects, the lymphocytes include T cells, e.g., CD4 + T cells or CD8 +They are T cells. In some embodiments, the lymphocytes express chimeric antigen receptors (e.g., CAR-expressing CD8+ T cells and / or CAR-expressing CD4+ T cells). In some embodiments, the lymphocytes express engineered TCRs (e.g., engineered TCR-expressing CD8+ T cells and / or engineered TCR-expressing CD4+ T cells). In some embodiments, the lymphocytes are tumor-infiltrating lymphocytes (TILs). In some embodiments, the TILs are CD8 + TILs. In some embodiments, the TILs are CD4 + TILs.

[0252] For any of the methods provided above in the present disclosure or described elsewhere, contacting a cell (e.g., an immune cell) with any of the following can be performed 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 the NR4A3 protein), (ii) a nucleotide sequence encoding the 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 embodiments, the contacting is performed in vivo (e.g., gene therapy). In some embodiments, the contacting is performed in vitro. In some embodiments, the contacting is performed ex vivo. In some embodiments, the cell is an autologous cell. In some embodiments, the cell is a heterologous cell.

[0253] As is apparent from the present disclosure, for the gene editing tools described herein to have their intended effects (e.g., reducing the levels of the NR4A3 gene and / or the NR4A3 protein), the gene editing tools must be able to enter cells and bind to the target gene. In some embodiments, any delivery vehicle known in the art for delivering a target molecule to a cell can be used. See, for example, U.S. Patent No. 10,047,355B2, which is hereby incorporated by reference in its entirety. Additional disclosure regarding vectors that can be used is provided elsewhere in the present disclosure.

[0254] In some embodiments, the gene editing tools useful for the present disclosure can remove the entire gene encoding the target protein (e.g., the NR4A3 protein). In some embodiments, the gene editing tool can remove a portion of the genome (e.g., one or more exons) encoding the target protein (e.g., the NR4A3 protein). In some embodiments, a gene editing tool, e.g., a base editor, modifies a specific nucleotide base without generating indels. As used herein, the term "indel" refers to an insertion or deletion of a nucleotide base in a nucleic acid that can result in a frameshift mutation within the coding region of a gene. Non-limiting examples of base editors are disclosed in U.S. Publication No. 2017 / 0121693, published May 4, 2017, which is hereby incorporated by reference in its entirety.

[0255] IV.A. Gene Editing Tools Exemplary gene editing tools that can be used in the present disclosure are provided below.

[0256] IV.A.1. CRISPR / Cas System In some aspects, gene editing tools that can be used in the present disclosure include the CRISPR / Cas system. Such systems can use, for example, Cas9 nuclease or a nucleic acid molecule encoding Cas9 nuclease, which in some examples is codon-optimized for the desired cell type in which it is to be expressed (e.g., T cells, e.g., CAR-expressing T cells). As further described herein, in some aspects, such systems can include a Cas9 nuclease protein.

[0257] The CRISPR / Cas system uses a Cas nuclease, e.g., Cas9 nuclease, which is complexed with a guide RNA (e.g., synthetic guide RNA) (gRNA) that hybridizes to a target DNA sequence immediately upstream of the NGG motif recognized by the Cas nuclease, e.g., Cas9, to be targeted to a genomic site. This results in a double-strand break 3 nucleotides upstream of the NGG motif. The unique ability of the CRISPR / Cas9 system is the ability to simultaneously target multiple distinguishable genomic loci by co-expressing a single Cas9 protein with two or more gRNAs (e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 gRNAs). Such systems can also use a guide RNA comprising two separate molecules. In some aspects, the two-molecule gRNA comprises a crRNA-like ("CRISPR RNA" or "targeter-RNA" or "crRNA" or "crRNA repeat") molecule and a corresponding tracrRNA-like ("trans-activating CRISPR RNA" or "activator-RNA" or "tracrRNA" or "scaffold") molecule.

[0258] The crRNA contains both the DNA targeting segment (single-stranded) of the gRNA and an extension of nucleotides that forms one half of the double-stranded RNA (dsRNA) duplex of the protein-binding segment of the gRNA. The corresponding tracrRNA (trans-activating crRNA) is an extension of nucleotides that forms the other half of the dsRNA duplex of the protein-binding segment of the gRNA. Thus, the nucleotide extension of the crRNA is complementary to and hybridizes with the nucleotide extension of the tracrRNA to form the dsRNA duplex of the protein-binding domain of the gRNA. Thus, each crRNA can be said to have a corresponding tracrRNA. The crRNA additionally provides a single-stranded DNA targeting segment. Thus, the gRNA contains a sequence that hybridizes to a target sequence (e.g., NR4A3 mRNA) and the tracrRNA. Thus, the crRNA and tracrRNA hybridize (as corresponding pairs) to form the gRNA. When 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., human) in which the RNA molecule will be used.

[0259] Naturally occurring genes encoding the three elements (Cas9, tracrRNA, and crRNA) are typically organized in an operon(s). Naturally occurring CRISPR RNAs vary depending on the Cas9 system and organism but often contain a targeting segment 21-72 nucleotides in length flanked by two direct repeats (DRs) 21-46 nucleotides in length (see, e.g., WO2014 / 131833). In the case of S. pyogenes, the DRs are 36 nucleotides in length and the targeting segment is 30 nucleotides in length. The DR located 3’ is complementary to and hybridizes with the corresponding tracrRNA, which in turn binds to the Cas9 protein.

[0260] Alternatively, the CRISPR systems used herein may further employ a fusion crRNA-tracrRNA construct (i.e., a single transcript) that functions with codon-optimized Cas9. This single RNA is often referred to as guide RNA or gRNA. Within the gRNA, the crRNA portion is identified as the "target sequence" for a given recognition site, and the tracrRNA is often referred to as the "scaffold". Briefly, a short DNA fragment containing the target sequence is inserted into the guide RNA expression plasmid. The guide RNA expression plasmid contains the target sequence (about 20 nucleotides in some embodiments), the form of the tracrRNA sequence (scaffold), as well as a suitable promoter that is active in the cell and the necessary elements for proper processing in eukaryotic cells. Many of the systems rely on custom complementary oligos that are annealed to form double-stranded DNA and then cloned into the guide RNA expression plasmid.

[0261] The guide RNA expression cassette and the Cas9 expression cassette are then introduced into the cell. See, for example, 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 W Y et al., Nat Biotechnol 2013 March;31(3):227-9; Jiang W et al., Nat Biotechnol 2013 March;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 also, for example, WO / 2013 / 176772A1, WO / 2014 / 065596A1, WO / 2014 / 089290A1, WO / 2014 / 093622A2, WO / 2014 / 099750A2, and WO / 2013142578A1 (each of which is incorporated herein by reference in its entirety).

[0262] In some embodiments, the Cas9 nuclease can be provided in protein form. For example, in some embodiments, cells useful for the present disclosure (e.g., CAR or TCR-expressing immune cells) can be modified by introducing nucleic acid molecules comprising the Cas9 nuclease protein and the gRNA (e.g., to have reduced levels of the NR4A gene and / or the NR4A protein). In some embodiments, the nucleic acid molecules comprising the Cas9 nuclease protein and the gRNA can be introduced into the cells sequentially. In some embodiments, the nucleic acid molecules comprising the Cas9 nuclease protein and the gRNA can be introduced into the cells simultaneously. For example, in some embodiments, co-administration comprises introducing the nucleic acid molecules comprising the Cas9 nuclease protein and the gRNA as separate compositions at the same time. In some embodiments, the Cas9 protein can be provided in the form of a complex with the nucleic acid molecule comprising the gRNA (i.e., as a single composition).

[0263] In some embodiments, the Cas9 nuclease can be provided in the form of a nucleic acid encoding the protein. Thus, in some embodiments, cells useful for the present disclosure (e.g., CAR or TCR-expressing immune cells) can be modified by introducing a first nucleic acid molecule encoding the Cas9 nuclease protein and a second nucleic acid molecule comprising the gRNA (e.g., to have reduced levels of the NR4A gene and / or the NR4A protein). In some embodiments, the first and second nucleic acid molecules can be introduced into the cells sequentially. In some embodiments, the first and second nucleic acid molecules can be introduced into the cells simultaneously. For example, in some embodiments, the first and second nucleic acid molecules can be introduced into the cells as separate compositions at the same time. In some embodiments, the first and second nucleic acid molecules can be part of a single polynucleotide, and the cells are modified to contain the single polynucleotide. In some embodiments, the nucleic acid molecule comprising the gene editing tool further comprises a guide RNA (e.g., a synthetic guide RNA disclosed herein) and a nucleic acid encoding a Cas nuclease, e.g., the Cas9 nuclease.

[0264] The nucleic acid encoding the Cas9 nuclease can be RNA (e.g., messenger RNA (mRNA)) or DNA. In some embodiments, the gRNA can be provided in the form of RNA. In some embodiments, the gRNA can be provided in the form of DNA encoding RNA. In some embodiments, the gRNA can be provided in the form of separate crRNA and tracrRNA molecules, or separate DNA molecules encoding crRNA and tracrRNA, respectively.

[0265] In some embodiments, the gRNA comprises a third nucleic acid sequence encoding clustered regularly interspaced short palindromic repeats (CRISPR) RNA (crRNA) and trans-activating CRISPR RNA (tracrRNA). In some embodiments, the Cas protein is a type I Cas protein. In some embodiments, the Cas protein is a type II Cas protein. In some embodiments, the type II Cas protein is Cas9. In some embodiments, the type II Cas, e.g., Cas9, is human codon-optimized Cas.

[0266] In some embodiments, the Cas protein is a "nickase" that can create a single-strand break (i.e., a "nick") within the target nucleic acid sequence without cleaving both strands of double-stranded DNA (dsDNA). Cas9, for example, contains two nuclease domains (the RuvC-like nuclease domain and the HNH-like nuclease domain), which are responsible for cleavage of opposing DNA strands. Mutations in either of these domains can create a nickase. Examples of mutations that create a nickase can be found, for example, in WO / 2013 / 176772A1 and WO / 2013 / 142578A1, each of which is incorporated herein by reference.

[0267] In some embodiments, two separate Cas proteins (e.g., nickases) specific for target sites on each strand of the dsDNA can create overhang sequences that are complementary to overhang sequences on another nucleic acid, or on separate regions on the same nucleic acid. The overhang ends created by contacting the nucleic acid with two nickases specific for target sites on both strands of the dsDNA can be either 5' or 3' overhang ends. For example, a first nickase can create a single-strand break in the first strand of the dsDNA, while a second nickase can create a single-strand break in the second strand of the dsDNA, thereby creating an overhang sequence. The target sites of each nickase that create the single-strand breaks can be selected such that the sequence of the overhang end created is complementary to the sequence of the overhang end of a nucleic acid molecule that is different. The complementary overhang ends of two different nucleic acid molecules can be annealed by the methods disclosed herein. In some embodiments, the target site of the nickase on the first strand is different from the target site of the nickase on the second strand.

[0268] In some embodiments, the expression of the NR4A3 gene, and the NR4A3 protein encoded thereby, is reduced by contacting the cell with, for example, a CRISPR specific for the NR4A3 gene (e.g., the CRISPR-Cas9 system). As further described elsewhere in the present disclosure, in some embodiments, the cells described herein (e.g., immune cells expressing a CAR or TCR and / or having an increased level of c-Jun protein) are further modified to reduce the levels of (i) the NR4A1 gene and / or protein, (ii) the NR4A2 gene and / or protein, or (iii) both (i) and (ii). Thus, in some embodiments, the CRISPR is specific for the NR4A1 gene. Thus, in some embodiments, after contacting with the CRISPR, the cell (e.g., a CAR or TCR-expressing immune cell) has (i) a reduced level of the NR4A1 gene and / or protein, (ii) an endogenous level of the NR4A2 gene and / or protein, and (iii) an endogenous level of the NR4A3 gene and / or protein. In some embodiments, the CRISPR is specific for the NR4A2 gene. Thus, in some embodiments, after contacting with the CRISPR, the cell (e.g., a CAR or TCR-expressing immune cell) has (i) an endogenous level of the NR4A1 gene and / or protein, (ii) a reduced level of the NR4A2 gene and / or protein, and (iii) an endogenous level of the NR4A3 gene and / or protein. In some embodiments, the CRISPR is specific for the NR4A3 gene. Thus, in some embodiments, after contacting with the CRISPR, the cell (e.g., a CAR or TCR-expressing immune cell) has (i) an endogenous level of the NR4A1 gene and / or protein, (ii) an endogenous level of the NR4A2 gene and / or protein, and (iii) a reduced level of the NR4A3 gene and / or protein.

[0269] In some embodiments, CRISPR targets multiple NR4A genes. For example, in some embodiments, CRISPR can target both the NR4A1 gene and the NR4A2 gene. Thus, in some embodiments, after contact with CRISPR, cells (e.g., CAR- or TCR-expressing immune cells) have (i) reduced levels of the NR4A1 gene and / or protein, (ii) reduced levels of the NR4A2 gene and / or protein, and (iii) endogenous levels of the NR4A3 gene and / or protein. In some embodiments, CRISPR can target both the NR4A1 gene and the NR4A3 gene. Thus, in some embodiments, after contact with CRISPR, cells (e.g., CAR- or TCR-expressing immune cells) have (i) reduced levels of the NR4A1 gene and / or protein, (ii) endogenous levels of the NR4A2 gene and / or protein, and (iii) reduced levels of the NR4A3 gene and / or protein. In some embodiments, CRISPR can target both the NR4A2 gene and / or the NR4A3 gene. In some embodiments, after contact with CRISPR, cells (e.g., CAR- or TCR-expressing immune cells) have (i) endogenous levels of the NR4A1 gene and / or protein, (ii) reduced levels of the NR4A2 gene and / or protein, and (iii) reduced levels of the NR4A3 gene and / or protein. In some embodiments, CRISPR can target the NR4A1 gene, the NR4A2 gene, and the NR4A3 gene. Thus, in some embodiments, after contact with CRISPR, cells (e.g., CAR- or TCR-expressing immune cells) have (i) reduced levels of the NR4A1 gene and / or protein, (ii) reduced levels of the NR4A2 gene and / or protein, and (iii) reduced levels of the NR4A3 gene and / or protein.

[0270] In some embodiments, gene editing using CRISPR reduces the NR4A3 gene level 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% compared to the NR4A3 gene level observed in a reference cell (e.g., a corresponding cell not subjected to gene editing using CRISPR). In some embodiments, the NR4A3 gene level can be measured, for example, by digital droplet PCR using any technique known in the art.

[0271] In some embodiments, the nucleic acids encoding the gRNA and / or Cas9 disclosed herein are RNA or DNA. In some embodiments, the RNA or DNA encoding the gRNA and / or Cas9 disclosed herein are synthetic RNA or synthetic DNA, respectively. In some embodiments, the synthetic RNA or DNA contains at least one unnatural nucleobase. In some embodiments, all nucleobases of a given class are replaced with unnatural nucleobases (e.g., all uridines in a polynucleotide disclosed herein can be replaced with an unnatural nucleobase, e.g., 5-methoxyuridine or pseudouridine). In some embodiments, a polynucleotide (e.g., synthetic RNA or synthetic DNA) contains only natural nucleobases, i.e., A, C, T, and U in the case of synthetic DNA, or A, C, T, and U in the case of synthetic RNA or synthetic DNA.

[0272] Generally, the CRISPR gene editing methods disclosed herein are performed on cells, e.g., immune cells, in vivo, in vitro, or ex vivo (i) Cas9 or a nucleic acid encoding Cas9; and, (ii) at least one NR4A3 gene guide RNA (gRNA) or a nucleic acid encoding the gRNA, comprising contacting with, the gRNA targets a sequence in the NR4A3 gene (e.g., intron and / or exon sequence), and contacting the cell with Cas9 and at least one gRNA results in a reduction in the expression of the NR4A3 gene and / or NR4A3 protein.

[0273] In some embodiments, the gRNA that can be used to reduce the level of the NR4A3 gene in a cell (e.g., an immune cell) includes any one or more of the gRNAs provided in Tables C and D. For example, in some embodiments, the gRNA that can be used to target the NR4A3 gene comprises, consists of, or consists essentially 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 embodiments, 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: 30. In some embodiments, the gRNA that can be used to target the NR4A3 gene comprises the sequence shown in SEQ ID NO: 30. In some embodiments, the gRNA that can be used to target the NR4A3 gene consists of the sequence shown in SEQ ID NO: 30. In some embodiments, the gRNA that can be used to target the NR4A3 gene consists essentially of the sequence shown in SEQ ID NO: 30. In some embodiments, 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: 52. In some embodiments, the gRNA that can be used to target the NR4A3 gene comprises the sequence shown in SEQ ID NO: 52. In some embodiments, the gRNA that can be used to target the NR4A3 gene consists of the sequence shown in SEQ ID NO: 52. In some embodiments, the gRNA that can be used to target the NR4A3 gene consists essentially of the sequence shown in SEQ ID NO: 52. In some embodiments, 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: 53. In some embodiments, the gRNA that can be used to target the NR4A3 gene comprises the sequence shown in SEQ ID NO: 53. In some embodiments, the gRNA that can be used to target the NR4A3 gene consists of the sequence shown in SEQ ID NO: 53. In some embodiments, the gRNA that can be used to target the NR4A3 gene consists essentially of the sequence shown in SEQ ID NO: 53.In some embodiments, 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: 54. In some embodiments, the gRNA that can be used to target the NR4A3 gene comprises the sequence shown in SEQ ID NO: 54. In some embodiments, the gRNA that can be used to target the NR4A3 gene consists of the sequence shown in SEQ ID NO: 54. In some embodiments, the gRNA that can be used to target the NR4A3 gene consists essentially of the sequence shown in SEQ ID NO: 54. In some embodiments, 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: 55. In some embodiments, the gRNA that can be used to target the NR4A3 gene comprises the sequence shown in SEQ ID NO: 55. In some embodiments, the gRNA that can be used to target the NR4A3 gene consists of the sequence shown in SEQ ID NO: 55. In some embodiments, the gRNA that can be used to target the NR4A3 gene consists essentially of the sequence shown in SEQ ID NO: 55. In some embodiments, 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: 56. In some embodiments, the gRNA that can be used to target the NR4A3 gene comprises the sequence shown in SEQ ID NO: 56. In some embodiments, the gRNA that can be used to target the NR4A3 gene consists of the sequence shown in SEQ ID NO: 56. In some embodiments, the gRNA that can be used to target the NR4A3 gene consists essentially of the sequence shown in SEQ ID NO: 56. In some embodiments, 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: 57. In some embodiments, the gRNA that can be used to target the NR4A3 gene comprises the sequence shown in SEQ ID NO: 57. In some embodiments, the gRNA that can be used to target the NR4A3 gene consists of the sequence shown in SEQ ID NO: 57.In some embodiments, the gRNA that can be used to target the NR4A3 gene consists essentially of the sequence shown in SEQ ID NO: 57. In some embodiments, 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: 58. In some embodiments, the gRNA that can be used to target the NR4A3 gene comprises the sequence shown in SEQ ID NO: 58. In some embodiments, the gRNA that can be used to target the NR4A3 gene consists of the sequence shown in SEQ ID NO: 58. In some embodiments, the gRNA that can be used to target the NR4A3 gene consists essentially of the sequence shown in SEQ ID NO: 58. In some embodiments, 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: 59. In some embodiments, the gRNA that can be used to target the NR4A3 gene comprises the sequence shown in SEQ ID NO: 59. In some embodiments, the gRNA that can be used to target the NR4A3 gene consists of the sequence shown in SEQ ID NO: 59. In some embodiments, the gRNA that can be used to target the NR4A3 gene consists essentially of the sequence shown in SEQ ID NO: 59. In some embodiments, 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 embodiments, the gRNA that can be used to target the NR4A3 gene comprises the sequence shown in SEQ ID NO: 60. In some embodiments, the gRNA that can be used to target the NR4A3 gene consists of the sequence shown in SEQ ID NO: 60. In some embodiments, the gRNA that can be used to target the NR4A3 gene consists essentially of the sequence shown in SEQ ID NO: 60. In some embodiments, 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: 61. In some embodiments, the gRNA that can be used to target the NR4A3 gene comprises the sequence shown in SEQ ID NO: 61.In some embodiments, the gRNA that can be used to target the NR4A3 gene consists of the sequence shown in SEQ ID NO: 61. In some embodiments, the gRNA that can be used to target the NR4A3 gene consists essentially of the sequence shown in SEQ ID NO: 61. In some embodiments, 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: 62. In some embodiments, the gRNA that can be used to target the NR4A3 gene comprises the sequence shown in SEQ ID NO: 62. In some embodiments, the gRNA that can be used to target the NR4A3 gene consists of the sequence shown in SEQ ID NO: 62. In some embodiments, the gRNA that can be used to target the NR4A3 gene consists essentially of the sequence shown in SEQ ID NO: 62. In some embodiments, 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: 63. In some embodiments, the gRNA that can be used to target the NR4A3 gene comprises the sequence shown in SEQ ID NO: 63. In some embodiments, the gRNA that can be used to target the NR4A3 gene consists of the sequence shown in SEQ ID NO: 63. In some embodiments, the gRNA that can be used to target the NR4A3 gene consists essentially of the sequence shown in SEQ ID NO: 63. In some embodiments, 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: 64. In some embodiments, the gRNA that can be used to target the NR4A3 gene comprises the sequence shown in SEQ ID NO: 64. In some embodiments, the gRNA that can be used to target the NR4A3 gene consists of the sequence shown in SEQ ID NO: 64. In some embodiments, the gRNA that can be used to target the NR4A3 gene consists essentially of the sequence shown in SEQ ID NO: 64. In some embodiments, 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: 65.In some embodiments, the gRNA that can be used to target the NR4A3 gene comprises the sequence shown in SEQ ID NO: 65. In some embodiments, the gRNA that can be used to target the NR4A3 gene consists of the sequence shown in SEQ ID NO: 65. In some embodiments, the gRNA that can be used to target the NR4A3 gene consists essentially of the sequence shown in SEQ ID NO: 65. In some embodiments, 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: 66. In some embodiments, the gRNA that can be used to target the NR4A3 gene comprises the sequence shown in SEQ ID NO: 66. In some embodiments, the gRNA that can be used to target the NR4A3 gene consists of the sequence shown in SEQ ID NO: 66. In some embodiments, the gRNA that can be used to target the NR4A3 gene consists essentially of the sequence shown in SEQ ID NO: 66. In some embodiments, 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: 67. In some embodiments, the gRNA that can be used to target the NR4A3 gene comprises the sequence shown in SEQ ID NO: 67. In some embodiments, the gRNA that can be used to target the NR4A3 gene consists of the sequence shown in SEQ ID NO: 67. In some embodiments, the gRNA that can be used to target the NR4A3 gene consists essentially of the sequence shown in SEQ ID NO: 67. In some embodiments, 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: 68. In some embodiments, the gRNA that can be used to target the NR4A3 gene comprises the sequence shown in SEQ ID NO: 68. In some embodiments, the gRNA that can be used to target the NR4A3 gene consists of the sequence shown in SEQ ID NO: 68. In some embodiments, the gRNA that can be used to target the NR4A3 gene consists essentially of the sequence shown in SEQ ID NO: 68.In some embodiments, 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: 69. In some embodiments, the gRNA that can be used to target the NR4A3 gene comprises the sequence shown in SEQ ID NO: 69. In some embodiments, the gRNA that can be used to target the NR4A3 gene consists of the sequence shown in SEQ ID NO: 69. In some embodiments, the gRNA that can be used to target the NR4A3 gene consists essentially of the sequence shown in SEQ ID NO: 69. In some embodiments, 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: 70. In some embodiments, the gRNA that can be used to target the NR4A3 gene is shown in SEQ ID NO: 70. It includes the sequences to be used. In some embodiments, the gRNA that can be used to target the NR4A3 gene consists of the sequence shown in SEQ ID NO: 70. In some embodiments, the gRNA that can be used to target the NR4A3 gene consists essentially of the sequence shown in SEQ ID NO: 70. In some embodiments, the gRNA that can be used to target the NR4A3 gene contains, consists of, or consists essentially of the sequence shown in SEQ ID NO: 71. In some embodiments, the gRNA that can be used to target the NR4A3 gene contains the sequence shown in SEQ ID NO: 71. In some embodiments, the gRNA that can be used to target the NR4A3 gene consists of the sequence shown in SEQ ID NO: 71. In some embodiments, the gRNA that can be used to target the NR4A3 gene consists essentially of the sequence shown in SEQ ID NO: 71. In some embodiments, the gRNA that can be used to target the NR4A3 gene contains, consists of, or consists essentially of the sequence shown in SEQ ID NO: 72. In some embodiments, the gRNA that can be used to target the NR4A3 gene contains the sequence shown in SEQ ID NO: 72. In some embodiments, the gRNA that can be used to target the NR4A3 gene consists of the sequence shown in SEQ ID NO: 72. In some embodiments, the gRNA that can be used to target the NR4A3 gene consists essentially of the sequence shown in SEQ ID NO: 72. In some embodiments, the gRNA that can be used to target the NR4A3 gene contains, consists of, or consists essentially of the sequence shown in SEQ ID NO: 73. In some embodiments, the gRNA that can be used to target the NR4A3 gene contains the sequence shown in SEQ ID NO: 73. In some embodiments, the gRNA that can be used to target the NR4A3 gene consists of the sequence shown in SEQ ID NO: 73. In some embodiments, the gRNA that can be used to target the NR4A3 gene consists essentially of the sequence shown in SEQ ID NO: 73. In some embodiments, the gRNA that can be used to target the NR4A3 gene contains, consists of, or consists essentially of the sequence shown in SEQ ID NO: 74.In some embodiments, the gRNA that can be used to target the NR4A3 gene comprises the sequence shown in SEQ ID NO: 74. In some embodiments, the gRNA that can be used to target the NR4A3 gene consists of the sequence shown in SEQ ID NO: 74. In some embodiments, the gRNA that can be used to target the NR4A3 gene consists essentially of the sequence shown in SEQ ID NO: 74. In some embodiments, 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: 75. In some embodiments, the gRNA that can be used to target the NR4A3 gene comprises the sequence shown in SEQ ID NO: 75. In some embodiments, the gRNA that can be used to target the NR4A3 gene consists of the sequence shown in SEQ ID NO: 75. In some embodiments, the gRNA that can be used to target the NR4A3 gene consists essentially of the sequence shown in SEQ ID NO: 75. In some embodiments, 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: 76. In some embodiments, the gRNA that can be used to target the NR4A3 gene comprises the sequence shown in SEQ ID NO: 76. In some embodiments, the gRNA that can be used to target the NR4A3 gene consists of the sequence shown in SEQ ID NO: 76. In some embodiments, the gRNA that can be used to target the NR4A3 gene consists essentially of the sequence shown in SEQ ID NO: 76. In some embodiments, 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: 77. In some embodiments, the gRNA that can be used to target the NR4A3 gene comprises the sequence shown in SEQ ID NO: 77. In some embodiments, the gRNA that can be used to target the NR4A3 gene consists of the sequence shown in SEQ ID NO: 77. In some embodiments, the gRNA that can be used to target the NR4A3 gene consists essentially of the sequence shown in SEQ ID NO: 77.In some embodiments, 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: 78. In some embodiments, the gRNA that can be used to target the NR4A3 gene comprises the sequence shown in SEQ ID NO: 78. In some embodiments, the gRNA that can be used to target the NR4A3 gene consists of the sequence shown in SEQ ID NO: 78. In some embodiments, the gRNA that can be used to target the NR4A3 gene consists essentially of the sequence shown in SEQ ID NO: 78. In some embodiments, 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: 79. In some embodiments, the gRNA that can be used to target the NR4A3 gene comprises the sequence shown in SEQ ID NO: 79. In some embodiments, the gRNA that can be used to target the NR4A3 gene consists of the sequence shown in SEQ ID NO: 79. In some embodiments, the gRNA that can be used to target the NR4A3 gene consists essentially of the sequence shown in SEQ ID NO: 79. In some embodiments, 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: 80. In some embodiments, the gRNA that can be used to target the NR4A3 gene comprises the sequence shown in SEQ ID NO: 80. In some embodiments, the gRNA that can be used to target the NR4A3 gene consists of the sequence shown in SEQ ID NO: 80. In some embodiments, the gRNA that can be used to target the NR4A3 gene consists essentially of the sequence shown in SEQ ID NO: 80. In some embodiments, 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: 81. In some embodiments, the gRNA that can be used to target the NR4A3 gene comprises the sequence shown in SEQ ID NO: 81. In some embodiments, the gRNA that can be used to target the NR4A3 gene consists of the sequence shown in SEQ ID NO: 81.In some embodiments, the gRNA that can be used to target the NR4A3 gene consists essentially of the sequence shown in SEQ ID NO: 81. In some embodiments, 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: 82. In some embodiments, the gRNA that can be used to target the NR4A3 gene comprises the sequence shown in SEQ ID NO: 82. In some embodiments, the gRNA that can be used to target the NR4A3 gene consists of the sequence shown in SEQ ID NO: 82. In some embodiments, the gRNA that can be used to target the NR4A3 gene consists essentially of the sequence shown in SEQ ID NO: 82. In some embodiments, 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: 83. In some embodiments, the gRNA that can be used to target the NR4A3 gene comprises the sequence shown in SEQ ID NO: 83. In some embodiments, the gRNA that can be used to target the NR4A3 gene consists of the sequence shown in SEQ ID NO: 83. In some embodiments, the gRNA that can be used to target the NR4A3 gene consists essentially of the sequence shown in SEQ ID NO: 83. In some embodiments, 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: 84. In some embodiments, the gRNA that can be used to target the NR4A3 gene comprises the sequence shown in SEQ ID NO: 84. In some embodiments, the gRNA that can be used to target the NR4A3 gene consists of the sequence shown in SEQ ID NO: 84. In some embodiments, the gRNA that can be used to target the NR4A3 gene consists essentially of the sequence shown in SEQ ID NO: 84. In some embodiments, 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: 85. In some embodiments, the gRNA that can be used to target the NR4A3 gene comprises the sequence shown in SEQ ID NO: 85.In some embodiments, the gRNA that can be used to target the NR4A3 gene consists of the sequence shown in SEQ ID NO: 85. In some embodiments, the gRNA that can be used to target the NR4A3 gene consists essentially of the sequence shown in SEQ ID NO: 85. In some embodiments, 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: 86. In some embodiments, the gRNA that can be used to target the NR4A3 gene comprises the sequence shown in SEQ ID NO: 86. In some embodiments, the gRNA that can be used to target the NR4A3 gene consists of the sequence shown in SEQ ID NO: 86. In some embodiments, the gRNA that can be used to target the NR4A3 gene consists essentially of the sequence shown in SEQ ID NO: 86. In some embodiments, 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: 87. In some embodiments, the gRNA that can be used to target the NR4A3 gene comprises the sequence shown in SEQ ID NO: 87. In some embodiments, the gRNA that can be used to target the NR4A3 gene consists of the sequence shown in SEQ ID NO: 87. In some embodiments, the gRNA that can be used to target the NR4A3 gene consists essentially of the sequence shown in SEQ ID NO: 87. In some embodiments, 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: 88. In some embodiments, the gRNA that can be used to target the NR4A3 gene comprises the sequence shown in SEQ ID NO: 88. In some embodiments, the gRNA that can be used to target the NR4A3 gene consists of the sequence shown in SEQ ID NO: 88. In some embodiments, the gRNA that can be used to target the NR4A3 gene consists essentially of the sequence shown in SEQ ID NO: 88. In some embodiments, 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: 89.In some embodiments, the gRNA that can be used to target the NR4A3 gene comprises the sequence shown in SEQ ID NO: 89. In some embodiments, the gRNA that can be used to target the NR4A3 gene consists of the sequence shown in SEQ ID NO: 89. In some embodiments, the gRNA that can be used to target the NR4A3 gene consists essentially of the sequence shown in SEQ ID NO: 89. In some embodiments, 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: 90. In some embodiments, the gRNA that can be used to target the NR4A3 gene comprises the sequence shown in SEQ ID NO: 90. In some embodiments, the gRNA that can be used to target the NR4A3 gene consists of the sequence shown in SEQ ID NO: 90. In some embodiments, the gR that can be used to target the NR4A3 gene. NA consists essentially of the sequence shown in SEQ ID NO: 90. In some embodiments, 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: 91. In some embodiments, the gRNA that can be used to target the NR4A3 gene comprises the sequence shown in SEQ ID NO: 91. In some embodiments, the gRNA that can be used to target the NR4A3 gene consists of the sequence shown in SEQ ID NO: 91. In some embodiments, the gRNA that can be used to target the NR4A3 gene consists essentially of the sequence shown in SEQ ID NO: 91. In some embodiments, 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: 92. In some embodiments, the gRNA that can be used to target the NR4A3 gene comprises the sequence shown in SEQ ID NO: 92. In some embodiments, the gRNA that can be used to target the NR4A3 gene consists of the sequence shown in SEQ ID NO: 92. In some embodiments, the gRNA that can be used to target the NR4A3 gene consists essentially of the sequence shown in SEQ ID NO: 92. In some embodiments, 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: 93. In some embodiments, the gRNA that can be used to target the NR4A3 gene comprises the sequence shown in SEQ ID NO: 93. In some embodiments, the gRNA that can be used to target the NR4A3 gene consists of the sequence shown in SEQ ID NO: 93. In some embodiments, the gRNA that can be used to target the NR4A3 gene consists essentially of the sequence shown in SEQ ID NO: 93. In some embodiments, 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: 94. In some embodiments, the gRNA that can be used to target the NR4A3 gene comprises the sequence shown in SEQ ID NO: 94. In some embodiments, the gRNA that can be used to target the NR4A3 gene consists of the sequence shown in SEQ ID NO: 94.In some embodiments, the gRNA that can be used to target the NR4A3 gene consists essentially of the sequence shown in SEQ ID NO: 94. In some embodiments, 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: 95. In some embodiments, the gRNA that can be used to target the NR4A3 gene comprises the sequence shown in SEQ ID NO: 95. In some embodiments, the gRNA that can be used to target the NR4A3 gene consists of the sequence shown in SEQ ID NO: 95. In some embodiments, the gRNA that can be used to target the NR4A3 gene consists essentially of the sequence shown in SEQ ID NO: 95. In some embodiments, 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: 96. In some embodiments, the gRNA that can be used to target the NR4A3 gene comprises the sequence shown in SEQ ID NO: 96. In some embodiments, the gRNA that can be used to target the NR4A3 gene consists of the sequence shown in SEQ ID NO: 96. In some embodiments, the gRNA that can be used to target the NR4A3 gene consists essentially of the sequence shown in SEQ ID NO: 96. In some embodiments, 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: 97. In some embodiments, the gRNA that can be used to target the NR4A3 gene comprises the sequence shown in SEQ ID NO: 97. In some embodiments, the gRNA that can be used to target the NR4A3 gene consists of the sequence shown in SEQ ID NO: 97. In some embodiments, the gRNA that can be used to target the NR4A3 gene consists essentially of the sequence shown in SEQ ID NO: 97. In some embodiments, 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: 98. In some embodiments, the gRNA that can be used to target the NR4A3 gene comprises the sequence shown in SEQ ID NO: 98.In some embodiments, the gRNA that can be used to target the NR4A3 gene consists of the sequence shown in SEQ ID NO: 98. In some embodiments, the gRNA that can be used to target the NR4A3 gene consists essentially of the sequence shown in SEQ ID NO: 98. In some embodiments, 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: 99. In some embodiments, the gRNA that can be used to target the NR4A3 gene comprises the sequence shown in SEQ ID NO: 99. In some embodiments, the gRNA that can be used to target the NR4A3 gene consists of the sequence shown in SEQ ID NO: 99. In some embodiments, the gRNA that can be used to target the NR4A3 gene consists essentially of the sequence shown in SEQ ID NO: 99.

[0274] As described herein, in some embodiments, the gene editing method may further comprise reducing the levels of (i) the NR4A1 gene and / or the NR4A1 protein, (ii) the NR4A2 gene and / or the NR4A2 protein, or (iii) both (i) and (ii). In some embodiments, the gRNA that can be used to target the NR4A1 gene comprises, consists of, or consists essentially of the sequence shown in SEQ ID NO: 25. In some embodiments, the gRNA that can be used to target the NR4A1 gene comprises the sequence shown in SEQ ID NO: 25. In some embodiments, the gRNA that can be used to target the NR4A1 gene consists of the sequence shown in SEQ ID NO: 25. In some embodiments, the gRNA that can be used to target the NR4A1 gene consists essentially of the sequence shown in SEQ ID NO: 25. In some embodiments, the gRNA that can be used to target the NR4A1 gene comprises, consists of, or consists essentially of the sequence shown in SEQ ID NO: 26. In some embodiments, the gRNA that can be used to target the NR4A1 gene comprises the sequence shown in SEQ ID NO: 26. In some embodiments, the gRNA that can be used to target the NR4A1 gene consists of the sequence shown in SEQ ID NO: 26. In some embodiments, the gRNA that can be used to target the NR4A1 gene consists essentially of the sequence shown in SEQ ID NO: 26. In some embodiments, the gRNA that can be used to target the NR4A2 gene comprises, consists of, or consists essentially of the sequence shown in SEQ ID NO: 27. In some embodiments, the gRNA that can be used to target the NR4A2 gene comprises the sequence shown in SEQ ID NO: 27. In some embodiments, the gRNA that can be used to target the NR4A1 gene consists of the sequence shown in SEQ ID NO: 27. In some embodiments, the gRNA that can be used to target the NR4A1 gene consists essentially of the sequence shown in SEQ ID NO: 27. In some embodiments, the gRNA that can be used to target the NR4A2 gene comprises, consists of, or consists essentially of the sequence shown in SEQ ID NO: 28.In some embodiments, the gRNA that can be used to target the NR4A2 gene comprises the sequence shown in SEQ ID NO: 28. In some embodiments, the gRNA that can be used to target the NR4A1 gene consists of the sequence shown in SEQ ID NO: 28. In some embodiments, the gRNA that can be used to target the NR4A1 gene consists essentially of the sequence shown in SEQ ID NO: 28. In some embodiments, the gRNA that can be used to target the NR4A2 gene comprises, consists of, or consists essentially of the sequence shown in SEQ ID NO: 29. In some embodiments, the gRNA that can be used to target the NR4A2 gene comprises the sequence shown in SEQ ID NO: 29. In some embodiments, the gRNA that can be used to target the NR4A1 gene consists of the sequence shown in SEQ ID NO: 29. In some embodiments, the gRNA that can be used to target the NR4A1 gene consists essentially of the sequence shown in SEQ ID NO: 29.

[0275] 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, together in a cell in vitro (e.g., adding a nucleic acid (s) encoding the gRNA and / or Cas protein, or gRNA(s) and / or Cas9 protein(s) to the cells in culture) or contacting the cell in vivo or ex vivo.

[0276] The step of contacting the NR4A3 gene target sequence with at least one gRNA and at least one Cas protein disclosed herein, such as Cas9 (or at least one nucleic acid encoding them), can be performed in any suitable manner. For example, cells, such as immune cells, can be treated under cell culture conditions. It is understood that the cells contacted with at least one gRNA and at least one Cas protein disclosed herein, such as Cas9 protein (or at least one nucleic acid encoding them), can also be contacted simultaneously or subsequently with another agent, such as a vector containing at least one nucleic acid sequence encoding a CAR or TCR. In some embodiments, after the cells are contacted in vitro or ex vivo, the method further includes introducing the cells into a subject, thereby treating or ameliorating the symptoms of a disease or condition, such as cancer.

[0277] For ex vivo methods, the cells can include autologous cells, i.e., immune cells (s) obtained from a subject in need of modifying a target polynucleotide sequence (e.g., the NR4A3 gene) in the cell (s) (i.e., the donor and recipient are the same individual). Autologous cells can have the advantage of avoiding any immune-based rejection of the cells. Alternatively, the cells can be allogeneic, e.g., obtained from a donor. Typically, when the cells are donor-derived, they are from a donor that is sufficiently immunologically compatible with the recipient, i.e., will not be subject to transplant rejection and will reduce or eliminate the need for immunosuppression. In some embodiments, the cells are obtained from a heterologous source, i.e., a non-human mammal that has been genetically engineered to be sufficiently immunologically compatible with the recipient, or the recipient's species. Methods for determining immunological compatibility are known in the art and include tissue typing to assess donor-recipient compatibility for HLA and ABO determinants. See, for example, Transplantation Immunology, Bach and Auchincloss, Eds. (Wiley, John & Sons, Incorporated 1994).

[0278] In some embodiments, the present disclosure provides a method for generating modified immune cells, comprising ex vivo modifying an NR4A3 gene sequence in a cell, such as an immune cell (e.g., a T cell), by contacting the cell with a Cas9 protein (or a nucleic acid encoding such a Cas9 protein) and one gRNA targeting a motif in the NR4A3 gene (e.g., a motif where the gRNA directs the Cas9 protein to the target gene, hybridizes to the target motif, the NR4A3 gene is partially or entirely cleaved, and the cleavage efficiency is from about 10% to about 100%). Non-limiting examples of such gRNAs are provided herein (see, e.g., Tables A, C, and D). As described herein, in some embodiments, the method for generating the modified immune cells described herein comprises modifying the NR4A gene sequence by contacting the cell with a first nucleic acid molecule encoding a Cas9 protein and a second nucleic acid molecule comprising a gRNA targeting one or more members of the NR4A gene family. In some embodiments, the first and nucleic acid molecules are contacted with the cell sequentially. In some embodiments, the first and nucleic acid molecules are contacted with the cell simultaneously. For example, in some embodiments, the 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.

[0279] In some embodiments, the cell is modified (e.g., transfected) with a nucleic acid (e.g., a vector) encoding a CAR or TCR before, after, or simultaneously with the modification step described above.

[0280] In some embodiments, 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%.

[0281] The CRISPR / Cas systems of the present disclosure can use gRNA spacer sequences of various lengths depending on the Cas used, e.g., Cas9. Cas9 from different species must pair with their corresponding gRNAs to form a functional ribonucleoprotein (RNP) complex. That is, chimeric gRNA frames engineered from different bacterial species can have different lengths due to differences in the spacer sequences and the chimeric frame sequences.

[0282] In some embodiments, the gRNA spacer sequence can be at least 18 nucleotides (e.g., 18, 19, 20, 21, or 22 nucleotides) in length. For example, the length of the S. pyogenes gRNA spacer sequence in a gRNA that binds to S. pyogenes Cas9 is 20 nucleotides, whereas the length of the S. aureus gRNA spacer sequence in a gRNA that binds to S. aureus Cas9 is 21 nucleotides. In some embodiments, the gRNA spacer sequence can comprise 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 certain embodiments, the gRNA comprises a spacer sequence consisting of 18-22 (e.g., 20) contiguous nucleotides corresponding to a subsequence of exon 3 of the NR4A3 gene. In some embodiments, the gRNA comprises a spacer sequence consisting of 18-22 (e.g., 20) contiguous nucleotides corresponding to a subsequence of exon 4 of the NR4A3 gene. In some embodiments, the gRNA comprises a spacer sequence consisting of 18-22 (e.g., 20) contiguous nucleotides corresponding to a subsequence of exon 3 or exon 4 of the NR4A3 gene.

[0283] A perfect match between the gRNA spacer sequence that binds on the NR4A3 gene and the DNA strand is preferred, but mismatches between the gRNA spacer sequence and the NR4A3 target sequence are also tolerated as long as they still result in a reduction in the NR4A3 gene level or a decrease in NR4A3 gene function. An 8-12 nucleotide "seed" sequence on the gRNA that is completely complementary to the target NR4A3 sequence is preferred for proper recognition of the target sequence on the NR4A3 gene. The remainder of the gRNA spacer sequence can contain one or more mismatches.

[0284] Generally, gRNA activity is inversely correlated with the number of mismatches. Preferably, the gRNA spacer sequences of the present disclosure contain less than 7 mismatches. In some embodiments, the gRNA spacer sequence contains 7 mismatches, 6 mismatches, 5 mismatches, 4 mismatches, 3 mismatches, more preferably 2 mismatches, or less, with the NR4A3 gene target sequence, and even more preferably contains no mismatches. The fewer the number of nucleotides in the gRNA, the fewer the number of tolerated mismatches. The binding affinity is thought to depend on the sum of the matching gRNA-DNA combinations.

[0285] The gRNA spacer sequences of the present disclosure can be selected to minimize the off-target effects of the CRISPR / Cas editing system. Thus, in some embodiments, the gRNA spacer sequence is selected to contain at least two mismatches when compared to all other genomic nucleotide sequences in the cell. In some embodiments, the gRNA spacer sequence is selected to contain at least one mismatch when compared to all other genomic nucleotide sequences in the cell. Those skilled in the art will understand that a variety of techniques can be used to select gRNA spacer sequences suitable for minimizing off-target effects (e.g., bioinformatics analysis).

[0286] In some embodiments, the gRNA spacer sequence comprises, consists of, or consists essentially of the spacer sequences of SEQ ID NOs: 31-42.

[0287] In some embodiments, the gRNA spacer sequence comprises, consists of, or consists essentially of a spacer sequence that contains at least 1, 2, 3, 4, or 5 nucleotide mismatches when compared to any one of the DNA sequences of SEQ ID NOs: 31-42.

[0288] In some embodiments, editing efficacy can be increased by targeting multiple positions. Thus, in some embodiments, the methods disclosed herein include using one gRNA that targets a position upstream from exon 1 of the NR4A3 gene. In some embodiments, the methods disclosed herein include using two, three, four, five, six, seven, eight, nine, or ten gRNAs that target a position upstream from exon 1 of the NR4A3 gene. Also, in some embodiments, the methods disclosed herein include using one gRNA that targets a position downstream from exon 4 of the NR4A3 gene. In some embodiments, the methods disclosed herein include using two, three, four, five, six, seven, eight, nine, or ten gRNAs that target a position downstream from exon 4 of the NR4A3 gene.

[0289] In some embodiments, two gRNAs are complementary to and / or hybridize to sequences on the same strand of the NR4A3 gene. In some embodiments, two gRNAs are complementary to and / or hybridize to sequences on opposite strands of the NR4A3 gene. In some embodiments, two gRNAs are not complementary to and / or do not hybridize to sequences on opposite strands of the NR4A3 gene. In some embodiments, two gRNAs are complementary to and / or hybridize to overlapping target motifs of the NR4A3 gene. In some embodiments, two gRNAs are complementary to and / or hybridize to offset target motifs of the NR4A3 gene.

[0290] Generally, the gRNAs of the disclosure can include any variant of their sequence or chemical modification as long as it enables binding to the corresponding Cas protein, e.g., Cas9 protein, to the target sequence and subsequent excision (whole or partial) of the NR4A3 gene.

[0291] In the methods disclosed herein, the Cas proteins used, e.g., Cas9, are endonucleases that cleave nucleic acids, are encoded by CRISPR loci in numerous bacterial genomes, and are involved in type II CRISPR systems. Cas9 proteins are produced by numerous species of bacteria, including Streptococcus pyogenes, Staphylococcus aureus, Streptococcus thermophilus, Neisseria meningitidis, etc. Thus, Cas9 proteins useful for the present disclosure can be derived from any suitable bacteria known in the art. Non-limiting examples of such bacteria include Streptococcus pyogenes, Streptococcus mutans, Streptococcus pneumonia, Staphylococcus aureus, Streptococcus thermophilus, Campylobacter jejuni, Neisseria meningitidis, Pasteurella multocida, Listeria innocua, and Francisella novicida. The methods disclosed herein can be carried out with any Cas9 known in the art. In some embodiments, Cas9 is wild-type Cas9. In some embodiments, Cas9 is a mutant Cas9 or a fusion protein containing a Cas9 moiety having improved enzymatic activity. In some embodiments, the Cas9 nuclease protein is the Cas9 protein of Streptococcus pyogenes.

[0292] Since Cas9 nuclease proteins are typically expressed in bacteria, it may be advantageous to modify their nucleic acid sequences for optimal expression in eukaryotic cells (e.g., mammalian cells) when designing and preparing Cas9 recombinant proteins. Thus, in some embodiments, the nucleic acids encoding Cas9 used in the methods disclosed herein are codon-optimized for expression in eukaryotic cells, e.g., for expression in cells of a human subject in need thereof.

[0293] In some embodiments, the Cas9 protein used in the methods disclosed herein comprises one or more amino acid substitutions or modifications. In some embodiments, the one or more amino acid substitutions comprise conservative amino acid substitutions. In some examples, the substitutions and / or modifications can prevent or reduce proteolysis and / or extend the half-life of the polypeptide in cells. In some embodiments, the Cas9 protein can comprise peptide bond replacements (e.g., urea, thiourea, carbamate, sulfonylurea, etc.). In some embodiments, the Cas9 protein can comprise naturally occurring amino acids. In some embodiments, the Cas9 protein can comprise alternative amino acids (e.g., D-amino acids, beta-amino acids, homocysteine, phosphoserine, etc.). In some embodiments, the Cas9 protein can comprise modifications for including heterologous moieties (e.g., PEGylation, glycosylation, lipidation, acetylation, end-capping, etc.).

[0294] The methods disclosed herein are typically carried out using the Cas9 protein, although in some embodiments, it is contemplated that the Cas protein can be Cas1, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, or Cas8. In some embodiments, the Cas protein is a Cas9 protein or a functional portion thereof from any bacterial species. In some specific embodiments, the Cas9 protein used in the methods disclosed herein is the Cas9 protein of Streptococcus pyogenes or Staphylococcus aureus or a functional portion thereof, or a nucleic acid encoding such Cas9 or a functional portion thereof. Non-limiting examples of other Cas nucleases that can be used are known in the art and are described, for example, in US9,970,001B2; US10,221,398B2; and US2020 / 0190487A1 (each of which is incorporated herein by reference in its entirety). In some embodiments, the Cas nucleases useful for 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 embodiments, the Cas nucleases useful for 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 embodiments, the Cas nucleases useful for the present disclosure include type III Cas proteins. Non-limiting examples include Cas10, Csm2, Cmr5, Csx10, Csx11, and variants thereof. In some embodiments, the Cas nucleases useful for the present disclosure include type IV Cas proteins. Non-limiting examples of such Cas proteins include Csf1. In some embodiments, the Cas nucleases useful for the present disclosure include type V Cas proteins.Non-limiting examples include Cas12, Cas12a (Cpf1), Cas12b (C2c1), Cas12c (C2c3), Cas12d (CasY), Cas12e (CasX), Cas12f (Cas14, C2c10), Cas12g, Cas12h, Cas12i, Cas12k (C2c5), C2c4, C2c8...

Claims

1. A method for reducing the level of the NR4A3 gene and / or NR4A3 protein in immune cells, comprising modifying the immune cells in vitro with a guide RNA (gRNA), wherein the gRNA comprises, essentially or consists of, the sequence shown in any one of SEQ ID NOs: 94, 52, 96, 53, 54, 86, 83, 55, 82, 56, 76, 57, 75, 58, 71, 61, 70, 65, 68, and 67, and after the modification, the level of the NR4A3 gene and / or NR4A3 protein in the immune cells is reduced compared to a reference immune cell (e.g., a corresponding immune cell not modified with the gRNA).

2. The method according to claim 1, further comprising modifying the immune cells to express (a) a reduced level of the NR4A1 gene and / or the NR4A1 protein; (b) a reduced level of the NR4A2 gene and / or the NR4A2 protein; (c) a ligand-binding protein; or (d) any combination of (a) to (c).

3. The method according to claim 1, wherein, after the modification, the immune cells, compared to the reference immune cells, (a) are more resistant to exhaustion, (b) exhibit increased persistence / survival, (c) exhibit increased growth / proliferation in response to persistent antigen stimulation, (d) exhibit increased effector function in response to persistent antigen stimulation, or (e) any combination of (a), (b), (c), and (d).

4. The method according to claim 3, wherein the effector function comprises (i) the ability to kill target cells (e.g., tumor cells), (ii) the ability to generate cytokines by further antigen stimulation, or (iii) both (i) and (ii).

5. A composition comprising or essentially comprising immune cells expressing a reduced level of the NR4A3 gene and / or NR4A3 protein, wherein the immune cells are modified with a guide RNA (gRNA), the gRNA comprising or essentially comprising the sequence shown in any one of SEQ ID NOs: 94, 52, 96, 53, 54, 86, 83, 55, 82, 56, 76, 57, 75, 58, 71, 61, 70, 65, 68, and 67.

6. The composition according to claim 5, wherein the immune cells are further modified to express (a) a reduced level of the NR4A1 gene and / or the NR4A1 protein; (b) a reduced level of the NR4A2 gene and / or the NR4A2 protein; (c) a ligand-binding protein; or (d) any combination of (a) to (c).

7. The composition according to claim 5, for treating tumors in subjects requiring treatment of tumors.

8. The composition according to claim 7, wherein the tumor is derived from cancer including 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, digestive tract cancer, ovarian cancer, cervical cancer, carcinoma, sarcoma, leukemia, lymphoma, myeloma, or a combination thereof.

9. The composition according to claim 7, which is suitable for administration in combination with additional therapeutic agents.

10. The composition according to claim 9, wherein the additional therapeutic agent comprises a chemotherapeutic agent, targeted anticancer therapy, oncolytic drug, cytotoxic agent, immune-based therapy, cytokine, surgical procedure, radiotherapy, activator of costimulatory molecules, immune checkpoint inhibitor, vaccine, cellular immunotherapy, or any combination thereof.

11. The method according to claim 2 or the composition according to claim 6, wherein the ligand-binding protein comprises 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.

12. The ligand-binding proteins mentioned above are CD19, TRAC, TCRβ, BCMA, CLL-1, CS1, CD38, CD19, TSHR, CD123, CD22, CD30, CD70, CD171, CD33, EGFRvIII, GD2, GD3, TnAg, PSMA, ROR1, ROR2, GPC1, GPC2, FLT3, FAP, TAG72, CD44v6, CEA, EPCAM, B7H3, KIT, IL-13Ra2, mesothelin, and IL-13Ra2. lRa, PSCA, PRSS21, VEGFR2, Lewis Y, CD24, PDGFR-beta, SSEA-4, CD20, folate receptor alpha, ERBB2 (Her2 / neu), MUC1, MUC16, EGFR, NCAM, prostase, PAP, ELF2M, ephrin B2, IGF-I receptor, CAIX, LMP2, gplOO, bcr-abl, tyrosinase, EphA2, fucosyl GM1, sLe, GM3, TGS5, HMWMAA, 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, WTl, NY-ESO-1, LAGE-la, MAGE-Al, Regmine, HPV E6,E7, MAGE Al, ETV6-AML, sperm protein 17, XAGE1, Tie2, MAD-CT-1, MAD-CT-2, Fos-related antigen 1, p53, p53 variant, prostain, sulbibin and telomerase, PCTA-1 / galectin 8, melan A / MARTl, Ras variant, hTERT, sarcoma translocation breakpoint, ML-IAP, ERG (TMPRSS2) The method or composition according to claim 11, which is capable of specifically binding to an antigen selected from 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, or any combination thereof.

13. The method according to any one of claims 1 to 4 or the composition according to any one of claims 5 to 10, wherein the immune cells include lymphocytes, neutrophils, monocytes, macrophages, dendritic cells, or a combination thereof.

14. The method or composition according to claim 13, wherein the lymphocytes include T cells, tumor-infiltrating lymphocytes (TILs), lymphokine-activated killer cells, natural killer (NK) cells, or a combination thereof.

15. A guide RNA (gRNA) comprising CRISPR RNA (crRNA) and transactivated crRNA (tracrRNA), wherein the crRNA consists of a sequence represented by any one of SEQ ID NOs: 94, 52, 96, 53, 54, 86, 83, 55, 82, 56, 76, 57, 75, 58, 71, 61, 70, 65, 68, and 67.

Images