Gene targets for t-cell immunotherapy to overcome t cell exhaustion

Abstract

Provided herein are genetically modified T cells that exhibit reduced T cell exhaustion upon repeated exposure to tumor cells compared to wild type T cells; and method of generating such T cells and using them for the treatment of cancer.

Description

Gene Targets for T-Cell Immunotherapy to Overcome T Cell ExhaustionCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claim priority benefit of U.S. Provisional Patent Application No. 63 / 594,925, filed October 31, 2023, which is incorporated by reference in its entirety for all purposes.BACKGROUND OF THE DISCLOSURE

[0002] T cell exhaustion generally refers to an acquired state of T cell dysfunction resulting from chronic antigen stimulation. It is a hallmark of chronic viral infection and response to tumors. Understanding pathways leading to exhaustion has important implications for therapeutic success of immunotherapies such as checkpoint inhibitor blockage and adoptive T cell transfer therapies.BRIEF SUMMARY OF CERTAIN ASPECTS OF THE DISCLOSURE

[0003] The present disclosure is based, in part, on the development of a genetic screen to identify genes that play a role in T-cell dysfunction that results from repeated antigen stimulation, and the targeting of such genes for inhibition to mitigate T cell exhaustion.

[0004] Thus, in one aspect, the disclosure features a genetically modified T cell that comprises a genetic modification to at least one T-cell exhaustion gene that inhibits expression or activity of the polypeptide product encoded by the T-cell exhaustion gene, wherein the gene is selected from the group consisting MED 4, KDM6A, AUP1, SATB1, STT3A, MED 16, PINLYP, MAPK14, UBAP2L, TAF3, BCL2L11, KCTD5,CCDC53, DDX47, ZNF671, TM2D1, TRIM58, TICRR, KRT26, MED15, B4GALT1, RFWD2, UBE2F, UBE2L3, DCUN1D3, wA MEF2D and expression or activity of the polypeptide product is inhibited by at least 25%, 30%, 35%, 40%, or 45%, or greater, e.g., at least 50% or greater, compared to a control wildtype T cell. In some embodiments, the gene is a gene selected from the group of genes listed in Table 2 and expression or activity of the polypeptide product is inhibited by at least 25%, 30%, 35%, 40%, or 45%, or greater, e.g., at least 50% or greater, compared to a control T cell. In some embodiments, the T-cell is a CD8+ T cell or CD4+ T cell. In some embodiments, the gene is a gene selected from the group of genes listed in Table 1. In someembodiments, the T-cell exhaustion gene is inhibited using a clustered, regularly interspaced, short palindromic repeats (CRISPR) system. In other embodiments, the T-cell exhaustion gene is inhibited using a transcription activator-like effector nuclease (TALEN) system, a zinc finger nuclease system, or a meganuclease system. In further embodiments, the T-cell exhaustion gene is inhibited using inhibitory RNA. In some embodiments, the T-cell exhuation gene is inhibited using shRNA, siRNA, microRNA, or an antisense RNA. In some embodiments, the T cell comprising genetic modifications to inhibit expression of at least two T-cell exhaustion genes expresses a TCR-based chimeric receptor, e.g., chimeric antigen receptor (CAR).

[0005] In a further aspect the disclosure provides a genetically modified T cell that comprises (i) a first genetic modification that inhibits expression or activity of a polypeptide product encoded by a first T-cell exhuation gene selected from the group consisting MED24, KDM6A, AUP1, SATB1, STT3A, MED 16, PINLYP, MAPK14, UBAP2L, TAF3, BCL2L11, KCTD5,CCDC53, DDX47, ZNF671, TM2D1, TRIM58, TICRR, KRT26, MED 15, B4GALT1, RFWD2, CUL5, ARIH2, RNF7, UBE2F, UBE2L3, CISH, DCUN1D3, and MEF2D; and (ii) a second genetic modification that inhibits expression or activity of the polypeptide product encoded by a second T-cell exhaustion gene selected from the group consisting MED24, KDM6A, AUP1, SATB1, STT3A, MED 16, PINLYP, MAPK14, UBAP2L, TAF3, BCL2L11, KCTD5,CCDC53, DDX47, ZNF671, TM2D1, TRIM58, TICRR, KRT26, MED 15, B4GALT1, RFWD2, CUL5, ARIH2, RNF7, UBE2F, UBE2L3, CISH, DCUN1D3, and MEF2D. In some embodiments three or more T-cell exhaustion genes as described herein are inhibited. In some embodiments, the first and / or the second T-cell exhaustion gene is a Cullin 5 complex gene selected from the group consisting of CUL5, ARIH2, RNF7, UBE2F, UBE2L3, CISH, and DCUN1D3. In some embodiments, the T cell is a CD8+ T cell or CD4+ T cell. In some embodiments, the first and / or the second T-cell modification gene, e.g., Cullin 5 complex gene, is inhibited using a clustered, regularly interspaced, short palindromic repeats (CRISPR) system. In other embodiments, the first and / or the second T-cell modification gene, e.g., Cullin 5 complex gene, is inhibited using a transcription activator-like effector nuclease (TALEN) system, a zinc finger nuclease system, or a meganuclease system. In some embodiments, the first and / or the second T-cell modification gene, e.g., Cullin 5 complex gene, is inhibited, e.g., knocked out, using a CRIPR gene editing system. In further embodiments, the first and / or the second T-cell modification gene, e.g., Cullin 5 complex gene, is inhibited using inhibitory RNA. In some embodiments, the first and / or the second T-cell modification gene, e.g., Cullin 5 complex gene, is inhibited using shRNA, siRNA, microRNA, or an antisense RNA. In some embodiments, the T cell comprising genetic modifications to inhibit expression of at least two T-cell exhuastion genes expresses a TCR- based chimeric receptor, e.g., chimeric antigen receptor (CAR).

[0006] In a further aspect, the disclosure features a population of cell comprising a genetically modified T cell as described herein, e.g., in the preceding to two paragraphs. In addition, the disclosure features a method of treating cancer comprising administering a population of cells comprising such a genetically modified T cell to a subject that has cancer.Definitions

[0007] As used herein, the singular forms "a", "an", and "the" are also intended to refer to the plural unless the context clearly dictates otherwise.

[0008] The terms “polynucleotide” and “nucleic acid” are used interchangeably to refer to a polymeric form of nucleotides of any length, either deoxyribonucleotides or ribonucleotides. The terms include RNA, DNA, and synthetic forms and mixed polymers of the above. In particular embodiments, a nucleotide refers to a ribonucleotide, deoxynucleotide or a modified form or analog of either type of nucleotide, and combinations thereof. In addition, a polynucleotide may include either or both naturally occurring and modified nucleotides linked together by naturally occurring and / or non-naturally occurring nucleotide linkages. The nucleic acid molecules may be modified chemically or biochemically or may contain non-natural or derivatized nucleotide bases. Such modifications include, for example, labels, substitution of one or more of the naturally occurring nucleotides with an analogue, internucleotide modifications such as uncharged linkages (e.g., methyl phosphonates, phosphotriesters, phosphoramidates, carbamates, etc.), charged linkages (e.g., phosphorothioates, phosphorodithioates, etc.), and modified linkages (e.g., alpha anomeric nucleic acids, etc.). The terms “polynucleotide” and “nucleic acid” are also intended to include any topological conformation, including single-stranded, double-stranded, partially duplexed, triplex, hairpinned, circular and padlocked conformations. A reference to a nucleic acid sequence encompasses its complement unless otherwise specified. Thus, a reference to a nucleic acid molecule having a particular sequence should be understood to encompass its complementary strand, with its complementary sequence. Reference to a “polynucleotide” or “nucleic acid” that encodes a polypeptide sequence also includes codon-optimized nucleicacids and nucleic acids that comprise alternative codons that encode the same polypeptide sequence.

[0009] As used herein, the term “complementary” or “complementarity” refers to specific base pairing between nucleotides or nucleic acids. Base pairing may be perfectly complementary or partially complementary.

[0010] The term “gene” can refer to the segment of DNA involved in producing or encoding a polypeptide chain. It may include regions preceding and following the coding region (leader and trailer) as well as intervening sequences (introns) between individual coding segments (exons). Genes disclosed herein, e.g., T-cell exhaustion genes, are defined by symbol and nomenclature for the human gene as assigned by the HUGO Gene Nomenclature Committee. Illustrative guide nucleotide sequences that target each of the T- cell exhaustion genes disclosed herein are provided in Table 3.

[0011] A “promoter” is defined as one or more a nucleic acid control sequences that direct transcription of a nucleic acid. As used herein, a promoter includes necessary nucleic acid sequences near the start site of transcription. A promoter also optionally includes distal enhancer or repressor elements, which can be located as much as several thousand base pairs from the start site of transcription.

[0012] The term “inhibiting expression” refers to inhibiting or reducing the expression of a gene or a protein. To inhibit or reduce the expression of a gene (i.e., a gene encoding a transcription factor, or a gene regulated by a transcription factor), the sequence and / or structure of the gene may be modified such that the gene would not be transcribed (for DNA) or translated (for RNA), or would not be transcribe or translated to produce a functional protein (e.g., a transcription factor). Various methods for inhibiting or reducing expression of a gene are described in detail further herein. Some methods may introduce nucleic acid substitutions, additions, and / or deletions into the wild-type gene. Some methods may also introduce single or double strand breaks into the gene. To inhibit or reduce the expression of a protein (e.g., a protein encoded by a T cell exhaustion gene), one may inhibit or reduce the expression of the gene or polynucleotide encoding the protein, as described above. In other embodiments, one may target the protein directly to inhibit or reduce the protein’s expression using, e.g., an antibody or a protease. “Inhibited” expression refers to a decrease by at least 20%, or at least 30%, or at least 40%, or at least 50%, or preferably at least 60%, or at least 70%, or at least 80%, or at least 90% or higher, up to and including a 100% decrease (i.e.absent level as compared to a reference sample). As used herein, the term "inactivated" refers to preventing expression of a polypeptide product encoded by the gene. Inactivation can occur at any stage or process of gene expression, including, but not limited to, transcription, translation, and protein expression, and inactivation can affect any gene or gene product including, but not limited to, DNA, RNA, such a mRNA, and polypeptides. In some embodiments, “inhibited expression” reflects inactivation in a percentage of cells that are modified, e.g., at least 20%, or at least 30%, or at least 40%, or at least 50%, or at least 60%, or at least 70%, or at least 80%, or at least 90% or greater of the cells in a population that also comprises cells in which the target gene is not inactivated.

[0013] The term “genetic modification” as used herein refers to any modification to a cell to alter expression of a gene. Such modifications include modifications to the genome as well as modifications to introduce inhibitory sequences, such as inhibitory RNAs, into the cell.

[0014] As used herein, the phrase “modifying” in the context of modifying a genome of a cell refers to inducing a structural change in the sequence of the genome at a target genomic region. For example, the modifying can take the form of inserting a nucleotide sequence into the genome of the cell. For example, a nucleotide sequence encoding a polypeptide can be inserted into the genomic sequence encoding an endogenous cell surface protein in the T cell. The nucleotide sequence can encode a functional domain or a functional fragment thereof. Such modifying can be performed, for example, by inducing a double stranded break within a target genomic region, or a pair of single stranded nicks on opposite strands and flanking the target genomic region. Methods for inducing single or double stranded breaks at or within a target genomic region include the use of a nuclease domain, e.g. Cas9, or a derivative thereof, and a guide, e.g, guide RNA, directed to the target genomic region.

[0015] The terms “patient,” “subject,” “individual,” and the like are used interchangeably herein, and refer to any animal, e.g, a mammal, such as a primate. In certain non-limiting embodiments, the patient, subject or individual is a human.

[0016] The terms "treatment", "treating", and the like are used herein to generally mean obtaining a desired pharmacologic and / or physiologic effect. The effect may be prophylactic, in terms of completely or partially preventing a disease, condition, or symptoms thereof, and / or may be therapeutic in terms of a partial or complete cure for a disease or condition and / or an adverse effect, such as a symptom, attributable to the disease or condition."Treatment" as used herein covers any treatment of a disease or condition of a subject and includes: (a) preventing the disease or condition from occurring in a subject which may be predisposed to the disease or condition but has not yet been diagnosed as having it; (b) inhibiting the disease or condition (e.g., arresting its development); or (c) relieving the disease or condition (e.g., causing regression of the disease or condition, providing improvement in one or more symptoms).BRIEF DESCRIPTION OF THE FIGURES

[0017] FIG. 1. Schematic of a genome-wide repetitive stimulation screen. A genome-wide exhaustion screen in T cells obtained from two human blood donors was performed. Pooled edited T cells were exposed to target tumor cells at a 1 : 1 ratio every 48 hours 9 times. Cells were then collected and next generation sequencing revealed the sgRNAs enriched in the T cells in the last time point compared to the first.

[0018] FIG. 2 identified various targets genes, including some genes previously identifed as affecting T cell fitness and in some cases, persistence.

[0019] FIG. 3 provide data showing seven members of the same complex, the Cullin 5 complex, that emerged as hits from screening.

[0020] FIG. 4 Arrayed knockout of Cullin-5 complex subunits improves persistent tumor killing. Multiple top target genes that are members of the Cul5 complex lead to enhanced tumor killing when knocked out.

[0021] FIG. 5A-5D T-cell killing of tumor killing using two guide RNAs to target Cul5 complex subunit genes. FIG. 5 A and FIG. 5B: killing activity prior to repeat exposure stimulation in T using two guide RNAs to target Cullin 5 complex members. FIG. 5C and FIG. 5D: killing activity after repeat exposure.

[0022] FIGS. 6A-6D show the results of in vivo experiments validating six target genes identified in the screen in an NALM6 xenograft model using CD19-CAR T cells. FIG. 6A: Tumor burden in mice with knockout of target gene MEF2D (▼), target gene UBAP2L (black octagon), or target gene BCL2L11 (♦) compared to control (*). FIG. 6B: Survival in mice with knockout of target gene MEF2D (▼), target gene UBAP2L (black octagon), or target gene BCL2L11 (♦) compared to control (*). FIG. 6C: Tumor burden in mice with knockout of target gene RFWD2 (■), target gene KDM6A (•), or target gene SATB1 (A)compared to control (*). FIG. 6D: Survival in mice with knockout of target gene RFWD2 (■), target gene KDM6A (•), or target gene SATB1 (A) compared to control (*).DETAILED DESCRIPTION OF THE DISCLOSURE

[0023] In one aspect, the disclosure provides engineered T cells that exhibit enhanced cytotoxicity to cells, e.g., tumor cells compared to counterpart unmodified T cells. Such engineered T cells are modified to inhibit expression or activity of a T-cell gene that plays a role in T-cell exhaustion. In the context of the present disclosure, such a gene is referred to herein as a T-cell exhaustion gene. Inhibition or knockout of one or more T-cell exhaustion genes confers persistent T cell function, i.e., T cell endurance, in the context of chronic tumor stimulation compared to control. Thus, T-cells harboring a genetic modification that knocks out a target T-cell exhaustion gene, or a genetic modification to the gene that inhibits function, have enhanced T cell effector function compared to unmodified controls for a longer period time when chronically exposed to a tumor.

[0024] Any T cell can be modified to inhibit expression of a T cell exhaustion gene. In some embodiments, a T cell modified in accordance with the invention is a CD8+ T cell. In some embodiments, the T cell is a CD4+ T-cell, or gamma delta T cell. In further embodiments, the T cell is a stem memory T cell, an effector memory T cell, a central memory T cell, or a naive T cell. Review of T cell subsets are provided, e.g., in Sallusto et al., Annual Rev. Immunol. 22745-763, 2004; Mueller et al., Annual Rev. Immunol 31 : 137- 161, 2013; and for memory stem T-cells, Gattinoni, et al., Nature Med. 23: 18-27, 2018. Descriptions of subsets by markers are available in the OMIP Wiley Online Library (see, e.g., Wingender and Kronenberg, OMIP-030: Characterization of human T cell subsets via surface markers Cytometry Part A 87A: 1067- 1069, 2015.

[0025] Expression of the target T cell exhaustion gene can be inhibited or, in some embodiments, inactivated, such that the gene does not express an active protein product. In some embodiments, a population of cells can be enriched for cells in which the gene is inactivated. In some embodiments, the gene is inactivated by a complete or partial deletion of the gene, e.g., using a gene editing systems such as CRISPR / Cas.

[0026] In some embodiments, a target T-cell is modified to inhibit expression of one or more of gene selected from the following genes: MED24, KDM6A, AUP1, SATB1, STT3A, MED 16, PINLYP, MAPK14, UBAP2L, TAF3, BCL2L11, KCTD5,CCDC53, DDX47,ZNF671, TM2D1, TRIM58, TICRR, KRT26, MED15, B4GALT1, RFWD2, UBE2F, UBE2L3, DCUN1D3, and MEF2D. In some embodiments, a T cell comprises a second modification to inhibit expression of a second T-cell exhaustion gene. In some embodiments, a T cell is modified to contain modifications to inhibit expression of a first and a second Cullin 5 complex gene. In some embodiments, the two Cullin 5 complex genes are selected from the group consisting of CUL5, ARIH2, RNF7, UBE2F, UBE2L3, CISH, and ZX7W / / J3. In some embodiments, the T cell is modified to knockout expression of two or more T-cell exhaustion genes as described herein, e.g., two or more o MED24, KDM6A, AUP1, SATB1, STT3A, MED 16, PINLYP, MAPK14, UBAP2L, TAF3, BCL2L11, KCTD5,CCDC53, DDX47, ZNF671, TM2D1, TRIM58, TICRR, KRT26, MED15, B4GALT1, RFWD2, CUL5, ARIH2, RNF7, UBE2F, UBE2L3, DCUN1D3, o MEF2D.

[0027] In some embodiments, a target T-cell is modified to inhibit expression of one or more of the following genes: CUI.5, ARIH2, RNF, CISH, UBE2F, UBE2L3, or DCUN ID 3

[0028] T cell function following inhibition of a T-cell exhaustion gene can be assessed using any number of assays to compare the activity of the genetically modified T-cell to a counterpart, e.g., control T-cell that does not contain the genetic modification(s) to inibit a T cell exhaustion gene. Illustrative assays include assays to evaluate cell killing by the genetically modified T cells compared to counterpart T cells that have become dysfunctional following repeated exposure to tumor cells, e.g., at least four times. The amount of cell killing by dysfunctional T cells that express a TCR for an HLA binding cancer antigen peptide and have at least one genetic modification to inhibit or knockout at least one T cell exhaustion gene can be quantified compared to that of the counterpart dysfunctional T cells that do not contain the genetic modification to target the T cell exhaustion gene. As used in this context “dysfunctional” refers to T cells that have reduced T cell killing following repeated exposure, e.g., at least four exposures or at least nine exposures, to tumor cells that express the cancer antigen. A genetically modified T cell is considered to have improved anti-tumor activity when there is an increase in tumor cell killing of at least 20% or at least 25%, or at least 30%, at least 35%, at least 40%, at least 45%, or at least 50% or greater compared to cell killing of the control cells with the genetic modification to one or more tumor exhaustion genes. T cell exhaustion (i.e., dsyfunction as used in this context), can also be monitored by assessing T cell exhaustion markers. Thus, in some embodiments, a T cell genetically modified to inhibit and / or knockout a T cell exhaustion gene as described herein that has “improved” function express lower levels of T cell exhaustion markers such as PD1,TIM3, LAG3, and CD39, e..g, at least 25%, or at least 30%, at least 35%, at least 40%, at least 45%, or at least 50% or lower levels of such markers compared to counterpart T cells without the genetic modification; and express higher levels of inflammatory cytokines (e.g., IFNgamma, TNF alpha), and activation markers (e.g., CD69, 4-1BB, CD125), e.g., at least 25%, or at least 30%, at least 35%, at least 40%, at least 45%, or at least 50% or higher levels compared to the counterpart T cells without the genetic modification

[0029] Another way to assess effects of gene edits on T cell function is through in vivo experiments. For example, gene editing can be performed with TCR-T and / or CAR-T cells to knock out or inhibit a target gene. The cells are then injected into tumor-bearing mice. Tumor burden can be measured, e.g., by caliper measure of tumor size or bioluminescent imaging, to assess effects on tumor growth over time, where a decrease in tumor burden by at least 20%, or at least 30% is indicative of improved function obtained by knockout / inhibition of the target gene. Survival in each cohort can also be tracked to assess for significant differences based on a Kaplan-Meier survival analysis.

[0030] In some embodiments, a T-cell exhaustion gene is inactivated by a gene deletion. As used herein, "gene deletion" refers to removal of at least a portion of a DNA sequence from, or in proximity to, a gene. In some embodiments, the sequence subjected to gene deletion comprises an exonic sequence of a gene. In some embodiments, the sequence subjected to gene deletion comprises a promoter sequence of the gene. In some embodiments, the sequence subjected to gene deletion comprises a flanking sequence of a gene. In some embodiments, a portion of a gene sequence is removed from a gene. In some embodiments, the complete gene sequence is removed from a chromosome. In some embodiments, the host cell comprises a gene deletion as described in the any of the embodiments herein. In some embodiments, the gene is inactivated by deletion of at least one nucleotide or nucleotide base pair in a gene sequence results in a non-functional gene product. In some embodiments, the gene is inactivated by a gene deletion, wherein deletion of at least one nucleotide to a gene sequence results in a gene product that no longer has the original gene product function or activity or exhibits aberrant function. In some embodiments, the gene is inactivated by a gene addition or substitution, wherein addition or substitution of at least one nucleotide or nucleotide base pair into the gene sequence results in a non-functional gene product. In some embodiments, the gene is inactivated by a gene inactivation, wherein incorporation or substitution of at least one nucleotide to the gene sequence results in a gene product that no longer has the original gene product function or activity; or exhibits aberrant function. Insome embodiments, the gene is inactivated by an addition or substitution, wherein incorporation or substitution of at least one nucleotide into the gene sequence results in a gene product having aberrant activity. In some embodiments, the host cell comprises a gene deletion as described in the any of the embodiments herein.

[0031] Methods and techniques for inactivating a T cell exhaustion gene in a host cell, or inactivating a target gene as described herein to suppress a T cell function associated with T cell exhaustion, include, but are not limited to, small interfering RNA (siRNA), small hairpin RNA (shRNA; also referred to as a short hairpin RNA), clustered, regularly interspaced, short palindromic repeats (CRISPR), transcription activator-like effector nuclease (TALEN), zinc- finger nuclease (ZFN), homologous recombination, non-homologous end-joining, and meganuclease. See, e.g., O'Keefe, Mater Methods, 3, 2013; Doench et al., Nat Biotechnol, 32, 2014; Gaj etal., Trends Biotechnol, 31, 2014; and Silva etal., Curr Gene Ther, 11, 2011.Inhibitory RNA

[0032] In some embodiments, the T-cell exhaustion gene is inactivated by a small interfering RNA (siRNA) system. siRNA sequences to inactivate a target gene can be identified using considerations such as length of siRNA, e.g., 21-23 nucleotides, or fewer; avoidance of regions with 50-100 nucleotides of the start codon and termination codon, avoidance of intron regions; avoidance of stretches of four or more of the same nucleotide; avoidance of regions with GC content that is less than 30% or greater than 60%; avoidance of repeats and low sequence complexity region; avoidance of single nucleotide polymorphic sites, and avoidance of sequences that are complementary to sequences in other off-target genes (see, e.g., Rules of siRNA design for RNA interference, Protocol Online, May 29, 2004; and Reynolds et al., Nat Biotechnol, 22:3236-330 2004).

[0033] In some embodiments, the siRNA system comprises a siRNA nucleotide sequence that is about 10 to 200 nucleotides in length, or about 10 to 100 nucleotides in length, or about 15 to 100 nucleotides in length, or about 10 to 60 nucleotides in length, or about 15 to 60 nucleotides in length, or about 10 to 50 nucleotides in length, or about 15 to 50 nucleotides in length, or about 10 to 30 nucleotides in length, or about 15 to 30 nucleotides in length. In some embodiments, the siRNA nucleotide sequence is approximately 10-25 nucleotides in length. In some embodiments, the siRNA nucleotide sequence is approximately 15-25 nucleotides in length. In some embodiments, the siRNA nucleotide sequence is at least about 10, at least about 15, at least about 20, or at least about 25nucleotides in length. In some embodiments, the siRNA system comprises a nucleotide sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, or 100% complementary to a region of the target mRNA molecule. In some embodiments, the siRNA system comprises a nucleotide sequence that is at least at least about 80%, at least about 85%, at least about 90%, at least about 95%, or 100% complementary to a region of the target pro-mRNA molecule. In some, embodiments, the siRNA system comprises a double stranded RNA molecule. In some embodiments, the siRNA system comprises a single stranded RNA molecule. In some embodiments, the host cell comprises a siRNA system as described in the any of the embodiments herein. In some embodiments, the host cell comprises a pro-siRNA nucleotide sequence that is processed into an active siRNA molecule as described in the any of the embodiments herein. In some embodiments, the host cell comprises a siRNA nucleotide sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, or 100% complementary to a region of the target mRNA molecule. In some embodiments, the host cell comprises an expression vector encoding a siRNA molecule as described in the any of the embodiments herein. In some embodiments, the host cell comprises an expression vector encoding a prosiRNA molecule as described in the any of the embodiments herein.

[0034] In some embodiments, the siRNA system comprises a delivery vector. In some embodiments, the host cell comprises a delivery vector. In some embodiments, the delivery vector comprises the pro-siRNA and / or siRNA molecule.

[0035] In some embodiments, the T-cell exhaustion gene is inactivated by a small hairpin RNA (shRNA; also referred to as a short hairpin RNA) system. Gene inactivation by shRNA systems are available. In some embodiments, the shRNA system comprises a nucleotide sequence that is about 10 to 200 nucleotides in length, or about 10 to 100 nucleotides in length, or about 15 to 100 nucleotides in length, or about 10 to 60 nucleotides in length, or about 15 to 60 nucleotides in length, or about 10 to 50 nucleotides in length, or about 15 to 50 nucleotides in length, or about 10 to 30 nucleotides in length, or about 15 to 30 nucleotides in length. In some embodiments, the shRNA nucleotide sequence is approximately 10-25 nucleotides in length. In some embodiments, the shRNA nucleotide sequence is approximately 15-25 nucleotides in length. In some embodiments, the shRNA nucleotide sequence is at least about 10, at least about 15, at least about 20, or at least about 25 nucleotides in length. In some embodiments, the shRNA system comprises a nucleotide sequence that is at least about 80%, at least about 85%, at least about 90%, at least about95%, or 100% complementary to a region of a T-cell exhusion gene mRNA molecule. In some embodiments, the shRNA system comprises a nucleotide sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, or 100% complementary to a region of a pro-mRNA molecule. In some, embodiments, the shRNA system comprises a double stranded RNA molecule. In some embodiments, the shRNA system comprises a single stranded RNA molecule. In some embodiments, the host cell comprises a shRNA system as described in the any of the embodiments herein. In some embodiments, the host cell comprises a pre-shRNA nucleotide sequence that is processed in an active shRNA nucleotide sequence as described in any of the embodiments herein. In some embodiments, the pro- shRNA molecule composed of DNA. In some embodiments, the pro-shRNA molecule is a DNA construct. In some embodiments, the host cell comprises a shRNA nucleotide sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, or 100% complementary to a region of the T-cell exhaustion gene mRNA molecule. In some embodiments, the host cell comprises an expression vector encoding a shRNA molecule as described in the any of the embodiments herein. In some embodiments, the host cell comprises an expression vector encoding a pro-shRNA molecule as described in the any of the embodiments herein.

[0036] In some embodiments, the shRNA system comprises a delivery vector. In some embodiments, the host comprises a delivery vector. In some embodiments, the delivery vector comprises the pro-shRNA and / or shRNA molecule. In some embodiments, the delivery vector is a virus vector. In some embodiments, the delivery vector is a lentivirus. In some embodiments, the delivery vector is an adenovirus. In some embodiments, the vector comprises a promoter.CRISPR

[0037] In some embodiments, inhibiting expression of a T cell exhaustion gene is accomplished using CRISPR / CAS methodology. Illustrative methods of using the CRISPR / Cas system to reduce gene expression are described in various publications, e.g., U.S. Patent Application Publication No. 2014 / 0170753. A CRISPR / Cas system includes a Cas protein and at least one to two ribonucleic acids that hybridize to a target motif in the T cell exhaustion gene and direct the Cas protein to the target motif. Any CRISPR / Cas system that is capable of altering a target polynucleotide sequence in a cell can be used. In some embodiments, the CRISPR Cas system is a CRISPR type I system, in some embodiments, theCRISPR / Cas system is a CRISPR type II system. In some embodiments, the CRISPR / Cas system is a CRISPR type V system.

[0038] The Cas protein used in the invention can be a naturally occurring Cas protein or a functional derivative thereof. A “functional derivative” includes, but are not limited to, fragments of a native sequence and derivatives of a native sequence polypeptide and its fragments, provided that they have a biological activity in common with a corresponding native sequence polypeptide. A biological activity contemplated herein is the ability of the functional derivative to hydrolyze a DNA substrate into fragments. The term “derivative” encompasses both amino acid sequence variants of polypeptide, covalent modifications, and fusions thereof such as derivative Cas proteins. Suitable derivatives of a Cas polypeptide or a fragment thereof include but are not limited to mutants, fusions, covalent modifications of Cas protein or a fragment thereof.

[0039] There are three main types of Cas nucleases (type I, type II, and type III), and 10 subtypes including 5 type I, 3 type II, and 2 type III proteins (see, e.g., Hochstrasser and Doudna, Trends Biochem Sci, 2015:40(l):58-66). Type II Cas nucleases include Casl, Cas2, Csn2, and Cas9. These Cas nucleases are known to those skilled in the art. For example, the amino acid sequence of the Streptococcus pyogenes wild-type Cas9 polypeptide is set forth, e.g., in NBCI Ref. Seq. No. NP_269215, and the amino acid sequence of Streptococcus thermophilus wild-type Cas9 polypeptide is set forth, e.g., in NBCI Ref. Seq. No.WP 011681470. Some CRISPR-related endonucleases that may be used in methods described herein are disclosed, e.g., in U.S. Application Publication Nos. 2014 / 0068797, 2014 / 0302563, and 2014 / 0356959. Non-limiting examples of Cas nucleases include 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, homologs thereof, variants thereof, mutants thereof, and derivatives thereof.

[0040] Cas9 homologs are found in a wide variety of eubacteria, including, but not limited to bacteria of the following taxonomic groups: Actinobacteria, Aquificae, Bacteroidetes- Chlorobi, Chlamydiae-Verrucomicrobia, Chlroflexi, Cyanobacteria, Firmicutes, Proteobacteria, Spirochaetes, and Thermotogae. An exemplary Cas9 protein is the Streptococcus pyogenes Cas9 protein. Additional Cas9 proteins and homologs thereof aredescribed in, e.g., Chylinksi, et al., RNA Biol. 2013 May 1; 10(5): 726-737; Nat. Rev. Microbiol. 2011 June; 9(6): 467-477; Hou, et al., Proc Natl Acad Sci USA. 2013 Sep 24;110(39): 15644-9; Sampson et al., Nature. 2013 May 9; 497(7448):254-7; and Jinek, et al., Science. 2012 Aug 17;337(6096):816-21. Variants of any of the Cas9 nucleases provided herein can be optimized for efficient activity or enhanced stability in the host cell. Thus, engineered Cas9 nucleases are also contemplated. Cas 9 from Streptococcus pyogenes contains 2 endonuclease domains, including an RuvC-like domain that cleaves target DNA that is noncomplementary to crRNA, and an HNH nuclease domain that cleave target DNA complementary to crRNA. The double-stranded endonuclease activity of Cas9 also involves a short conserved sequence, (2-5 nucleotides), known as a protospacer-associated motif (PAM), which follows immediately 3 '- of a target motif in the target sequence

[0041] Additionally, Cas nucleases, e.g., Cas9 polypeptides, can be derived from a variety of bacterial species including, but not limited to, Veillonella atypical, Fusobacterium nucleatum, Filifactor alocis, Solobacterium moorei, Coprococcus catus, Treponema denticola, Peptoniphilus duerdenii, Catenibacterium mitsuokai, Streptococcus mutans, Listeria innocua, Staphylococcus pseudintermedius, Acidaminococcus intestine, Olsenella uli, Oenococcus kitaharae, Bifidobacterium bifidum, Lactobacillus rhamnosus, Lactobacillus gasseri, Finegoldia magna, Mycoplasma mobile, Mycoplasma gallisepticum, Mycoplasma ovipneumoniae, Mycoplasma canis, Mycoplasma synoviae, Eubacterium rectale, Streptococcus thermophilus, Eubacterium dolichum, Lactobacillus coryniformis subsp.Torquens, Ilyobacter polytropus, Ruminococcus albus, Akkermansia muciniphila, Acidothermus cellulolyticus, Bifidobacterium longum, Bifidobacterium dentium, Corynebacterium diphtheria, Elusimicrobium minutum, Nitratifractor salsuginis, Sphaerochaeta globus, Fibrobacter succinogenes subsp. Succinogenes, Bacteroides fragilis, Capnocytophaga ochracea, Rhodopseudomonas palustris, Prevotella micans, Prevotella ruminicola, Flavobacterium columnare, Aminomonas paucivorans, Rhodospirillum rubrum, Candidatus Puniceispirillum marinum, Verminephrobacter eiseniae, Ralstonia syzygii, Dinoroseobacter shibae, Azospirillum, Nitrobacter hamburgensis, Bradyrhizobium, Wolinella succinogenes, Campylobacter jejuni subsp. Jejuni, Helicobacter mustelae, Bacillus cereus, Acidovorax ebreus, Clostridium perfringens, Parvibaculum lavamentivorans, Roseburia intestinalis, Neisseria meningitidis, Pasteurella multocida subsp. Multocida, Sutterella wadsworthensis, proteobacterium, Legionella pneumophila, Parasutterella excrementihominis, Wolinella succinogenes, and Francisella novicida.

[0042] Other RNA-mediated nucleases include Cpfl (See, e.g., Zetsche et al., Cell, Volume 163, Issue 3, p759-771, 22 October 2015) and homologs thereof.

[0043] As used herein, the term “Cas9 ribonucleoprotein” complex and the like refers to a complex between the Cas9 protein and a guide RNA, the Cas9 protein and a crRNA, the Cas9 protein and a trans-activating crRNA (tracrRNA), or a combination thereof (e.g., a complex containing the Cas9 protein, a tracrRNA, and a crRNA guide RNA). It is understood that in any of the embodiments described herein, a Cas9 nuclease can be subsitututed with another RNA-mediated nuclease, e.g., an alternative Cas protein or a Cpfl nuclease.

[0044] In some embodiments, the Cas protein is introduced into T-cells in polypeptide form. Thus, for example, in certain embodiments, the Cas proteins can be conjugated to or fused to a cell-penetrating polypeptide or cell-penetrating peptide that is well known in the art. Non-limiting examples of cell-penetrating peptides include those provided in Milletti F, “Drug Discov. Today 17: 850-860, 2012, the relevant disclosure of which is hereby incorporated by reference in its entirety. In some cases, T cells may be genetically engineered to produce the Cas protein.

[0045] In some embodiments, a Cpfl nuclease or the Cas9 nuclease and the gRNA are introduced into the cell as a ribonucleoprotein (RNP) complex.

[0046] In some embodiments, the RNP complex may be introduced into about 1 x 105to about 2 * 106cells e.g., 1 x 105cells to about 5 x io5cells, about 1 x io5cells to about 1 x 106cells, 1 x 105cells to about 1.5 x io6cells, 1 x io5cells to about 2 x io6cells, about 1 x 106cells to about 1.5 x io6cells, or about 1 x io6cells to about 2 x io6cells). In some embodiments, the cells are cultured under conditions effective for expanding the population of modified cells. Also disclosed herein is a population of cells, in which the genome of at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99% or greater of the cells comprises a genetic modification or heterologous polynucleotide that inhibits expression of a T cell exhaustion gene s as described herein. In some embdoiments, the population comprises subpopulations of cells each of which subpopulations have a differnet genetic modification to inhibit expression of a T cell exhaustion gene as described herein.

[0047] In some embodiments, the RNP complex is introduced into the T cells by electroporation. Methods, compositions, and devices for electroporating cells to introduce a RNP complex are available in the art, see, e.g., WO 2016 / 123578, WO / 2006 / 001614, and Kim, J. A. et al. Biosens. Bioelectron. 23, 1353-1360 (2008). Additional or alternativemethods, compositions, and devices for electroporating cells to introduce a RNP complex can include those described in U.S. Patent Appl. Pub. Nos. 2006 / 0094095; 2005 / 0064596; or 2006 / 0087522; Li, L.H. et al. Cancer Res. Treat. 1, 341-350 (2002); U.S. Patent Nos.: 6,773,669; 7,186,559; 7,771,984; 7,991,559; 6,485,961; 7,029,916; and U.S. Patent Appl. Pub. Nos: 2014 / 0017213; and 2012 / 0088842; Geng, T. et al., J. Control Release 144, 91-100 (2010); and Wang, J., et al. Lab. Chip 10, 2057-2061 (2010).

[0048] In some embodiments, the Cas9 protein can be in an active endonuclease form, such that when bound to target nucleic acid as part of a complex with a guide RNA or part of a complex with a DNA template, a double strand break is introduced into the target nucleic acid. In the methods provided herein, a Cas9 polypeptide or a nucleic acid encoding a Cas9 polypeptide can be introduced into the T cell. The double strand break can be repaired by HDR to insert the DNA template into the genome of the T cell. Various Cas9 nucleases can be utilized in the methods described herein. For example, a Cas9 nuclease that requires an NGG protospacer adjacent motif (PAM) immediately 3’ of the region targeted by the guide RNA can be utilized. Such Cas9 nucleases can be targeted to a region in exon 1 of the TRAC or exon 1 of the TRAB that contains an NGG sequence. As another example, Cas9 proteins with orthogonal PAM motif requirements can be used to target sequences that do not have an adjacent NGG PAM sequence. Exemplary Cas9 proteins with orthogonal PAM sequence specificities include, but are not limited to those described in Esvelt et al., Nature Methods 10: 1116-1121 (2013).

[0049] In some cases, the Cas9 protein is a nickase, such that when bound to target nucleic acid as part of a complex with a guide RNA, a single strand break or nick is introduced into the target nucleic acid. A pair of Cas9 nickases, each bound to a structurally different guide RNA, can be targeted to two proximal sites of a target genomic region and thus introduce a pair of proximal single stranded breaks into the target genomic region, for example exon 1 of a TRAC gene or exon 1 of a TRBC gene. Nickase pairs can provide enhanced specificity because off-target effects are likely to result in single nicks, which are generally repaired without lesion by base-excision repair mechanisms. Illustrative Cas9 nickases include Cas9 nucleases having a D10A or H840A mutation (See, for example, Jinek et al., Science 337:816-821, 2012; Qi et al., Cell, 152(5): 1173-1183, 2012; Ran et a / ., Cell 154: 1380-1389, 2013). In one embodiment, the Cas9 polypeptide from Streptococcus pyogenes comprises at least one mutation at position D10, G12, G17, E762, H840, N854, N863, H982, H983, A984, D986, A987 or any combination thereof. Descriptions of such dCas9 polypeptides andvariants thereof are provided in, for example, International Patent Publication No. WO 2013 / 176772. The Cas9 enzyme may contain a mutation at DIO, E762, H983, or D986, as well as a mutation at H840 or N863. In some instances, the Cas9 enzyme may contain a D10A or DION mutation. In further embodiments, the Cas9 enzyme may contain a H840A, H840Y, or H840N. In some embodiments, the Cas9 enzyme may contain D10A and H840A; D10A and H840Y; D10A and H840N; DION and H840A; DION and H840Y; or DION and H840N substitutions. The substitutions can be conservative or non-conservative substitutions to render the Cas9 polypeptide catalytically inactive and able to bind to target DNA.

[0050] In some embodiments, the Cas nuclease can be a high-fidelity or enhanced specificity Cas9 polypeptide variant with reduced off-target effects and robust on-target cleavage. Non-limiting examples of Cas9 polypeptide variants with improved on-target specificity include the SpCas9 (K855A), SpCas9 (K810A / K1003A / R1060A) (also referred to as eSpCas9(1.0)), and SpCas9 (K848A / K1003A / R1060A) (also referred to as eSpCas9(l.l)) variants described in Slaymaker et al. , Science, 351(6268): 84-8 (2016), and the SpCas9 variants described in Kleinstiver et al., Nature, 529(7587):490-5 (2016) containing one, two, three, or four of the following mutations: N497A, R661 A, Q695A, and Q926A (e.g., SpCas9-HFl contains all four mutations).

[0051] In some embodiments, the target motifs can be selected to minimize off-target effects of the CRISPR / Cas systems of the present invention. For example, in some embodiments, the target motif is selected such that it contains at least two mismatches when compared with all other genomic nucleotide sequences in the cell. In some embodiments, the target motif is selected such that it contains at least one mismatch when compared with all other genomic nucleotide sequences in the cell. Those skilled in the art will appreciate that a variety of techniques can be used to select suitable target motifs for minimizing off-target effects (e.g., bioinformatics analyses).

[0052] In some embodiments, CRISPRi is employed for sequence-specific repression of gene expression of a T-cell exhaution gene described herein. Description of CRISPRi methods is provided, e.g., in Engreitz et al., Cold Spring Harb Perspect Biol, 2019, 1 l:a035386. In some embodiments, the CRISPRi system includes a dCas9 polypeptide or a dCasl2 polypeptide operably linked to a repression domain. In some embodiments, the repression domain is selected from the group consisting of a Kriippel-associated box (KRAB) repressor domain, a NuE repressor domain, a NcoR repressor domain, a SID repressordomain, a SID4X repressor domain, an EZH2 repressor domain, a FOG repressor domain, a DNMT3 A repressor domain, and a DNMT3L repressor domain.

[0053] In some embodiments, CRISPRoff is employed to silence a T-cell exhaustion gene (see, e.g., Nunez JK, Chen J, Pommier GC, et al. Genome-wide programmable transcriptional memory by CRISPR-based epigenome editing. Cell, 2021;0(0). doi:10.1016 / j.cell.2021.03.02.)

[0054] In some embodiments, base editing to introduce point mutations into a T-cell exhaustion gene. For example, DNA base editors comprise fusions between a catalytically impaired Cas nuclease and a base-modification enzyme, such as a cytosine deaminase that operates on single-stranded DNA (ssDNA), but not double-stranded DNA (dsDNA). Upon binding to its target locus in DNA, base pairing between a guide RNA and target DNA strand leads to displacement of a small segment of single-stranded DNA in an R loop. DNA bases within this single-stranded DNA bubble are modified by the deaminase enzyme. To improve efficiency in eukaryotic cells, the catalytically disabled nuclease also generates a nick in the non-edited DNA strand, inducing cells to repair the non-edited strand using the edited strand as a template. DNA base editors are available that can mediate all four possible transition mutations (C to T, A to G, T to C, and G to A). See, for example Rees & Liu, Nat. Rev.Genet. 19:770-788, 2008 and references cited therein.

[0055] As used throughout, a guide nucleic acid sequence, typically a guide RNA (gRNA) sequence, is a sequence that interacts with a site-specific or targeted nuclease and specifically binds to or hybridizes to a target nucleic acid within the genome of a cell, such that the gRNA and the targeted nuclease co-localize to the target nucleic acid in the genome of the cell. Each gRNA includes a DNA targeting sequence or protospacer sequence of about 10 to 50 nucleotides in length that specifically binds to or hybridizes to a target DNA sequence in the genome. For example, the targeting sequence may be about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 nucleotides in length. In some embodiments, the gRNA comprises a crRNA sequence and a transactivating crRNA (tracrRNA) sequence. In some embodiments, the gRNA does not comprise a tracrRNA sequence.

[0056] The sgRNAs can be selected depending on the particular CRISPR / Cas system employed, and the sequence of the target polynucleotide, as will be appreciated by those skilled in the art. As indicated above, in some embodiments, the one to two ribonucleic acidscan also be selected to minimize hybridization with nucleic acid sequences other than the target polynucleotide sequence. In some embodiments, the one to two ribonucleic acids hybridize to a target motif that contains at least two mismatches when compared with all other genomic nucleotide sequences in the cell. In some embodiments, the one to two ribonucleic acids hybridize to a target motif that contains at least one mismatch when compared with all other genomic nucleotide sequences in the cell. In some embodiments, the one to two ribonucleic acids are designed to hybridize to a target motif immediately adjacent to a deoxyribonucleic acid motif recognized by the Cas protein. In some embodiments, each of the one to two ribonucleic acids are designed to hybridize to target motifs immediately adjacent to deoxyribonucleic acid motifs recognized by the Cas protein which flank a mutant allele located between the target motifs. Guide RNAs can also be designed using software that are readily available, for example, at the website crispr.mit.edu. The one or more sgRNAs can be transfected into T cells in which Cas protein is present by transfection, according to methods known in the art.

[0057] In some cases, the DNA targeting sequence can incorporate wobble or degenerate bases to bind multiple genetic elements. In some cases, the 19 nucleotides at the 3’ or 5’ end of the binding region are perfectly complementary to the target genetic element or elements. In some cases, the binding region can be altered to increase stability. For example, nonnatural nucleotides, can be incorporated to increase RNA resistance to degradation. In some cases, the binding region can be altered or designed to avoid or reduce secondary structure formation in the binding region. In some cases, the binding region can be designed to optimize G-C content. In some cases, G-C content is preferably between about 40% and about 60% (e.g., 40%, 45%, 50%, 55%, 60%).

[0058] In some embodiments, the sequence of the gRNA, or a portion thereof is designed to complement (e.g., perfectly complement) or substantially complement (e.g., 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94% 95%, 96%, 97%, 98%, or 99% complement) the target region in the T-cell exhaustion gene. In some embodiments, the portion of the gRNA that complements and binds the targeting region in the polynucleotide is, or is about, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39 or 40 or more nucleotides in length. In some cases, the portion of the gRNA that complements and binds the targeting region in the polynucleotide is between about 19 and about 21 nucleotides in length. In some cases, the gRNA may incorporate wobble or degenerate bases to bind target regions. In some cases, the gRNA canbe altered to increase stability. For example, non-natural nucleotides, can be incorporated to increase RNA resistance to degradation. In some cases, the gRNA can be altered or designed to avoid or reduce secondary structure formation. In some cases, the gRNA can be designed to optimize G-C content. In some cases, G-C content is between about 40% and about 60% (e.g., 40%, 45%, 50%, 55%, 60%). In some cases, the binding region can contain modified nucleotides such as, without limitation, methylated or phosphorylated nucleotides.

[0059] In some embodiments, the gRNA can be optimized for expression by substituting, deleting, or adding one or more nucleotides. In some cases, a nucleotide sequence that provides inefficient transcription from an encoding template nucleic acid can be deleted or substituted. For example, in some cases, the gRNA is transcribed from a nucleic acid operably linked to an RNA polymerase III promoter. In such cases, gRNA sequences that result in inefficient transcription by RNA polymerase III, such as those described in Nielsen et al., Science. 2013 Jun 28;340(6140): 1577-80, can be deleted or substituted. For example, one or more consecutive uracils can be deleted or substituted from the gRNA sequence. In some cases, if the uracil is hydrogen bonded to a corresponding adenine, the gRNA sequence can be altered to exchange the adenine and uracil. This “A-U flip” can retain the overall structure and function of the gRNA molecule while improving expression by reducing the number of consecutive uracil nucleotides.

[0060] In some embodiments, the gRNA can be optimized for stability. Stability can be enhanced by optimizing the stability of the gRNAmuclease interaction, optimizing assembly of the gRNAmuclease complex, removing or altering RNA destabilizing sequence elements, or adding RNA stabilizing sequence elements. In some embodiments, the gRNA contains a 5’ stem -loop structure proximal to, or adjacent to, the region that interacts with the gRNA- mediated nuclease. Optimization of the 5’ stem-loop structure can provide enhanced stability or assembly of the gRNAmuclease complex. In some cases, the 5’ stem-loop structure is optimized by increasing the length of the stem portion of the stem-loop structure.

[0061] gRNAs can be modified by methods known in the art. In some cases, the modifications can include, but are not limited to, the addition of one or more of the following sequence elements: a 5’ cap (e.g., a 7-methylguanylate cap); a 3’ polyadenylated tail; a riboswitch sequence; a stability control sequence; a hairpin; a subcellular localization sequence; a detection sequence or label; or a binding site for one or more proteins.Modifications can also include the introduction of non-natural nucleotides including, but not limited to, one or more of the following: fluorescent nucleotides and methylated nucleotides.

[0062] Also provided herein are expression cassettes and vectors for producing gRNAs in a host cell. The expression cassettes can contain a promoter (e.g., a heterologous promoter) operably linked to a polynucleotide encoding a gRNA. The promoter can be inducible or constitutive. The promoter can be tissue specific. In some cases, the promoter is a U6, Hl, or spleen focus-forming virus (SFFV) long terminal repeat promoter. In some cases, the promoter is a weak mammalian promoter as compared to the human elongation factor 1 promoter (EFl A). In some cases, the weak mammalian promoter is a ubiquitin C promoter or a phosphoglycerate kinase 1 promoter (PGK). In some cases, the weak mammalian promoter is a TetOn promoter in the absence of an inducer. In some cases, when a TetOn promoter is utilized, the host cell is also contacted with a tetracycline transactivator. In some embodiments, the strength of the selected gRNA promoter is selected to express an amount of gRNA that is proportional to the amount of Cas9 or dCas9. The expression cassette can be in a vector, such as a plasmid, a viral vector, a lentiviral vector, etc. In some cases, the expression cassette is in a host cell. The gRNA expression cassette can be episomal or integrated in the host cell.Modifications using alternative targeted nuclease systems

[0063] In some embodiments, a targeted nuclease that is employed in modifying a T cell to inhibit expression of a T-cell exhaustion gene a transcription activator-like effector nuclease (TALEN), a zinc finger nuclease (ZFN) or a megaTAL (See, for example, Merkert and Martin “Site-Specific Genome Engineering in Human Pluripotent Stem Cells,” Int. J. Mol. Sci. 18(7): 1000 (2016)).Zinc-fmger nuclease to inhibit T-cell exhaustion gene expression

[0064] In some embodiments, modified T cells comprising a T-cell exhaustion gene- targeted alteration are produced by inhibiting expression using ZFN. Methods of using the ZFNs to reduce gene expression are described, e.g., in U.S. Patent No. 9,045,763, and also in Durai et al., Nucleic Acid Research 33 :5978-5990, 2005; Carroll el al. Genetics Society of America 188: 773-782, 2011; and Kim et al. Proc. Natl. Acad. Sci. USA 93: 1156-1160.

[0065] A ZFN comprises a FokI nuclease domain (or derivative thereof) fused to a DNA- binding domain. In the case of a ZFN, the DNA-binding domain comprises one or more zincfingers. A zinc finger is a small protein structural motif stabilized by one or more zinc ions. A zinc finger can comprise, for example, Cys2His2, and can recognize an approximately 3-bp sequence. Various zinc fingers of known specificity can be combined to produce multi-finger polypeptides which recognize about 6, 9, 12, 15 or 18-bp sequences. Various selection and modular assembly techniques are available to generate zinc fingers (and combinations thereof) recognizing specific sequences, including phage display, yeast one-hybrid systems, bacterial one-hybrid and two-hybrid systems, and mammalian cells.

[0066] A ZFN dimerizes to cleave DNA. Thus, a pair of ZFNs are used to target non- palindromic DNA sites. The two individual ZFNs bind opposite strands of the DNA with their nucleases properly spaced apart (see, e.g., Bitinaite et al. , Proc. Natl. Acad. Sci. USA 95: 10570-5, 1998). A ZFN can create a double-stranded break in the DNA, which can create a frame-shift mutation if improperly repaired, leading to a decrease in the expression and level of expression of the target gene in a cell in a cell.TALENs to inhibit T-cell exhaustion genes

[0067] In some embodiments, T-cells that comprise a targeted alteration are produced by inhibiting the desired T-cell exhaustion gene with transcription activator-like effector nucleases (TALENS). TALENs are similar to ZFNs in that they bind as a pair around a genomic site and direct a non-specific nuclease, e.g., FoKI, to cleave the genome at a specific site, but instead of recognizing DNA triplets, each domain recognizes a single nucleotide. Methods of using TALENS to reduce gene expression are disclosed, e.g., in U.S. Patent No. 9,005,973; Christian et al. “Genetics 186(2): 757-761, 2010; Zhang et al. 2011 Nature Biotech. 29: 149-53, 2011; Geibler et al. 2011 PLoS ONE 6: el9509, 2011; Boch et al. 2009 Science 326: 1509-12; Moscou et al. 2009 Science 326: 3501.

[0068] To produce a TALEN, a TALE protein is typically fused to a FokI endonuclease, which can be a wild-type or mutated FokI endonuclease. Several mutations to FokI have been made for its use in TALENs; these, for example, improve cleavage specificity or activity. Cermak et al., NucL Acids Res. 39:e82, 2011; Miller et al., Nature Biotech. 29: 143- 8, 2011; Hockemeyer et al., Nature Biotech. 29:731-734, 2011; Wood et al., Science 333:307, 2011; Doyon et al., Nature Methods 8:74-79, 2010; Szczepek et al., Nature Biotech. 25:786-793, 2007; and Guo et al., J. Mol. Biol. 200:96, 2010.

[0069] The FokI domain functions as a dimer and typically employ two constructs with unique DNA binding domains for sites in the target genome with proper orientation andspacing. Both the number of amino acid residues between the TALE DNA binding domain and the FokI cleavage domain and the number of bases between the two individual TALEN binding sites appear to be important parameters for achieving high levels of activity, (e.g., Miller et aL, 2011, supra).Meganucleases

[0070] “Meganucleases” are rare-cutting endonucleases or homing endonucleases that can be highly specific, recognizing DNA target sites ranging from at least 12 base pairs in length, e.g., from 12 to 40 base pairs or 12 to 60 base pairs in length. Meganucleases can be modular DNA-binding nucleases such as any fusion protein comprising at least one catalytic domain of an endonuclease and at least one DNA binding domain or protein specifying a nucleic acid target sequence. The DNA-binding domain can contain at least one motif that recognizes single- or double-stranded DNA. The meganuclease can be monomeric or dimeric.

[0071] In some embodiments of the methods described herein, meganucleases may be used to inhibit the expression of a T-cell exhaustion gene as described herein. In some instances, the meganuclease is naturally-occurring (found in nature) or wild-type, and in other instances, the meganuclease is non-natural, artificial, engineered, synthetic, or rationally designed. In certain embodiments, the meganucleases that may be used in methods described herein include, but are not limited to, an LCrel meganuclease, LCeuI meganuclease, I-Msol meganuclease, LScel meganuclease, variants thereof, mutants thereof, and derivatives thereof.

[0072] Detailed descriptions of useful meganucleases and their application in gene editing are found, e.g., in Silva et al., Curr Gene Ther, 2011, 11(1): 11-27; Zaslavoskiy et al., BMC Bioinformatics, 2014, 15: 191; Takeuchi et al., Proc Natl Acad Sci USA, 2014, 111(11):4061- 4066, and U.S. Patent Nos. 7,842,489; 7,897,372; 8,021,867; 8,163,514; 8,133,697; 8,021,867; 8,119,361; 8,119,381; 8,124,36; and 8,129,134.

[0073] Efficiency of the inhibition of expression of any T-cell exhaustion gene using a method as described herein can be assessed by measuring the amount of mRNA or protein using methods well known in the art, for example, quantitative PCR, western blot, flow cytometry, etc and the like. In some embodiments, the level of protein is evaluated to assess efficiency of inhibition efficiency. In certain embodiments, the efficiency of reduction of target gene expression is at least 5%, at least 10%, at least 20% , at least 30%, at least 50%, at least 60%, or at least 80%, or at least 90%, or greater, as compared to corresponding cells thatdo not have the targeted modification. In certain embodiments, the efficiency of reduction is from about 10% to about 90%. In certain embodiments, the efficiency of reduction is from about 30% to about 80%. In certain embodiments, the efficiency of reduction is from about 50% to about 80%. In some embodiments, the efficiency of reduction is greater than or equal to about 80%.

[0074] In some embodiments, a T cell modified as described herein to inhibit a T-cell exhaustion gene comprises one or more additional genetic modifications to tailor T cell activity. In some embodiments, a T-cell that is modified to inhibit expression of a T-cell exhaustion gene expresses, or is further modified to express, a synthetic chimeric receptor construct construct containing an extracellular binding domain, e.g., a variable region from an antibody that binds to an antigen and an intracellular signaling domain.. In some embodiments, the synthetic T-cell receptor-based construct is a CAR (chimeric antigen receptor), which comprises an extracelluar binding domain that targets an antigen and an intracellular signaling domain that can activate or stimulate an immunoresponsive cell upon engagement of the extracellular domain with a target antigen.. In certain embodiments, the CAR also comprises a transmembrane domain. In some embodimetns, the chimeric receptor is a TCR like fusion molecule. Examples of such TCR fusion molecules include an HLA- Independent TCR-based Chimeric Antigen Receptor (also known as “HIT-CAR”, e.g., those disclosed in International Patent Application No. PCT / US19 / 017525), T cell receptor fusion constructs (TRuCs) (e.g., those disclosed in Baeuerle et al., “Synthetic TRuC receptors engaging the complete T cell receptor for potent anti-tumor response,” Nature Comm. 10: 2087 (2019), synthetic T cell receptor and antigen receptors (STARs) (e.g., those disclosed in Liu et al. Science Translational Medicine 13(586):eabb5191, 2021), antibody-T-cell receptor (AbTCR) (e.g., those disclosed in Xu et al. Cell Discovery (2018) 4:62), and T cell antigen coupler (TAC) (e.g., those disclosed in Helsen et al. Nature Communications (2018);9:3049).Methods of Identifying T Cell Exhaustion Genes

[0075] In another aspect, the present disclosure provides a method of identifying T-Cell exhaustion genes, the method comprising: providing a library of gene-edited T cells comprising a population of activated T cells that express a TCR for an HLA peptide from a cancer antigen from a target tumor type, wherein T cells in the population comprise sgRNAs for a target gene that is inactived by gene editing. The library is generated to provide an average of one sgRNA per T cells. A pool (also referred to as a subpopoulation) of cellsfrom the library is co-cultured with tumor cells of the target tumor type that presents the HL A peptide, e.g., for at least 48 hours. The co-culture time can vary. For example, an initial exposure of T cells to tumor tissue may require only 24 hours for tumor clearance to be observed, whereas exposures later in the repetition cycle may require longer incubation periods, e.g., 48 hours of exposure. Following incubation, T-cells are collected, counted and then incubated with fresh tumor cells from the target tumor type. The collection and incubating steps are repeated at least at at least 2 times, preferably at least 3, 4, 5, 6, 7, or 8, or more times. In some instances, gene-edited T cells are incubated with fresh tumor cells for a total of 3 times. In some instances, gene-edited T cells are incubated with fresh tumor cells for a total of 4 times. In some instances, gene-edited T cells are incubated with fresh tumor cells for a total of 5 time or 6 times. In some instances, gene-edited T cells are incubated with fresh tumor cells for a total of 7 or 8 times. In some instances gene-edited T cells are incubated with fresh tumor cells for a total of 9 times. Following the final exposure of T cells to tumor tissue, genomic DNA is isolated from the cells and sgRNAs are amplified, e.g., PCR amplified, and size-selected for next-generation sequencing to identify target genes of the sgRNA sequences. The data is then analyzed to determine log-fold changes, P-values and false discovery rates. In some embodiments, the library of gene edited T cells is generated usinga Cas nuclease, e.g., Cas9 and 1, 2, 3, or 5 gRNAs per target gene.

[0076] Candidate genes exhibiting about 1.3 log-fold or greater positive change of levels or representation of sgRNA sequences in the pool of T cells from the T cell library subjected to repetitive rounds of tumor cell exposure, e.g., at least 4 or 5 rounds or more, or at least 8 or 9 rounds of exposure, compared to a control pool of T cells from the T cell library not exposed to tumor cells, are selected for further evaluation. In such an evaluation, a target gene is inhibited, preferably knocked out, in activated T cells from a donor that expresses a TCR to the HLA peptide from the cancer antigen. Multiple guide RNAs for each target gene can then be evaluated to validate persistant T-cell function after repeated exposure to tumor compared to control cells that do not contain a genetic modification to inactivate or inhibit the target gene.Treatment Methods and Compositions

[0077] Any of the methods described herein may be used to modify T cells, e.g., CD8+ T cells, obtained from a human subject. T-cells modified in accordance with the invention may be used to treat any number of cancers, including solid tumors.Methods of Treating Cancer

[0078] In some embodiments, T cells are modified to decrease expression of one or more T-cell exhaustion genes as described herein. In some embodiments, a T-cell exhaustion gene that is modified sMED24, KDM6A, AUP1, SATB1, STT3A, MED 16, PINLYP, MAPK14, UBAP2L, TAF3, BCL2L11, KCTD5,CCDC53, DDX47, ZNF671, TM2D1, TRIM58, TICRR, KRT26, MED15, B4GALT1, RFWD2, UBE2F, UBE2L3, MEF2D, OX DCUN1D3. Thus, in some embodiments, provided herein is a method of treating cancer in a human subject comprising: a) obtaining T cells, e.g., CD8+ T cells, from the subject; b) modifying the T cells using any of the methods provided herein to decrease expression of a T cell exhaustion gene, e.g., a gene disclosed in this paragraph; and c) administering the modified T cells to the subject.

[0079] In some embodiments, T cells, e.g., CD8+ T cells, obtained from a subject that has cancer may be expanded ex vivo. The characteristics of the subject’s cancer may determine a set of tailored cellular modifications (e.g., selection of one or more T cell exhaustion gene targets), and these modifications may be applied to the T cells using any of the methods described herein. Modified Tcells may then be reintroduced to the subject. This strategy capitalizes on and enhances the function of the subject’s natural repertoire of cancer specific T cells, providing a diverse arsenal to eliminate mutagenic cancer cells quickly.

[0080] Any cancer can be treated with genetically modified T cells as described herein. In some embodiments, the cancer is a carcinoma or a sarcoma. In some embodiments, the cancer is a hematological cancer. In some embodiments, the cancer is breast cancer, prostate cancer, testicular cancer, renal cell cancer, bladder cancer, liver cancer, ovarian cancer, cervical cancer, endometrial cancer, lung cancer, colorectal cancer, anal cancer, pancreatic cancer, gastric cancer, esophageal cancer, hepatocellular cancer, kidney cancer, head and neck cancer, glioblastoma, mesothelioma, melanoma, a chondrosarcoma, or a bone or soft tissue sarcoma. In some embodiments, the cancer is adrenocortical carcinoma, anal cancer, appendix cancer, astrocytoma, basal-cell carcinoma, bile duct cancer, bone tumor, brainstem glioma, brain cancer, cerebellar astrocytoma, cerebral astrocytoma, ependymoma, medulloblastoma, supratentorial primitive neuroectodermal tumors, visual pathway and hypothalamic glioma, or bronchial adenomas. In some emodiments, the cancer is acute lymphoblastic leukemia, acute myeloid leukemia, Burkitt's lymphoma, central nervous system lymphoma, chronic lymphocytic leukemia, chronic myelogenous leukemia, hairy cellleukemia, chronic myeloproliferative disorders, a myelodysplastic syndrome, an adult acute myeloproliferative disorder, multiple myeloma, cutaneous T-cell lymphoma, Hodgkin lymphoma, or non-Hodgkin lymphoma. In some embodiments, the cancer is desmoplastic small round cell tumor, ependymoma, epitheliod hemangioendothelioma (EHE), Ewing's sarcoma, extracranial germ cell tumor, extragonadal germ cell tumor, extrahepatic bile duct cancer, intraocular melanoma, retinoblastoma, gallbladder cancer, gastrointestinal carcinoid tumor, gastrointestinal stromal tumor (GIST), germ cell tumor, gestational trophoblastic tumor, gastric carcinoid, heart cancer, hypopharyngeal cancer, hypothalamic and visual pathway glioma, childhood, intraocular melanoma, islet cell carcinoma, Kaposi sarcoma, laryngeal cancer, lip and oral cavity cancer, liposarcoma, non-small cell lung cancer, smallcell lung cancer, macroglobulinemia, male breast cancer, malignant fibrous histiocytoma of bone, medulloblastoma, melanoma, Merkel cell cancer, mesothelioma, metastatic squamous neck cancer, mouth cancer, multiple endocrine neoplasia syndrome, mycosis fungoides, chronic, myxoma, nasal cavity and paranasal sinus cancer, nasopharyngeal carcinoma, neuroblastoma, oligodendroglioma, oral cancer, oropharyngeal cancer, osteosarcoma, ovarian epithelial cancer, ovarian germ cell tumor, ovarian low malignant potential tumor, paranasal sinus and nasal cavity cancer, parathyroid cancer, penile cancer, pharyngeal cancer, pheochromocytoma, pineal astrocytoma, pineal germinoma, pineoblastoma, supratentorial primitive neuroectodermal tumors, pituitary adenoma, plasma cell neoplasia, pleuropulmonary blastoma, primary central nervous system lymphoma, renal cell carcinoma, retinoblastoma, rhabdomyosarcoma, salivary gland cancer, uterine sarcoma, Sezary syndrome, non-melanoma skin cancer, melanoma Merkel cell skicinoma, small intestine cancer, squamous cell carcinoma, squamous neck cancer, throat cancer, thymoma, thyroid cancer, transitional cell cancer of the renal pelvis and ureter, trophoblastic tumor, gestational, urethral cancer, uterine cancer, vaginal cancer, vulvar cancer, Waldenstrom macroglobulinemia, or Wilms tumor.

[0081] In certain embodiments, the genetically modified T cells, or individual populations of sub-types of the genetically modified T cells, are administered to the subject at a range of about one million to about 100 billion cells, such as, e.g., 1 million to about 50 billion cells (e.g., about 5 million cells, about 25 million cells, about 500 million cells, about 1 billion cells, about 5 billion cells, about 20 billion cells, about 30 billion cells, about 40 billion cells, or a range defined by any two of the foregoing values), such as about 10 million to about 100 billion cells (e.g., about 20 million cells, about 30 million cells, about 40 million cells, about60 million cells, about 70 million cells, about 80 million cells, about 90 million cells, about 10 billion cells, about 25 billion cells, about 50 billion cells, about 75 billion cells, about 90 billion cells, or a range defined by any two of the foregoing values), and in some cases about 100 million cells to about 50 billion cells (e.g., about 120 million cells, about 250 million cells, about 350 million cells, about 450 million cells, about 650 million cells, about 800 million cells, about 900 million cells, about 3 billion cells, about 30 billion cells, about 45 billion cells) or any value in between these ranges.

[0082] In some embodiments, the dose of total cells and / or dose of individual subpopulations of cells is within a range of between at or about 104and at or about 109cells / kilograms (kg) body weight, such as between 105and 106cells / kg body weight, for example, at least about 1 x 105cells / kg, 1.5 x 105cells / kg, 2 x 105cells / kg, 5 x 105cells / kg, or 1 x 106cells / kg body weight.

[0083] The appropriate dosage may depend on the type of cancer to be treated, the severity and course of the disease, previous therapy, the subject's clinical history and response to the cells, and the discretion of the attending physician. The compositions and cells are in some embodiments suitably administered to the subject at one time or over a series of treatments.

[0084] The cells can be administered by any suitable means, for example, by bolus infusion, by injection, e.g., intravenous or subcutaneous injections, intraocular injection, periocular injection, subretinal injection, intravitreal injection, trans-septal injection, subscleral injection, intrachoroidal injection, intracameral injection, subconjectval injection, subconjuntival injection, sub-Tenon's injection, retrobulbar injection, peribulbar injection, or posterior juxtascleral delivery. In some embodiments, they are administered by parenteral, intrapulmonary, and intranasal, and, if desired for local treatment, intralesional administration. Parenteral infusions include intramuscular, intravenous, intraarterial, intraperitoneal, or subcutaneous administration. In some embodiments, a given dose is administered by a single bolus administration of the cells. In some embodiments, it is administered by multiple bolus administrations of the cells, for example, over a period of no more than 3 days, or by continuous infusion administration of the cells.

[0085] In some embodiments, the cells are administered as part of a combination treatment, such as simultaneously with or sequentially with, in any order, another therapeutic intervention, such as an antibody or engineered cell or receptor or agent, such as a cytotoxic or therapeutic agent. The cells in some embodiments are co-administered with one or moreadditional therapeutic agents or in connection with another therapeutic intervention, either simultaneously or sequentially in any order. In some contexts, the cells are co-administered with another therapy sufficiently close in time such that the cell populations enhance the effect of one or more additional therapeutic agents, or vice versa. In some embodiments, the cells are administered prior to the one or more additional therapeutic agents. In some embodiments, the cells are administered after the one or more additional therapeutic agents.

[0086] Publications cited herein and the material for which they are cited are hereby specifically incorporated by reference in their entireties.EXAMPLES

[0087] The following examples are offered to illustrate, but not to limit the claimed invention.Example 1. Identification of genes that play a role in T cell exhaustion following repeated exposure to target tumor cells

[0088] A genome-wide CRISPR screen in primary human T cells was performed to identify genes that regulate exhaustion in T cells. Primary human T cells were transduced with an NY-ESO-1 -specific TCR as well as a genome-wide pooled sgRNA library (Brunello library (Doesnch et al, Nat. Biotechnol., 2016 Feb:34(l): 184-191), Addgene). A SLICE protocol (Shifrut and Carnevale et al., Cell, 2018) was performed to edit the cells by nucleofecting them with Cas9 protein. Two human T cell donors were used in this screen.

[0089] Twelve days after producing pooled edited cells, co-culturing was initiated with tumor cells that naturally express NY-ESO-1 peptide on cognate MHCI. Every 48 hours the T cells were collected, counted, and then incubated again at 1 : 1 ratios with fresh target tumor cells. We previously showed that this “repetitive stimulation” method, when repeated at least 3 times, but typically over 5 times or more, induces clear dysfunction and exhaustion in the T cells and thus can be used to model the effects of chronic antigen stimulation causing exhaustion / dysfunction in T cells. Given that we wanted to have strong log-fold change values for this screen, we stressed these cells to an extreme. Cells were repetitively stimulated nine times, to the point that pronounced failure to clear the tumor cells was observed.

[0090] We harvested cells from both donors in each arm of the screen at key time points, including after transduction with the library, after initial expansion and before initiation of tumor co-culture (time zero), after 4 (total) stimulations, and after 9 total stimulations. Additionally, we included arms of the screen where the same pooled edited cells were cultured without any tumor cell exposure so we could compare the datasets. Once the cells were collected, genomic DNA was isolated from the cells, and then the sgRNAs were PCR amplified and size selected for next-generation sequencing on an Illumina HiSeq instrument. MAGeCK analysis was used to analyze the data to determine log fold changes, P-values, and false discovery rates.

[0091] We observed that a number of the top-scoring genes, i.e, genes showing the highest positive log-fold changes, were genes that had been previously described in the literature as loss-of-function gene editing targets that confer enhanced T cell fitness, and in some cases, persistence (FIG. 2). This observation provides evidence that the data obtained in this screen is robust, as these previously described gene targets serve in essence as positive controls for the screen. Next, high-scoring genes that are not well-described in the literature as being associated with T cell exhaustion or persistence were reviewed. Seven members of the Cullin 5 complex were among the top scoring genes (Cullin5, ARIH2, RNF7, UBE2F, UBE2L3, CISH, DCUN1D3) (FIG. 3). One hundred of the top-scoring genes after 9 stimulations are shown in Table 1. Top-scoring genes after 4 stimulations are shown in Table 2.

[0092] We then performed analyses demonstrating knocking out any of the seven Cullin5 members can induce a tumor killing advantage, which is enhanced when these cells are exposed to repeated tumor co-cultures to exhaust them (FIG. 4). In other words, knocking out any of the seven Cullin 5 complex members appear to confer resistance to exhaustion induced by chronic tumor antigen exposure. The strongest phenotype with knocking out a single gene was observed when we knocked out Cullin 5, which serves as the major scaffold holding the complex together. We repeated this finding of enhanced killing persistence using six separate human T cell donors with the NY-ESO-1 TCR specificity. In addition to evaluating the killing capacity, we also performed FACS-based phenotyping of these cells, and found that the Cul5 KO cells after repeated tumor stimulations expressed lower levels of canonical exhaustion markers (PD1, TIM3, LAG3, CD39), as well as higher inflammatory cytokines (IFNgamma, TNF alpha) and higher activation markers (CD69, 4-1BB, CD125).

[0093] FIG. 5 A-5D provides further data validating the Cullin 5 complex members using two guides for each target gene. Primary human T cells from one donor were transduced with an NYESO-TCR via lentiviral delivery one day post T cell activation. SgRNAs targeting seven different CUL5 complex members as well as a control sgRNA targeting the AAVS1 locus were delivered on day 2 via RNP (electroporation). T cells were co-cultured at an effector to target (E:T) ratio of 1: 1 on melanoma cell line A375 (target cells) starting day 9 post T cell activation. After 48 hours of co-culture, T cell numbers were normalized and cells re-cultured on fresh target cells at an E:T of 1 : 1 for a total of 4 rounds.

[0094] Before and after 4 rounds of tumor stimulation, tumor killing capacity of all conditions was evaluated via Incucyte on RFP+ A375 cells. The results showed that knockout of all tested sgRNAs targeting seven CUL5 complex members increased tumor killing compared to control upon first tumor exposure (FIGS. 5A and 5B). After 4 rounds of tumor exposure, all tested sgRNAs and genes (besides DCUN1D3 gl and g3, which did show as dramatic an effect) showed vastly increased tumor killing and persistence compared to control cells when co-cultured with RFP+ cell line A375 (FIGS. 5C and 5D).

[0095] In view of our observations in the NY-ESO-1 TCR system, validated that these findings are also found in the CAR context. Specifically, we demonstarted that in three different CAR contexts, including CD 19 CAR, BCMA CAR, and HER2 CAR, the same persistent killing advantage in the setting of repeated tumor stimulations in vitro was observed. In addition, we tested the BCMA CAR-T cells in an n vivo model of multiple myeloma, and we found an advantage in terms of tumor control in this model for the Cul5 kO BMCA CAR-T cells.

[0096] Knocking out Cul5 was also tested in a 1XX BCMA CAR-T cell model. 1XX is a CAR that contains only one ITAM (the other two have been mutated to become nonfunctional) which is hypothesized to help T cell differentiation and prevent exhaustion. This study confirmed that CUL5 KO cells showed increased tumor killing also in this model in vitro.

[0097] The Cul5 KO 1XX BCMA CAR was also evaluated in vivo. Specifically, we first injected mice with luciferase positive OPM2 multiple myeloma cells, and then injected them with BCMA-specific 1XX CAR-T cells with and without Cul5 deletion. We observed that both CAR-T cell conditions showed dramatic cancer clearance based on BLI imaging, and that neither of these cohorts relapsed when rechallenged with up to five extra doses of up tofive million OPM2 cancer cells. This was in line with the expected extremely enhanced potency and persistence of the 1XX CAR model. We additionally isolated these CAR-T cells from the spleens and tumors of these mice over 90 days after injection. Here we saw that there were significantly higher frequencies of the Cul5 KO CAR-T cells than the control edited CAR-T cells in the bone marrow, while there were equivalent frequencies in the spleens. This suggested that Cul5 deletion confers an antigen-dependent expansion advantage, as the multiple myeloma cells would be expected to home to the bone marrow but would not remain in the spleens. In addition, we T cells obtained from the spleens of these mice were co-cultured with multiple myeloma cells in vitro (of note, these CAR-T cells were over 100 days old by the time of this experiment), and observed dramatically higher levels of killing of the target tumor cells in the Cul5 KO condition. This suggests that while this 1XX model is very hard to improve upon in terms of enhancing the already very potent and persistent killing advantage, there are clear advantages to the Cul5 KO BCMA 1XX CAR-T cells when we perform these ex vivo analyses at a very late time point. These data also strongly support the Cullin5 complex as an important gene editing target to enhance the potency and persistence of adoptive T-cell therapies.

[0098] We performed single cell RNAseq and ATACseq analyses that indicate that after repeated stimulation, the Cul5 KO T cells are in an extremely different cell state than the control exhausted T cells. In addition, seahorse analyses demonstrated that Cul5 KO CAR-T cells appear to have higher levels of oxidative phosphorylation. In addition, functional genomics experiments are employed to knockout each of the 40 unique substrate receptors that are known to be substrate receptors for Cul5, using Cas9 RNP electroporation. For those substrate receptors that exhibit an enhanced persistence killing phenotype, different combinations substrate receptors that shown an enhanced persistence killing phenotype are tested to determine which combinations can produce the Cul5 KO killing phenotype. In addition, a variety of proteomic approaches are evaluated to determine which Cul5 targets are differentially ubiquitinated in the acute and chronic antigen exposure contexts. Specifically, a flag-tagged Cul5 construct will be expressed in human T cells, exposing them to a single versus multiple tumor stimulations, and followed by Immunoprecipitation-Mass Spectrometry (IP -MS) to identify all of the binding targets of the Cullin5 complex in these conditions.

[0099] In addition to Cullin 5, we also have a number of different high-scoring targets of interest from our genome-wide repeated stimulation screen, many of which have not been studied in the T cell exhaustion context. Examples of these include the following:MED24KDM6A AUP1 SATB1 STT3A MED16 PINLYP MAPK14 UBAP2L TAF3 BCL2L11 KCTD5 CCDC53 DDX47 ZNF671 TM2D1 TRIM58 TICRR KRT26 MED15 B4GALT1 RFWD2 MEF2D

[0100] IncuCyte tumor killing validation data demonstrated enhanced persistent killing by NY-ESO-1 TCR-T cells that have been knocked out for Mediator24, Mediatorl5, and TAF3. These are all members of the pre-initiation complex, suggesting a role for this complex in inducing T cell exhaustion. Further, it was recently shown that knocking out members of the mediator kinase module can enhance CAR-T cell fitness (Freitas et al., Science, 2022), although the proposed mechanism was that the mediator kinase module inhibits the main mediator complex activity (mediator complex is made up of a head, middle, and tail complex in addition to the kinase module). The data provided in the present diclosure supports the findings that knocking out the kinase module enhances fitness acutely in T cells (ie: KO of med 12 was a top hit in our screen data after 4 rep stims), however when we look at the late rep stim data (after 9 total stimulations), the top hits are involve the tail and middle of the mediator complex, which would actually be expected to reduce or redirect, rather than enhance, mediator complex activity. In addition, our data includes the TAF3 component,suggesting the entire PIC-complex may serve an important role in restraining T cell function in the context of chronic stimulation.Example 2. Validation in a NALM6 xenograft model

[0101] Six of the top hits that emerged the in vitro repetitive stimulation screen were evaluated in vivo in a NALM6 xenograft model utilizing CD19-CAR T cells. Results are shown in FIG. 6A-6D. The results evalutating knockout of target genes MEF2D, UBAP2L, and BCL2L11 showed that the knockout mice for each target exhibited a much lower tumor burden and prolonged survival compared to control mice (FIG. 6A and 6B, respectively).

[0102] The results evalutating knockout of target genes RF2D2, KDM6A, and SATB1, showed that the knockout mice for each target exhibited prolonged survival (FIG. 6D) and a trend toward lower tumor burden compared to control mice (FIG. 6C).MethodologyIsolation of primary T cells from healthy donors

[0103] Leukopaks from deidentified healthy donors with Institutional Review Board- approved consent forms and protocols were purchased from StemCell Technologies. Primary Human T cells were isolated using EasySep Human T cell isolation kit according to the manufacturer’s protocol using the EasySep magnets. The cells were seeded in appropriate culture vessels and activated with Dynabeads Human T-Activator CD3 / CD28 (Gibco) at a 1 : 1 bead to cell ratio. Cells were kept in culture at a density of 106cells per ml throughout, and cultured with IL-2 at 50 IU ml-1, unless otherwise specified. Cells were cultured in X- Vivo-15 medium, which was supplemented with 5% fetal calf serum, 50 pM 2- mercaptoethanol, and lO mM N-acetyl-L-cysteine.Pooled CRISPR-KO screens

[0104] In brief, isolated T cells from two human donors were stimulated as above, and 24 h later were transduced with a lentiviral pool to express the genome-wide Brunello sgRNA library as well as transduced with an NY-ESO1 -specific TCR (IG4). Twenty four hours after transduction, T cells were washed once with PBS, electroporated with Cas9 protein and expanded in culture as above. On Day 12, co-culture of the pooled edited T cells with tumor cells that naturally express NY-ESO-1 peptide on cognate MHCI was initiated. At 48 hour intervals, the T cells were collected, counted, and then incubated again at 1 : 1 ratios with freshtarget tumor cells. We have shown that this “repetitive stimulation” method, when repeated at least 3 times, and typically 5 times or more, induces clear dysfunction and exhaustion in the T cells and thus can be used to model the effects of chronic antigen stimulation causing exhaustion / dysfunction in T cells. In order to obtain strong log-fold change values for this screen, the cells were stressed to an extreme. Cells were repetitively stimulated nine times, to the point that unambiguous failure to clear the tumor cells was observed. Cells from both donors in each arm of the screen were harvested at key time points, including after transduction with the library, after initial expansion and before initiation of tumor co-culture (time zero), after 4 total stimulations, and after 9 total stimulations. Additionally, arms of the screen were included where the same pooled edited cells were cultured without any tumor cell exposure so the datasets could be compared. Once the cells were collected, genomic DNA was isolated from the cells and sgRNAs were then PCR amplified and size-selected for next-generation sequencing on an Illumina HiSeq instrument. MAGeCK analysis was used to analyze the data to determine log fold changes, P-values, and false discovery rates.CRISPR KO in primary human T cells using Cas9—RNP electroporation

[0105] T cells were isolated and stimulated as above and transduced with either the NY- ESO-1 TCR, or the CD19, BCMA, or HER2 CAR. Forthy eight hours later, Cas9-sgRNA- RNP electroporation was performed using the Amaxa P3 Primary Cell 96-well 4D- Nucleofector Kit (Lonza). Lyophilized crRNA and tracrRNAs (Dharmacon) were resuspended in nuclease-free duplex buffer at a concentration of 160 pM. Unless otherwise stated, control-edited T cells were targeted with the AAVS1 sequence GGGCCACTAGGGACAGGAT. The crRNAs and tRNAs were complexed at 1 : 1 v / v ratio for 30 min at 37 °C. sgRNAs were mixed with Cas9 (Stock 40 pM) at a 1 : 1 v / v ratio and incubated at 37 °C for 15 min to form the RNP complex. T cells were counted, resuspended in P3 buffer at 1 x 106cells per 20 pl, mixed with 3 pl of RNPs and added to a 96-well electroporation plate. The cells were electroporated using the EH115 protocol and immediately recovered by adding 80 pl T cell medium (X-Vivo-15, Lonza) at 37 °C for 15 min. Once recovered, cells were transferred to appropriate culture vessels in X-Vivo-15 medium with IL-2 at 50 IU ml-1.Repetitive stimulation assay

[0106] Tumour cells were seeded in complete RPMI medium one day prior to co-culture. Complete RPMI medium includes RPMI (Gibco 21870076), 10% fetal bovine serum, 1% L-glutamine, 1% penicillin-streptomycin. The next day, RPMI medium was replaced with T cell medium and antigen-specific T cells were seeded on top of the tumor cells at a 1 : 1 E:T ratio with IL-2 at 50 IU ml-1. Subsequent repeated co-cultures were set up every 48 h. The T cells were counted and replated onto fresh tumor cells at a 1 : 1 ratio every 48 hours until we saw evidence of failure of tumor control (typically beyond 4 repeated tumor stimulations). For each co-culture, T cells were collected and counted using the Vi-CELL XR cell counter and viability analyzer and replated onto fresh target tumor cells at a 1 : 1 E:T ratio. Before using the T cells for any assays, T cells were collected, counted and purified using EasySep Release Human CD45 positive selection kit (Stem Cell 100-0105) or purified by flow sorting.Lentiviral production and T cell transduction of TCR

[0107] Lenti-X 293T cell line (Takara Bio 632180) cells were seeded at 18-20 million cells per 15 cm dish pre-coated with poly-L-lysine 16 h before transfection and cultured in DMEM + 5% FBS + 1% penicillin-streptomycin. Cells were transfected with the sgRNA transfer plasmids and second-generation lentiviral packaging plasmids, pMD2.G (Addgene 12259) and psPAX2 (Addgene 12260) using the Lipofectamine 3000 transfection reagent per manufacturer’s protocol. Six hours after transfection, the transfection medium was replaced with DMEM + 5% FBS + 1% penicillin-streptomycin containing viral boost reagent at 500 / per the manufacturer’s instructions (Alstem). Twenty-four- and forty-eight-hour viral supernatants were collected and centrifigured at 300g for 10 min at 4 °C to remove the cell debris. The lentiviral particles were concentrated using Lenti-X Concentrator (Takara Bio) and stored overnight at 4 °C. The virus was centrifuged at 1,500g for 45 min at 4 °C and resuspended at 100 / of the original volume in ice-cold PBS and stored at -80 °C until further use. For T cell transduction, 24 h after TCR stimulation, the concentrated lentivirus was directly added to T cells at 1 :25 v / v ratio with X-Vivo-15 medium and gently mixed by tilting.CRISPR knock-in of CD 19 or BCMA CAR into TRAC using adeno-associated virus

[0108] Adeno-associated virus (AAV)-ITR plasmids containing the BCMA or CD 19 1928z CAR and TRAC -targeting homology arms for homology directed repair were used as previously described46. The AAV-ITR containing plasmid was packaged to AAV6 by transfection of HEK293T cells together with pHelper and pAAV Rep-Cap plasmids using Polyethylenimine. The AAVs were further purified using iodixanol gradient ultracentrifugation. The titration of the AAV was performed by quantitative PCR on DNasel(NEB) treated, proteinase K (Qiagen)-digested AAV samples, using primers against the left homology arm (forward: CTTTGCTGGGCCTTTTTCCC, reverse: CCTGCCACTCAAGGAAACCT). The quantitative PCR was performed with SsoFast EvaGreen Supermix (Bio-Rad 1725201) on a StepOnePlus Real-Time PCR System (Applied Biosystems).

[0109] T cells were isolated and activated as previously described46. After 48 h of T cell activation, cells were transfected by electroporation of RNP using a 4D Nucleofector 96 well unit (Lonza). One reaction of RNP was generated by incubating 60 pmol of Cas9 protein with 120 pmol sgRNA (Synthego, TRAC guide RNA (gRNA): ACAGGGUUCUGGAUAUCUGU) at 37 °C. Two million cells were electroporated and diluted into culture medium and incubated at 37 °C, 5% CO2. Recombinant AAV6 donor vector was added to the culture 30 to 60 min after electroporation, at the indicated multiplicity of infection (105), and incubated with the cells overnight. The day after the electroporation, edited cells were resuspended in T cell growth medium and expanded using standard culture conditions and kept at a density of 106cells per ml. Knock-in efficiency was evaluated by flow cytometry by staining the CAR with a goat anti-mouse Fab (Jackson ImmunoResearch, 115-606-003).In vitro cancer killing assay by TCR T and CAR-T cells

[0110] Antigen-specific T cells were co-cultured with pre-plated RFP+ A375 or GFP+ Nalm6 tumour cells in a 96-well flat bottom plate starting at a 2: 1 E:T ratio then with a log2 serial dilution in triplicates. For target cancer cells, A375 cells (ATCC) were used for TCR T assays. For CAR T assays, CD19-expressing RFP+ A375 melanoma cells were generated by targeted non-viral knock-in of an SFFV promoter in front of the endogenous CD 19 gene (guide targeting CATGGTGGTCAGACTCTCCG) as previously described45. For experiments with annexin detection, Annexin V Dyes (Essen Bioscience) Red (4641) and Green (4642) were used according to the manufacturer’s instructions. The plates were imaged every 2-3 h for 72-96 h using IncuCyte Zoom live-cell imaging (Essen Bioscience). The RFP+ or GFP+ object counts per well was recorded over time. Cancer cell growth was calculated as the count at any given time point, normalized by the count at t = 0.Flow cytometry assays[OHl] For cell surface activation markers, 2 * 105to 5 * 105TCR T cells were seeded per well in a round bottom 96-well plate. The TCR T cells were stimulated by Immunocult at12.5 [il ml1at 37 °C for 4-6 h. CAR T cells were stimulated by co-culturing with CD19+ Nalm6 leukaemia cells at 1 : 1 E:T ratio at 37 °C for 4-6 h. Next, the cells were centrifuged, washed once with 200 pl of cell staining buffer and stained with antibodies (5 pl antibody per in 100 pl staining buffer) for 30 min at 4 °C in the dark. Samples were read using Attune NXT Cytometer (Invitrogen) and analyzed by FlowJo. For exhaustion and differentiation markers, cells were not stimulated, but stained as described above. Antibodies used: Brilliant Violet 421 CD69 (Biolegend 310930), FITC anti-human CD154 (Biolegend 310804), PE anti-human CD25 (Biolegend 302606), FITC anti-human CD279 (PD-1) (Biolegend 621612), Brilliant Violet 711 CD223 (LAG-3) (Biolegend 369320), Brilliant Violet 421 antihuman CD366 (Tim-3) (Biolegend 345008), PE anti-human CD39 (Biolegend 328208), PE anti-human CD62L (Biolegend 304806), PE CD19 (Beckman Coulter IM1285U) and APC CD 19 (Beckman Coulter IM2470U).

[0112] For intracellular cytokine staining, TCR T cells were stimulated by Immunocult at 12.5 pl ml-land brefeldin A (eBioscience 00-4506-51) at 37 °C for 4-6 h. For CAR T cells stimulation, T cells and CD19+ Nalm6 leukemia cells were co-cultured at 1 : 1 E:T ratio and incubated with brefeldin A at 37 °C for 4-6 h. Next, cells were fixed and permeabilized with Fix & Perm Cell Permeabilization Kit (Thermo Fisher Gas004) and incubated with fluorochrome-conjugated antibodies (5 pl antibody per in 100 pl staining buffer) for 20 min at room temperature in the dark. Samples were read using Attune NXT Cytometer (Invitrogen) and analysed using FlowJo. Antibodies used: PE mouse anti-human IFNy (BD Biosciences 554701), BV711 mouse anti-human IL-2 (BD Biosciences 563946), Pacific Blue anti-human TNF (Biolegend 502920).

[0113] Antibodies and reagents used for flow cytometry experiments on cells isolated from bone marrow included: PE-Cyanine7 anti-human CD8a (eBioscience 25-0087-42), APC-Cy7 mouse anti-human CD45 (BD Biosciences 557833), BUV395 mouse anti-human CD4 (BD Biosciences 563550), BV421 mouse anti-human CD62L (BD Biosciences 563862), BV650 mouse anti-human CD45RA (BD Biosciences 563963), BV480 mouse anti-human CD279 (PD-1) (BD Biosciences 566112), PerCP-eFluor 710 anti-human CD223 (LAG-3) (eBioscience 46-2239-42), BUV737 mouse anti-human CD19 (BD Biosciences 564303), BV785 anti-human CD366 (Tim-3) (Biolegend 345032), PE CD127 (IL7RA) (Biolegend 351304), PE anti-human EGFR (Biolegend 352904), 7-AAD (Invitrogen A1310), Counting Beads (Invitrogen C36995). For these antibody stains, cells were resuspended in 2 plantibody in 100 pl total staining buffer volume (see ‘Bone Marrow CAR T cell isolation, processing and staining’).Bone marrow CAR T cell isolation, processing and staining

[0114] OPM2 multiple myeloma cells were injected via tail-vein into NSG mice in an amount of 1 x 106. OPM2 -bearing mice were treated with 3 * 1051-XX TRAC CAR T cells and monitored for BLI levels over time. Once the BLI levels were cleared, after 50 days, the mice were rechallenged 5 different times with more OPM2 cells (up to 5 million cells per injection). Mice were euthanized at day 90 after infusion. For bone marrow extraction, long bones of each leg were isolated by dissection, and then crushed with PBS in a mortar and pestle, followed by PBS washes through a cell strainer. Spleens were processed similarly. Cells were then centrifuged and treated for 2 min with ACK lysing buffer (118-156-721, Quality Biological) for red blood cell lysis, the reaction was quenched with FACS buffer. Remaining cells per each mouse were then resuspended in 300 pl FACS buffer. After addition of Fc block (10 pl per sample) (130-092-575, Miltenyi Biotec), cells were stained for the CAR (1 pl per sample) (115-606-07, Jackson ImmunoResearch) and incubated for 30 min at room temperature. After a wash, cells were resuspended in 2% normal mouse serum (Millipore- Sigma) and Fc block (10 pl) and incubated for 20 min at room temperature. Next, cells were stained with the relevant antibody mix (100 pl per tube staining volume; specific antibodies found above in the flow cytometry methods section) and incubated for 45 min at room temperature. After staining, cells were washed and resuspended in 300 pl FACS buffer as well as counting beads (50 pl per sample) (ThermoFisher Scientific) and then analysed by flow cytometry. Some of these extracted cells were left unstained, and instead were put into co-culture with OPM2 cells in vitro to quantify killing capacity.NALM6 xenograft model utilizing CD19-CAR T cells

[0115] Male mice from 8-12 week-old NOD / SCID / IL-2Ry-null (NSG) were obtained through Jax Labs or in-house breeding. Mice were intravenously injected with 0.5 x 106effLuc-GFP NALM-6, followed four days later by intravenous injection of 0.1 x 106CD 19- targeting CAR T cells. Tumor burden was monitored via bioluminescence imaging on a IVIS® Lumina™ S5 Imaging System. Mice were randomized based on bioluminescence imaging results to distribute tumor burden equally in each arm before T cell injections.

[0116] Mice were followed over time with bioluminescence imaging, results are shown on the graphs on the next two pages. Six gene targets were tested on their ability to eradicate thishighly aggressive type of blood cancer versus a safe harbor edited control KO condition (AAVS1):

[0117] It is understood that the examples and embodiments described herein are for illustrative purposes only and that various modifications or changes in light thereof will be suggested to persons skilled in the art and are to be included within the spirit and purview of this application and scope of the appended claims. All publications, patents, and patent applications cited herein are hereby incorporated by reference for the contents for which they are cited.Table 1. Positive-fold change for candidate genes identified from Screens that ModelTumor Exhaustion after 9 exposuresPos. fold change Gene 5.3929 RNF75.3864 MED243.99 TNFAIP33.73 ARIH22.9581 CUL52.8905 CISH2.8814 RASA22.8083 KDM6A2.5071 AUP12.4751 MEF2D2.3318 SATB12.2499 STT3A2.2429 BRD92.1989 DCUN1D32.1274 UBE2L32.1238 MED162.0556 PINLYP1.9818 MAPK141.9727 UBAP2L1.9426 TAF31.9334 DNMT3A1.9265 BCL2L111.9175 KCTD51.9029 CCDC531.8958 DDX471.8893 ZNF6711.8872 TM2D11.8674 TRIM581.8607 TICRR1.8315 KRT261.8219 MED151.8009 SMARCD11.7958 MAP3K41.776 NBPF121.7524 HP1BP31.7518 CCDC221.7339 SUPT6H1.7316 YWHAZ1.7228 UXS11.7008 TMEM19Table 2. Positive-fold change for candidate genes identified from Screens that Model Tumor Exhaustion 4 exposures.Positive fold¬Gene change MED12 2.5843 ARIH2 2.5271 TCTN3 2.2008 MED19 2.1756 RPP38 2.1151 TRIM58 1.9153 Clorfl41 1.8935 B3GNT2 1.7836 ATP6V0D1 1.757 CISH 1.7509 RNF7 1.7034 ITGAM 1.6724 XP01 1.6554 ARFRP1 1.6513 OR5D13 1.6504 DCN 1.644 CCT5 1.5763 OR2AT4 1.5571 HSPB1 1.5453 GRIP1 1.5446 ZNF775 1.5391 HIST1H2BA 1.5385 JUP 1.5369 PIK3CD 1.5279 DMPK 1.5166 CELSR1 1.5136 TMEM19 1.5001 SPATA21 1.495 TMEM17 1.4873 PHF2OL1 1.4756 APOBEC3A 1.4725 ENDOU 1.4648 POLRMT 1.4647 ACTR3B 1.4623 LRRC75A 1.4566 FOXH1 1.4515 RPL38 1.4487DHX57 1.4436 NDST1 1.4371 FOXK1 1.4314 SLC5A10 1.4292 WNT4 1.4273 OR2A5 1.4221 TAF3 1.417 C22orf24 1.4134 ATG12 1.4121 CENPN 1.4098 TNFAIP3 1.405 NBPF15 1.4041 PAX5 1.3946 LIN7A 1.3844 KLC4 1.3675 GPD1 1.349 DGKZ 1.3424 FSIP1 1.3298 RPL22 1.3197 MORC2 1.3171 ABCB1 1.3154 RBX1 1.3144 SYBU 1.3119 VPS53 1.3071Table 3. Guide Sequences for Hits (Table 1) from Screen that Models Tumor ExhaustionGene Guide sequence # 1 Guide sequence # 2 Guide sequence # 3 Guide sequence # 4 RNF7 AACAAACAAGAGGACTGTGT AGCTCAGGCTCCAAGTCGGG CCTCAAGAAGTGGAACGCGG CGATACGTGCGCCATCTGCA MED24 CCTCGAGCAGGATCACGGCG GAAGTACTCTCATGGAGACA GCCCGGAAATTCATCAAGTG GCTGCACATCGCCAAACTAG TNFAI P3 CCACTTGTTAACAGAGACCG CTTGTGGCGCTGAAAACGAA TATGCCATGAGTGCTCAGAG TGAGAGACTCCAGTTGCCAG ARIH2 ATATCTCTGAAACTTGCCAG CCTGTACCCGAATAACCATG GCACTGCTCAGTTCTCGTCA GGACATAGAGGACTATTACG CUL5 AG CTTGTTTAC ATAATCCG C ATTGGAGTAAGAGAATCCTA GAGGAACATATCATTAGTGC GGAAGACAGTATTGTTCGAA CISH AAGGCTGACCACATCCGGAA AC AAG CAGTTG G AGTCCAG A CTCACC AG ATTCCCG AAG GT CTGTCAGTGAAAACCACTCG RASA2 AGATATCACACATTACAGTG AGGATCGACTTGTGGAACAA GTGCAGCAGCAGTCGTACAA TTAG CATC A AG G C ATG CC AT KDM6A CAATTGTCAGAAGTATTCTG CCAACTATCTAACTCCACTC CTGGTAAGTCTCACCTTCCG TCTTTGTATGAACAGCTGGG AUP1 C AG G G GTCTCTG A ACTTG C A G ACCAG CAC AC AAAG CTGG G GTGTCCCCAGATTCGTAGTG TCCTGTTCATCGCCAACTAG MEF2D CAAGTACCGACGCGCCAGCG CATCATCCCTCACGGACCCG G ATG ACTG CACTC ACC AAC A GGTGAGCGAATGAGTAGACT SATB1 ATGCTAAGTACCTGTGAAAG CATTGAATATGATTGCAAGG TAGGTGTTGATACGAGCCCA TATTCATAGATCTACTGACA STT3A ACAGACATTCCGAATGTCGA G CTG CG C AG GT A ATCC AC A A TACCTGGAAACATGAAGACG TCATAGGTCTCGTCATGGGG BRD9 ACTCCAGTTACTATGATGAC AGAGAGGGAGCACTGTGACA AGATACCGTGTACTACAAGT CTTGACGGACAGTACCGCAG DCUN1D3 GCAGCACTCGAAATTCTGTG GCATTGACGGAATCTGTGCA GGAGTCCAAGTCCAATGCCG TTGACGAGGATATCTCCACC UBE2L3 AAGGCTCCCTTATCATATGG ACCCAAACATCGACGAAAAG GAAAAACTTCCGTAACATCC TTGAAGAAATCCGCAAATGT MED16 CGACGGTGAGAACTTGACTC GGTGACCGGCTACGACTGGT GGTGTG GTAGTCG CAC ACG C TAGCCTTTAAGTGGACGGCG PI NLYP ATTCTCAGTAAGATTGGTCA CGGCGTTATATCCACCACCA CTGCTGATACACACACCATG GCGAAATATGTACGGCGGCG MAPK14 AAGTAACCG C AGTTCTCTGT CAAGGCGAGTAATACCTGTC CAC A A A A ACG G G GTTACGTG TGATGAAATGACAGGCTACG UBAP2L AAGTAGCCTGTATACCAGCA ACCGGGACAGAGACTATAGT GCAATAGCAGCGGCAATACG TCTGCCAAGCAAATCCACAT TAF3 CCTTTAGTGCTTAATAGCCG G AAATG ACG G AATTTG GTGT GAATGTGAGTCGTGACCTTG GCTTTGGGAGTAGTACACTC DNMT3A CCGGGAACAGCTTCCCCGCG CGATGACGAGCCAGAGTACG GCGGGCACAAGGGTACCTAC GCGTACCAGTACGACGACGA BCL2L11 AGGTAGACAATTGCAGCCTG CTTGGGCGATCCATATCTCT GTTCTGATGCAGCTTCCATG TATGGATCGCCCAAGAGTTG KCTD5 ACGAGACAGCAAAACATCGC CTGGCATAAGCGGTACAGGA ACGAGACAGCAAAACATCGC TGGGTCCGACTCAACGTCGG CCDC53 CCTCACAAACTGTAGAAAAG TCTCAGTTGTCATCTATCCC TCTTAGAACTCACTTACCTG TTTGTTGGATACGAAGTGAA DDX47 AACTAAAACATTT AAAG ACC ATTCATTTAGAATATAAACC TAGGGCAAACAAACGCTGCG TCCTCTATTG G AGTG CAG AG ZNF671 AAGAGCACTAAGCTTGTGAG CACCTGGCTAAATACCATGG CGGAGCTAACGGACTCCGCG GCAGTCATGAAACTAGAGCGTM2D1 AAATGGCTATTCCTACAAAG ATAACTTGTAAGGATTCCAG CGAGGAGTCGCTTAAGTGCG GCCGTGACGGCCAGACTCGT TRI M58 CAGCATGCTGCATCCCTGGG CGGTTGGGGCGAAAGCCCGA GGAGGACGCCTTGACTCAGG TTGGAGTTTGAGAAGCATCG TICRR ACTGGGTGGATACCACCGAA GACGCTGCTAGACTACCAGT GGATAGAGTCAATATACCTG TCAAAGAGATCACTAAAGCG KRT26 AGCATGCACATGGTCCAGGT GCCTGGTTCAACGAGAGGGT GCTTCTGCAATAGCGGTGGA GTGGAGATGAATGCAACCCC MED15 GGAATTGGCATGCCTCCTCG GTACTCACAAATTTCAGTGG GTGACGGCGAGGACAGCATG TGTGTTTAAAGGTCCAGGTG SMARCD1 CCTG GT A ATCC AG C ATC AGT GAAACGGCTAGATATCCAAG G AG CG GTACAG CCCTTG ACC TTTGTCCAGTTCAATCACCA MAP3K4 ACATAATCTGAACCCCACAT AGGACGGACAATTAACGACC ATAACTCATTACATACGGGG GCATGTCAACCAAACATCAG NBPF12 AGACAAAGTCAACTCAACTG AGAGCAGCTGAAACAAGCTG GAATGAGCTACAGTTCAAGG GCTGCTGTAAGACTGGTATG HP1BP3 AAAGGCGAGTAAAGGCCAGT CCACGCGGACAAGTTAGGTG GATGGATGCAATCTTAACTG TCCACCTGCTACTTCGAGTG CCDC22 ACTTCTCTG AGTG CGTG AAG CCGCAGGGTTGATCACACGC CTGC AC AAAG CTG ACG CCCA GCTGCAGCTTGGGAGTGCGA SUPT6H GCTGTGATTACCTAGACCGA GGAATACGATGAATCAGCCG GTTCGCCGTTTGGTAAAATG TG AATGTCC ACAG G AG CATG YWHAZ AAAATGTTGTAGGAGCCCGT GCCGCTGGTGATGACAAGAA GGCTGAGCGATATGATGACA TCTCTGCTTGTGAAGCATTG UXS1 CCTCAAAGTGAGGATTACTG CGAGTTGATTAACCACGACG GTAGGGCTGGCAAAACGAGT GTCCGGATGCAGATAACAGG TMEM19 ACTGCTTGGAAAAATCGACT ACTTACGACCTATTTCTCCG GAAAAGTCTAGATCACAGTG TCCAAGACTGATAACAACCT YME1L1 AGGTCCCTTAAGTTAAGTGA ATGATGTCGATACAAGCAAG GCACTCACACAAAAAACCAA TGTTTC AG CT A ATCTCTCG G BCAR1 ACTGCGTGGTGGTTGGACGG GATGGTGGCGTGGTCGACAG GGGGCAGGGCTATGTATACG GTG G ACG GC ACTCTG G AC AG SYNM AAAAGCCGGAGATACAAGGG CTGTGATTGAAACTTGCCAA GAGAGCAAAGGTCGTCAACG GGCAGGATGAAATCGTGCAG P0C1B- GALNT4 ATTAGGACCAATAAGCGAGA GATCCACCCGTGTTCCCACG GCAATGATAACAGAGGTGGT TACCTTGGAACGTATGACAC ALG5 AGCAGTTGTAAATGCAACGA ATC ACTTCATAAGTG AACG C CAAAGTACGTGTGATAACCC GTAGGTGAGTCCCATATGCT CD48 AAACTGTCATGTGTGATACC ATGTACAGTGCGCCACTCTG CTGGTCGAAAGTATAAAACC TCACTTGGTACATATGACCG FAM71D AAATTGCTCTCTAACATGGG GCTG G AAAAAC AG ATG CCTA GGCCACCCATTGAGAAAACA TGTACCTGCATACATGTACG ARFRP1 CGTCATTGACTCCACCGACG CTTACTGTTTAGGCCCACGG GATTTAACAAGAACTACAAG TGCTTCCTCCCAGTATTATG B4GALT1 CACGTCACTAAACACAAAGC CGAGTCCTTACCAAGCAGCG G ACCG AG GTC AAGTTG CTAG TCTATGTTATCAACCAGGTG UBE2F AGACAGGGGAAATATGTCTG AGCGTCCGACTCGACTCGGA AGTAAACTGAAGCGTGACGA TCCCGATGCGTACAACATGG AKR1C3 AATGAGCAGAATCTATATGG AGAAATCTAGCAATTTACTC GGATCTCTGTACCACCTGGG GGGTGTCAAACTTCAACCGC LYPLA1 ACCTGCCAGTTTCTGCTGTG GCCTACG CCACTC ACCG CAG TGGATTGGGAGATACTGGGT TGTTACATTAAATATGAACG NPC1L1 AGAACTCACTACGGACCCCG AGAGCCATACACGCCACACA CCTCAACAATTACCCTGCCG CTTCGGGCGACCATACCTGGSH2B3 ACTACCGGGACACAGGCCGT GCAGCAGCTGAATTCATGGA GGAGCTCTTCGACCCACCCA TGAGTTGCACGCCGTAGCGGIKZF1 GAAAATGAATGGCTCCCACA GATGGCTTGGTCCATCACGT GGGGCCTCATTCACCCAGAA TCCAAGAGTGACAGAGTCGT RFWD2 ATCACTAGCATATGACAATG G ATTCTTATG G AATTCCTCA GCTAACGTGCTATTATACCA TACCAATCTAGACAACTCAG C16orf62 CATTACCTGGTAAGACGCTG CTG ATC ATG CATGTCAACCG GAGCCTTGGACCAACAAACG GGATTACGTGAACCGCATAG SH3GL1 GCACCACCTGAAGAAACTGG GCCATCCTATCCCAGCCTCG GTCCAGCTGTGCATCCACCA GTGCATGATCCGCCACGGGA KRTAP5-5 AGCCCCCACAGCCGGAGCCA CTCCGGCTGTGGAGGCTGTG GCCCCCCTTGGACCCCCCAC TCTCCTGTGGGGTGTCCAAG LRRC75A AAGTCCGGCCGTTCGCGTCG ATACAGAACGTCGTCTAGCG CTACATCCACTGTCCCAAGC CTACCACCGGCGAGTCGGCA SEC14L1 ATGGAAGGCGGACTCCAACG CTCCCGCGTAGTAATCCTGA TATTAAAATCCCGTGCACGG TC AC ACCAG GTCTCC AC AAG TARBP1 AAGAAGCCCATCTATACCCA ACACCCTTATACCAACCACA GTCTATGGCCTCACACACCA TCCAATGGAGCTACACGTAG RBM4 ACCTCAACAAAGTTGCATGT ACGCGGCCTGAACGATCTAT GCCTTACACCATGAGCTATG TGTGTTATCAAGGCCCCTGA ATHL1 CCCCGACGAGTACCATTCAG CCTCTCC AATGG G AG CCGTG CGTGAGCGGCGTGTACAATG GCGATGCGCATAGATGCACT URM1 AGTCACTTTGCCTGGACAGG CATGGCTGCGCCCTTGTCAG CCTCCTTTGCAGAGGTGGTG TCTCCCTG G ATG AAC AACTC FRS2 AAATAATAGTATAAATGTGG GCATGTTCCATTGGAGGCGA TACACCAGTTTGTTAACAGA TCTCCAAATGAGGGATATCG NOSIP CCACAGATGATGTCCAACCT CTCACGTG AC AACAG G ATCG GCCCCG CAC ATG GTCCTG CG GTCAGGTCCGACATGCGCAG KCNRG AAAAAGTTACCATTCCAGGT CAATTGAGATGCTAACAGGG CAGAACCACACTGGAAAACC GAGGGAAGATATTCACGACA TAS2R43 GAGCAAATAAAATATGCTGA GGGTACTAAGTTTGCTACCA TAAACATGAATGAGATTGTG TCATTCTGGTGATGTTGTTG STARD9 ACAGTGTGGACATCAACAGG GAGGACGTCGGACTGACCAG TGAGCCCAAAGATGTTAACA TGGGGTTGGAGCTCACAATG CBLB CTTC ATCTCTTG G ATCAAAG TGCACAGAACTATCGTACCA TGTGGGATGTCGACTCCTAG TTCCGCAAAATAGAGCCCCA TRIM2 AGAGGCGCGTTAAGTCCCCG AG CCACTTAC ATTCCC ATCG CAACCATGGCACGGAGACCG TGGTGGTTAAGATCGTCCCG SRRD AGGGAAACGGAGTATTCGCG GCATCTTGACTCATTGCCAG GCGGAGTTCGAGTCTGACAG GTTTCTAG CT ACG ATG C AG G CHIC2 ACCTTCCTGTTAATGTACGT CGAAATCTATGAGGAAGAGG CGGTGACGTGACCGGAGCCG GCACATTAGGTTGCAGTATG NINJ2 CAGGGTGGTGTAGTAGTGAG CATGG CGTTG G AC ATG AAC A GTGCCGGGAACAGACTGCGT TTACGCCACCAAGAAGAGCG GRN ATCGACCATAACACAGCACG CCCTGCCCAGAGGACTAACA CTGCTG CCGTCTAC AGTCG G TTTACGTGTGACACGCAGAA EFNA4 CAATGTCTAG GTAATCGTTG CAG GTAAG AACTC AAAG CCG CATGTACAAAGCAAACGTCT TCTTCTCTGAGAATTGAACA ERF CGGAAGTGGCTACCACCCGA CTGAGCCCGATGTACCCCAG GACCGGCCAGAGGCGACACA GGAAGGCACCCAGATCCGGA MSL1 CCACTCAGTAGAGCCTCTAA CCT ATT AAAG AG G AACCCTG GAGGATTCCCGTAATGGTAA GGGGTGTCCACTGAAGACCG CCL27 CACTCTCAGACAAGCTACTG CCAAGTACTCACCTGCTGTA TCGGTAGAGCTGAGTACAGC TGCAGATGCTGCGTTGAGCC DET1 AATCAGTGACCCCCAACCCA ACG CACGTTC AAGTGTG AC A CAGCGGTCAGTGAATATCCG TAGTGTACTTGATAAACAGGDNASE2 CCAGCCAGAAGCCCCCATCG CTGGTGTTG CTCCG GTACAG GCTGGTAGTTATAGACCCAG TCCAGGGTTCTTGGCTAACGEP300 ATGGTGAACCATAAGGATTG CTGTAATAAGTGGCATCACG GGTACGACTAGGTACAGGCG GTGGCACGAAGATATTACTCERVV-1 AACTTATTCAACCCCCCCTG CATAAAAATACATAGCCAGG GAAAAGAGCTAATCACATGG GACACCAGTGAGTCAAGTTAHLA-A CACGCACGTGCCATCCAGGT CCAGTCACAGACTGACCGAG GGATGTGAAGAAATACCTCA TACCGGCAGGACGCCTACGAOR2H2 AATCATCCACCTGCCGATCG CACAACCAAGATGAAGACAC CATTTGGGGAACACAACTCG TTCCTACCTCCTAACCCTAGC0MMD7 ATGGCCCATCGAGCAAGGGT GCCAAGACTGATCTGATTGG GTGGAAAAGCACCTCTGTCA TTCTCTTATAGGTCTTAGTGZNF282 CAGGGAAGAACCTTGTGTGT CGACTGTGAAAAGACGGCCG CTTGTCCCGGATTAAACAGG TCTCCGTCCATTGAGTCGCGZSWI M8 CAAGTGAGATGAGTACCATG GGAACAGGAGAGTCGCATGG GTGATTGAGAACGTCAAGCG TCCTAAAGGGGCCTACAACGRAB21 AATAGGTAATAAAATAGACT AGTAAATTGGACCCAATGCA CCACCTTGAACGAGTAGGCT GTTCACTTACCCATATGGCASHANK1 ACTCTTCCGGCATTATACCG GTGAACATGATCCGCCAAGG TACCATGACTCGGATTCGGG TGTCGGCACCTCGATACAGGUBALD2 AAGGGGCTGAAGTTTGCAGG AGAACATGGCCAGCGCATCG CATGATCAACCAGTTCGTGC TTG G AAG AACGTG CTC AG CGMED1 CCAACCAACACCTTTCCGGG CCAGTAATAGTACTCTCTCG GAAGGTTATACAGATTAACA GAGCTTGAAGAATCTCGAAGLOC643355 AAGGTCAGCATGTGCCCACA ATCC AG G ATTCCG GG C ACCG CCAGATTCCCACCCTCAGGG GCTCCGTGCCTCGTCGACCCDON SON AGGTACAAACGTAGAAATAG CCCGCCTGGACAACCGACCG GAAGGTACAACGGAGCTCAG TGAGGACGGAATATCAGGCTTXNDC8 AACGGTGTGGTCCCTGCAAA GGTACAGATTATTAAAGACA GTTGTACTCACATGGAAAAC TGCCGGACACAAACTCGCAGUNC13D ACTGAGACCTACCCAGACCG CATCACCCACAACCGTCGTG GCAGCAGCACCATCAACCCA TGAAGGTCTCGTCCCAGACGEPC2 GAAAACGTAAAAACTGCAGG GATGTATAGGATTTGCAAGG TACCATTTGGGAGACTATGG TATTACAATCGCTTGTACAAGOLGA7 AACAACCTTCGAGATATGAC AGTTTCCATGCATAGGAAGA ATGAACACCTTTCCGGACAC CATGGAAACTCATTATGAGAAGAP6 CACCCACGCCCGTTTGCAAG CGTGACCGGGAGATGCCTGA GAG CATGCC ATTG G AAC AC A TTATTAGCATGAGGAGAGGG

Claims

WHAT IS CLAIMED IS:

1. A genetically modified T cell that comprises a genetic modification to a T-cell exhaustion gene that inhibits expression or activity of the polypeptide product encoded by the T-cell exhaustion gene, wherein the gene is selected from the group consisting of UBAP2L, MED24, KDM6A, RFWD2, AUP1, SATB1, STT3A, MED16, PINLYP, MAPK14, TAF3, BCL2L11, KCTD5,CCDC53, DDX47, ZNF671, TM2D1, TRIM58, TICRR, KRT26, MED 15, B4GALT1, UBE2F, UBE2L3, DCUN1D3 and MEF2D, and expression or activity of the polypeptide product is inhibited by at least 50% compared to a control wildtype T cell cell.

2. A genetically modified T cell that comprisesat least one genetic modification to a T-cell exhaustion gene that inhibits expression or activity of the polypeptide product encoded by the T-cell exhaustion gene, wherein the gene is selected from the genes set forth in Table 2.

3. The genetically modified T cell of claim 1 or 2, wherein the T-cell is a CD8+ T cell or CD4+ T cell.

4. The genetically modified T cell of claim 1, 2, or 3, wherein the T-cell exhaustion gene is inhibited using a clustered, regularly interspaced, short palindromic repeats (CRISPR) system.

5. The genetically modified T cell of claim 1, 2, or 3, wherein the T-cell exhaustion gene is inhibited using a transcription activator-like effector nuclease (TALEN) system.

6. The genetically modified T cell of claim 1, 2, or 3, wherein the T-cell exhaustion gene is inhibited using a zinc finger nuclease system.

7. The genetically modified T cell of claim 1, 2, or 3, wherein the T-cell exhaustion gene is inhibited using a meganuclease system.

8. The genetically modified T cell of claim 1, 2, or 3, wherein the T-cell exhaustion gene is inhibited using inhibitory RNA.

9. The genetically modified T cell of claim 1, 2, or 3, wherein the T-cell exhaustion gene is inhibited using shRNA, siRNA, microRNA, or an antisense RNA.

10. A genetically modified T cell that comprises (i) a first genetic modification that inhibits expression or activity of the polypeptide product encoded by a first gene selected from the group consisting of MED24, KDM6A, AUP1, SATB1, STT3A, MED 16, PINLYP, MAPK14, UBAP2L, TAF3, BCL2L11, KCTD5,CCDC53, DDX47, ZNF671, TM2D1, TR1M58, TICRR, KRT26, MED15, B4GALT1, RFWD2, CUL5, ARIH2, RNF, CISH, UBE2F, UBE2L3, MEF2D, and DCUN1D3, and (ii) a second genetic modification that inhibits expression or activity of the polypeptide product encoded by a second gene selected from the group consisting of MED24, MEF2D, KDM6A, AUP1, SATB1, STT3A, MED16, PINLYP, MAPK14, UBAP2L, TAF3, BCL2L11, KCTD5,CCDC53, DDX47, ZNF671, TM2D1, TB1M58, TICRR, KRT26, MED15, B4GALT1, RFWD2, CUL5, ARIH2, RNF, CISH, UBE2F, UBE2L3, and DCUN1D3.

11. The genetically modified T cell of claim 10, wherein the T-cell is a CD8+ T cell or CD4+ T cell.

12. The genetically modified T cell of claim 10 or 11, wherein the first and / or the second T-cell exhaustion gene is inhibited using a clustered, regularly interspaced, short palindromic repeats (CRISPR) system.

13. The genetically modified T cell of claim 10 or 11, wherein the first and / or the second T-cell exhaustion gene is inhibited using a transcription activator-like effector nuclease (TALEN) system.

14. The genetically modified T cell of cclaim 10 or 11, wherein the first and / or the second T-cell exhaustion gene is inhibited using a zinc finger nuclease system.

15. The genetically modified T cell of claim 10 or 11, wherein the first and / or the second T-cell exhaustion gene is inhibited using a meganuclease system.

16. The genetically modified T cell of cclaim 10 or 11, wherein the first and / or the second T-cell exhaustion gene is inhibited using inhibitory RNA.

17. The genetically modified T cell of claim 10 or 11, wherein the first and / or the second T-cell exhaustion gene is inhibited using shRNA, siRNA, microRNA, or an antisense RNA.

18. The genetically modified T cell of any one of claims 1-17, wherein the T cells express a CAR.

19. The genetically modified T cell of any one of claims 1-17, wherein the T cells express an HLA-Independent TCR-based Chimeric Antigen Receptor, a T cell receptor fusion construct (TRuC), a synthetic T cell receptor and antigen receptor (STAR), an antibody-T-cell receptor (AbTCR) or a T cell antigen coupler (TAC).

20. A population of cell comprising the genetically modified T cell of any one of claims 1-19.

21. A method of treating cancer comprising administering a population of cells comprising a genetically modified T cell of any any one of claims 1-17 to a subject that has cancer.

22. A method of identifying a gene that plays a role in T cell exhaustion, the method comprising:(a) providing a library of gene-edited T cells comprising a population of activated T cells that express a TCR that binds an HLA peptide from a cancer antigen from a target tumor type, wherein the library comprises T cells comprising sgRNAs for target genes that are inactivated by gene editing;(b) co-culturing the library of T cells with tumor cells from the target tumor type that presents the HLA peptide;(c) collecting T cells and re-incubating with fresh tumor cells from the target tumor type;(d) repeating steps (b)-(c) at least 2 times; and(e) collecting and processing T cells for next generation sequencing for sequence analysis to quantify the level of sgRNAs in (i) a population of T cells repeatedly exposed to tumor compared to (ii) a control subpopulation of T cells not repeatedly exposed to tumor cells to identify target genes; and(f) selecting a target gene that exhibit a 1.3 log-fold or greater positive change in (i) compared to (ii).

23. The method of claim 22, wherein the library of T cells is exposed to the target tumor for a total of at least 4 times.

24. The method of claim 23, wherein the library of T cells is exposed to the target tumor for a total of at least 9 times.

25. The method of any one of claims 22-24, wherein each gene is targeted by at least four sgRNAs

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