Compositions and methods for overcoming t-cell exhaustion

Abstract

The present invention provides methods and compositions for treating cancer in a subject comprising the use of therapeutic cells and compositions to prevent or reduce T cell exhaustion. Modified hematopoietic cells comprising a genomic modification in an enhancer surround the GAB3 gene are also provided.

Description

TITLE OF THE INVENTIONCOMPOSITIONS AND METHODS FOR OVERCOMING T CELL EXHAUSTIONCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the priority of U.S. Provisional Appl. Ser. No. 63 / 672,315, filed July 17, 2024, the entire disclosure of which is incorporated herein by reference.INCORPORATION OF SEQUENCE LISTING

[0002] A sequence listing containing the file named “MDCC019WO_ST26.xml” which is 308,437 bytes (measured in MS-Windows®) and created on July 13, 2025, and comprises 59 sequences, is incorporated herein by reference in its entirety.FIELD OF THE INVENTION

[0003] The present disclosure relates to the field of cancer therapy, and more specifically to compositions and methods for overcoming T cell exhaustion.BACKGROUND OF THE INVENTION

[0004] Adoptive cellular therapy (ACT) is a promising form of immunotherapy that involves ex vivo isolation and expansion of antigen- specific T cells for infusion. Adoptive T cell therapy produces a long-lasting response in only a small subgroup of cancer patients. One significant hurdle to achieving effective and long-term results is the limited cytotoxicity and persistence of the transferred T cells due to progressive exhaustion. This leads to high rates of relapse. As observed in patients who have received CAR-T cell based treatments, relapse rates exceed 80% at the 6-month mark. It has further been observed that exhausted T cells hinder the effectiveness of the immune response following immune checkpoint therapy. Methods and compositions that prevent or reverse T cell exhaustion remain an important unmet need in the art. The present disclosure provides such methods and compositions for preventing or reversing T cell exhaustion.SUMMARY OF THE INVENTION

[0005] In one aspect, the present disclosure provides a modified hematopoietic cell comprising at least one genomic modification in a nucleotide sequence having at least about 85% sequence identity to SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, or SEQ ID NO: 10. In one embodiment, the genomic modification alters the expression level or activity of at least one protein selected from the group consisting of GAB3, LAGE3, FAM50A, UBL4A, GD11, SLC10A3, G6PD, CTAG1A, TP6AP1, PLXNA3, FAM3A, IKBKG, CTAG1B, CTAG2, DKC1, MeCP2, EMD, and TAZ. In another embodiment, the genomic modification decreases the expression level or activity of at least one protein selected from the group consisting of GAB3, LAGE3, FAM50A, UBL4A, GDI1 , SLC10A3, G6PD, CTAG1A, TP6AP1, PLXNA3, FAM3A, IKBKG, CTAG1B, CTAG2, DKC1, MeCP2, EMD, and TAZ. In yet another embodiment, the genomic modification increases the expression level or activity of at least one protein selected from the group consisting of LAGE3, FAM50A, UBL4A, GDI1, SLC10A3, G6PD, CTAG1A, TP6AP1, PLXNA3, FAM3A, IKBKG, CTAG1B, CTAG2, DKC1, MeCP2, EMD, and TAZ. In still yet another embodiment, the modified hematopoietic cell is selected from the group consisting of a T cell, a CD4+ T cell, a CD8+ T cell, a CAR T cell, a tumor-infiltrating lymphocyte, a T cell expressing an engineered TCR, an endogenous T cell, a monocyte, a granulocyte, and an NK cell. The hematopoietic cell, in one embodiment, comprises at least one genomic modification in a nucleotide sequence having at least about 90%, at least about 95%, at least about 99%, or 100% sequence identity to SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, or SEQ ID NO: 10. The hematopoietic cell, in another embodiment, comprises at least two genomic modifications in at least two nucleotide sequences having at least about 85% sequence identity to SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, or SEQ ID NO: 10. The genomic modification, in yet another embodiment, comprises a deletion, an insertion, a substitution, an inversion, a duplication, or a combination of any thereof. The hematopoietic cell, in still yet another embodiment, is heterozygous for the genomic modification. In one embodiment, the hematopoietic cell is homozygous for the genomic modification. In another embodiment, the hematopoietic cell is a T cell, and the genomic modification decreases or eliminates the development of T cell exhaustion. In yet another embodiment, the modifiedhematopoietic cell further comprises at least one genomic modification of an endogenous gene selected from the group consisting of GAB3, LAGE3, FAM50A, UBL4A, GDI1, SLC10A3, G6PD, CTAG1A, TP6AP1, PLXNA3, FAM3A, IKBKG, CTAG1B, CTAG2, DKC1, MeCP2, EMD, and TAZ. In still yet another embodiment, the GAB3 gene encodes a polypeptide having at least about 85% sequence identity to SEQ ID NO:21, the LAGE3 gene encodes a polypeptide having at least about 85% sequence identity to SEQ ID NO:22, the FAM50A gene encodes a polypeptide having at least about 85% sequence identity to SEQ ID NO:23, the UBL4A gene encodes a polypeptide having at least about 85% sequence identity to SEQ ID NO:24, the GDI1 gene encodes a polypeptide having at least about 85% sequence identity to SEQ ID NO:25, the SEC10A3 gene encodes a polypeptide having at least about 85% sequence identity to SEQ ID NO:26, the G6PD gene encodes a polypeptide having at least about 85% sequence identity to SEQ ID NO:27, the CTAG1A gene encodes a polypeptide having at least about 85% sequence identity to SEQ ID NO:28, the TP6AP1 gene encodes a polypeptide having at least about 85% sequence identity to SEQ ID NO:29, the PLXNA3 gene encodes a polypeptide having at least about 85% sequence identity to SEQ ID NO:30, the FAM3A gene encodes a polypeptide having at least about 85% sequence identity to SEQ ID NO:31, the IKBKG gene encodes a polypeptide having at least about 85% sequence identity to SEQ ID NO:32, the CTAG1B gene encodes a polypeptide having at least about 85% sequence identity to SEQ ID NO:33, the CTAG2 gene encodes a polypeptide having at least about 85% sequence identity to SEQ ID NO:34, the DKC1 gene encodes a polypeptide having at least about 85% sequence identity to SEQ ID NO:35, the MeCP2 gene encodes a polypeptide having at least about 85% sequence identity to SEQ ID NO:54, the EMD gene encodes a polypeptide having at least about 85% sequence identity to SEQ ID NO:55, or the TAZ gene encodes a polypeptide having at least about 85% sequence identity to SEQ ID NO:56. In one embodiment, the GAB3 gene comprises a sequence having at least about 85% sequence identity to SEQ ID NO:36, the LAGE3 gene comprises a sequence having at least about 85% sequence identity to SEQ ID NO:37, the FAM50A gene comprises a sequence having at least about 85% sequence identity to SEQ ID NO:38, the UBL4A gene comprises a sequence having at least about 85% sequence identity to SEQ ID NO:39, the GDI1 gene comprises a sequence having at least about 85% sequence identity to SEQ ID NO:40, the SLC10A3 gene comprises a sequence having at least about 85% sequence identity to SEQ ID NO:41, the G6PD gene comprises a sequence having at least about 85% sequence identity to SEQ ID NO:42, the CTAG1A gene comprises asequence having at least about 85% sequence identity to SEQ ID NO:43, the TP6AP1 gene comprises a sequence having at least about 85% sequence identity to SEQ ID NO:44, the PLXNA3 gene comprises a sequence having at least about 85% sequence identity to SEQ ID NO:45, the FAM3A gene comprises a sequence having at least about 85% sequence identity to SEQ ID NO:46, the IKBKG gene comprises a sequence having at least about 85% sequence identity to SEQ ID NO:47, the CTAG1B gene comprises a sequence having at least about 85% sequence identity to SEQ ID NO:48, the CTAG2 gene comprises a sequence having at least about 85% sequence identity to SEQ ID NO:49, the DKC1 gene comprises a sequence having at least about 85% sequence identity to SEQ ID NQ:50, the MeCP2 gene comprises a sequence having at least about 85% sequence identity to SEQ ID NO:57, the EMD gene comprises a sequence having at least about 85% sequence identity to SEQ ID NO:58, or the TAZ gene comprises a sequence having at least about 85% sequence identity to SEQ ID NO:59. The genomic modification, in yet another embodiment, alters the expression level or activity of at least one protein selected from the group consisting of GAB3, LAGE3, FAM50A, UBL4A, GDI1, SLC10A3, G6PD, CTAG1A, TP6AP1, PLXNA3, FAM3A, IKBKG, CTAG1B, CTAG2, DKC1, MeCP2, EMD, and TAZ.

[0006] In another aspect, the present disclosure provides a method of treating cancer in a subject in need thereof, the method comprising administering a modified hematopoietic cell of the present disclosure to the subject. In one embodiment, the cancer is selected from the group consisting of lung cancer, colorectal cancer, pancreatic cancer, appendiceal cancer, small bowel adenocarcinoma, hepatobiliary cancer, gynecological cancer, hematopoietic cancer, breast cancer, bladder cancer, prostate cancer, skin cancer, head and neck squamous cell carcinoma, melanoma, and genitourinary cancer. In another embodiment, the methods of the present disclosure may further comprise administering a second therapy to the subject. In yet another embodiment, the second therapy is selected from the group consisting of a chemotherapy, a radiotherapy, an immunotherapy, and a surgery. The subject, in still yet another embodiment, is a mammalian subject. The subject, in one embodiment, is a human subject.

[0007] In yet another aspect, the present disclosure provides a method for producing a modified hematopoietic cell, the method comprising introducing into a hematopoietic cell at least one genomic modification into at least one target site of a nucleotide sequence comprising at least about 85% sequence identity to SEQ ID NO:1, SEQ ID NO:2, SEQ ID NOG, SEQ ID NO:4, SEQ ID NOG, SEQ ID NO:6, SEQ ID NOG, SEQ ID NOG, SEQ ID NO:9, SEQ ID NO: 10, SEQ IDNO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID N0:41 , SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:57, SEQ ID NO:58, or SEQ ID NO:59. Introducing the genomic modification, in one embodiment, comprises introducing a sitespecific nuclease into the hematopoietic cell, wherein the site-specific nuclease is capable of specifically binding to and cleaving the target site. The site-specific nuclease, in another embodiment, is selected from the group consisting of an RNA-guided nuclease, a zinc finger nuclease, and a TALEN. The site-specific nuclease, in yet another embodiment, is an RNA-guided nuclease. In still yet another embodiment, the methods of the present disclosure may further comprise introducing into the hematopoietic cell at least one guide polynucleotide molecule comprising a nucleotide sequence that is substantially complementary to the target site, wherein the guide polynucleotide and the RNA-guided nuclease form a complex that is capable of specifically binding to and cleaving the target site. The guide polynucleotide molecule, in one embodiment, comprises a nucleotide sequence having at least about 85%, at least about 90%, or at least about 95% sequence identity to SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO:20, SEQ ID NO:51, SEQ ID NO:52, or SEQ ID NO:53.

[0008] In still yet another aspect, the present disclosure provides a guide polynucleotide molecule comprising a nucleotide sequence having at least about 85%, at least about 90%, or at least about 95% sequence identity to SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO: 16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:51, SEQ ID NO:52, or SEQ ID NO:53. In one embodiment, the present disclosure provides a composition comprising a guide polynucleotide molecule of the present disclosure. The composition, in yet another embodiment, may further comprise a site-specific nuclease.

[0009] In one aspect, the present disclosure provides a modified hematopoietic cell comprising at least one genomic modification that modulates the expression of an endogenous gene selected from the group consisting of LAGE3, FAM50A, UBL4A, GDI1, SEC10A3, G6PD, CTAG1A, TP6AP1, PLXNA3, FAM3A, IKBKG, CTAG1B, CTAG2, DKC1, MeCP2, EMD, and TAZ. In one embodiment, the LAGE3 gene encodes a polypeptide having at least about 85% sequence identity to SEQ ID NO:22, the FAM50A gene encodes a polypeptide having at least about 85% sequence identity to SEQ ID NO:23, the UBL4A gene encodes a polypeptide having at least about 85%sequence identity to SEQ ID NO:24, the GD11 gene encodes a polypeptide having at least about 85% sequence identity to SEQ ID NO:25, the SLC10A3 gene encodes a polypeptide having at least about 85% sequence identity to SEQ ID NO:26, the G6PD gene encodes a polypeptide having at least about 85% sequence identity to SEQ ID NO:27, the CTAG1A gene encodes a polypeptide having at least about 85% sequence identity to SEQ ID NO:28, the TP6AP1 gene encodes a polypeptide having at least about 85% sequence identity to SEQ ID NO:29, the PLXNA3 gene encodes a polypeptide having at least about 85% sequence identity to SEQ ID NO:30, the FAM3A gene encodes a polypeptide having at least about 85% sequence identity to SEQ ID NO:31, the IKBKG gene encodes a polypeptide having at least about 85% sequence identity to SEQ ID NO:32, the CTAG1B gene encodes a polypeptide having at least about 85% sequence identity to SEQ ID NO:33, the CTAG2 gene encodes a polypeptide having at least about 85% sequence identity to SEQ ID NO:34, the DKC1 gene encodes a polypeptide having at least about 85% sequence identity to SEQ ID NO:35, the MeCP2 gene encodes a polypeptide having at least about 85% sequence identity to SEQ ID NO:54, the EMD gene encodes a polypeptide having at least about 85% sequence identity to SEQ ID NO:55, or the TAZ gene encodes a polypeptide having at least about 85% sequence identity to SEQ ID NO:56. In another embodiment, the LAGE3 gene comprises a sequence having at least about 85% sequence identity to SEQ ID NO:37, the FAM50A gene comprises a sequence having at least about 85% sequence identity to SEQ ID NO:38, the UBE4A gene comprises a sequence having at least about 85% sequence identity to SEQ ID NO:39, the GDI1 gene comprises a sequence having at least about 85% sequence identity to SEQ ID NO:40, the SLC10A3 gene comprises a sequence having at least about 85% sequence identity to SEQ ID NO:41, the G6PD gene comprises a sequence having at least about 85% sequence identity to SEQ ID NO:42, the CTAG1A gene comprises a sequence having at least about 85% sequence identity to SEQ ID NO:43, the TP6AP1 gene comprises a sequence having at least about 85% sequence identity to SEQ ID NO:44, the PLXNA3 gene comprises a sequence having at least about 85% sequence identity to SEQ ID NO:45, the FAM3A gene comprises a sequence having at least about 85% sequence identity to SEQ ID NO:46, the IKBKG gene comprises a sequence having at least about 85% sequence identity to SEQ ID NO:47, the CTAG1B gene comprises a sequence having at least about 85% sequence identity to SEQ ID NO:48, the CTAG2 gene comprises a sequence having at least about 85% sequence identity to SEQ ID NO:49, the DKC1 gene comprises a sequence having at least about 85% sequence identity to SEQ ID NO:50, the MeCP2 genecomprises a sequence having at least about 85% sequence identity to SEQ ID NO:57, the EMD gene comprises a sequence having at least about 85% sequence identity to SEQ ID NO:58, or the TAZ gene comprises a sequence having at least about 85% sequence identity to SEQ ID NO:59. In yet another embodiment, the genomic modification alters the expression level or activity of at least one protein selected from the group consisting of LAGE3, FAM50A, UBL4A, GDI1, SLC10A3, G6PD, CTAG1A, TP6AP1, PLXNA3, FAM3A, IKBKG, CTAG1B, CTAG2, DKC1, MeCP2, EMD, and TAZ. The modified hematopoietic cell, in still yet another embodiment, may comprise at least one genomic modification of an endogenous GAB3 gene. In one embodiment, the GAB3 gene encodes a polypeptide having at least about 85% sequence identity to SEQ ID NO:21. In another embodiment, the GAB3 gene comprises a sequence having at least about 85% sequence identity to SEQ ID NO:36. In yet another embodiment, the modified hematopoietic cell is selected from the group consisting of a T cell, a CD4+ T cell, a CD8+ T cell, a CAR T cell, a tumor-infiltrating lymphocyte, a T cell expressing an engineered TCR, an endogenous T cell, a monocyte, a granulocyte, and an NK cell. In still yet another embodiment, the hematopoietic cell is a T cell, and the genomic modification decreases or eliminates the development of T cell exhaustion.

[0010] In still yet another aspect, the present disclosure provides a method of increasing expansion of a hematopoietic cell, the method comprising: a) introducing into the hematopoietic cell at least one genomic modification into at least one target site of a nucleotide sequence comprising at least about 85% sequence identity to SEQ ID NO:1, SEQ ID NO:2, SEQ ID NOG, SEQ ID NO:4, SEQ ID NOG, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO: 10, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:57, SEQ ID NO:58, or SEQ ID NO:59; and b) culturing the hematopoietic cell.BRIEF DESCRIPTION OF THE DRAWINGS

[0011] The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present invention. The invention may be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein.

[0012] FIG. 1 is an IGV plot showing EnHubl enhancers in melanoma patients before (pre-) or after (post-) treatment with immune checkpoint inhibitor therapy in responders and non-rcspondcrs (RECIST criteria).

[0013] FIG.2 demonstrates that targeting EnHubl (an enhancer hub surrounding GAB 3) induces tumor cell killing. FIG. 2, Panel A is a schematic of the co-culture tumor cell killing experiment. FIG. 2, Panel B is a graph demonstrating killing of HLA-matched cognate melanoma cells (M562, HLA-matched) by TILs harboring control or EnHubl CRISPRi. Y-axis shows caspase-positive cells, which is a surrogate marker for apoptotic cells. Enhanced toxicity was observed at a higher TIL:MEL ratio for all TILs. Surprisingly, however, TIL3162B and TIL3183 demonstrated more than a three-fold increase in tumor cell killing at the lower 1 : 1 TIL:MEL ratio following inhibition of EnHubl.

[0014] FIG. 3 demonstrates that GAB3 knockout reduces T cell exhaustion. FIG. 3, Panel A shows a western blot demonstrating GAB3 knockout in CD8+T cells. FIG. 3, Panel B shows CD39 and TIGIT expression by FACS in GAB3K0and GAB3WTCD8+T cells subjected to the in vitro T cell exhaustion model.

[0015] FIG. 4 demonstrates that GAB3 regulates the T cell activation response. GAB3KOand GAB3WTCD8+T cells were subjected to an in vitro T cell exhaustion protocol followed by reverse phase protein array (RPPA). Expression of key proteins She (FIG. 4, Panel A), PKM2 (FIG. 4, Panel B), CDK1 (FIG. 4, Panel C), and STING (FIG. 4, Panel D) are shown.

[0016] FIG. 5 demonstrates that EnHubl suppression augments CD8+T cell function and tumor clearance. FIG. 5, Panel A shows a workflow of the experiment and the resulting tumor growth curve. Mice bearing M526 xenograft tumors were injected with TILs (n=5 per group; NO TILs, control (unmodified) TILs, and EnHubl_edited_TILs) on Day 35, followed by intermittent tumor measurement and harvesting on Day 64. EnHubl_edit TILs show significant delay in M526 progression compared to respective controls. FIG. 5, Panel B shows increased intra-tumoral CD8+T cell infiltration upon EnHubl modulation. Flow cytometry analysis of intra-tumoral CD8+T cells in xenografted tumors from NO TIL, Control TIL and EnHubl_edit TIL-injected mice. EnHubl modulation significantly increases CD8 ' T cell accumulation within the tumor microenvironment. ****p <0.0001. FIG. 5, Panel C demonstrates that EnHubl modification promotes effector memory phenotype in CD8+T cells. Percentage of effector memory (CD45RO+)CD8+T cells within tumors across groups. EnHubl -modified TILs exhibit a higher frequency of effector memory phenotype, indicative of enhanced cytotoxic potential. ****p < 0.0001. FIG. 5, Panel D demonstrates reduced exhaustion in intra-tumoral CD8+T cells following EnHubl inhibition. Frequency of exhausted TIGIT+CD8+T cells in tumors from NO TIL, Control TIL and EnHubl_edit TIL groups. A significant reduction in exhausted CD8+T cells is observed in the enhancer-modified group. ****p < 0.0001.

[0017] FIG. 6 demonstrates that GAB3 KO enhances T cell expansion. FIG. 6, Panel A shows fold expansion of control or GAB3 KO tumor-derived T cells in a TIL expansion protocol from a patient biopsy that utilizes low dose IL-2. FIG. 6, Panel B shows co-culture assay results with varying effector (tumor-derived T cells from FIG. 6, Panel A) and cognate tumor cells ratios.BRIEF DESCRIPTION OF THE SEQUENCES

[0018] SEQ ID NO:1 is a representative sequence of the + strand of enhancer El in the enhancer hub surrounding GAB3.

[0019] SEQ ID NO:2 is a representative sequence of the + strand of enhancer E2 in the enhancer hub surrounding GAB3.

[0020] SEQ ID NOG is a representative sequence of the + strand of enhancer E3 in the enhancer hub surrounding GAB3.

[0021] SEQ ID NO:4 is a representative sequence of the + strand of enhancer E4 in the enhancer hub surrounding GAB3.

[0022] SEQ ID NOG is a representative sequence of the + strand of enhancer E5 in the enhancer hub surrounding GAB3.

[0023] SEQ ID NOG is a representative sequence of the + strand of enhancer E6 in the enhancer hub surrounding GAB3.

[0024] SEQ ID NO:7 is a representative sequence of the + strand of enhancer E7 in the enhancer hub surrounding GAB 3.

[0025] SEQ ID NO:8 is a representative sequence of the + strand of enhancer E8 in the enhancer hub surrounding GAB 3.

[0026] SEQ ID NO:9 is a representative sequence of the + strand of enhancer E9 in the enhancer hub surrounding GAB3.

[0027] SEQ ID NO: 10 is a representative sequence of the + strand of enhancer E10 in the enhancer hub surrounding GAB3.

[0028] SEQ ID NO: 11 is a representative sequence of an El targeting gRNA.

[0029] SEQ ID NO: 12 is a representative sequence of an E2 targeting gRNA.

[0030] SEQ ID NO: 13 is a representative sequence of an E3 targeting gRNA.

[0031] SEQ ID NO: 14 is a representative sequence of an E4 targeting gRNA.

[0032] SEQ ID NO: 15 is a representative sequence of an E5 targeting gRNA.

[0033] SEQ ID NO: 16 is a representative sequence of an E6 targeting gRNA.

[0034] SEQ ID NO: 17 is a representative sequence of an E7 targeting gRNA.

[0035] SEQ ID NO: 18 is a representative sequence of an E8 targeting gRNA.

[0036] SEQ ID NO: 19 is a representative sequence of an E9 targeting gRNA.

[0037] SEQ ID NO:20 is a representative sequence of an E10 targeting gRNA.

[0038] SEQ ID NO:21 is a representative amino acid sequence encoded by GAB3.

[0039] SEQ ID NO:22 is a representative amino acid sequence encoded by LAGE3.

[0040] SEQ ID NO:23 is a representative amino acid sequence encoded by FAM50A.

[0041] SEQ ID NO:24 is a representative amino acid sequence encoded by UBL4A.

[0042] SEQ ID NO:25 is a representative amino acid sequence encoded by GDI1.

[0043] SEQ ID NO:26 is a representative amino acid sequence encoded by SLC10A3.

[0044] SEQ ID NO:27 is a representative amino acid sequence encoded by G6PD.

[0045] SEQ ID NO:28 is a representative amino acid sequence encoded by CTAG1A.

[0046] SEQ ID NO:29 is a representative amino acid sequence encoded by TP6AP1.

[0047] SEQ ID NO:30 is a representative amino acid sequence encoded by PLXNA3.

[0048] SEQ ID NO:31 is a representative amino acid sequence encoded by FAM3A.

[0049] SEQ ID NO:32 is a representative amino acid sequence encoded by IKBKG.

[0050] SEQ ID NO:33 is a representative amino acid sequence encoded by CTAG1B.

[0051] SEQ ID NO:34 is a representative amino acid sequence encoded by CTAG2.

[0052] SEQ ID NO:35 is a representative amino acid sequence encoded by DKC1.

[0053] SEQ ID NO:36 is a representative sequence of a GAB 3 gene.

[0054] SEQ ID NO:37 is a representative sequence of a LAGE3 gene.

[0055] SEQ ID NO:38 is a representative sequence of a FAM50A gene.

[0056] SEQ ID NO:39 is a representative sequence of a UBL4A gene.

[0057] SEQ ID NO:40 is a representative sequence of a GDI1 gene.

[0058] SEQ ID NO:41 is a representative sequence of a SLC10A3 gene.

[0059] SEQ ID NO:42 is a representative sequence of a G6PD gene.

[0060] SEQ ID NO:43 is a representative sequence of a CTAG1A gene.

[0061] SEQ ID NO:44 is a representative sequence of a TP6AP1 gene.

[0062] SEQ ID NO:45 is a representative sequence of a PLXNA3 gene.

[0063] SEQ ID NO:46 is a representative sequence of a FAMS A gene.

[0064] SEQ ID NO:47 is a representative sequence of an IKBKG gene.

[0065] SEQ ID NO:48 is a representative sequence of a CTAG1B gene.

[0066] SEQ ID NO:49 is a representative sequence of a CTAG2 gene.

[0067] SEQ ID NO:50 is a representative sequence of a DKC1 gene.

[0068] SEQ ID NO:51 is a representative sequence of a gRNA targeting the enhancer hub surrounding the GAB3 gene.

[0069] SEQ ID NO:52 is a representative sequence of a gRNA targeting the enhancer hub surrounding the GABS gene.

[0070] SEQ ID NO:53 is a representative sequence of a gRNA targeting the enhancer hub surrounding the GAB3 gene.

[0071] SEQ ID NO:54 is a representative amino acid sequence encoded by MeCP2.

[0072] SEQ ID NO:55 is a representative amino acid sequence encoded by EMD.

[0073] SEQ ID NO:56 is a representative amino acid sequence encoded by TAZ.

[0074] SEQ ID NO:57 is a representative sequence of a MeCP2 gene.

[0075] SEQ ID NO:58 is a representative sequence of an EMD gene.

[0076] SEQ ID NO:59 is a representative sequence of a TAZ gene.DETAILED DESCRIPTION OF THE INVENTION

[0077] The present disclosure provides methods and compositions for the treatment of cancer. In particular, the present disclosure provides methods and compositions for preventing exhaustion in cell-based therapies. Adoptive T cell therapy produces a long-lasting response in only a small subgroup of cancer patients. One significant hurdle to achieving effective and long-term results is the limited cytotoxicity and persistence of the transferred T cells due to progressive exhaustion. This leads to high rates of relapse. As observed in patients who have received CAR-T cell based treatments relapse rates exceed 80% at the 6-month mark. It has further been observed that exhausted T cells hinder the effectiveness of the immune response following immune checkpoint therapy. Methods and compositions that prevent or reverse T cell exhaustion remain an important unmet need in the art. The present disclosure provides such methods and compositions for preventing or reversing T cell exhaustion.A. Modified Hematopoietic Cells

[0078] Certain aspects of the present disclosure provide a modified hematopoietic cell comprising at least one genomic modification in a nucleotide sequence having at least about 85% sequence identity to SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, or SEQ ID NO: 10. As described in the brief description of sequences and Table 2, each of SEQ ID NOs:l-10 is an enhancer sequence of the enhancer hub surrounding GAB3. As used herein the term “enhancer” or “enhancer sequence” refers to DNA sequence that regulates the transcription of certain genes. As used herein the term “enhancer hub” refers to a region of DNA that comprises at least two enhancer sequences. In certain embodiments, an enhancer sequence may increase the transcription of certain genes. Infurther embodiments, an enhancer sequence may increase the transcription of certain genes but decrease the transcription of other genes. In other aspects the present disclosure provides a modified hematopoietic cell comprising at least one genomic modification of an endogenous gene selected from the group consisting of GAB3, LAGE3, FAM50A, UBL4A. GDI1, SLC10A3, G6PD. CTAG1A, TP6AP1, PLXNA3. FAM3A, IKBKG. CT AG IB. CTAG2. DKC1, MeCP2, EMD, and TAZ.

[0079] As used herein the term “modified” in the context of a cell genome refers to a cell genome comprising an engineered change in the endogenous sequence of at least one enhancer or gene of interest or comprising at least one engineered change in the expression level of one of more genes. In some embodiments, a modified cell or cell genome may comprise one or more nucleotide deletions, insertions, or substitutions introduced through genome editing or any known mutagenesis technique. In particular embodiments, a modified cell may comprise one or more transgenes. A modified cell of the present disclosure may comprise, in some embodiments, a genomic modification that alters the expression level or activity of at least one protein selected from the group consisting of GAB3, LAGE3, FAM50A, UBL4A, GDI1, SLC10A3, G6PD, CTAG1A, TP6AP1, PLXNA3, FAM3A, IKBKG, CTAG1B, CTAG2, DKC1, MeCP2, EMD, and TAZ.

[0080] As used herein the term “hematopoietic cell” refers to a cell associated with the hematopoietic lineage. Non-limiting examples of hematopoietic cells include hematopoietic stem cells, pluripotent hematopoietic stem cells, myeloid progenitor cells, macronuclear cells, primordial cells, erythroid primordial cells, lymphoid primordial cells, monocytes, macrophages, neutrophils, eosinophils, erythrocytes, megakaryocytes, platelets, dendritic cells, NK cells, T cells, and B-cells.

[0081] Modified cells, in certain embodiments, may have been subjected to mutagenesis, genome editing, site-directed integration, genetic transformation, or any combination thereof. Modified cells of the present disclosure include progeny cells and cells derived from modified cells as described herein. In some embodiments, modified cells may comprise a genome edit, a DNA construct, or a vector as described herein.

[0082] As used herein, the term “activity” refers to the biological function of a gene or protein. A gene or a protein may provide one or more distinct functions. A reduction, disruption, or alterationin “activity” thus refers to a lowering, reduction, or elimination of one or more functions of a gene or a protein in a cell of the present disclosure. Additionally, an increase in “activity” thus refers to an elevation of one or more functions of a gene or a protein in a cell of the present disclosure. According to some embodiments, a modified hematopoietic cell comprising at least one genomic modification in a nucleotide sequence having at least about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or 100% sequence identity to SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NOG, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, or SEQ ID NO: 10 may comprise altered expression or activity of at least one protein selected from the group consisting of GAB3, LAGE3, FAM50A, UBL4A, GDI1, SLC10A3, G6PD, CTAG1A, TP6AP1, PLXNA3, FAM3A, IKBKG, CTAG1B, CTAG2, DKC1, MeCP2, EMD, and TAZ. In certain embodiments, a modified hematopoietic cell comprising at least one genomic modification in a nucleotide sequence having at least about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or 100% sequence identity to SEQ ID NO:1, SEQ ID NOG, SEQ ID NOG, SEQ ID NO:4, SEQ ID NOG, SEQ ID NO:6, SEQ ID NOG, SEQ ID NOG, SEQ ID NO:9, or SEQ ID NO: 10 may comprise reduced, disrupted, or altered activity of at least one protein selected from the group consisting of GAB3, LAGE3, FAM50A, UBL4A, GDI1, SLC10A3, G6PD, CTAG1A, TP6AP1, PLXNA3, FAM3A, IKBKG, CTAG1B, CTAG2, DKC1, MeCP2, EMD, and TAZ by at least about 1%, about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 99%, about 5% to about 20%, about 5% to about 25%, about 5% to about 30%, about 5% to about 40%, about 5% to about 50%, about 5% to about 60%, about 5% to about 70%, about 5% to about 75%, about 5% to about 80%, about 5% to about 85%, about 5% to about 90%, about 5% to about 95%, about 5% to about 100%, about 75% to about 100%, about 50% to about 100%, about 50% to about 90%, about 50% to about 75%, about 25% to about 75%, about 30% to about 80%, or about 10% to about 75% compared to a control cell, including all ranges and values derivable therebetween. In particular embodiments, a modified hematopoietic cell comprising at least one genomic modification in a nucleotide sequence having at least about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or 100% sequence identity to SEQ ID NO:1, SEQ ID NOG, SEQ ID NOG, SEQ ID NO:4, SEQ ID NOG,SEQ ID N0:6, SEQ ID N0:7, SEQ ID N0:8, SEQ ID N0:9, or SEQ ID NO: 10 may comprise reduced, disrupted, or altered expression of an mRNA molecule encoding at least one protein selected from the group consisting of GAB3, LAGE3, FAM50A, UBL4A, GDI1, SLC10A3, G6PD, CTAG1A, TP6AP1, PLXNA3, FAM3A, IKBKG, CTAG1B, CTAG2, DKC1, MeCP2, EMD, and TAZ by at least about 1%, about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 99%, about 5% to about 20%, about 5% to about 25%, about 5% to about 30%, about 5% to about 40%, about 5% to about 50%, about 5% to about 60%, about 5% to about 70%, about 5% to about 75%, about 5% to about 80%, about 5% to about 85%, about 5% to about 90%, about 5% to about 95%, about 5% to about 100%, about 75% to about 100%, about 50% to about 100%, about 50% to about 90%, about 50% to about 75%, about 25% to about 75%, about 30% to about 80%, or about 10% to about 75% compared to a control cell, including all ranges and values derivable therebetween.

[0083] The present disclosure relates to hematopoietic cells, in some embodiments, T cells comprising a genomic modification that reduces or eliminates the development of T cell exhaustion. T cell exhaustion is a state of T cell dysfunction that can arise during certain disease states such as chronic infection or cancer. T cell exhaustion, in some embodiments, may be defined by poor effector function, sustained expression of inhibitory receptors, and / or a transcriptional state distinct from that of functional effector or memory T cells. In some embodiments, exhausted T cells may express increased or altered levels of one or more of the following: PD1, CTLA4, TIM3, LAG3, 2B4, CD 160, TIGIT, and BATF. In some aspects, the present disclosure relates to hematopoietic cells, in certain embodiments, T cells that comprise a genomic modification that increases expression of sternness or memory markers when exposed to conditions that may result in exhaustion in control T cells. Non-limiting examples of markers associated with sternness or memory include LEF1, TCF7, and BACH2.

[0084] In particular embodiments, the present disclosure provides a polynucleotide molecule comprising or encoding a sequence having at least about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or 100% sequence identity to any one of SEQ ID NOs:l-59, or fragments thereof, including all ranges and values derivable therebetween. The present disclosure further provides modified hematopoietic cells comprising such polynucleotide molecules.B. Genome Editing

[0085] The present disclosure provides, in certain embodiments, hematopoietic cells produced through genome modification using site- specific integration or genome editing. Genome editing can be used to make one or more edit(s) or mutation(s) at a desired target site in the genome of a cell, such as to change expression and / or activity of one or more genes, or to integrate an insertion sequence or transgene at a desired location in a cell genome. Any site or locus within the genome of a hematopoietic cell may potentially be chosen for making a genomic edit (or gene edit) or site- directed integration of a transgene, construct, or transcribable DNA sequence. As used herein, a “target site” for genome editing or site-directed integration refers to the location of a polynucleotide sequence within a hematopoietic cell genome that is bound and cleaved by a sitespecific nuclease to introduce a double-stranded break (DSB) or single- stranded nick into the nucleic acid backbone of the polynucleotide sequence and / or its complementary DNA strand within the genome. A target site may comprise, for example, at least 10, at least 11 , at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 26, at least 27, at least 28, at least 29, or at least 30 consecutive nucleotides. A “target site” for an RNA-guided nuclease may comprise the sequence of either complementary strand of a double-stranded nucleic acid (DNA) molecule or chromosome at the target site. A site-specific nuclease may bind to a target site, such as via a noncoding guide RNA (e.g., without being limiting, a CRISPR RNA (crRNA) or a single-guide RNA (sgRNA) as described further herein). A non-coding guide RNA provided herein may be complementary to a target site (e.g., complementary to either strand of a double-stranded nucleic acid molecule or chromosome at the target site). It will be appreciated that perfect identity or complementarity may not be required for a non-coding guide RNA to bind or hybridize to a target site. For example, at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, or at least 8 mismatches (or more) between a target site and a non-coding RNA may be tolerated. A “target site” also refers to the location of a polynucleotide sequence within a genome that is bound and cleaved by any other site-specific nuclease that may not be guided by a non-coding RNA molecule, such as a zinc finger nuclease (ZFN), a transcription activator-like effector nuclease (TALEN), a meganuclease, etc., to introduce a DSB or single-stranded nick into the polynucleotide sequence and / or its complementary DNA strand. As used herein, a “target region” or a “targeted region” refers to a polynucleotide sequence or region that is flanked by two or more target sites.Without being limiting, in some embodiments a target region may be subjected to a mutation, deletion, insertion, substitution, inversion, or duplication. As used herein, “flanked” when used to describe a target region of a polynucleotide sequence or molecule, refers to two or more target sites of the polynucleotide sequence or molecule surrounding the target region, with one target site on each side of the target region.

[0086] As used herein, a “targeted genome editing technique” refers to any method, protocol, or technique that allows the precise and / or targeted editing of a specific location in a genome of a hematopoietic cell (z.e., the editing is largely or completely non-random) using a site-specific nuclease, such as a meganuclease, a zinc-finger nuclease (ZFN), an RNA-guided endonuclease (e.g., the CRISPR / Cas9 or Casl2a system), a TALE (transcription activator-like effector)- endonuclease (TALEN), a recombinase, or a transposase. In particular embodiments, a “targeted genome editing technique” refers to an RNA-guided dCas9-KRAB system. As used herein, “editing” or “genome editing” refers to generating a targeted mutation, deletion, insertion, substitution, inversion or duplication of at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 75, at least 100, at least 250, at least 500, at least 1000, at least 2500, at least 5000, at least 10,000, or at least 25,000 nucleotides of an endogenous hematopoietic cell genome nucleic acid sequence. As used herein, “editing” or “genome editing” may also encompass the targeted insertion or site-directed integration of at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 75, at least 100, at least 250, at least 500, at least 750, at least 1000, at least 1500, at least 2000, at least 2500, at least 3000, at least 4000, at least 5000, at least 10,000, or at least 25,000 nucleotides into the endogenous genome of a hematopoietic cell. An “edit” or “genomic edit” in the singular refers to one such targeted mutation, deletion, insertion, substitution, inversion, or duplication, whereas “edits” or “genomic edits” refers to two or more targeted mutation(s), deletion(s), insertion(s), substitution(s), inversion(s), and / or duplication(s), with each “edit” being introduced via a targeted genome editing technique.

[0087] According to some embodiments, a site-specific nuclease may be co-delivered with a donor template molecule to serve as a template for making a desired edit, mutation, or insertion into the genome at the desired target site through repair of the double strand break (DSB) or nick createdby the site-specific nuclease. According to some embodiments, a site-specific nuclease may be co-dclivcrcd with a DNA molecule comprising a selectable or scrccnablc marker gene.

[0088] A site-specific nuclease provided herein may be selected from the group consisting of a zinc-finger nuclease (ZFN), a TALE-endonuclease (TALEN), a meganuclease, an RNA-guided endonuclease (e.g., Cas9 and Cpfl), a recombinase, a transposase, or any combination thereof. Zinc finger nucleases (ZFN) are synthetic proteins consisting of an engineered zinc finger DNA- binding domain fused to a cleavage domain (or a cleavage half-domain), which may be derived from a restriction endonuclease (e.g., FokF). The DNA binding domain may be canonical (C2H2) or non-canonical (e.g., C3H or C4). The DNA-binding domain can comprise one or more zinc fingers (e.g., 2, 3, 4, 5, 6, 7, 8, 9 or more zinc fingers) depending on the target site but may typically be composed of 3-4 (or more) zinc-fingers. Multiple zinc fingers in a DNA-binding domain may be separated by linker sequence(s). ZFNs can be designed to cleave almost any stretch of doublestranded DNA by modification of the zinc finger DNA-binding domain. ZFNs form dimers from monomers composed of a non-specific DNA cleavage domain (e.g., derived from the FokI nuclease) fused to a DNA-binding domain comprising a zinc finger array engineered to bind a target site DNA sequence. The amino acids at positions -1, +2, +3, and +6 relative to the start of the zinc finger a-helix, which contribute to site-specific binding to the target site, can be changed and customized to fit specific target sequences. The other amino acids may form a consensus backbone to generate ZFNs with different sequence specificities.

[0089] Methods and rules for designing ZFNs for targeting and binding to specific target sequences are known in the art. See, e.g., U.S. Patent App. Pub. Nos. 2005 / 0064474, 2009 / 0117617, and 2012 / 0142062. The FokI nuclease domain may require dimerization to cleave DNA and therefore two ZFNs with their C-terminal regions are needed to bind opposite DNA strands of the cleavage site (separated by 5-7 bp). The ZFN monomer can cut the target site if the two-ZF-binding sites are palindromic. A ZFN, as used herein, is broad and includes a monomeric ZFN that can cleave double stranded DNA without assistance from another ZFN. The term ZFN may also be used to refer to one or both members of a pair of ZFNs that are engineered to work together to cleave DNA at the same site. Because the DNA-binding specificities of zinc finger domains can be re-engineered using one of various methods, customized ZFNs can theoretically be constructed to target nearly any target sequence (e.g., at or near a gene in a genome). Publiclyavailable methods for engineering zinc finger domains include Context-dependent Assembly (CoDA), Oligomerized Pool Engineering (OPEN), and Modular Assembly.

[0090] Transcription activator-like effectors (TALEs) can be engineered to bind practically any DNA sequence, such as at or near the genomic locus of an enhancer or a gene in a hematopoietic cell. TALE has a central DNA-binding domain composed of 13-28 repeat monomers of 33-34 amino acids. The amino acids of each monomer are highly conserved, except for hypervariable amino acid residues at positions 12 and 13. The two variable amino acids are called repeat-variable diresidues (RVDs). The amino acid pairs NI, NG, HD, and NN of RVDs preferentially recognize adenine, thymine, cytosine, and guanine / adenine, respectively, and modulation of RVDs can recognize consecutive DNA bases. This simple relationship between amino acid sequence and DNA recognition has allowed for the engineering of specific DNA binding domains by selecting a combination of repeat segments containing the appropriate RVDs.

[0091] TALENs are artificial restriction enzymes generated by fusing the TALE DNA binding domain to a nuclease domain. In some aspects, the nuclease is selected from a group consisting ot PvuII, MutH, TevI, FokI, Alwl, Mlyl, Sbfl, Sdal, StsI, CleDORF, Clo051, and Pept071. When each member of a TALEN pair binds to the DNA sites flanking a target site, the FokI monomers dimerize and cause a double- stranded DNA break at the target site. The term TALEN, as used herein, is broad and includes a monomeric TALEN that can cleave double stranded DNA without assistance from another TALEN. The term TALEN also refers to one or both members of a pair of TALENs that work together to cleave DNA at the same site.

[0092] Besides the wild-type FokI cleavage domain, variants of the FokI cleavage domain with mutations have been designed to improve cleavage specificity and cleavage activity. The FokI domain functions as a dimer, requiring two constructs with unique DNA binding domains for sites in the target genome with proper orientation and spacing. Both the number of amino acid residues between the TALEN DNA binding domain and the FokI cleavage domain and the number of bases between the two individual TALEN binding sites are parameters for achieving high levels of activity. PvuII, MutH, and TevI cleavage domains are useful alternatives to FokI and FokI variants for use with TALEs. PvuII functions as a highly specific cleavage domain when coupled to a TALE (see Yank et al., PLoS One 8:e82539, 2013). MutH is capable of introducing strand-specific nicks in DNA (see Gabsalilow et al., Nucleic Acids Research. 41:e83, 2013). TevI introducesdouble-stranded breaks in DNA at targeted sites (see Beurdeley et al., Nature Communications 4:1762, 2013).

[0093] The relationship between amino acid sequence and DNA recognition of the TALE binding domain allows for designable proteins. Software programs such as DNAWorks can be used to design TALE constructs. Other methods of designing TALE constructs are known to those of skill in the ail. See Doyle et al. (Nucleic Acids Research 40:W 117-122, 2012); Cermak et al. (Nucleic Acids Research 39:c82. 2011); and tale-nt.cac.comell.edu / about. In another aspect, a TALEN provided herein is capable of generating a targeted DSB .

[0094] A site-specific nuclease may be a meganuclease. Meganucleases, which are commonly identified in microbes, such as the LAGLID ADG family of homing endonucleases, are unique enzymes with high activity and long recognition sequences (> 14 bp) resulting in site-specific digestion of target DNA. Engineered versions of naturally occurring meganucleases typically have extended DNA recognition sequences (for example, 14 to 40 bp). The engineering of meganucleases can be more challenging than ZFNs and TALENs because the DNA recognition and cleavage functions of meganucleases are intertwined in a single domain. Specialized methods of mutagenesis and high-throughput screening have been used to create novel meganuclease variants that recognize unique sequences and possess improved nuclease activity.

[0095] A site-specific nuclease may be an RNA-guided nuclease. According to some embodiments, an RNA-guided endonuclease may be selected from the group consisting of Casl, CaslB, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9 (also known as Csnl and Csxl2), CaslO, Csyl, Csy2, Csy3, Csel, Cse2, Cscl, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmrl, Cmr3, Cmr4, Cmr5, Cmr6, Csbl, Csb2, Csb3, Csxl7, Csxl4, CsxlO, Csxl6, CsaX, Csx3, Csxl, Csxl5, Csfl, Csf2, Csf3, Csf4, Cpfl, CasX, CasY, and homologs or modified versions of any thereof, as well as Argonaute proteins (non-limiting examples of Argonaute proteins include Thermus thermophilus Argonaute (TtAgo), Pyrococcus furiosus Argonaute (PfAgo), Natronobacterium gregoryi Argonaute (NgAgo), and homologs or modified versions of any thereof). According to some embodiments, an RNA-guided endonuclease is a Cas9 or Cpfl (also referred to herein as Casl 2a) enzyme. The RNA-guided nuclease may be delivered as a protein or a recombinant DNA construct comprising a polynucleotide sequence encoding the nuclease,with or without a guide RNA; or the guide RNA may be complexed with the RNA-guided nuclease enzyme and delivered as a ribonuclcoprotcin (RNP).

[0096] For RNA-guided endonucleases, a guide RNA molecule may be further provided to direct the endonuclease to a target site in the genome of a hematopoietic cell via base-pairing or hybridization to cause a DSB or nick at or near the target site. As described herein, the guide RNA may be transformed or introduced into a cell or tissue as a gRNA molecule, or as a recombinant DNA molecule, construct or vector comprising a transcribable DNA sequence encoding one or more guide RNAs operably linked to a single promoter or individual promoters. As understood in the art, a guide RNA may comprise, for example, a CRISPR RNA (crRNA), a single-chain guide RNA (sgRNA), or any other RNA molecule that may guide or direct an endonuclease to a specific target site in the genome. A prototypical CRISPR associated protein, Cas9 from .S'. pyogenes, naturally binds two RNAs, a CRISPR RNA (crRNA) guide and a trans-acting CRISPR RNA (tracrRNA), to assemble a CRISPR ribonucleoprotein (crRNP). In comparison, the CRISPR- Cas 12a system does not require a trans-activating crispr RNA (tracrRNA) for biogenesis of mature crRNA. Instead, the RuvC endonuclease domain of Casl2a processes its mature crRNA directly. A “single-chain guide RNA” (or “sgRNA”) is an RNA molecule comprising a crRNA covalently linked a tracrRNA by a linker sequence, which may be expressed as a single RNA transcript or molecule. The guide RNA comprises a guide or targeting sequence (also referred to herein as a “spacer sequence”) that is identical or complementary to a target site within the cell genome, such as at or near a gene. The guide RNA is typically a non-coding RNA molecule that does not encode a protein. The guide sequence of the guide RNA may be at least 10 nucleotides in length, such as 12-40 nucleotides, 12-30 nucleotides, 12-20 nucleotides, 12-35 nucleotides, 12-30 nucleotides, 15- 30 nucleotides, 17-30 nucleotides, or 17-25 nucleotides in length, or about 12, 13, 14, 15, 16, 17,18, 19, 20, 21, 22, 23, 24, 25 or more nucleotides in length. The guide sequence may be at least 95%, at least 96%, at least 97%, at least 99% or 100% identical or complementary to at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, or more consecutive nucleotides of a DNA sequence at the genomic target site. In some embodiments, the target site may comprise at least 10, at least 11 , at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, or more consecutive nucleotides of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ IDN0:4, SEQ ID N0:5, SEQ ID N0:6, SEQ ID N0:7, SEQ ID N0:8, SEQ ID N0:9, SEQ ID NO: 10, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID N0:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:57, SEQ ID NO:58, or SEQ ID NO:59. A gRNA of the present disclosure may comprise, in specific embodiments, a sequence selected from the group consisting of SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:51, SEQ ID NO:52, and SEQ ID NO:53.

[0097] In addition to the guide sequence, a guide RNA may further comprise one or more other structural or scaffold sequence(s), which may bind or interact with an RNA-guided endonuclease. Such scaffold or structural sequences may further interact with other RNA molecules (e.g., tracrRNA). Methods and techniques for designing targeting constructs and guide RNAs for genome editing and site-directed integration at a target site within the genome of a cell using an RNA-guided endonuclease are known in the art.

[0098] As mentioned above, a target gene for genome editing may be any gene of interest present in a hematopoietic cell. Non-limiting examples of such genes of interest include GAB3. LAGE3, FAM50A, UBL4A. GDI1, SLC10A3. G6PD. CTAG1A, TP6AP1, PLXNA3, FAM3A, IKBKG, CTAG1B, CTAG2, DKC1, MeCP2, EMD, and TAZ. For knockdown mutations of the gene of interest through genome editing, an RNA-guided endonuclease may be targeted to an upstream or downstream sequence, such as a promoter and / or enhancer sequence, or an intron, 5'UTR, and / or 3'UTR sequence of the gene to mutate one or more promoter and / or regulatory sequences of the gene to affect or reduce its level of expression. Similarly, mutations of the gene of interest through genome editing, an RNA-guided endonuclease may be targeted to a transcribable DNA sequence i.e., a transcribable region) of the gene, such as a region of the gene comprising a coding sequence, a specific DNA sequence encoding a protein domain, an exon region, an intron region, or a combination thereof. For example, in certain embodiments a transcribable DNA sequence targeted for genome editing may comprise an exon / intron boundary or may be in close proximity to an exon / intron boundary. If the resulting modification spans an exon / intron boundary, the modification may be re I erred to as a modification in an exon region and an intron region. In certain embodiments, for genetic modification of the gene of interest, a guide RNA may be used, which comprises a guide sequence that is at least 90%, at least 95%, at least 96%, at least 97%, at least99% or 100% identical or complementary to at least 10, at least 11 , at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, or more consecutive nucleotides of the sequence of the GAB3, LAGE3, FAM50A. UBL4A, GDI], SLC10A3, G6PD, CTAG1A, TP6AP1, PLXNA3, FAM3A, IKBKG, CTAG1B, CTAG2, DKC1, MeCP2, EMD, or TAZ gene or a sequence complementary thereto, although alternative splicing and different exon / intron boundaries may occur. As used herein, the term “consecutive” in reference to a polynucleotide or protein sequence means without deletions or gaps in the sequence.

[0099] As used herein, with respective to a given sequence, a “complement,” a “complementary sequence” and a “reverse complement” are used interchangeably. All three terms refer to the inversely complementary sequence of a nucleotide sequence, i.e., to a sequence complementary to a given sequence in reverse order of the nucleotides.

[0100] Antisense RNA molecules are single- stranded nucleic acids which can combine with a sense RNA strand, sequence, or mRNA to form duplexes due to complementarity of the sequences. The term “antisense strand” refers to a nucleic acid strand that is complementary to the “sense” strand. The “sense strand” of a gene or locus is the strand of DNA or RNA that has the same sequence as an RNA molecule transcribed from the gene or locus (with the exception of uracil in RNA and thymine in DNA).

[0101] A protospacer-adjacent motif (PAM) may be present in the genome immediately adjacent and upstream to the 5’ end of the genomic target site sequence complementary to the targeting sequence of the guide RNA - i.e., immediately downstream (3’) to the sense (+) strand of the genomic target site (relative to the targeting sequence of the guide RNA) as known in the art. See, e.g., Wu etal. Quant Biol. 2(2):59-70, 2014). The genomic PAM sequence on the sense (+) strand adjacent to the target site (relative to the targeting sequence of the guide RNA) may comprise 5’- NGG-3’ for Cas9; or 5’-TTTN-3’ for Casl2a. However, the corresponding sequence of the guide RNA i.e., immediately downstream (3’) to the targeting sequence of the guide RNA) may generally not be complementary to the genomic PAM sequence.

[0102] In some embodiments, a site-specific nuclease is a recombinase. Non-limiting examples of recombinases that may be used include a serine recombinase attached to a DNA recognition motif, a tyrosine recombinase attached to a DNA recognition motif, or any recombinase enzymeknown in the art attached to a DNA recognition motif. In certain embodiments, the site-specific nuclease is a recombinase or transposasc, which may be a DNA transposasc or recombinase attached or fused to a DNA binding domain. Non-limiting examples of recombinases include a tyrosine recombinase selected from the group consisting of a Cre recombinase, a Gin recombinase, a Flp recombinase, and a Tnpl recombinase attached to a DNA recognition motif provided herein. In one aspect of the present disclosure, a Cre recombinase or a Gin recombinase provided herein is tethered to a zine-finger DNA-binding domain, a TALE DNA-binding domain, or a Cas9 nuclease. In another aspect, a serine recombinase selected from the group consisting of a PhiC31 integrase, an R4 integrase, and a TP-901 integrase may be attached to a DNA recognition motif provided herein. In yet another aspect, a DNA transposase selected from the group consisting of a TALE-piggyBac and TALE- Mutator may be attached to a DNA binding domain provided herein.

[0103] Several site-specific nucleases, such as recombinases, zinc finger nucleases (ZFNs), meganucleases, and TALENs, are not RNA-guided and instead rely on their protein structure to determine their target site for causing the DSB or nick, or they are fused, tethered, or attached to a DNA-binding protein domain or motif. The protein structure of the site-specific nuclease (or the fused / attached / tethered DNA binding domain) may target the site-specific nuclease to the target site. According to many of these embodiments, non-RNA-guided site-specific nucleases, such as recombinases, zinc finger nucleases (ZFNs), meganucleases, and TALENs, may be designed, engineered and constructed according to known methods to target and bind to a target site at or near the genomic locus of an enhancer sequence or an endogenous gene of a hematopoietic cell to create a DSB or nick at such a genomic locus. The DSB or nick created by the non-RNA-guided site- specific nuclease may lead to knockdown of gene expression, or a change in the activity of the protein encoded by the endogenous gene, via repair of the DSB or nick, which may result in a mutation or insertion of a sequence at the site of the DSB or nick through cellular repair mechanisms. Such cellular repair mechanism may be guided by a donor template molecule.

[0104] As used herein, a “donor molecule”, “donor template”, or “donor template molecule” (collectively a “donor template”), which may be a recombinant polynucleotide, DNA or RNA donor template or sequence, is defined as a nucleic acid molecule having a homologous nucleic acid template or sequence (e.g., homology sequence) and / or an insertion sequence for site-directed, targeted insertion or recombination into the genome of a hematopoietic cell via repair of a nick or DSB in the genome of a hematopoietic cell. A donor template may be a separate DNA moleculecomprising one or more homologous sequence(s) and / or an insertion sequence for targeted integration, or a donor template may be a sequence portion (i.e.. a donor template region) of a DNA molecule further comprising one or more other expression cassettes, genes / transgenes, and / or transcribable DNA sequences. For example, a “donor template” may be used for site- directed integration of a transgene or construct, or as a template to introduce a mutation, such as an insertion, deletion, substitution, etc., into a target site within the genome of a hematopoietic cell. A targeted genome editing technique provided herein may comprise the use of one or more, two or more, three or more, four or more, or five or more donor molecules or templates. A donor template provided herein may comprise at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, or at least ten gene(s) or transgene(s) and / or transcribable DNA sequence(s). Alternatively, a donor template may comprise no genes, transgenes, or transcribable DNA sequences.

[0105] An “insertion sequence” of a donor template is a sequence designed for targeted insertion into the genome of a hematopoietic cell, which may be of any suitable length. For example, the insertion sequence of a donor template may be between 2 and 50,000, between 2 and 10,000, between 2 and 5000, between 2 and 1000, between 2 and 500, between 2 and 250, between 2 and 100, between 2 and 50, between 2 and 30, between 15 and 50, between 15 and 100, between 15 and 500, between 15 and 1000, between 15 and 5000, between 18 and 30, between 18 and 26, between 20 and 26, between 20 and 50, between 20 and 100, between 20 and 250, between 20 and 500, between 20 and 1000, between 20 and 5000, between 20 and 10,000, between 50 and 250, between 50 and 500, between 50 and 1000, between 50 and 5000, between 50 and 10,000, between 100 and 250, between 100 and 500, between 100 and 1000, between 100 and 5000, between 100 and 10,000, between 250 and 500, between 250 and 1000, between 250 and 5000, or between 250 and 10,000 nucleotides or base pairs in length. A donor template may also have at least one homology sequence or homology arm, such as two homology arms, to direct the integration of a mutation or insertion sequence into a target site within the genome of a cell via homologous recombination, wherein the homology sequence or homology arm(s) are identical or complementary, or have a percent identity or percent complementarity, to a sequence at or near the target site within the genome of the cell. When a donor template comprises homology arm(s) and an insertion sequence, the homology arm(s) will flank or surround the insertion sequence of the donor template. Each homology arm may be at least 70%, at least 75%, at least 80%, at least85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 99% or 100% identical or complementary to at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 150, at least 200, at least 250, at least 500, at least 1000, at least 2500, or at least 5000 consecutive nucleotides of a target DNA sequence within the genome of a cell.

[0106] Any method known in the ail for site-directed integration may be used with the present disclosure. In the presence of a donor template molecule with an insertion sequence, the DSB or nick can be repaired by homologous recombination between homology arm(s) of the donor template and the genome, or by non-homologous end joining (NHEJ), resulting in site-directed integration of the insertion sequence into the genome to create the targeted insertion event at the site of the DSB or nick. Thus, site-specific insertion or integration of a transgene, transcribable DNA sequence, construct, or sequence may be achieved if the transgene, transcribable DNA sequence, construct or sequence is located in the insertion sequence of the donor template.

[0107] The introduction of a DSB or nick may also be used to introduce targeted mutations in the genome of a hematopoietic cell. According to this approach, mutations, such as deletions, insertions, substitutions, inversions, and / or duplications may be introduced at a target site via imperfect repair of the DSB or nick to produce a genetic modification within a gene or enhancer sequence. Such mutations may be generated by imperfect repair of the targeted locus even without the use of a donor template molecule. A modification of a gene or enhancer may be achieved by inducing a DSB or nick at or near the endogenous locus of the gene or enhancer that results in expression of a non-functional protein, interfering protein, or a protein having reduced, disrupted, or altered activity as compared to a protein expressed from the gene lacking the modification.

[0108] As used herein, the term “insertion” as it relates to a mutation, refers to the addition of one or more extra nucleotides into the DNA.

[0109] As used herein, the term “deletion” as it relates to a mutation refers to the removal of one or more nucleotides from the DNA.

[0110] As used herein, the term “substitution” as it relates to a mutation refers to an exchange of at least one single nucleotide for another.

[0111] As used herein, the term “inversion” refers to reversing the orientation of a chromosomal segment. An inversion can be accompanied by a loss of nucleotides flanking cither one or both sites of the inversion due to DNA repair mechanisms occurring at the cut and ligation sites during the formation of an inversion.

[0112] As used herein, the term “duplication” refers to the creation of multiple copies of chromosomal regions, increasing the dosage of the genes located within them.

[0113] As used herein, a “missense mutation” refers to a single nucleotide change that results in a codon that codes for a different amino acid. For example, the codon “CGU” encodes an arginine amino acid. If a missense mutation changes the G to a U, producing a “CUU” codon, the codon now encodes a leucine amino acid. Missense mutations can be caused by an insertion, deletion, substitution, duplication, or inversion.

[0114] A “loss-of-function mutation” is a mutation that causes the function of the gene product, usually a protein, to be either reduced or completely absent. A loss-of-function mutation can, for instance, be caused by the truncation of the gene product or by disruption of an enhancer sequence resulting in reduced expression of a gene product.

[0115] Similarly, such targeted mutations of a gene may be generated with a donor template molecule to direct a particular or desired mutation at or near the target site via repair of the DSB or nick. The donor template molecule may comprise a homologous sequence with or without an insertion sequence and comprising one or more mutations, such as one or more deletions, insertions, substitutions, inversions, and / or duplications, relative to the targeted genomic sequence at or near the site of the DSB or nick. For example, targeted mutations of a gene may be achieved by deleting, inserting, substituting, inverting, or duplicating at least a portion of the gene, such as by introducing a frame shift or premature stop codon into the coding sequence of the gene or introducing a modification into a transcribable DNA sequence. A deletion of a portion of a gene may also be introduced by generating DSBs or nicks at two target sites and causing a deletion of the intervening target region flanked by the target sites. A modification of a targeted gene or enhancer may result in expression of a non-functional protein, interfering protein, or a protein having reduced, disrupted, or altered activity as compared to a protein expressed from the gene lacking the modification.T1C. Therapeutic Compositions and Methods

[0116] In certain aspects, the present disclosure provides pharmaceutical and therapeutic compositions comprising the polynucleotide molecules and / or cells of the present disclosure. In some embodiments, the polynucleotide molecules and / or cells of the present disclosure may be combined with a pharmaceutically acceptable carrier. As used herein, a “pharmaceutically acceptable carrier,” “pharmaceutically acceptable adjuvant,” or “adjuvant” refers to reagents, cells, compounds, materials, compositions, and / or dosage forms that are not only compatible with the polynucleotide molecules, cells, and / or or other agents to be administered therapeutically, but also are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other complication commensurate with a reasonable benefit / risk ratio. Also included may be an agent that modifies the effect of other agents and is useful in preparing a therapeutic compound or composition that is generally safe, non-toxic, and neither biologically nor otherwise undesirable. Such an agent may be added to a therapeutic composition to modify the immune response of a subject by boosting the response such as to give a higher amount of polynucleotide molecules and or cells and longer-lasting protection from degradation. Such an agent may include any excipient, diluent, carrier, or adjuvant that is acceptable for pharmaceutical use. Such an agent may be non- naturally occurring, or may be naturally occurring, but not naturally found in combination with other agents in the immunogenic composition.

[0117] As used herein, a “therapeutic compound” or “therapeutic composition” refers to a composition comprising a polynucleotide molecule or a cell of the present disclosure. In some embodiments, a therapeutic composition has the activity of altering or disrupting the activity of an enhancer comprising a sequence having at least about 85% sequence identity to any one of SEQ ID NOs:l-10. In certain embodiments, a therapeutic composition of the present disclosure has the activity of altering or disrupting the activity of a gene having at least about 85% sequence identity to any one of SEQ ID NOs:36-50 and 57-59. In particular embodiments, the composition comprises a modified hematopoietic cell. In one embodiment the composition comprises a modified T cell. In other embodiments, the composition may comprise a gRNA of the present disclosure. In certain embodiments, a gRNA of the present disclosure may comprise a nucleotide sequence having at least about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or 100% identity to at least about 10,about 20, about 30, about 40, about 50, about 60, or about 70 consecutive nucleotides of SEQ ID NO:11, SEQ ID NO: 12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO:20, SEQ ID NO:51, SEQ ID NO:52, or SEQ ID NO:53. The composition, in certain embodiments, may comprise a gRNA of the present disclosure and a site-specific nuclease. Such a compound or composition is meant to encompass a composition suitable for administration to a subject, such as a mammal, particularly a human subject. In general, a therapeutic composition is sterile, and preferably free of contaminants that are capable of eliciting an undesirable response within the subject (e.g., the compound(s) in the immunogenic composition is pharmaceutical grade). Therapeutic compositions may be designed for administration to subjects in need thereof via a number of different routes of administration including oral, intravenous, buccal, rectal, parenteral, intraperitoneal, topical, intradermal, intratracheal, intramuscular, subcutaneous, inhalational, and the like. The appropriate dosage of a composition, as described herein, may be determined based on the type of disease to be treated, the severity and course of the disease, the clinical condition of the individual, clinical history, response to the treatment, and the discretion of the attending physician. In some embodiments, therapeutic compositions provided by the present disclosure may include various “unit doses.” A unit dose is defined as containing a predetermined quantity of the therapeutic composition. The quantity to be administered, and the particular route and formulation, is within the skill of determination of those in the clinical ails. A unit dose need not be administered as a single injection but may comprise continuous infusion over a set period of time. In some aspects, a unit dose comprises a single administrable dose.

[0118] Precise amounts of the therapeutic composition also depend on the judgment of the practitioner and are peculiar to each individual. Factors affecting dose include physical and clinical state of the patient, the route of administration, the intended goal of treatment (alleviation of symptoms versus cure) and the potency, stability and toxicity of the particular therapeutic substance or other therapies a subject may be undergoing.

[0119] As used herein, “subject” or “patient” refers to animals, including humans, who are treated with the therapeutic compounds or compositions or in accordance with the methods described herein. For diagnostic or research applications, a wide variety of mammals may be suitable subjects, including rodents (e.g., mice, rats, hamsters), rabbits, primates, and swine, such as inbred pigs and the like. In particular embodiments, a subject in need of therapy may be any subject whocomprises a cancer cell. In another embodiment, the cancer cell may be selected from the group consisting of: a lung cancer cell, a non-small cell lung cancer (NSCLC) cell, a colorectal cancer cell, a pancreatic cancer cell, an appendiceal cancer cell, a small bowel adenocarcinoma cell, an hepatobiliary cancer cell, a gynecological malignancy cell, a hematopoietic cancer cell, a breast cancer cell, a bladder cancer cell, a prostate cancer cell, a skin cancer cell, a head and neck squamous cell carcinoma cell, a melanoma cell, and a genitourinary cancer cell.

[0120] A composition, as described herein, may include, in particular embodiments, a combination of therapeutic agents. In some embodiments, a composition as described here may be administered as a single composition or as more than one composition. Different compositions as provided herein, in certain embodiments, may be administered by the same route of administration or by different routes of administration.

[0121] In certain embodiments, the compositions and methods for treating an individual described herein may be combined with any other composition or method of treatment known in the art. The compositions and methods may be administered in any suitable manner known in the art. For example, a first and a second cancer treatment may be administered sequentially (at different times) or concurrently (at the same time). In some aspects, a first and a second cancer treatment may be administered in separate compositions. In certain embodiments, a first and a second cancer treatment may be administered in the same composition.

[0122] Non-limiting examples of additional treatment modalities that may be included in combination with the compositions and methods provided herein include immunotherapy, chemotherapy, radiation therapy, and surgery. Types of immunotherapy that may be used in combination with the compositions and methods provided by the present disclosure include, but are not limited, to: (a) checkpoint inhibitors, for example, inhibitors of PD-1, PDL1, PDL2, and CTLA-4; (b) inhibitors of co-stimulatory molecules, for example, inhibitors of B7-1, B7-2 CD28, ICOS, 0X40, 4-1BB, CD137, CD40L, and GITR; (c) dendritic cell therapy; (d) other T cell therapies directed to the same or to a different antigen; and (e) cytokine therapy. Non-limiting types of chemotherapy that may be used in combination with the compositions and methods provided by the present disclosure include (a) alkylating agents; (b) antimetabolites; (c) natural products, for example, vinca alkaloids, enzymes (e.g., L-asparaginase), and biological responsemodifiers (e.g., interferon -a); and (d) other agents, for example, platinum coordination complexes, substituted ureas, and taxols.

[0123] Therapeutic compounds or compositions may be provided to a subject in a single dose or multiple doses and as such provided in single-dose or multi-dose containers, such as sealed ampules or vials. Such containers may be sealed to preserve sterility of the composition until use. In general, compositions as described herein may be stored as suspensions, solutions, or emulsions in oily or aqueous vehicles. Alternatively, such a composition may be stored in a freeze-dried condition requiring only the addition of a sterile liquid carrier immediately prior to use.

[0124] Such compositions may also comprise buffers (e.g., neutral buffered saline or phosphate buffered saline), carbohydrates (e.g., glucose, mannose, sucrose or dextrans), mannitol, proteins, polypeptides or amino acids such as glycine, antioxidants, bacteriostats, chelating agents such as EDTA or glutathione, adjuvants (e.g., aluminum hydroxide), solutes that render the formulation isotonic, hypotonic, or weakly hypertonic with the blood of a subject, suspending agents, thickening agents, and / or preservatives. Alternatively, compositions of the present disclosure may be formulated as a lyophilizate. Compounds may also be encapsulated within liposomes using methods known in the ail.

[0125] For administration, compounds of the present disclosure can be administered at a rate determined by the LD-50 of the molecule or therapeutic compound, and the side-effects thereof at various concentrations, as applied to the mass and overall health of the subject. Administration may be accomplished via single, multiple, or divided doses.

[0126] The term “isolated compound” means a compound which has been substantially separated from, or enriched relative to, other compounds with which it occurs in nature. Isolated compounds are usually at least about 80% pure, at least 90% pure, at least 98% pure, or at least about 99% pure, by weight.

[0127] The term “unit dosage form,” as used herein, refers to physically discrete units suitable as unitary dosages for animal subjects, each unit containing a predetermined quantity of a compound calculated in an amount sufficient to produce the desired effect in association with a pharmaceutically acceptable diluent, carrier, or vehicle. The specifications for unit dosage forms depend on the particular compound employed, the route and frequency of administration, the effect to be achieved, and the pharmacodynamics associated with each compound in the host.

[0128] The phrase “effective amount” refers to a concentration or amount of a therapeutic compound or composition as described herein, reagent, or other agent, which is effective for producing an intended result, including treatment of cancer as described herein. With respect to the administration of a therapeutic compound as disclosed herein, an effective amount may be any effective range or concentration. The exact dose will depend on the purpose of the treatment, and one of skill in the art will be able to determine such a dose using techniques known in the art.

[0129] As used herein, “expression” refers to the combination of intracellular processes, including transcription and translation undergone by a coding DNA molecule such as a structural gene to produce a polypeptide or functional nucleic acid (e.g., an RNAi, gRNA, antisense molecule, ribozyme, aptamer, etc.).D. Gene Suppression

[0130] As used herein the phrase “gene suppression,” is intended to refer to any of the well- known methods for reducing the levels of protein produced as a result of gene transcription to mRNA and subsequent translation of the mRNA. Gene suppression is also intended to mean the reduction of protein expression from a gene or a coding sequence including posttranscriptional gene suppression and transcriptional suppression. Post-transcriptional gene suppression is mediated by the homology between of all or a part of a mRNA transcribed from a gene or coding sequence targeted for suppression and the corresponding double stranded RNA used for suppression and refers to the substantial and measurable reduction of the amount of available mRNA available in the cell for binding by ribosomes. The transcribed RNA can be in the sense orientation to effect what is called co-suppression, in the anti-sense orientation to effect what is called anti-sense suppression, or in both orientations producing a double stranded RNA (dsRNA) to effect what is called RNA interference (RNAi). Transcriptional suppression is mediated by the presence in the cell of a dsRNA, a gene suppression agent, exhibiting substantial sequence identity to a promoter DNA sequence or the complement thereof to effect what is referred to as promoter trans suppression. Gene suppression may be effective against a native gene associated with a trait, e.g., to suppress expression of a GAB3, LAGE3, FAM50A, UBL4A, GDI1, SLC10A3, G6PD, CTAG1A, TP6AP1, PLXNA3, FAM3A, IKBKG, CTAG1B, CTAG2, DKC1, MeCP2, EMD, or TAZ protein. Sequences of the GAB3, LAGE3, FAM50A, UBE4A, GDI1, SLC10A3, G6PD, CTAG1A, TP6AP1, PLXNA3, FAM3A, IKBKG, CTAG1B, CTAG2, DKC1, MeCP2, EMD, andTAZ genes and sequences encoded by the GAB3, LAGE3, FAM50A, UBL4A, GDI I , SLC10A3, G6PD, CTAG1A, TP6AP1, PLXNA3. FAM3A, IKBKG. CTAG1B, CTAG2. DKC1, MeCP2, EMD, and TAZ genes are known in the art and any such gene, mRNA, or protein sequence may be targeted according to the present disclosure. One of skill the art would understand that due to, for example, certain genetic polymorphisms or mutations that may be present in the human population, the gene, encoded mRNA molecule, or encoded polypeptide sequence may comprise a sequence having at least about 70%, about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or 100% sequence identity to a GABS, LAGE3, FAM50A, UBL4A, GDI1, SLC10A3, G6PD, CTAG1A, TP6AP1, PLXNA3, FAM3A, IKBKG, CTAG1B, CTAG2, DKC1, MeCP2, EMD, or TAZ gene, a sequence encoded thereby, or a fragment thereof.

[0131] Post-transcriptional gene suppression may employ both sense-oriented and anti-sense- oriented, transcribed RNA, which is stabilized, e.g., as a hairpin and stem and loop structure. A preferred DNA construct for effecting post transcriptional gene suppression one in which a first segment encodes an RNA exhibiting an anti-sense orientation exhibiting substantial identity to a segment of a gene targeted for suppression, which is linked to a second segment encoding an RNA exhibiting substantial complementarity to the first segment. Such a construct would be expected to form a stem and loop structure by hybridization of the first segment with the second segment and a loop structure from the nucleotide sequences linking the two segments.

[0132] The present disclosure provides dsRNA or siRNA molecules for reduction or elimination of GAB3, LAGE3, FAM50A, UBL4A, GDI1, SLC10A3, G6PD, CTAG1A, TP6AP1, PLXNA3, FAM3A, IKBKG, CTAG1B, CTAG2, DKC1, MeCP2, EMD, or TAZ expression. siRNA technology is known in the art and used to study inhibition of, for example, apolipoprotein receptors such as ApoCIII (Gaudet et al., N Engl J Med 373(5):438-47, 2015; Graham et al., Circulation Res 112:1479-1490, 2013) ApoA (Tsimikas et al., Lancet 2015), or chemicals useful in accordance with the disclosure, such as PCSK9 (Miao et al., Arterioscler Thromb Vase Biol 35(7): 1589-96, 2015). The dsRNA or siRNA nucleotide sequences comprise double strands of polymerized ribonucleotide and may include modifications to either the phosphate-sugar backbone or the nucleoside. Modifications in RNA structure may be tailored to allow specific genetic inhibition. In one embodiment, the dsRNA molecules may be modified through an enzymatic process so the siRNA molecules may be generated. The siRNA can efficiently mediate the down-regulation effect for some target genes. This enzymatic process may be accomplished by utilizing an RNAsc III enzyme or a DICER enzyme, present in the cells of an individual in the eukaryotic RNAi pathway. Both the DICER enzyme and RNAse III, naturally occurring in an individual or made through recombinant DNA techniques, cleave larger dsRNA strands into smaller oligonucleotides. The DICER enzymes specifically cut the dsRNA molecules into siRNA pieces each of which is about 19-25 nucleotides in length while the RNAse III enzymes normally cleave the dsRNA molecules into 12-15 base-pair siRNA. The siRNA molecules produced by the either of the enzymes have 2 to 3 nucleotide 3' overhangs, and 5' phosphate and 3' hydroxyl termini. The siRNA molecules generated by RNAse III enzyme are the same as those produced by Dicer enzymes in the eukaryotic RNAi pathway and are hence then targeted and degraded by an inherent cellular RNA-degrading mechanism after they are subsequently unwound, separated into singlestranded RNA, and hybridized with the RNA sequences transcribed by the target gene. This process results in the effective degradation or removal of the RNA sequence encoded by the nucleotide sequence of the target gene. The outcome is the silencing of a particularly targeted nucleotide sequence within the individual.

[0133] Inhibition of a target gene using the stabilized dsRNA technology of the present disclosure is sequence-specific in that nucleotide sequences corresponding to the duplex region of the RNA are targeted for genetic inhibition. RNA containing a nucleotide sequence identical to a portion of the target gene is preferred for inhibition. RNA sequences with insertions, deletions, and single point mutations relative to the target sequence have also been found to be effective for inhibition. In performance of the present disclosure, it is preferred that the inhibitory dsRNA and the portion of the target gene share at least from about 80% sequence identity, about 85% identity, about 90% sequence identity, about 91% identity, about 92% identity, about 93% identity, about 94% identity, about 95% sequence identity, about 96% identity, about 97% identity, about 98% identity, about 99% sequence identity, or about 100% sequence identity. Alternatively, the duplex region of the RNA may be defined functionally as a nucleotide sequence that is capable of hybridizing with a portion of the target gene transcript. A less than full length sequence exhibiting a greater homology compensates for a longer less homologous sequence. The length of the identical nucleotide sequences may be at least about 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, or at least about 1000 bases. Normally, a sequence of greater than 15-100 nucleotides may be used, or a sequence of greater than about 200-300 nucleotides, ora sequence of greater than about 500-1000 nucleotides, depending on the size of the target gene. A dsRNA or siRNA as described herein may be able to tolerate sequence variations that might be expected due to genetic mutation, polymorphism, or evolutionary divergence. The introduced nucleic acid molecule may not need to be absolute homology, may not need to be full length, relative to either the primary transcription product or fully processed mRNA of the target gene. Therefore, those skilled in the art will understand that 100% sequence identity between the RNA and the target gene is not required to practice the present disclosure.

[0134] The dsRNA or siRNA molecules may be synthesized either in vivo or in vitro. The dsRNA may be formed by a single self-complementary RNA strand or from two complementary RNA strands. Endogenous RNA polymerase of the cell may mediate transcription in vivo, or cloned RNA polymerase can be used for transcription in vivo or in vitro. Inhibition may be targeted by specific transcription in an organ, tissue, or cell type; stimulation of an environmental condition (e.g., infection, stress, temperature, chemical inducers); and / or engineering transcription at a developmental stage or age. The RNA strands may or may not be polyadenylated; the RNA strands may or may not be capable of being translated into a polypeptide by a cell's translational apparatus.

[0135] The RNA, dsRNA, siRNA, or miRNA of the present disclosure may be produced chemically or enzymatically by one skilled in the art through manual or automated reactions or in vivo in another organism. RNA may also be produced by partial or total organic synthesis; any modified ribonucleotide can be introduced by in vitro enzymatic or organic synthesis. The use and production of an expression construct are known in the art. If synthesized chemically or by in vitro enzymatic synthesis, the RNA may be purified prior to introduction into the cell. For example, RNA can be purified from a mixture by extraction with a solvent or resin, precipitation, electrophoresis, chromatography, or a combination thereof. Alternatively, the RNA may be used with no or minimum of purification to avoid losses due to sample processing. The RNA may be dried for storage or dissolved in an aqueous solution. The solution may contain buffers or salts to promote annealing, and / or stabilization of the duplex strands.

[0136] As used herein, the term “nucleic acid” refers to a single or double-stranded polymer of deoxyribonucleotide or ribonucleotide bases read from the 5’ to the 3’ end. The “nucleic acid” may also optionally contain non-naturally occurring or altered nucleotide bases that permit correct read through by a polymerase and do not reduce expression of a polypeptide encoded by thatnucleic acid. The term “nucleotide sequence” or “nucleic acid sequence” refers to both the sense and antisense strands of a nucleic acid as cither individual single strands or in the duplex. The term “ribonucleic acid” (RNA) is inclusive of RNAi (inhibitory RNA), dsRNA (double stranded RNA), siRNA (small interfering RNA), mRNA (messenger RNA), miRNA (micro-RNA), tRNA (transfer RNA, whether charged or discharged with a corresponding acylated amino acid), and cRNA (complementary RNA) and the term “deoxyribonucleic acid” (DNA) is inclusive of cDNA and genomic DNA and DNA-RNA hybrids. The words “nucleic acid segment”, “nucleotide sequence segment”, or more generally “segment” will be understood by those in the art as a functional term that includes both genomic sequences, ribosomal RNA sequences, transfer RNA sequences, messenger RNA sequences, operon sequences and smaller engineered nucleotide sequences that express or may be adapted to express, proteins, polypeptides or peptides.

[0137] The term "about" is used to indicate that a value includes the standard deviation of the mean for the device or method being employed to determine the value. The use of the term "or" in the claims is used to mean "and / or" unless explicitly indicated to refer to alternatives only or the alternatives are mutually exclusive. When used in conjunction with the word "comprising" or other open language in the claims, the words "a" and "an" denote "one or more," unless specifically noted otherwise. The terms "comprise," "have," and "include" are open-ended linking verbs. Any forms or tenses of one or more of these verbs, such as "comprises," "comprising," "has," "having," "includes," and "including," are also open-ended. For example, any method that "comprises," "has," or "includes" one or more steps is not limited to possessing only those one or more steps and also covers other unlisted steps. Similarly, any system or method that "comprises," "has," or "includes" one or more components is not limited to possessing only those components and covers other unlisted components.

[0138] Other objects, features, and advantages of the present disclosure are apparent from the detailed description provided herein. It should be understood, however, that the detailed description and any specific examples provided, while indicating specific embodiments of the disclosure, are given by way of illustration only, since various changes and modifications within the spirit and scope of the disclosure will become apparent to those skilled in the art from this detailed description. Any embodiment of the present disclosure may be used in combination with any other embodiment described herein.

[0139] All references herein are incorporated herein by reference in their entirety.EXAMPLES

[0140] The following examples are included to demonstrate preferred embodiments of the invention. It should be appreciated by those of skill in the art that the techniques disclosed in the examples which follow represent techniques discovered by the inventors to function well in the practice of the invention, and thus can be considered to constitute preferred modes for its practice. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the concept, spirit and scope of the invention. More specifically, it will be apparent that certain agents which arc both chemically and physiologically related may be substituted for the agents described herein while the same or similar results would be achieved. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the invention as defined by the appended claims.

[0141] Adoptive T cell therapy exhibits long-lasting response in a small subgroup of cancer patients. One significant hurdle in achieving effective and enduring results lies in the limited cytotoxicity and persistence of the transferred T cells due to their progressive exhaustion, leading to high rates of relapse, as observed in patients who receive CAR-T cell-based treatments, with relapse rates exceeding 80% at the 6-month mark. In the realm of immune checkpoint therapy, exhausted T cells hinder the effectiveness of the immune response. Consequently, it becomes imperative to develop optimized approaches aimed at reversing or resisting T cell exhaustion.

[0142] To address the issue of T cell exhaustion, a study focused on the epigenetic aspects of T cell exhaustion within the context of the altered epigenetic profiles of T cells infiltrating tumors was conducted. This encompassed an analysis of the epigenomes of tumor infiltrating lymphocytes (TILs) from several melanoma patients. This information was then integrated with another epigenetic dataset comprising patients who either responded or did not respond to anti- PD1 treatment. Additionally, information from an independent dataset containing open chromatin information from PD1 -expressing exhausted CD8+T cells derived from melanoma patients was incorporated. The results of these studies demonstrate that the epigenome is a key regulator of Tcell exhaustion, and that non-coding epigenetic elements, such as enhancers, can be modified to produce exhaustion resistant T cells for therapy.EXAMPLE 1CRISPR-dCAS9 KARB Screening with gRNAs Targeting Aberrant Enhancer Programming in Tumor Infiltrating Lymphocytes

[0143] A previous study conducted by Maitituoheti et al., (doi.org / 10.1101 / 2022.08.31.506051, 2022,) identified distinct chromatin states associated with anti-PDl resistance in non-responsive melanoma patients. Enhancers marked with the H3K27Ac histone modification were identified as playing a crucial role in distinguishing between anti-PDl responsive and non-responsive patient cohorts.

[0144] Highly significant enhancers were aggregated (with a p-value < 10-5) from eight melanoma patients, resulting in a total of 82,564 enhancers. TILs are in close proximity to tumor cells within the tumor microenvironment, facilitating communication through various mechanisms like cell signaling and extracellular vesicles. This suggests the potential for a shared epigenetic profile within the tumor microenvironment.

[0145] With this in mind, enhancers exclusive to TILs were identified by collecting 83,062 enhancers associated with eight short-term cultures (STC) derived from matching tumors and overlapped them with TIL enhancers. This process yielded 39,164 unique enhancers associated specifically with TILs. These exclusive enhancers play a role in regulating the diverse phenotype of TILs within the tumor.

[0146] Furthermore, how to identify enhancers related to TIL dysfunctionality was investigated by integrating 40,890 enhancers associated with anti-PD 1 resistance. 9,629 enhancers within TILs were identified that potentially contribute to ICT resistance. As one of the key reasons behind ICT resistance lies in the progressive increase in the number of exhausted CD8+T cells within the tumor microenvironment, 42,131 accessible chromatin regions from PD1 -expressing exhausted CD8+T cells of a melanoma patient were integrated with the previously identified 9,629 enhancers associated with ICT resistance in TILs. This analysis narrowed the focus of subsequent experiments to 561 enhancers that are exclusively associated with exhaustion and ICT resistance in CD8+T cells.

[0147] Next methods to assess the functionality of the identified enhancers were devised. To achieve this, an approach was adopted that involved targeted introduction of the repressive mark H3K9Me3 at the loci of these enhancers. To accomplish this, a fusion product known as dCas9KRAB, where KRAB is fused with dCAS9, was used. This approach allowed for the effective deactivation of the enhancer-regulated central dogma in exhausted CD8+T cells within TILs.

[0148] Individual guide RNAs (gRNAs) designed to target specific enhancers were cloned into a lentiviral plasmid termed pLV hU6-sgRNA hUbC-dCas9-KRAB-T2a-Puro. This plasmid enables the simultaneous expression of sgRNAs and dCas9KRAB. Through this process, a CRISPRi (CRISPR interference) library capable of targeting a total of 553 enhancers in exhausted CD8+T cells was developed with 500X strength.

[0149] To assess reversal of exhaustion in TILs following incorporation of the CRISPRi library, three distinct readouts were employed: 1) low expression of CD39 on the cell surface, 2) high secretion of IFN-y, and 3) high mitochondrial potential. TIL #2765 was specifically chosen for this study, as these TILs were derived from a patient and were readily available in significant numbers. Given that TILs constitute a heterogeneous population of immune cells, fluorescence- activated cell sorting (FACS) was employed to isolate three independent subsets of exhausted CD8+T cells that demonstrated a reversal of exhaustion within their respective categories. Surprisingly, exhaustion reversal was observed in a range of 4% to 18% of CD8+T cells. As a result, three distinct CD8+T cell populations were obtained, each comprising gRNAs responsible for targeting enhancers associated with T cell exhaustion.

[0150] For each sequenced library, both end adapters were removed using custom in-house scripts, and indexed libraries were generated using the “makeblastdb” tool. Simultaneously, the same input settings were applied to trim the designed sgRNA dictionary. Subsequently, the trimmed sgRNA dictionary was aligned against the BLAST-indexed sequencing libraries using Blastn. Three specific alignment criteria were enforced during the Blast sequence alignment process: 1) 'max_hsps' was set to 1, 2) 'max_target_seqs' was limited to 1, and 3) no more than one mismatched nucleotide was allowed. From the resulting matching hits, read counts were calculated to generate a count matrix. To standardize the data, the identified read counts were initially normalized to one million reads. This was achieved by calculating reads per gRNAdivided by the total reads for all gRNAs, multiplied by 1 ,000,000, plus 1 . Subsequently, the log fold change (LFC) at base 2 was calculated for each sgRNA by comparing it to the control reads.

[0151] To prioritize sgRNAs based on their differential effects, a ranking system was implemented using the Z-Score method. For each sgRNA the LFC was determined, including the average LFC and the standard deviation for LFC. The sgRNAs were then ranked with a minimum Z-Score threshold set at 1.5.EXAMPLE 2Targeting an Enhancer Hub Surrounding the GAB3 gene Improves Interferon gamma Production in Tumor Infiltrating Lymphocytes

[0152] Exemplary gRNAs targeting the enhancer hub surrounding GAB3 as well as exemplary enhancer sequences comprised within the hub are provided in Table 1. Suppression of an enhancer hub surrounding the gene GAB3 (on Chr 12) using dCas9-KRAB (dead Cas9 coupled with KRAB suppressor) and any of the gRNAs shown in Table 2 led to increase in interferon gamma production when introduced into tumor infiltrating lymphocytes (TILs) isolated from melanoma patients (TIL2765). Table 3 shows the percentage of TILs that were positive for IFNy activation following gRNA treatment.Table 1. Exemplary enhancer sequences of the enhancer hub surrounding GAB3 (on Chr X) and exemplary gRNAs targeting the hub.Table 2. Exemplary gRNAs targeting an enhancer hub surrounding GAB 3 (on Chr X).Table 3. IFNy activation in TILs following gRNA treatment.

[0153] The enhancers surrounding the GAB3 gene demonstrated interaction with multiple different genes through Hi-ChIP experiments performed in the same TIL, including GAB3, LAGE3, FAM50A, UBL4A, GDI], SLC10A3, G6PD, CTAG1A, TP6AP1, PLXNA3, FAM3A, IKBKG, CTAG1B, CTAG2, DKC1, MeCP2, EMD, and TAZ. The enhancer hub was upregulated in both pre-treatment and post-treatment samples from patients who were not responsive to immune checkpoint inhibitor therapy (FIG. 1).EXAMPLE 3Targeting an Enhancer Hub Surrounding the GAB3 Gene and Targeting the GAB 3 gene Improves T cell Mediated Killing of HLA-matched Melanoma Cells

[0154] A co-culture experiment was performed with TIL3.0, which are currently used in aclinical trial in melanoma at MD Anderson Cancer Center. TILs were grown in the presence of high dose IL-2 (6000 U / ml) and agnostic antibodies targeting CD3 and CD 137. TILs (TIL3196A,TIL3162B, and TIL3183) were activated with plate bound anti-CD3 (300 ng / ml) and subjected to knockout of EnHubl (an enhancer surrounding the GAB3 gene) using the Synthego gene KO approach. Cytotoxic potential of EnHubl deficient TILs was analyzed by co-culturing TILs with Cell Trace Violet labeled HLA matched melanoma cell line M526 at effector to target ratios of 1:1, 5:1, and 10:1 for 3 hours (FIG. 2, Panel A). At the end of the assay, target cells were stained with a Caspase 3 / 7 staining kit to analyze the percent of target cells undergoing effector mediated apoptosis. A significant increase in TIL mediated killing of M526 cells (up to 37%) was demonstrated in each of these experiments (FIG. 2, Panel B). Enhanced toxicity was observed at a higher TIL:MEL ratio for all TILs. Surprisingly, however, TIL3162B and TIL3183 demonstrated more than a three-fold increase in tumor cell killing at the lower 1:1 TIL:MEL ratio following inhibition of EnHubl.

[0155] To confirm the functional role of EnHubl as well as the GAB3 gene itself, it was next investigated whether GAB3 knockout in primary T cells will reduce the exhaustion induced by repeated antigen stimulation and TGFp exposure under hypoxic conditions. CD8+T cells from PBMCs of healthy donors activated using anti-CD3 / 28 and expanded on IL-2 were used. Control or GAB3 KO cell lines were made using CRISPR. FIG. 3, Panel A confirms GAB3 knockdown. To induce exhaustion, T cells were re-stimulated with antigen for three rounds using plate-bound anti-CD3 and TGFP inside a hypoxic chamber. Cells with GAB3 KO showed strong reduction in exhaustion markers CD39 and TIGIT when compared to control cells (FIG. 3, Panel B). These data demonstrate that epigenetic editing of enhancers can be used to reduce T cell exhaustion and improve T cell mediated killing of cognate tumor cells.EXAMPLE 4GAB3 Knockout Increases Sternness Markers and Reduces Exhaustion Markers in T cells

[0156] Single cell multiomic sequencing (single cell RNA-seq and single cell ATAC-seq from the same cells) was performed in GAB3K0and GAB3WTCD8+T cells subjected to the in vitro T cell exhaustion protocol as described in Example 3. Single cell data analysis was completed by CellRanger and Seurat v5 UMAP of cells that passed stringent quality control parameters. The results of this analysis demonstrated that control resting (CtrlRes) cells separated from GAB3K0resting cells (KO_RES). Resting cells were not subjected to the in vitro T cell exhaustion protocol. Furthermore, control exhausted cells (CtrlEx) significantly separated from GAB3KOexhaustedcells (KO_Ex). In this experiment “exhausted cells” were those that have been subjected to the in vitro T cell exhaustion protocol. Clustering analysis followed by examination of known markers of exhaustion demonstrated loss of TIGIT, BATF, and ENTPD1 in GAB3KOexhausted cells compared to control exhausted cells. Furthermore, GAB3KOexhausted cells demonstrated increased expression of sternness or memory markers LEF1, TCF7, and BACH2 compared to control exhausted cells. Epigenetically, GAB3KOcells show increase in API programs, which are associated with T cell activation. GABK0cells also demonstrate a reduction in IRF and BATF programs, which are associated with exhaustion. Pathway analysis of BATF programs that are shut down by GAB3KO, showed enrichment of TGF0 signaling, demonstrating that GAB3KOreduces TGFP signaling promoted by the BATF program. This data confirms that GAB3K0decreases exhaustion and increases memory or sternness phenotypes in T cells.EXAMPLE 5GAB3 Regulates the T cell Activation Response

[0157] A reverse phase protein array (RPPA) was performed to determine signaling pathways that are impacted by GAB3 knockout. RPPA was performed in GAB3KOand GAB3WTCD8+T cells subjected to the in vitro T cell exhaustion protocol described above. Proteins that were upregulated in control exhaustion conditions and downregulated in GAB3 knockout exhaustion conditions included the Y317 phosphorylated form of She (FIG. 4, Panel A). This phosphorylation event occurs upon TCR signaling. The TCR is activated and activates the downstream RAS signaling required for T cell proliferation. Downregulation of CDK1 phosphorylation, PKM2 levels, and STING levels was also observed in GAB3KOT cells (FIG. 4, Panel B; FIG. 4, Panel C; FIG. 4, Panel D). CDK1 phosphorylation is critical for cell cycle progression, PKM2 is critical for activation of glycolysis, and STING induces an interferon response. The data demonstrate that these key signaling molecules increase allowing for hyperactivation of T cells (due to repeated antigen stimulation under harsh tumor microenvironmental conditions), which leads to eventual T cell exhaustion. GAB3 contributes to this process. Thus, knockout of GAB3 balances hyperactivation of the T cell response and prevents cells from undergoing complete exhaustion.EXAMPLE 6EnHubl Suppression Augments CD8+ T Cell Function and Tumor Clearance

[0158] To further investigate the role of EnHubl modulation in enhancing T cell-mediated antitumor responses, in vivo xenograft studies were conducted using M526 melanoma cells and tumorinfiltrating lymphocytes (TILs). Mice receiving enhancer-modified TILs demonstrated a substantial delay in tumor progression relative to control and WT TIL-treated groups, underscoring the improved tumor-clearing capacity of the modified T cells (FIG. 5, Panel A). Flow cytometric analysis of tumor-infiltrating lymphocytes showed that EnHubl inhibition markedly increased the accumulation of intra-tumoral CD8+T cells (FIG. 5, Panel B). Further phenotypic characterization indicated a significant skewing toward an effector memory phenotype (CD45RCF) among these CD8+T cells (FIG. 5, Panel C), demonstrating enhanced functionality and persistence. Importantly, the frequency of exhausted TIGIT+CD8+T cells was significantly reduced in the EnHubl modified group, pointing to an epigenetically driven reversal of T cell exhaustion (FIG. 5, Panel D). Together, these findings demonstrate that EnHubl inhibition reprograms TILs toward a cytotoxic, memory-enriched, and less exhausted state, thereby significantly improving antitumor efficacy both in vitro and in vivo.EXAMPLE 7GAB3 KO Improves Mitochondrial Function in T Cells

[0159] Metabolic reprogramming is an important feature of different T cell phenotypes. For example, CD8+memory T cells show increased oxygen consumption rate (OCAR) in seahorse assays. To determine if GAB3 KO shows improved mitochondrial activity, Agilent seahorse XF was performed on control and GAB3KOCD8+T cells. Briefly, oxygen consumption rate was measured before and after adding pharmacological agents to respiring cells. For the first time point total cellular oxygen consumption was measured for 18 min. Next oligomycin, an inhibitor of adenosine-5’ -triphosphate (ATP) respiration, was added and a measurement of this was made over the next 18 min. This quantity was subtracted from the total cellular oxygen consumption to determine ATP-linked respiration and non-mitochondrial respiration can be subtracted from this quantity to obtain proton-leak respiration. Next carbonyl cyanide-p-trifluoromethoxyphenyl- hydrazon, a protonophore, was added and a measurement of this was made over the next 18 min.The protonophore collapses the inner membrane gradient by making the inner membrane permeable to protons. This drives the electron transport chain to function at its maximum rate. Subtracting non-mitochondrial respiration from this quantity produces a measure of maximum respiratory capacity. Finally, antimycin A, a complex III inhibitor, and rotenone, a complex-I inhibitor, are added to shut down electron transport chain function. The resulting measurement over the next 18 min represents non-mitochondrial respiration, a measurement that can be used with the other measurements to calculate respiratory parameters. Finally, reserve capacity was calculated by subtracting basal respiration from maximum respiratory capacity. Similar to key features of central memory population, GAB3KOshowed increased OCAR in this assay.EXAMPLE 8GAB3 KO Enhances T cell Expansion

[0160] The ability of the T cells with GAB3KOto be expanded from tumor fragments was analyzed. CD8+T cells were expanded in T cell culture media using the Rapid Expansion Protocol (REP) with low-dose IL-2. GAB3 KO cells demonstrated a 4-5 fold better expansion than control T cells (FIG. 6, Panel A). To measure their tumor-killing potential, post-REP T cells were collected 14 days and co-cultured with target tumor cells. Caspase- 3 / 7 staining showed much higher ability of GAB3KOT cells to kill cognate tumor cells (FIG. 6, Panel B).* * J

[0161] All of the methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. While the compositions and methods of this invention have been described in terms of preferred embodiments or aspects, it will be apparent to those of skill in the ai that variations may be applied to the methods and in the steps or in the sequence of steps of the method described herein without departing from the concept, spirit, and scope of the invention. More specifically, it will be apparent that certain agents which are both chemically and physiologically related may be substituted for the agents described herein while the same or similar results would be achieved. All such similar substitutes and modifications apparent to those skilled in the ail are deemed to be within the spirit, scope and concept of the invention as defined by the appended claims.

Claims

CLAIMS1. A modified hematopoietic cell comprising at least one genomic modification in a nucleotide sequence having at least about 85% sequence identity to SEQ ID NO:1, SEQ ID NO:2, SEQ ID NOG, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, or SEQ ID NO: 10.

2. The modified hematopoietic cell of claim 1, wherein said genomic modification alters the expression level or activity of at least one protein selected from the group consisting of GAB3, LAGE3, FAM50A, UBL4A, GD11, SLC10A3, G6PD, CTAG1A, TP6AP1, PLXNA3, FAM3A, IKBKG, CTAG1B, CTAG2, DKC1, MeCP2, EMD, and TAZ.

3. The modified hematopoietic cell of claim 2, wherein said genomic modification decreases the expression level or activity of at least one protein selected from the group consisting of GAB3, LAGE3, FAM50A, UBL4A, GDI1, SLC10A3, G6PD, CTAG1A, TP6AP1, PLXNA3, FAM3A, IKBKG, CTAG1B, CTAG2, DKC1, MeCP2, EMD, and TAZ.

4. The modified hematopoietic cell of claim 2, wherein said genomic modification increases the expression level or activity of at least one protein selected from the group consisting of LAGE3, FAM50A, UBL4A, GDI1, SLC10A3, G6PD, CTAG1A, TP6AP1, PLXNA3, FAM3A, IKBKG, CTAG1B, CTAG2, DKC1 , MeCP2, EMD, and TAZ.

5. The modified hematopoietic cell of claim 1, wherein said modified hematopoietic cell is selected from the group consisting of a T cell, a CD4+T cell, a CD8+T cell, a CAR T cell, a tumorinfiltrating lymphocyte, a T cell expressing an engineered TCR, an endogenous T cell, a monocyte, a granulocyte, and an NK cell.

6. The modified hematopoietic cell of claim 1, wherein the hematopoietic cell comprises at least one genomic modification in a nucleotide sequence having at least about 90%, at least about 95%, at least about 99%, or 100% sequence identity to SEQ ID NO:1, SEQ ID NO:2, SEQ ID NOG, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, or SEQ ID NO: 10.

7. The modified hematopoietic cell of claim 1, wherein the hematopoietic cell comprises at least two genomic modifications in at least two nucleotide sequences having at least about 85%sequence identity to SEQ ID N0:1 , SEQ ID N0:2, SEQ ID N0:3, SEQ ID N0:4, SEQ ID N0:5, SEQ ID N0:6, SEQ ID N0:7, SEQ ID N0:8, SEQ ID N0:9, or SEQ ID NO: 10.

8. The modified hematopoietic cell of claim 1, wherein the genomic modification comprises a deletion, an insertion, a substitution, an inversion, a duplication, or a combination of any thereof.

9. The modified hematopoietic cell of claim 1 , wherein the hematopoietic cell is heterozygous for the genomic modification.

10. The modified hematopoietic cell of claim 1, wherein the hematopoietic cell is homozygous for the genomic modification.

11. The modified hematopoietic cell of claim 1 , wherein the hematopoietic cell is a T cell and said genomic modification decreases or eliminates the development of T cell exhaustion.

12. The modified hematopoietic cell of claim 1, wherein the modified hematopoietic cell further comprises at least one genomic modification of an endogenous gene selected from the group consisting of GABS, LAGES, FAM50A, UBL4A, GDIS, SLC10A3, G6PD, CTAG1A, TP6AP1, PLXNA3, FAM3A, IKBKG, CTAG1B, CTAG2, DKC1, MeCP2, EMD, and TAZ.

13. The modified hematopoietic cell of claim 12, wherein: a) the GAB3 gene encodes a polypeptide having at least about 85% sequence identity to SEQ ID NO:21, the LAGE3 gene encodes a polypeptide having at least about 85% sequence identity to SEQ ID NO:22, the FAM 50 A gene encodes a polypeptide having at least about 85% sequence identity to SEQ ID NO:23, the UBL4A gene encodes a polypeptide having at least about 85% sequence identity to SEQ ID NO:24, the GDI1 gene encodes a polypeptide having at least about 85% sequence identity to SEQ ID NO:25, the SLC10A3 gene encodes a polypeptide having at least about 85% sequence identity to SEQ ID NO:26, the G6PD gene encodes a polypeptide having at least about 85% sequence identity to SEQ ID NO:27, the CTAG1A gene encodes a polypeptide having at least about 85% sequence identity to SEQ ID NO:28, the TP6AP1 gene encodes a polypeptide having at least about 85% sequence identity to SEQ ID NO:29, the PLXNA3 gene encodes a polypeptide having at least about 85% sequence identity to SEQ ID NQ:30, the FAM3A gene encodes a polypeptide having at least about 85% sequence identity to SEQ ID NO:31,the IKBKG gene encodes a polypeptide having at least about 85% sequence identity to SEQ ID NO:32, the CTAG1B gene encodes a polypeptide having at least about 85% sequence identity to SEQ ID NO:33, the CTAG2 gene encodes a polypeptide having at least about 85% sequence identity to SEQ ID NO:34, the DKC1 gene encodes a polypeptide having at least about 85% sequence identity to SEQ ID NO:35, the MeCP2 gene encodes a polypeptide having at least about 85% sequence identity to SEQ ID NO:54, the EMD gene encodes a polypeptide having at least about 85% sequence identity to SEQ ID NO:55, or the TAZ gene encodes a polypeptide having at least about 85% sequence identity to SEQ ID NO:56; or b) the GAB3 gene comprises a sequence having at least about 85% sequence identity to SEQ ID NO:36, the LAGE3 gene comprises a sequence having at least about 85% sequence identity to SEQ ID NO:37, the FAM50A gene comprises a sequence having at least about 85% sequence identity to SEQ ID NO:38, the UBL4A gene comprises a sequence having at least about 85% sequence identity to SEQ ID NO:39, the GDI1 gene comprises a sequence having at least about 85% sequence identity to SEQ ID NO:40, the SLC10A3 gene comprises a sequence having at least about 85% sequence identity to SEQ ID NO:41, the G6PD gene comprises a sequence having at least about 85% sequence identity to SEQ ID NO:42, the CTAG1A gene comprises a sequence having at least about 85% sequence identity to SEQ ID NO:43, the TP6AP1 gene comprises a sequence having at least about 85% sequence identity to SEQ ID NO:44, the PEXNA3 gene comprises a sequence having at least about 85% sequence identity to SEQ ID NO:45, the FAM3A gene comprises a sequence having at least about 85% sequence identity to SEQ ID NO:46, the IKBKG gene comprises a sequence having at least about 85% sequence identity to SEQ ID NO:47, the CTAG1B gene comprises a sequence having at least about 85% sequence identity to SEQ ID NO:48, the CTAG2 gene comprises a sequence having at least about 85% sequence identity to SEQ ID NO:49, the DKC1 gene comprises a sequence having at least about 85% sequence identity to SEQ ID NO:50, the MeCP2 gene comprises a sequence having at least about 85% sequence identity to SEQ ID NO:57, the EMD gene comprises a sequence having at least about 85% sequence identity to SEQ ID NO:58, or the TAZ gene comprises a sequence having at least about 85% sequence identity to SEQ ID NO:59.

14. The modified hematopoietic cell of claim 12, wherein said genomic modification alters the expression level or activity of at least one protein selected from the group consisting of GAB3, LAGE3, FAM50A, UBL4A, GDI1, SLC10A3, G6PD, CTAG1A, TP6AP1, PLXNA3, FAM3A, IKBKG, CTAG1B, CTAG2, DKC1, MeCP2, EMD, and TAZ.

15. A method of treating cancer in a subject in need thereof, the method comprising administering the modified hematopoietic cell of claim 1 to said subject.

16. The method of claim 15, wherein said cancer is selected from the group consisting of lung cancer, colorectal cancer, pancreatic cancer, appendiceal cancer, small bowel adenocarcinoma, hepatobiliary cancer, gynecological cancer, hematopoietic cancer, breast cancer, bladder cancer, prostate cancer, skin cancer, head and neck squamous cell carcinoma, melanoma, and genitourinary cancer.

17. The method of claim 15, the method further comprising administering a second therapy to said subject.

18. The method of claim 17, wherein the second therapy is selected from the group consisting of a chemotherapy, a radiotherapy, an immunotherapy, and a surgery.

19. The method of claim 15, wherein the subject is a mammalian subject.

20. The method of claim 19, wherein the subject is a human subject.

21. A method for producing a modified hematopoietic cell, the method comprising introducing into a hematopoietic cell at least one genomic modification into at least one target site of a nucleotide sequence comprising at least about 85% sequence identity to SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, or SEQ ID NO: 10.

22. The method of claim 21, wherein said introducing the genomic modification comprises introducing a site-specific nuclease into the hematopoietic cell, wherein the site-specific nuclease is capable of specifically binding to and cleaving said target site.

23. The method of claim 22, wherein the site-specific nuclease is selected from the group consisting of an RNA-guided nuclease, a zinc finger nuclease, and a TALEN.

24. The method of claim 23, wherein the site-specific nuclease is an RNA-guided nuclease.

25. The method of claim 24, further comprising introducing into the hematopoietic cell at least one guide polynucleotide molecule comprising a nucleotide sequence that is substantially complementary to the target site, wherein the guide polynucleotide and the RNA-guided nuclease form a complex that is capable of specifically binding to and cleaving the target site.

26. The method of claim 25, wherein the guide polynucleotide molecule comprises a nucleotide sequence having at least about 85%, at least about 90%, or at least about 95% sequence identity to SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO:20, SEQ ID NO:51, SEQ ID NO:52, or SEQ ID NO:53.

27. A guide polynucleotide molecule comprising a nucleotide sequence having at least about 85%, at least about 90%, or at least about 95% sequence identity to SEQ ID NO: 11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:51, SEQ ID NO:52, or SEQ ID NO:53.

28. A composition comprising the guide polynucleotide molecule of claim 27.

29. The composition of claim 28, further comprising a site-specific nuclease.

30. A modified hematopoietic cell comprising at least one genomic modification that modulates the expression of an endogenous gene selected from the group consisting of LAGES, FAM50A, UBL4A, GDI], SLC10A3, G6PD, CTAG1A, TP6AP1, PLXNA3, FAM3A, IKBKG, CTAG1B, CTAG2, DKC1, MeCP2, EMD, and TAZ.

31. The modified hematopoietic cell of claim 30, wherein: a) the LAGE3 gene encodes a polypeptide having at least about 85% sequence identity to SEQ ID NO:22, the FAM 50 A gene encodes a polypeptide having at least about 85% sequence identity to SEQ ID NO:23, the UBL4A gene encodes a polypeptide having at least about 85% sequence identity to SEQ ID NO:24, the GDI1 gene encodes a polypeptide having at least about 85% sequence identity to SEQ ID NO:25, the SLC10A3 gene encodes a polypeptide having at least about 85% sequence identity to SEQ ID NO:26, the G6PD gene encodes a polypeptide having at least about 85% sequence identity to SEQ ID NO:27,the CTAG1A gene encodes a polypeptide having at least about 85% sequence identity to SEQ ID NO:28, the TP6AP1 gene encodes a polypeptide having at least about 85% sequence identity to SEQ ID NO:29, the PLXNA3 gene encodes a polypeptide having at least about 85% sequence identity to SEQ ID NO:30, the FAM3A gene encodes a polypeptide having at least about 85% sequence identity to SEQ ID NO:31, the IKBKG gene encodes a polypeptide having at least about 85% sequence identity to SEQ ID NO:32, the CTAG1B gene encodes a polypeptide having at least about 85% sequence identity to SEQ ID NO:33, the CTAG2 gene encodes a polypeptide having at least about 85% sequence identity to SEQ ID NO: 34, the DKC1 gene encodes a polypeptide having at least about 85% sequence identity to SEQ ID NO:35, the MeCP2 gene encodes a polypeptide having at least about 85% sequence identity to SEQ ID NO:54, the EMD gene encodes a polypeptide having at least about 85% sequence identity to SEQ ID NO:55, or the TAZ gene encodes a polypeptide having at least about 85% sequence identity to SEQ ID NO:56; or b) the LAGE3 gene comprises a sequence having at least about 85% sequence identity to SEQ ID NO:37, the FAM50A gene comprises a sequence having at least about 85% sequence identity to SEQ ID NO:38, the UBE4A gene comprises a sequence having at least about 85% sequence identity to SEQ ID NO:39, the GDI1 gene comprises a sequence having at least about 85% sequence identity to SEQ ID NO:40, the SEC10A3 gene comprises a sequence having at least about 85% sequence identity to SEQ ID NO:41, the G6PD gene comprises a sequence having at least about 85% sequence identity to SEQ ID NO:42, the CTAG1A gene comprises a sequence having at least about 85% sequence identity to SEQ ID NO:43, the TP6AP1 gene comprises a sequence having at least about 85% sequence identity to SEQ ID NO:44, the PEXNA3 gene comprises a sequence having at least about 85% sequence identity to SEQ ID NO:45, the FAM3A gene comprises a sequence having at least about 85% sequence identity to SEQ ID NO:46, the IKBKG gene comprises a sequence having at least about 85% sequence identity to SEQ ID NO:47, the CTAG1B gene comprises a sequence having at least about 85% sequence identity to SEQ ID NO:48, the CTAG2 gene comprises a sequence having at least about 85% sequence identity to SEQ ID NO:49, the DKC1 gene comprises a sequence having at least about 85% sequence identity to SEQ ID NQ:50, the MeCP2 gene comprises a sequence having at leastabout 85% sequence identity to SEQ ID NO:57, the EMD gene comprises a sequence having at least about 85% sequence identity to SEQ ID NO:58, or the TAZ gene comprises a sequence having at least about 85% sequence identity to SEQ ID NO:59.

32. The modified hematopoietic cell of claim 30, wherein said genomic modification alters the expression level or activity of at least one protein selected from the group consisting of LAGE3, FAM50A, UBL4A, GDI1, SLC10A3, G6PD, CTAG1A, TP6AP1, PLXNA3, FAM3A, IKBKG, CTAG1B, CTAG2, DKC1, MeCP2, EMD, and TAZ.

33. The modified hematopoietic cell of claim 30, further comprising at least one genomic modification of an endogenous GABS gene.

34. The modified hematopoietic cell of claim 33, wherein: a) the GAB3 gene encodes a polypeptide having at least about 85% sequence identity to SEQ ID NO:21; or b) the GAB3 gene comprises a sequence having at least about 85% sequence identity to SEQ ID NO:36.

35. The modified hematopoietic cell of claim 30, wherein said modified hematopoietic cell is selected from the group consisting of aT cell, a CD4+T cell, a CD8+T cell, a CAR T cell, a tumorinfiltrating lymphocyte, a T cell expressing an engineered TCR, an endogenous T cell, a monocyte, a granulocyte, and an NK cell.

36. The modified hematopoietic cell of claim 30, wherein the hematopoietic cell is a T cell and said genomic modification decreases or eliminates the development of T cell exhaustion.

37. A method of treating cancer in a subject in need thereof, the method comprising administering the modified hematopoietic cell of claim 30 to said subject.

38. The method of claim 37, wherein said cancer is selected from the group consisting of lung cancer, colorectal cancer, pancreatic cancer, appendiceal cancer, small bowel adenocarcinoma, hepatobiliary cancer, gynecological cancer, hematopoietic cancer, breast cancer, bladder cancer, prostate cancer, skin cancer, head and neck squamous cell carcinoma, melanoma, and genitourinary cancer.

39. The method of claim 37, further comprising administering a second therapy to said subject.

40. The method of claim 39, wherein the second therapy is selected from the group consisting of a chemotherapy, a radiotherapy, an immunotherapy, and a surgery.

41. The method of claim 37, wherein the subject is a mammalian subject.

42. The method of claim 41, wherein the subject is a human subject.

43. A method of for producing a modified hematopoietic cell, the method comprising introducing into a hematopoietic cell at least one genomic modification into at least one target site of a nucleotide sequence comprising at least about 85% sequence identity to SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:57, SEQ ID NO:58, or SEQ ID NO:59.

44. The method of claim 43, wherein said introducing the genomic modification comprises introducing a site-specific nuclease into the hematopoietic cell, wherein the site-specific nuclease is capable of specifically binding to and cleaving said target site.

45. The method of claim 44, wherein the site-specific nuclease is selected from the group consisting of an RNA-guided nuclease, a zinc finger nuclease, and a TALEN.

46. The method of claim 45, wherein the site-specific nuclease is an RNA-guided nuclease.

47. The method of claim 46, further comprising introducing into the hematopoietic cell at least one guide polynucleotide molecule comprising a nucleotide sequence that is substantially complementary to the target site, wherein the guide polynucleotide and the RNA-guided nuclease form a complex that is capable of specifically binding to and cleaving the target site.

48. A method of increasing expansion of a hematopoietic cell, the method comprising: a) introducing into the hematopoietic cell at least one genomic modification into at least one target site of a nucleotide sequence comprising at least about 85% sequence identity to SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO: 10, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41,SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:57, SEQ ID NO:58, or SEQ ID NO:59; and b) culturing the hematopoietic cell.

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