WO2024178397A2

Movatterモバイル変換

Info

Publication number: WO2024178397A2
Application number: PCT/US2024/017171
Authority: WO
Inventors: Elena Geretti; Iulia Diaconu; Sandra MILASTA; Hasan MAHMUD; Pei-Yun Tsai
Original assignee: Elevatebio Technologies, Inc.
Priority date: 2023-02-24
Filing date: 2024-02-23
Publication date: 2024-08-29
Also published as: WO2024178397A3

Abstract

The present disclosure provides methods and compositions related to the modification of cells to increase therapeutic efficacy. In some embodiments, immune effector cells modified to reduce expression of at least two endogenous target genes, or to reduce two or more functions of an endogenous protein to enhance effector functions of the immune cells are provided. In some embodiments, immune effector cells further modified by introduction of transgenes conferring antigen specificity, such as exogenous T cell receptors (TCRs) or chimeric antigen receptors (CARs) are provided. Methods of treating a cell proliferative disorder, such as a cancer, using the modified cells described herein, are also provided.

Description

the modified cells described herein comprise 2, 3, 4, 5, 6, 7, 8, 9, 10, or more modified endogenous target genes encoding 2, 3, 4, 5, 6, 7, 8, 9, 10, or more modified endogenous proteins. In some embodiments, the modified endogenous protein demonstrates reduced or altered binding affinity for another protein expressed by the modified cell or expressed by another cell; reduced or altered signaling capacity; reduced or altered enzymatic activity; reduced or altered DNA-binding activity; or reduced or altered ability to function as a scaffolding protein. [0086] In some embodiments, the modified endogenous target gene comprises one or more dominant negative mutations. As used herein, a “dominant-negative mutation” refers to a substitution, deletion, or insertion of one or more nucleotides of a target gene such that the encoded protein acts antagonistically to the protein encoded by the unmodified target gene. The mutation is dominant-negative because the negative phenotype confers genic dominance over the positive phenotype of the corresponding unmodified gene. A gene comprising one or more dominant-negative mutations and the protein encoded thereby are referred to as a “dominant-negative mutants”, e.g. dominant-negative genes and dominant-negative proteins. In some embodiments, the dominant negative mutant protein is encoded by an exogenous transgene inserted at one or more locations in the genome of the immune effector cell. [0087] Various mechanisms for dominant negativity are known. Typically, the gene product of a dominant negative mutant retains some functions of the unmodified gene product but lacks one or more crucial other functions of the unmodified gene product. This causes the dominant-negative mutant to antagonize the unmodified gene product. For example, as an illustrative embodiment, a dominant-negative mutant of a transcription factor may lack a functional activation domain but retain a functional DNA binding domain. In this example, the dominant-negative transcription factor cannot activate transcription of the DNA as the unmodified transcription factor does, but the dominant-negative transcription factor can indirectly inhibit gene expression by preventing the unmodified transcription factor from binding to the transcription-factor binding site. As another illustrative embodiment, dominant- negative mutations of proteins that function as dimers are known. Dominant-negative mutants of such dimeric proteins may retain the ability to dimerize with unmodified protein but be unable to function otherwise. The dominant-negative monomers, by dimerizing with unmodified monomers to form heterodimers, prevent formation of functional homodimers of the unmodified monomers. [0088] In some embodiments, the modified cells comprise a gene-regulating system capable of reducing the expression or function of one or more endogenous target genes. In some embodiments, the modified cells comprise a gene-regulating system capable of reducing the expression or function of at least two endogenous target genes. The gene-regulating system can reduce the expression and/or function of the endogenous target genes modifications by a variety of mechanisms including by modifying the genomic DNA sequence of the endogenous target gene (e.g., by insertion, deletion, or mutation of one or more nucleic acids in the genomic DNA sequence); by regulating transcription of the endogenous target gene (e.g., inhibition or repression of mRNA transcription); and/or by regulating translation of the endogenous target gene (e.g., by mRNA degradation). [0089] In some embodiments, the modified cells described herein comprise a gene- regulating system (e.g., a nucleic acid-based gene-regulating system, a protein-based gene- regulating system, or a combination protein/nucleic acid-based gene-regulating system). In some embodiments, the gene-regulating system comprised in the modified cell is capable of modifying one or more endogenous target genes. In some embodiments, the gene-regulating system comprised in the modified cell is capable of modifying at least two endogenous target genes. In some embodiments, the modified cells described herein comprise a gene-regulating system comprising: (a) one or more nucleic acid molecules capable of reducing the expression or modifying the function of a gene product encoded by one or more endogenous target genes; (b) one or more polynucleotides encoding a nucleic acid molecule that is capable of reducing the expression or modifying the function of a gene product encoded by one or more endogenous target genes; (c) one or more proteins capable of reducing the expression or modifying the function of a gene product encoded by one or more endogenous target genes; (d) one or more polynucleotides encoding a protein that is capable of reducing the expression or modifying the function of a gene product encoded by one or more endogenous target genes; (e) one or more guide RNAs (gRNAs) capable of binding to a target DNA sequence in an endogenous gene; (f) one or more polynucleotides encoding one or more gRNAs capable of binding to a target DNA sequence in an endogenous gene; (g) one or more site-directed modifying polypeptides capable of interacting with a gRNA and modifying a target DNA sequence in an endogenous gene; (h) one or more polynucleotides encoding an effector protein capable of interacting with a gRNA and modifying a target DNA sequence in an endogenous gene; (i) one or more guide DNAs (gDNAs) capable of binding to a target DNA sequence in an endogenous gene; (j) one or more polynucleotides encoding one or more gDNAs capable of binding to a target DNA sequence in an endogenous gene; (k) one or more site-directed modifying polypeptides capable of interacting with a gDNA and modifying a target DNA sequence in an endogenous gene; (l) one or more polynucleotides encoding an effector protein capable of interacting with a gDNA and modifying a target DNA sequence in an endogenous gene; (m) one or more gRNAs capable of binding to a target mRNA sequence encoded by an endogenous gene; (n) one or more polynucleotides encoding one or more gRNAs capable of binding to a target mRNA sequence encoded by an endogenous gene; (o) one or more site-directed modifying polypeptides capable of interacting with a gRNA and modifying a target mRNA sequence encoded by an endogenous gene; (p) one or more polynucleotides encoding an effector protein capable of interacting with a gRNA and modifying a target mRNA sequence encoded by an endogenous gene; or (q) any combination of the above. [0090] In some embodiments, one or more polynucleotides encoding the gene- regulating system are inserted into the genome of the modified cell. In some embodiments, one or more polynucleotides encoding the gene-regulating system are expressed episomaly and are not inserted into the genome of the modified cell. [0091] In some embodiments, the modified cells described herein comprise reduced expression and/or function of one or more endogenous target genes and further comprise one or more exogenous transgenes inserted at one or more genomic loci (e.g., a genetic “knock- in”). In some embodiments, the one or more exogenous transgenes encode detectable tags, safety-switch systems, chimeric switch receptors, and/or engineered antigen-specific receptors. [0092] In some embodiments, the modified cells described herein further comprise an exogenous transgene encoding a detectable tag. Examples of detectable tags include but are not limited to, FLAG tags, poly-histidine tags (e.g. 6xHis), SNAP tags, Halo tags, cMyc tags, glutathione-S-transferase tags, avidin, enzymes, fluorescent proteins, luminescent proteins, chemiluminescent proteins, bioluminescent proteins, and phosphorescent proteins. In some embodiments the fluorescent protein is selected from the group consisting of blue/UV proteins (such as BFP, TagBFP, mTagBFP2, Azurite, EBFP2, mKalama1, Sirius, Sapphire, and T- Sapphire); cyan proteins (such as CFP, eCFP, Cerulean, SCFP3A, mTurquoise, mTurquoise2, monomeric Midoriishi-Cyan, TagCFP, and mTFP1); green proteins (such as: GFP, eGFP, meGFP (A208K mutation), Emerald, Superfolder GFP, Monomeric Azami Green, TagGFP2, mUKG, mWasabi, Clover, and mNeonGreen); yellow proteins (such as YFP, eYFP, Citrine, Venus, SYFP2, and TagYFP); orange proteins (such as Monomeric Kusabira-Orange, mKOκ, mKO2, mOrange, and mOrange2); red proteins (such as RFP, mRaspberry, mCherry, mStrawberry, mTangerine, tdTomato, TagRFP, TagRFP-T, mApple, mRuby, and mRuby2); far-red proteins (such as mPlum, HcRed-Tandem, mKate2, mNeptune, and NirFP); near- infrared proteins (such as TagRFP657, IFP1.4, and iRFP); long stokes shift proteins (such as mKeima Red, LSS-mKate1, LSS-mKate2, and mBeRFP); photoactivatible proteins (such as PA-GFP, PAmCherry1, and PATagRFP); photoconvertible proteins (such as Kaede (green), Kaede (red), KikGR1 (green), KikGR1 (red), PS-CFP2, PS-CFP2, mEos2 (green), mEos2 (red), mEos3.2 (green), mEos3.2 (red), PSmOrange, and PSmOrange); and photoswitchable proteins (such as Dronpa). In some embodiments, the detectable tag can be selected from AmCyan, AsRed, DsRed2, DsRed Express, E2-Crimson, HcRed, ZsGreen, ZsYellow, mCherry, mStrawberry, mOrange, mBanana, mPlum, mRasberry, tdTomato, DsRed Monomer, and/or AcGFP, all of which are available from Clontech. [0093] In some embodiments, the modified cells described herein further comprise an exogenous transgene encoding a safety-switch system. Safety-switch systems (also referred to in the art as suicide gene systems) comprise exogenous transgenes encoding for one or more proteins that enable the elimination of a modified cell after the cell has been administered to a subject. Examples of safety-switch systems are known in the art. For example, safety-switch systems include genes encoding for proteins that convert non-toxic pro-drugs into toxic compounds such as the Herpes simplex thymidine kinase (Hsv-tk) and ganciclovir (GCV) system (Hsv-tk/GCV). Hsv-tk converts non-toxic GCV into a cytotoxic compound that leads to cellular apoptosis. As such, administration of GCV to a subject that has been treated with modified cells comprising a transgene encoding the Hsv-tk protein can selectively eliminate the modified cells while sparing endogenous immune effector cells. (See e.g., Bonini et al., Science, 1997, 276(5319):1719-1724; Ciceri et al., Blood, 2007, 109(11):1828-1836; Bondanza et al., Blood 2006, 107(5):1828-1836). [0094] Additional safety-switch systems include genes encoding for cell-surface markers, enabling elimination of modified cells by administration of a monoclonal antibody specific for the cell-surface marker via ADCC. In some embodiments, the cell-surface marker is CD20 and the modified cells can be eliminated by administration of an anti-CD20 monoclonal antibody such as Rituximab (See e.g., Introna et al., Hum Gene Ther, 2000, 11(4):611-620; Serafini et al., Hum Gene Ther, 2004, 14, 63-76; van Meerten et al., Gene Ther, 2006, 13, 789-797). Similar systems using EGF-R and Cetuximab or Panitumumab are described in International PCT Publication No. WO 2018006880. Additional safety-switch systems include transgenes encoding pro-apoptotic molecules comprising one or more binding sites for a chemical inducer of dimerization (CID), enabling elimination of modified cells by administration of a CID which induces oligomerization of the pro-apoptotic molecules and activation of the apoptosis pathway. In some embodiments, the pro-apoptotic molecule is Fas (also known as CD95) (Thomis et al., Blood, 2001, 97(5), 1249-1257). In some embodiments, the pro-apoptotic molecule is caspase-9 (Straathof et al., Blood, 2005, 105(11), 4247-4254). [0095] In some embodiments, the modified cells described herein further comprise an exogenous transgene encoding a chimeric switch receptor. Chimeric switch receptors are engineered cell-surface receptors comprising an extracellular domain from an endogenous cell- surface receptor and a heterologous intracellular signaling domain, such that ligand recognition by the extracellular domain results in activation of a different signaling cascade than that activated by the wild type form of the cell-surface receptor. In some embodiments, the chimeric switch receptor comprises the extracellular domain of an inhibitory cell-surface receptor fused to an intracellular domain that leads to the transmission of an activating signal rather than the inhibitory signal normally transduced by the inhibitory cell-surface receptor. In particular embodiments, extracellular domains derived from cell-surface receptors known to inhibit immune effector cell activation can be fused to activating intracellular domains. Engagement of the corresponding ligand will then activate signaling cascades that increase, rather than inhibit, the activation of the immune effector cell. For example, in some embodiments, the modified cells described herein comprise a transgene encoding a PD1-CD28 switch receptor, wherein the extracellular domain of PD1 is fused to the intracellular signaling domain of CD28 (See e.g., Liu et al., Cancer Res 76:6 (2016), 1578-1590 and Moon et al., Molecular Therapy 22 (2014), S201). In some embodiments, the modified cells described herein comprise a transgene encoding the extracellular domain of CD200R and the intracellular signaling domain of CD28 (See Oda et al., Blood 130:22 (2017), 2410-2419). [0096] In some embodiments, the modified cells described herein further comprise an antigen-specific receptor recognizing a protein target expressed by a target cell, such as a tumor cell or an antigen presenting cell (APC), referred to herein as “modified receptor-engineered cells”. In some embodiments, the antigen-specific receptor is an engineered antigen receptor. In some embodiments, the antigen-specific receptor is an exogenous TCR, i.e., a TCR that is not endogenously expressed by the modified cell but is a naturally occurring TCR derived from another cell. In some embodiments, the modified cell expresses an engineered TCR. [0097] The term “engineered antigen receptor” refers to a non-naturally occurring antigen-specific receptor such as a chimeric antigen receptor (CAR) or a recombinant T cell receptor (TCR). In some embodiments, the engineered antigen receptor is a CAR comprising an extracellular antigen binding domain fused via hinge and transmembrane domains to a cytoplasmic domain comprising an intracellular signaling domain. In some embodiments, the CAR extracellular domain binds to an antigen expressed by a target cell in an MHC- independent manner leading to activation and proliferation of the receptor-engineered cell. In some embodiments, the extracellular domain of a CAR recognizes a tag fused to an antibody or antigen-binding fragment thereof. In such embodiments, the antigen-specificity of the CAR is dependent on the antigen-specificity of the labeled antibody, such that a single CAR construct can be used to target multiple different antigens by substituting one antibody for another (See e.g., US Patent Nos.9,233,125 and 9,624,279; US Patent Application Publication Nos. 20150238631 and 20180104354). In some embodiments, the extracellular domain of a CAR may comprise an antigen binding fragment derived from an antibody. Antigen binding domains that are useful in the present disclosure include, for example, scFvs; antibodies; antigen binding regions of antibodies; variable regions of the heavy/light chains; and single chain antibodies. [0098] In some embodiments, the intracellular signaling domain of a CAR may be derived from the TCR complex zeta chain (such as CD3ξ signaling domains), FcγRIII, FcεRI, or the T-lymphocyte activation domain. In some embodiments, the intracellular signaling domain of a CAR further comprises a costimulatory domain, for example a 4-1BB, CD28, CD40, MyD88, or CD70 domain. In some embodiments, the intracellular signaling domain of a CAR comprises two costimulatory domains, for example any two of 4-1BB, CD28, CD40, MyD88, or CD70 domains. Exemplary CAR structures and intracellular signaling domains are known in the art (See e.g., WO 2009/091826; US 20130287748; WO 2015/142675; WO 2014/055657; and WO 2015/090229, incorporated herein by reference). [0099] CARs specific for a variety of tumor antigens are known in the art, for example CD171-specific CARs (Park et al., Mol Ther (2007) 15(4):825-833), EGFRvIII-specific CARs (Morgan et al., Hum Gene Ther (2012) 23(10):1043-1053), EGF-R-specific CARs (Kobold et al., J Natl Cancer Inst (2014) 107(1):364), carbonic anhydrase K-specific CARs (Lamers et al., Biochem Soc Trans (2016) 44(3):951-959), FR-α-specific CARs (Kershaw et al., Clin Cancer Res (2006) 12(20):6106-6015), HER2-specific CARs (Ahmed et al., J Clin Oncol (2015) 33(15)1688-1696;Nakazawa et al., Mol Ther (2011) 19(12):2133-2143; Ahmed et al., Mol Ther (2009) 17(10):1779-1787; Luo et al., Cell Res (2016) 26(7):850-853; Morgan et al., Mol Ther (2010) 18(4):843-851; Grada et al., Mol Ther Nucleic Acids (2013) 9(2):32), CEA- specific CARs (Katz et al., Clin Cancer Res (2015) 21(14):3149-3159), IL13Rα2-specific CARs (Brown et al., Clin Cacner Res (2015) 21(18):4062-4072), GD2-specific CARs (Louis et al., Blood (2011) 118(23):6050-6056; Caruana et al., Nat Med (2015) 21(5):524-529), ErbB2-specific CARs (Wilkie et al., J Clin Immunol (2012) 32(5):1059-1070), VEGF-R- specific CARs (Chinnasamy et al., Cancer Res (2016) 22(2):436-447), FAP-specific CARs (Wang et al., Cancer Immunol Res (2014) 2(2):154-166), MSLN-specific CARs (Moon et al, Clin Cancer Res (2011) 17(14):4719-30), NKG2D-specific CARs (VanSeggelen et al., Mol Ther (2015) 23(10):1600-1610), CD19-specific CARs (Axicabtagene ciloleucel (Yescarta^®) and Tisagenlecleucel (Kymriah^®). See also¸ Li et al., J Hematol and Oncol (2018) 11(22), reviewing clinical trials of tumor-specific CARs. [00100] In some embodiments, the engineered antigen receptor is an engineered TCR. Engineered TCRs comprise TCRα and/or TCRβ chains that have been isolated and cloned from T cell populations recognizing a particular target antigen. For example, TCRα and/or TCRβ genes (i.e., TRAC and TRBC) can be cloned from T cell populations isolated from individuals with particular malignancies or T cell populations that have been isolated from humanized mice immunized with specific tumor antigens or tumor cells. Engineered TCRs recognize antigen through the same mechanisms as their endogenous counterparts (e.g., by recognition of their cognate antigen presented in the context of major histocompatibility complex (MHC) proteins expressed on the surface of a target cell). This antigen engagement stimulates endogenous signal transduction pathways leading to activation and proliferation of the TCR-engineered cells. [00101] Engineered TCRs specific for tumor antigens are known in the art, for example WT1-specific TCRs (JTCR016, Juno Therapeutics; WT1-TCRc4, described in US Patent Application Publication No. 20160083449), MART-1 specific TCRs (including the DMF4T clone, described in Morgan et al., Science 314 (2006) 126-129); the DMF5T clone, described in Johnson et al., Blood 114 (2009) 535-546); and the ID3T clone, described in van den Berg et al., Mol. Ther. 23 (2015) 1541-1550), gp100-specific TCRs (Johnson et al., Blood 114 (2009) 535-546), CEA-specific TCRs (Parkhurst et al., Mol Ther. 19 (2011) 620-626), NY- ESO and LAGE-1 specific TCRs (1G4T clone, described in Robbins et al., J Clin Oncol 26 (2011) 917-924; Robbins et al., Clin Cancer Res 21 (2015) 1019-1027; and Rapoport et al., Nature Medicine 21 (2015) 914-921), and MAGE-A3-specific TCRs (Morgan et al., J Immunother 36 (2013) 133-151) and Linette et al., Blood 122 (2013) 227-242). (See also, Debets et al., Seminars in Immunology 23 (2016) 10-21). [00102] In some embodiments, the engineered antigen receptor is directed against a target antigen selected from a cluster of differentiation molecule, such as CD3, CD4, CD8, CD16, CD24, CD25, CD33, CD34, CD45, CD64, CD70, CD71, CD78, CD80 (also known as B7-1), CD86 (also known as B7-2), CD96, , CD116, CD117, CD123, CD133, and CD138, CD371 (also known as CLL1); a tumor-associated surface antigen, such as 5T4, alphafetoprotein (AFP), lectin-reactive AFP, β-human chorionic gonadotropin, BCMA (also known as CD269 and TNFRSF17, UniProt # Q02223), carcinoembryonic antigen (CEA), carbonic anhydrase 9 (CAIX or MN/CAIX), CD19, CD20, CD22, CD30, CD40, claudin-6 (CLDN6), claudin 18.2 (CLDN18.2), disialogangliosides such as GD2, delta-like ligand 3 (DLL3), ELF2M, ductal-epithelial mucin, EPH receptor A2 (EPHA2), ephrin B2, epithelial cell adhesion molecule (EpCAM), ErbB2 (HER2/neu), FCRL5 (UniProt# Q68SN8), FKBP11 (UniProt# Q9NYL4), fms related receptor tyrosine kinase 3 (FLT3), glioma-associated antigen, glycosphingolipids, gp36, GPRC5D (UniProt# Q9NZD1), guanylate cyclase 2C (GUCU2C), mut hsp70-2, intestinal carboxyl esterase, insulin growth factor (IGF)-I, IGF-II, IGF-I receptor, ITGA8 (UniProt# P53708), KAMP3, KRAS, LAGE-1a, MAGE, MAGE-A1, MAGE-A4, M- CSF, mesothelin, mucin 1 (MUC1), mucin 16 (MUC16), neutrophil elastase, NKG2D, Nkp30, NY-ESO-1, p53, PAP, Preferentially Expressed Antigen in Melanoma (PRAME), prostase, prostate-carcinoma tumor antigen-1 (PCTA-1), prostate specific antigen (PSA), prostate stem cell antigen (PSCA), prostate specific membrane antigen (PSMA), prostein, RAGE-1, ROR1, RU1 (SFMBT1), RU2 (AS), SLAMF7 (UniPro t# Q9NQ25), survivin, TAG-72, telomerase, telomerase reverse transcriptase (hTERT), and thyroglobulin; a major histocompatibility complex (MHC) molecule presenting a tumor-specific peptide epitope; tumor stromal antigens, such as the extra domain A (EDA) and extra domain B (EDB) of fibronectin; the A1 domain of tenascin-C (TnC A1) and fibroblast associated protein (FAP); cytokine receptors, such as epidermal growth factor receptor (EGFR), EGFR variant III (EGFRvIII), TFGβ-R or components thereof such as endoglin; a major histocompatibility complex (MHC) molecule; a virus-specific surface antigen such as an HIV-specific antigen (such as HIV gp120); an EBV- specific antigen, a CMV-specific antigen, a HPV-specific antigen, a Lassa virus-specific antigen, an Influenza virus-specific antigen as well as any derivate or variant of these surface antigens. [00103] In some embodiments, the transmembrane domain of the engineered antigen receptor (e.g., a CAR) comprises a sequence from or a sequence derived from one or more of the following nonlimiting transmembrane domains: 4-1BB/CD137, an Immunoglobulin protein, B7-H3, CD2, CD276 (B7-H3), CD28, CD3 delta (CD3δ), CD3 epsilon (CD3ε), CD3 gamma (CD3γ), CD3 zeta (CD3ζ), CD4, CD40, CD7, CD84, CD8 alpha, CD8 beta, a cytokine receptor, an Fc gamma receptor, IL-2R beta, IL-2R gamma, IL-7R alpha, inducible T cell costimulator (ICOS), an integrin, a ligand that specifically binds with CD83, lymphocyte function-associated antigen-1 (LFA-1; CD11a/CD18), a MHC class 1 molecule, OX-40, programmed death-1 (PD-1), TNF receptor proteins, or a fragment, truncation, or a combination thereof. A. Effector functions [00104] In some embodiments, the present disclosure provides a modified cell comprising reduced expression of at least two endogenous genes (e.g., DNMT3A and ACAT1) relative to the expression of the at least two endogenous genes in a non-modified cell, wherein the modified cell is a modified cell and wherein the reduced expression of the endogenous genes results in an increase in one or more immune cell effector functions. Herein, the term “effector function” refers to functions of an immune cell related to the generation, maintenance, and/or enhancement of an immune response against a target cell or target antigen. In some embodiments, the modified cells described herein demonstrate one or more of the following characteristics compared to an unmodified cell: increased infiltration or migration in to a tumor, increased proliferation and/or expansion, increased or prolonged cell viability, increased accumulation at the site of a tumor, increased resistance to inhibitory factors in the surrounding microenvironment such that the activation state of the cell is prolonged or increased, increased production of pro-inflammatory immune factors (e.g., pro-inflammatory cytokines, chemokines, and/or enzymes), increased cytotoxicity, and/or increased resistance to exhaustion. In some embodiments, the modified cells demonstrate increased resistance to exhaustion. In some embodiments, the modified cells demonstrate increased resistance to exhaustion and increased production of pro-inflammatory immune factors (e.g. pro- inflammatory cytokines, chemokines, and/or enzymes). [00105] In some embodiments, the modified cells described herein demonstrate increased infiltration into a tumor compared to an unmodified cell. In some embodiments, increased tumor infiltration by modified cells refers to an increase the number of modified cells infiltrating into a tumor during a given period of time compared to the number of unmodified cells that infiltrate into a tumor during the same period of time. In some embodiments, the modified cells demonstrate a 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.5, 3, 3.5, 4, 4.5, 5, 6, 7, 8, 9, 10, 15, 20,25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, or more fold increase in tumor filtration compared to an unmodified immune cell. Tumor infiltration can be measured by isolating one or more tumors from a subject and assessing the number of modified immune cells in the sample by flow cytometry, immunohistochemistry, and/or immunofluorescence. [00106] In some embodiments, the modified cells described herein demonstrate an increase in cell proliferation compared to an unmodified cell. In these embodiments, the result is an increase in the number of modified cells present compared to unmodified cells after a given period of time. For example, in some embodiments, modified cells demonstrate increased rates of proliferation compared to unmodified cells, wherein the modified cells divide at a more rapid rate than unmodified cells. In some embodiments, the modified cells demonstrate a 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.5, 3, 3.5, 4, 4.5, 5, 6, 7, 8, 9, 10, 15, 20,25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, or more fold increase in the rate of proliferation compared to an unmodified immune cell. In some embodiments, modified cells demonstrate prolonged periods of proliferation compared to unmodified cells, wherein the modified cells and unmodified cells divide at similar rates, but wherein the modified cells maintain the proliferative state for a longer period of time. In some embodiments, the modified cells maintain a proliferative state for 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.5, 3, 3.5, 4, 4.5, 5, 6, 7, 8, 9, 10, 15, 20,25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, or more times longer than an unmodified immune cell. [00107] In some embodiments, the modified cells described herein demonstrate increased or prolonged cell viability compared to an unmodified cell. In such embodiments, the result is an increase in the number of modified cells or present compared to unmodified cells after a given period of time. For example, in some embodiments, modified cells described herein remain viable and persist for 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.5, 3, 3.5, 4, 4.5, 5, 6, 7, 8, 9, 10, 15, 20,25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, or more times longer than an unmodified immune cell. [00108] In some embodiments, the modified cells described herein demonstrate increased resistance to inhibitory factors compared to an unmodified cell. Exemplary inhibitory factors include signaling by immune checkpoint molecules (e.g., PD1, PDL1, CTLA4, LAG3, IDO) and/or inhibitory cytokines (e.g., IL-10, TGFβ). [00109] In some embodiments, the modified T cells described herein demonstrate increased resistance to T cell exhaustion compared to an unmodified T cell. T cell exhaustion is a state of antigen-specific T cell dysfunction characterized by decreased effector function and leading to subsequent deletion of the antigen-specific T cells. In some embodiments, exhausted T cells lack the ability to proliferate in response to antigen, demonstrate decreased cytokine production, and/or demonstrate decreased cytotoxicity against target cells such as tumor cells. In some embodiments, exhausted T cells are identified by altered expression of cell surface markers and transcription factors, such as decreased cell surface expression of CD122 and CD127; increased expression of inhibitory cell surface markers such as PD1, LAG3, CD244, CD160, TIM3, and/or CTLA4; and/or increased expression of transcription factors such as Blimp1, NFAT, and/or BATF. In some embodiments, exhausted T cells demonstrate altered sensitivity cytokine signaling, such as increased sensitivity to TGFβ signaling and/or decreased sensitivity to IL-7 and IL-15 signaling. T cell exhaustion can be determined, for example, by co-culturing the T cells with a population of target cells and measuring T cell proliferation, cytokine production, and/or lysis of the target cells. In some embodiments, the modified cells described herein are co-cultured with a population of target cells (e.g., autologous tumor cells or cell lines that have been engineered to express a target tumor antigen) and effector cell proliferation, cytokine production, and/or target cell lysis is measured. These results are then compared to the results obtained from co-culture of target cells with a control population of immune cells (such as unmodified cells or immune effector cells that have a control modification). [00110] In some embodiments, resistance to T cell exhaustion is demonstrated by increased production of one or more cytokines (e.g., IFNγ, TNFα, or IL-2) from the modified cells compared to the cytokine production observed from the control population of immune cells. In some embodiments, a 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5, 6, 7, 8, 9, 10, 15, 20, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100 or more fold increase in cytokine production from the modified cells compared to the cytokine production from the control population of immune cells is indicative of an increased resistance to T cell exhaustion. In some embodiments, resistance to T cell exhaustion is demonstrated by increased proliferation of the modified cells compared to the proliferation observed from the control population of immune cells. In some embodiments, a 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5, 6, 7, 8, 9, 10, 15, 20, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100 or more fold increase in proliferation of the modified cells compared to the proliferation of the control population of immune cells is indicative of an increased resistance to T cell exhaustion. In some embodiments, resistance to T cell exhaustion is demonstrated by increased target cell lysis by the modified cells compared to the target cell lysis observed by the control population of immune cells. In some embodiments, a 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5, 6, 7, 8, 9, 10, 15, 20, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100 or more fold increase in target cell lysis by the modified cells compared to the target cell lysis by the control population of immune cells is indicative of an increased resistance to T cell exhaustion. [00111] In some embodiments, exhaustion of the modified cells compared to control populations of immune cells is measured during the in vitro or ex vivo manufacturing process. For example, in some embodiments, receptor engineered immune cells are modified according to the methods described herein and then expanded in one or more rounds of expansion to produce a population of modified receptor engineered immune cells. In such embodiments, the exhaustion of the modified receptor engineered immune cells can be determined immediately after harvest and prior to a first round of expansion, after the first round of expansion but prior to a second round of expansion, and/or after the first and the second round of expansion. In some embodiments, exhaustion of the modified cells compared to control populations of immune cells is measured at one or more time points after antigen stimulation or co-culture of the modified cells with target cells. For example, in some embodiments, the modified cells are produced according to the methods described herein and subjected to multiple, sequential cycles of in vitro cytotoxicity assays. Samples can then be taken from the assay at various time points to determine exhaustion of the modified cells in vitro over time. [00112] In some embodiments, the modified cells described herein demonstrate increased expression or production of one or more pro-inflammatory immune factors compared to an unmodified cell. Examples of pro-inflammatory immune factors include cytolytic factors, such as granzyme B, perforin, and granulysin; and pro-inflammatory cytokines such as interferons (IFNα, IFNβ, IFNγ), TNFα, IL-1β, IL-12, IL-2, IL-17, CXCL8, and/or IL-6. In some embodiments, the modified cells demonstrate a 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, or more fold increase in expression or production of one or more pro- inflammatory immune factors compared to an unmodified immune cell. [00113] In some embodiments, the modified cells described herein demonstrate increased cytotoxicity against a target cell compared to an unmodified cell. In some embodiments, the modified cells demonstrate a 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, or more fold increase in cytotoxicity against a target cell compared to an unmodified immune cell. [00114] Assays for measuring immune effector function are known in the art. For example, tumor infiltration can be measured by isolating tumors from a subject and determining the total number and/or phenotype of the lymphocytes present in the tumor by flow cytometry, immunohistochemistry, and/or immunofluorescence. Cell-surface receptor expression can be determined by flow cytometry, immunohistochemistry, immunofluorescence, Western blot, and/or qPCR. Cytokine and chemokine expression and production can be measured by flow cytometry, immunohistochemistry, immunofluorescence, Western blot, ELISA, and/or qPCR. Responsiveness or sensitivity to extracellular stimuli (e.g., cytokines, inhibitory ligands, or antigen) can be measured by assaying cellular proliferation and/or activation of downstream signaling pathways (e.g., phosphorylation of downstream signaling intermediates) in response to the stimuli. Cytotoxicity can be measured by target-cell lysis assays known in the art, including in vitro or ex vivo co-culture of the modified cells with target cells and in vivo murine tumor models, such as those described throughout the Examples. B. Regulation of endogenous genes [00115] In some embodiments, the modified cells described herein demonstrate a reduced expression or function of at least two endogenous target genes. In some embodiments, the modified cells described herein comprise reduced expression and/or function of the ACAT1 gene. Herein, ACAT1 refers to acetyl-CoA acetyltransferase 1. ACAT1 encodes a mitochondrially localized enzyme that catalyzes the reversible formation of acetoacetyl-CoA from two molecules of acetyl-CoA. Defects in this gene are associated with 3-ketothiolase deficiency, an inborn error of isoleucine catabolism characterized by urinary excretion of 2- methyl-3-hydroxybutyric acid, 2-methylacetoacetic acid, tiglylglycine, and butanone. Additional sequence information (nucleic acid and protein) for ACAT1 is provided in Table 1. Table 1: ACAT1 Gene and Protein Information Gene Name acetyl-CoA acetyltransferase 1 Aliases T2; MAT; ACAT; THIL

[00116] In some embodiments, the modified effector cells described herein comprise reduced expression and/or function of the DNMT3A gene. Herein, DNMT3A refers to DNA methyltransferase 3 alpha. This gene encodes a DNA methyltransferase that is thought to function in de novo methylation, rather than maintenance methylation. The protein localizes to the cytoplasm and nucleus and its expression is developmentally regulated. Additional sequence information (nucleic acid and protein) for DNMT3A is provided in Table 2. Table 2: DNMT3A Gene and Protein Information Gene Name DNA methyltransferase 3 alpha Aliases TBRS; HESJAS; DNMT3A2; M.HsaIIIA

[00117] In some embodiments, the modified cells described herein comprise reduced expression and/or function of ACAT1 and/or DNMT3A. In some embodiments, the modified cells comprise reduced expression and/or function of ACAT1. In some embodiments, the modified cells comprise reduced expression and/or function of DNMT3A. In some embodiments, the modified cells comprise reduced expression and/or function of both ACAT1 and DNMT3A. In some embodiments, the modified cells comprise reduced expression and/or function of MCJ. In some embodiments, the modified cells comprise reduced expression and/or function of MCJ and/or DNMT3A. In some embodiments, the modified cells comprise reduced expression and/or function of both MCJ and DNMT3A. [00118] In some embodiments, the modified cells exhibiting reduced expression and/or function of ACAT1 exhibit increased cytokine production compared to an unmodified cell. In some embodiments, the modified cells exhibiting reduced expression and/or function of DNMT3a exhibit increased persistence and/or decreased cell exhaustion compared to an unmodified cell. In some embodiments, the modified cells exhibiting reduced expression and/or function of both ACAT1 and DNMT3a exhibit increased cytokine production and increased persistence and/or decreased cell exhaustion compared to an unmodified cell. [00119] In some embodiments, the modified effector cells comprise reduced expression and/or function of ACAT1 and DNMT3A and one or more additional endogenous genes related to expression of MHC class I or MHC class II receptors or the TCR. In some embodiments, the modified effector cells comprise reduced expression and/or function of MCJ and DNMT3A and one or more additional endogenous genes related to expression of MHC class I or MHC class II receptors or the TCR. Genes related to the expression of MHC class I or MHC class II receptors include, but are not limited to, B2M, TAP1, TAPBP, CIITA, HLA-A, HLA-B, HLA-C, HLA-DR, HLA-DQ, HLA-DP. Genes related to the expression of the TCR include, but are not limited to, TRA, TRB, TRD, TRG, TRAC, TRBC1, TRBC2. [00120] In some embodiments, the modified effector cells comprise reduced expression and/or function of ACAT1 and DNMT3A and one or more additional endogenous immune checkpoint genes. In some embodiments, the modified effector cells comprise reduced expression and/or function of MCJ and DNMT3A and one or more additional endogenous one or more additional endogenous immune checkpoint genes. Immune checkpoint genes include, but are not limited to, PDCD1, CD274, CTLA4, IDO, LAG3, HAVCR2, TIGIT, A2AR, A2BR, B7-H3, BTLA, KIR, NOX2, VISTA, and SIGLEC7. [00121] In some embodiments, the modified effector cells comprise reduced expression and/or function of ACAT1 and DNMT3A and one or more additional endogenous genes selected from PDCD1, CD274, CTLA4, IDO, LAG3, HAVCR2, TIGIT, B2M, TRAC, CD52, MCJ, TRBC1, TRBC2, PDCD1, CD274, CTLA4, IDO, LAG3, HAVCR2, TIGIT, A2AR, A2BR, B7- H3, BTLA, KIR, NOX2, VISTA, SIGLEC7, B2M, TAP1, TAPBP, TRA, TRB, TRD, TRG, CD52, HLA-A, HLA-B, HLA-C, HLA-DR, HLA-DQ, HLA-DP, and CIITA. In some embodiments, the modified effector cells comprise reduced expression and/or function of ACAT1, DNMT3A B2M, TRAC, TRBC1, and TRBC2. In some embodiments, the modified effector cells comprise reduced expression and/or function of MCJ and DNMT3A and one or more additional endogenous genes selected from PDCD1, CD274, CTLA4, IDO, LAG3, HAVCR2, TIGIT, B2M, TRAC, CD52, ACAT1, TRBC1, TRBC2, PDCD1, CD274, CTLA4, IDO, LAG3, HAVCR2, TIGIT, A2AR, A2BR, B7-H3, BTLA, KIR, NOX2, VISTA, SIGLEC7, B2M, TAP1, TAPBP, TRA, TRB, TRD, TRG, CD52, HLA-A, HLA-B, HLA-C, HLA-DR, HLA-DQ, HLA-DP, and CIITA. In some embodiments, the modified effector cells comprise reduced expression and/or function of MCJ, DNMT3A, B2M, TRAC, TRBC1, and TRBC2. Table 3: Exemplary Endogenous Genes Gene Human Human Murine Murine S_ymbol^Aliases UniProt NCBI UniProt NCBI : 1 8 6 6

Gene Human Human Murine Murine S_ymbol^Aliases UniProt NCBI UniProt NCBI Rf Gn ID Rf Gn ID:

. g g y [00122] Herein, the term “gene-regulating system” refers to a protein, nucleic acid, or combination thereof that is capable of modifying an endogenous target DNA sequence when introduced into a cell, thereby regulating the expression or function of the encoded gene product. Numerous gene editing systems suitable for use in the methods of the present disclosure are known in the art including, but not limited to, shRNAs, siRNAs, zinc-finger nuclease systems, TALEN systems, and RNA-guided nuclease (RGN) systems. [00123] As used herein, “regulate,” when used in reference to the effect of a gene- regulating system on an endogenous target gene encompasses any change in the sequence of the endogenous target gene, any change in the epigenetic state of the endogenous target gene, and/or any change in the expression or function of the protein encoded by the endogenous target gene. [00124] In some embodiments, the gene-regulating system may mediate a change in the sequence of the endogenous target gene, for example, by introducing one or more mutations into the endogenous target sequence, such as by insertion or deletion of one or more nucleic acids in the endogenous target sequence. Exemplary mechanisms that can mediate alterations of the endogenous target sequence include, but are not limited to, non-homologous end joining (NHEJ) (e.g., classical or alternative), microhomology-mediated end joining (MMEJ), homology-directed repair (e.g., endogenous donor template mediated), SDSA (synthesis dependent strand annealing), single strand annealing or single strand invasion. [00125] In some embodiments, the gene-regulating system may mediate a change in the epigenetic state of the endogenous target sequence. For example, in some embodiments, the gene-regulating system may mediate covalent modifications of the endogenous target gene DNA (e.g., cytosine methylation and hydroxymethylation) or of associated histone proteins (e.g. lysine acetylation, lysine and arginine methylation, serine and threonine phosphorylation, and lysine ubiquitination and sumoylation). [00126] In some embodiments, the gene-regulating system may mediate a change in the expression of the protein encoded by the endogenous target gene. In such embodiments, the gene-regulating system may regulate the expression of the encoded protein by modifications of the endogenous target DNA sequence, or by acting on the mRNA product encoded by the DNA sequence. In some embodiments, the gene-regulating system may result in the expression of a modified endogenous protein. In such embodiments, the modifications to the endogenous DNA sequence mediated by the gene-regulating system result in the expression of an endogenous protein demonstrating a reduced function as compared to the corresponding endogenous protein in an unmodified cell. In such embodiments, the expression level of the modified endogenous protein may be increased, decreased or may be the same, or substantially similar to, the expression level of the corresponding endogenous protein in an unmodified immune cell. A. Nucleic acid-based gene-regulating systems [00127] As used herein, a nucleic acid-based gene-regulating system is a system comprising one or more nucleic acid molecules that is capable of regulating the expression of an endogenous target gene without the requirement for an exogenous protein. In some embodiments, the nucleic acid-based gene-regulating system comprises an RNA interference molecule or antisense RNA molecule that is complementary to a target nucleic acid sequence. [00128] An “antisense RNA molecule” refers to an RNA molecule, regardless of length, that is complementary to an mRNA transcript. Antisense RNA molecules refer to single stranded RNA molecules that can be introduced to a cell, tissue, or subject and result in decreased expression of an endogenous target gene product through mechanisms that do not rely on endogenous gene silencing pathways, but rather rely on RNaseH-mediated degradation of the target mRNA transcript. In some embodiments, an antisense nucleic acid comprises a modified backbone, for example, phosphorothioate, phosphorodithioate, or others known in the art, or may comprise non-natural internucleoside linkages. In some embodiments, an antisense nucleic acid can comprise locked nucleic acids (LNA). [00129] “RNA interference molecule” as used herein refers to an RNA polynucleotide that mediates the decreased the expression of an endogenous target gene product by degradation of a target mRNA through endogenous gene silencing pathways (e.g., Dicer and RNA-induced silencing complex (RISC)). Exemplary RNA interference agents include micro RNAs (also referred to herein as “miRNAs”), short hair-pin RNAs (shRNAs), small interfering RNAs (siRNAs), RNA aptamers, and morpholinos. [00130] In some embodiments, the nucleic acid-based gene-regulating system comprises one or more miRNAs. miRNAs refers to naturally occurring, small non-coding RNA molecules of about 21-25 nucleotides in length. miRNAs are at least partially complementary to one or more target mRNA molecules. miRNAs can downregulate (e.g., decrease) expression of an endogenous target gene product through translational repression, cleavage of the mRNA, and/or deadenylation. [00131] In some embodiments, the nucleic acid-based gene-regulating system comprises one or more shRNAs. shRNAs are single stranded RNA molecules of about 50-70 nucleotides in length that form stem-loop structures and result in degradation of complementary mRNA sequences. shRNAs can be cloned in plasmids or in non-replicating recombinant viral vectors to be introduced intracellularly and result in the integration of the shRNA-encoding sequence into the genome. As such, an shRNA can provide stable and consistent repression of endogenous target gene translation and expression. [00132] In some embodiments, nucleic acid-based gene-regulating system comprises one or more siRNAs. siRNAs refer to double stranded RNA molecules typically about 21-23 nucleotides in length. The siRNA associates with a multi protein complex called the RNA- induced silencing complex (RISC), during which the “passenger” sense strand is enzymatically cleaved. The antisense “guide” strand contained in the activated RISC then guides the RISC to the corresponding mRNA because of sequence homology and the same nuclease cuts the target mRNA, resulting in specific gene silencing. Optimally, an siRNA is 18, 19, 20, 21, 22, 23 or 24 nucleotides in length and has a 2 base overhang at its 3’ end. siRNAs can be introduced to an individual cell and/or culture system and result in the degradation of target mRNA sequences. siRNAs and shRNAs are further described in Fire et al., Nature, 391:19, 1998 and US Patent Nos.7,732,417; 8,202,846; and 8,383,599. [00133] In some embodiments, the nucleic acid-based gene-regulating system comprises one or more morpholinos. “Morpholino” as used herein refers to a modified nucleic acid oligomer wherein standard nucleic acid bases are bound to morpholine rings and are linked through phosphorodiamidate linkages. Similar to siRNA and shRNA, morpholinos bind to complementary mRNA sequences. However, morpholinos function through steric-inhibition of mRNA translation and alteration of mRNA splicing rather than targeting complementary mRNA sequences for degradation. [00134] In some embodiments, the nucleic acid-based gene-regulating system comprises an siRNA molecule or an shRNA molecule selected from those known in the art, such as the siRNA and shRNA constructs available from commercial suppliers such as Sigma Aldrich, Dharmacon, ThermoFisher, and the like. [00135] In some embodiments, the nucleic acid-based gene-regulating system comprises a nucleic acid molecule (e.g., an siRNA, an shRNA, an RNA apatamer, or a morpholino) that binds to a target RNA sequence that is at least 90% identical to an RNA sequence encoded by the ACAT1 and/or DNMT3A gene. In some embodiments, the nucleic acid-based gene- regulating system comprises a nucleic acid molecule (e.g., an siRNA, an shRNA, an RNA apatamer, or a morpholino) that binds to a target RNA sequence that is at least 95%, 96%, 97%, 98%, 99%, or 100% identical to an RNA sequence encoded by a DNA sequence the ACAT1 and/or DNMT3A gene. B. Protein-based gene-regulating systems [00136] In some embodiments, a protein-based gene-regulating system is a system comprising one or more proteins capable of regulating the expression of an endogenous target gene in a sequence specific manner without the requirement for a nucleic acid guide molecule. In some embodiments, the protein-based gene-regulating system comprises a protein comprising one or more zinc-finger binding domains and an enzymatic domain. In some embodiments, the protein-based gene-regulating system comprises a protein comprising a Transcription activator-like effector nuclease (TALEN) domain and an enzymatic domain. Such embodiments are referred to herein as “TALENs”. Zinc finger systems [00137] Zinc finger-based systems comprise a fusion protein comprising two protein domains: a zinc finger DNA binding domain and an enzymatic domain. A “zinc finger DNA binding domain”, “zinc finger protein”, or “ZFP” is a protein, or a domain within a larger protein, that binds DNA in a sequence-specific manner through one or more zinc fingers, which are regions of amino acid sequence within the binding domain whose structure is stabilized through coordination of a zinc ion. The zinc finger domain, by binding to a target DNA sequence, directs the activity of the enzymatic domain to the vicinity of the sequence and, hence, induces modification of the endogenous target gene in the vicinity of the target sequence. A zinc finger domain can be engineered to bind to virtually any desired sequence. Accordingly, after identifying a target genetic locus containing a target DNA sequence at which cleavage or recombination is desired (e.g., a target locus in a target gene referenced in Tables 2 or 3), one or more zinc finger binding domains can be engineered to bind to one or more target DNA sequences in the target genetic locus. Expression of a fusion protein comprising a zinc finger binding domain and an enzymatic domain in a cell, effects modification in the target genetic locus. [00138] In some embodiments, a zinc finger binding domain comprises one or more zinc fingers. Miller et al. (1985) EMBO J. 4:1609-1614; Rhodes (1993) Scientific American Febuary:56-65; U.S. Pat. No. 6,453,242. Typically, a single zinc finger domain is about 30 amino acids in length. An individual zinc finger binds to a three-nucleotide (i.e., triplet) sequence (or a four-nucleotide sequence which can overlap, by one nucleotide, with the four- nucleotide binding site of an adjacent zinc finger). Therefore the length of a sequence to which a zinc finger binding domain is engineered to bind (e.g., a target sequence) will determine the number of zinc fingers in an engineered zinc finger binding domain. For example, for ZFPs in which the finger motifs do not bind to overlapping subsites, a six-nucleotide target sequence is bound by a two-finger binding domain; a nine-nucleotide target sequence is bound by a three- finger binding domain, etc. Binding sites for individual zinc fingers (i.e., subsites) in a target site need not be contiguous, but can be separated by one or several nucleotides, depending on the length and nature of the amino acids sequences between the zinc fingers (i.e., the inter- finger linkers) in a multi-finger binding domain. In some embodiments, the DNA-binding domains of individual ZFNs comprise between three and six individual zinc finger repeats and can each recognize between 9 and 18 basepairs. [00139] Zinc finger binding domains can be engineered to bind to a sequence of choice. See, for example, Beerli et al. (2002) Nature Biotechnol.20:135-141; Pabo et al. (2001) Ann. Rev. Biochem. 70:313-340; Isalan et al. (2001) Nature Biotechnol. 19:656-660; Segal et al. (2001) Curr. Opin. Biotechnol.12:632-637; Choo et al. (2000) Curr. Opin. Struct. Biol.10:411- 416. An engineered zinc finger binding domain can have a novel binding specificity, compared to a naturally-occurring zinc finger protein. Engineering methods include, but are not limited to, rational design and various types of selection. [00140] Selection of a target DNA sequence for binding by a zinc finger domain can be accomplished, for example, according to the methods disclosed in U.S. Pat. No. 6,453,242. It will be clear to those skilled in the art that simple visual inspection of a nucleotide sequence can also be used for selection of a target DNA sequence. Accordingly, any means for target DNA sequence selection can be used in the methods described herein. A target site generally has a length of at least 9 nucleotides and, accordingly, is bound by a zinc finger binding domain comprising at least three zinc fingers. However binding of, for example, a 4-finger binding domain to a 12-nucleotide target site, a 5-finger binding domain to a 15-nucleotide target site or a 6-finger binding domain to an 18-nucleotide target site, is also possible. As will be apparent, binding of larger binding domains (e.g., 7-, 8-, 9-finger and more) to longer target sites is also possible. [00141] In some embodiments, the zinc finger binding domains bind to a target DNA sequence that is at least 90% identical to a target DNA sequence of the ACAT1 and/or DNMT3A gene. In some embodiments, the zinc finger binding domains bind to a target DNA sequence that is at least 95%, 96%, 97%, 98%, 99%, or 100% identical to a target DNA sequence of the ACAT1 and/or DNMT3A gene. [00142] The enzymatic domain portion of the zinc finger fusion proteins can be obtained from any endo- or exonuclease. Exemplary endonucleases from which an enzymatic domain can be derived include, but are not limited to, restriction endonucleases and homing endonucleases. See, for example, 2002-2003 Catalogue, New England Biolabs, Beverly, Mass.; and Belfort et al. (1997) Nucleic Acids Res.25:3379-3388. Additional enzymes which cleave DNA are known (e.g., 51 Nuclease; mung bean nuclease; pancreatic DNaseI; micrococcal nuclease; yeast HO endonuclease; see also Linn et al. (eds.) Nucleases, Cold Spring Harbor Laboratory Press, 1993). One or more of these enzymes (or functional fragments thereof) can be used as a source of cleavage domains. [00143] Exemplary restriction endonucleases (restriction enzymes) suitable for use as an enzymatic domain of the ZFPs described herein are present in many species and are capable of sequence-specific binding to DNA (at a recognition site), and cleaving DNA at or near the site of binding. Certain restriction enzymes (e.g., Type IIS) cleave DNA at sites removed from the recognition site and have separable binding and cleavage domains. For example, the Type IIS enzyme FokI catalyzes double-stranded cleavage of DNA, at 9 nucleotides from its recognition site on one strand and 13 nucleotides from its recognition site on the other. See, for example, U.S. Pat. Nos. 5,356,802; 5,436,150 and 5,487,994; as well as Li et al. (1992) Proc. Natl. Acad. Sci. USA 89:4275-4279; Li et al. (1993) Proc. Natl. Acad. Sci. USA 90:2764- 2768; Kim et al. (1994a) Proc. Natl. Acad. Sci. USA 91:883-887; Kim et al. (1994b) J. Biol. Chem. 269:31,978-31,982. Thus, in one embodiment, fusion proteins comprise the enzymatic domain from at least one Type IIS restriction enzyme and one or more zinc finger binding domains. [00144] An exemplary Type IIS restriction enzyme, whose cleavage domain is separable from the binding domain, is FokI. This particular enzyme is active as a dimer. Bitinaite et al. (1998) Proc. Natl. Acad. Sci. USA 95: 10,570-10,575. Thus, for targeted double-stranded DNA cleavage using zinc finger-FokI fusions, two fusion proteins, each comprising a FokI enzymatic domain, can be used to reconstitute a catalytically active cleavage domain. Alternatively, a single polypeptide molecule containing a zinc finger binding domain and two FokI enzymatic domains can also be used. Exemplary ZFPs comprising FokI enzymatic domains are described in US Patent No.9,782,437. TALEN systems [00145] TALEN-based systems comprise a protein comprising a TAL effector DNA binding domain and an enzymatic domain. They are made by fusing a TAL effector DNA- binding domain to a DNA cleavage domain (a nuclease which cuts DNA strands). The FokI restriction enzyme described above is an exemplary enzymatic domain suitable for use in TALEN-based gene-regulating systems. [00146] TAL effectors are proteins that are secreted by Xanthomonas bacteria via their type III secretion system when they infect plants. The DNA binding domain contains a repeated, highly conserved, 33–34 amino acid sequence with divergent 12th and 13th amino acids. These two positions, referred to as the Repeat Variable Diresidue (RVD), are highly variable and strongly correlated with specific nucleotide recognition. Therefore, the TAL effector domains can be engineered to bind specific target DNA sequences by selecting a combination of repeat segments containing the appropriate RVDs. The nucleic acid specificity for RVD combinations is as follows: HD targets cytosine, NI targets adenenine, NG targets thymine, and NN targets guanine (though, in some embodiments, NN can also bind adenenine with lower specificity). [00147] In some embodiments, the TAL effector domains bind to a target DNA sequence that is at least 90% identical to a target DNA sequence of the ACAT1 and/or DNMT3A gene. In some embodiments, the TAL effector domains bind to a target DNA sequence that is at least 95%, 96%, 97%, 98%, 99%, or 100% identical to a target DNA sequence of the ACAT1 and/or DNMT3A gene. [00148] Methods and compositions for assembling the TAL-effector repeats are known in the art. See e.g., Cermak et al, Nucleic Acids Research, 39:12, 2011, e82. Plasmids for constructions of the TAL-effector repeats are commercially available from Addgene. C. Combination nucleic acid/protein-based gene-regulating systems [00149] Combination gene-regulating systems comprise an effector protein and a nucleic acid guide molecule. Herein, an “effector protein” refers to a polypeptide that binds to a nucleic acid guide molecule to form a complex which is targeted to a target nucleic acid sequence by the nucleic acid guide molecule and modifies the target nucleic acid sequence (e.g., cleavage, mutation, methylation, transition, or transversion of a target nucleic acid sequence). [00150] An effector protein comprises a portion that binds the nucleic acid guide and an activity portion. In some embodiments, an effector protein comprises an activity portion that exhibits site-directed enzymatic activity (e.g., DNA methylation, DNA or RNA cleavage, histone acetylation, histone methylation, etc.), wherein the site of enzymatic activity is determined by the guide nucleic acid. In some cases, an effector protein comprises an activity portion that has enzymatic activity that modifies the endogenous target nucleic acid sequence (e.g., nuclease activity, methyltransferase activity, demethylase activity, DNA repair activity, DNA damage activity, deamination activity, dismutase activity, alkylation activity, depurination activity, oxidation activity, pyrimidine dimer forming activity, integrase activity, transposase activity, recombinase activity, polymerase activity, ligase activity, helicase activity, photolyase activity or glycosylase activity). In other cases, an effector protein comprises an activity portion that has enzymatic activity that modifies a polypeptide (e.g., a histone) associated with the endogenous target nucleic acid sequence (e.g., methyltransferase activity, demethylase activity, acetyltransferase activity, deacetylase activity, kinase activity, phosphatase activity, ubiquitin ligase activity, deubiquitinating activity, adenylation activity, deadenylation activity, SUMOylating activity, deSUMOylating activity, ribosylation activity, deribosylation activity, myristoylation activity or demyristoylation activity). In some embodiments, an effector protein comprises an activity portion that modulates transcription of a target DNA sequence (e.g., to increase or decrease transcription). In some embodiments, an effector protein comprises an activity portion that modulates expression or translation of a target RNA sequence (e.g., to increase or decrease transcription). [00151] In some embodiments, the effector protein is a fusion protein, comprising a first domain with a first effector function (e.g., DNA-binding activity, cleavage activity) and a second domain with a second effector function (e.g., deaminase activity, transposase activity, recombinase activity, or polymerase activity). In some embodiments, the effector protein is a base editor fusion protein. [00152] The nucleic acid guide comprises two portions: a first portion that is complementary to, and capable of binding with, an endogenous target nucleic sequence (referred to herein as a “nucleic acid-binding segment”), and a second portion that is capable of interacting with the effector protein (referred to herein as a “protein-binding segment”). In some embodiments, the nucleic acid-binding segment and protein-binding segment of a nucleic acid guide are comprised within a single polynucleotide molecule. In some embodiments, the nucleic acid-binding segment and protein-binding segment of a nucleic acid guide are each comprised within separate polynucleotide molecules, such that the nucleic acid guide comprises two polynucleotide molecules that associate with each other to form the functional guide. [00153] The nucleic acid guide mediates the target specificity of the combined protein/nucleic acid gene-regulating systems by specifically hybridizing with a target nucleic acid sequence. In some embodiments, the target nucleic acid sequence is an RNA sequence, such as an RNA sequence comprised within an mRNA transcript of a target gene. In some embodiments, the target nucleic acid sequence is a DNA sequence comprised within the DNA sequence of a target gene. Reference herein to a target gene encompasses the full-length DNA sequence for that particular gene which comprises a plurality of target genetic loci (i.e., portions of a particular target gene sequence (e.g., an exon or an intron)). Within each target genetic loci are shorter stretches of DNA sequences referred to herein as “target DNA sequences” that can be modified by the gene-regulating systems described herein. Further, each target genetic loci comprises a “target modification site,” which refers to the precise location of the modification induced by the gene-regulating system (e.g., the location of an insertion, a deletion, or mutation, the location of a DNA break, or the location of an epigenetic modification). [00154] In some embodiments, the combined protein/nucleic acid gene-regulating systems comprise site-directed modifying polypeptides derived from Argonaute (Ago) proteins (e.g., T. thermophiles Ago or TtAgo). In such embodiments, the effector protein is a T. thermophiles Ago DNA endonuclease and the nucleic acid guide is a guide DNA (gDNA) (See, Swarts et al., Nature 507 (2014), 258-261). In some embodiments, the present disclosure provides a polynucleotide encoding a gDNA. In some embodiments, a gDNA-encoding nucleic acid is comprised in an expression vector, e.g., a recombinant expression vector. In some embodiments, the present disclosure provides a polynucleotide encoding a TtAgo site-directed modifying polypeptide or variant thereof. In some embodiments, the polynucleotide encoding a TtAgo site-directed modifying polypeptide is comprised in an expression vector, e.g., a recombinant expression vector. Effector proteins [00155] In some embodiments, the effector protein is an RNA-guided nuclease. The term RNA-guided nuclease (RGN) refers to a polypeptide that binds to a particular target nucleotide sequence in a sequence-specific manner and is directed to the target nucleotide sequence by a guide RNA molecule that is complexed with the polypeptide and hybridizes with the target sequence. Although an RNA-guided nuclease can be capable of cleaving the target sequence upon binding, the term RNA-guided nuclease also encompasses nuclease-dead RNA- guided nucleases that are capable of binding to, but not cleaving, a target sequence. Cleavage of a target sequence by an RNA-guided nuclease can result in a single- or double -stranded break. RNA-guided nucleases only capable of cleaving a single strand of a double- stranded nucleic acid molecule are referred to herein as nickases. [00156] RNA-guided nucleases (RGNs) allow for the targeted manipulation of specific site(s) within a genome and are useful in the context of gene targeting for therapeutic and research applications. In a variety of organisms, including mammals, RNA-guided nucleases have been used for genome engineering by stimulating non-homologous end joining and homologous recombination, for example. RGNs are useful for creating single- or double - stranded breaks in polynucleotides, modifying polynucleotides, detecting a particular site within a polynucleotide, or modifying the expression of a particular gene. [00157] In specific embodiments, the RNA-guided nucleases are directed to the target sequence by a guide RNA (gRNA) as part of a Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) RNA-guided nuclease system. The RGNs are considered “RNA-guided” because guide RNAs form a complex with the RNA-guided nucleases to direct the RNA-guided nuclease to bind to a target sequence and in some embodiments, introduce a single-stranded or double-stranded break at the target sequence. After the target sequence has been cleaved, the break can be repaired such that the DNA sequence of the target sequence is modified during the repair process. Thus, provided herein are methods for using the RNA- guided nucleases to modify a target sequence in the DNA of host cells. For example, RNA- guided nucleases can be used to modify a target sequence at a genomic locus of eukaryotic cells or prokaryotic cells. [00158] In some embodiments, the RGN is catalytically inactivated into a nickase and introduces a single-stranded break at the target sequence. In some embodiments, the RGN introduces a double-stranded break at the target sequence. In some embodiments, after the target sequence has been cleaved, the break can be repaired, by non-homologous end joining and homologous recombination, for example, such that the DNA sequence of the target sequence is modified during the repair process. [00159] RNA-guided nucleases that lack nuclease activity can be used to deliver a fused polypeptide, polynucleotide, or small molecule payload to a particular genomic location. In some of these embodiments, the RGN polypeptide or guide RNA can be fused to a detectable label to allow for detection of a particular sequence. As a non-limiting example, a nuclease- dead RGN can be fused to a detectable label (e.g., fluorescent protein) and targeted to a particular sequence associated with a disease to allow for detection of the disease-associated sequence. [00160] Alternatively, nuclease-dead RGNs can be targeted to particular genomic locations to alter the expression of a desired sequence. In some embodiments, the binding of a nuclease-dead RNA-guided nuclease to a target sequence results in the repression of expression of the target sequence or a gene under transcriptional control by the target sequence by interfering with the binding of RNA polymerase or transcription factors within the targeted genomic region. In other embodiments, the RGN (e.g., a nuclease-dead RGN) or its complexed guide RNA further comprises an expression modulator that, upon binding to a target sequence, serves to either repress or activate the expression of the target sequence or a gene under transcriptional control by the target sequence. In some of these embodiments, the expression modulator modulates the expression of the target sequence or regulated gene through epigenetic mechanisms. [00161] In some embodiments, the RGN is a CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)/Cas (CRISPR Associated) nuclease. In some embodiments, the RGN is a CRISPR/Cas Class 2 RGN. Class 2 CRISPR/Cas systems are divided into three types: Type II, Type V, and Type VI systems. In some embodiments, the CRISPR/Cas RGN is a Cas9 protein, a Cas12 proteins (e.g., Cas12a (also known as Cpf1), Cas12b (also known as C2c1), Cas12c (also known as C2c3), Cas12d (also known as CasY), Cas12e (also known as CasX)), or a Cas13 protein (e.g., Cas13a (also known as C2c2), Cas13b, and Cas13c). (See, Pyzocha et al., ACS Chemical Biology, 13(2), 347-356). [00162] Cas molecules of a variety of species can be used in the methods and compositions described herein, including Cas molecules derived from S. pyogenes, S. aureus, N. meningitidis, S. thermophiles, Acidovorax avenae, Actinobacillus pleuropneumoniae, Actinobacillus succinogenes, Actinobacillus suis, Actinomyces sp., Cycliphilusdenitrificans, Aminomonas paucivorans, Bacillus cereus, Bacillus smithii, Bacillus thuringiensis, Bacteroides sp., Blastopirellula marina, Bradyrhizobium sp., Brevibacillus laterospoxus, Campylobacter coli, Campylobacter jejuni, Campylobacter lari, Candidatus puniceispirillum, Clostridium cellulolyticum, Clostridium perfringens, Corynebacterium accolens, Corynebacterium diphtheria, Corynebacterium matruchotii, Dinoroseobacter shibae, Eubacterium dolichum, Gammaproteobacterium, Gluconacetobacter diazotrophicus, Haemophilus parainfluenzae, Haemophilus sputomm, Helicobacter canadensis, Helicobacter cinaedi, Helicobacter mustelae, Ilyobacter polytropus, Kingella kingae, Lactobacillus crispatus, Listeria ivanovii, Listeria monocytogenes, Listeriaceae bacterium, Methylocystis sp., Methylosinus trichosporium, Mobiluncus mulieris, Neisseria bacilliformis, Neisseria cinerea, Neisseria flavescens, Neisseria lactamica, Neisseria meningitidis, Neisseria sp., Neisseria wadsworthii, Nitrosomonas sp., Parvibaculum lavamentivorans, Pasteurella multocida, Phascolarctobacterium succinatutens, Ralstonia syzygii, Rhodopseudomonas palustris, Rhodovulum sp., Simonsiella muelleri, Sphingomonas sp., Sporolactobacillus vineae, Staphylococcus aureus, Staphylococcus lugdunensis, Streptococcus sp., Subdoligranulum sp., Tistrella mobilis, Treponema sp., or Verminephrobacter eiseniae. [00163] In some embodiments, the Cas endonuclease is selected from the group consisting of C2C1, C2C3, Cpf1 (also referred to as Cas12a), Cas12b, Cas12c, Cas12d, Cas12e, Cas13a, Cas13b, Cas13c, Cas13d, Casl, CaslB, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9 (also known as Csnl and Csx12), Cas10, Csyl, Csy2, Csy3, Csel, Cse2, Cscl, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmrl, Cmr3, Cmr4, Cmr5, Cmr6, Csbl, Csb2, Csb3, Csxl7, Csxl4, Csx10, Csx16, CsaX, Csx3, Csxl, Csxl5, Csfl, Csf2, Csf3, and Csf4. [00164] In some embodiments, the Cas polypeptide is fused to heterologous proteins that recruit DNA-damage signaling proteins, exonucleases, or phosphatases to further increase the likelihood or the rate of repair of the target sequence by one repair mechanism or another. In some embodiments, a WT Cas polypeptide is co-expressed with a nucleic acid repair template to facilitate the incorporation of an exogenous nucleic acid sequence by homology- directed repair. [00165] In some embodiments, different Cas proteins (i.e., Cas9 proteins from various species) may be advantageous to use in the various provided methods in order to capitalize on various enzymatic characteristics of the different Cas proteins (e.g., for different PAM sequence preferences; for increased or decreased enzymatic activity; for an increased or decreased level of cellular toxicity; to change the balance between NHEJ, homology-directed repair, single strand breaks, double strand breaks, etc.). [00166] In some embodiments, the RGN can be any one of the RGNs disclosed in any one of International Patent Publication Nos. WO 2019/236566, WO 2021/030344, WO 2020/139783, WO 2021/217002, WO 2021/138247, and WO 2021/231437, WO2023139557, and PCT International Appl. No. PCT/IB2023/058160 filed August 12, 2023, each of which is incorporated by reference in its entirety. [00167] In some embodiments, the RGNs are engineered to alter one or more properties of the RGN polypeptide. For example, in some embodiments, the RGN polypeptide comprises altered enzymatic properties, e.g., altered nuclease activity, (as compared with a naturally occurring or other reference RGN molecules) or altered helicase activity. In some embodiments, an engineered RGN polypeptide can have an alteration that alters its size, e.g., a deletion of amino acid sequence that reduces its size without significant effect on another property of the RGN polypeptide. In some embodiments, an engineered RGN polypeptide comprises an alteration that affects PAM recognition. For example, an engineered RGN polypeptide can be altered to recognize a PAM sequence other than the PAM sequence recognized by the corresponding wild-type RGN protein. [00168] In an embodiment, a mutant RGN polypeptide comprises a cleavage property that differs from a naturally occurring Cas polypeptide. In some embodiments, the RGN is enzymatically dead (e.g., deactivated or dead). In such embodiments, the RGN polypeptide does not comprise any intrinsic enzymatic activity and is unable to mediate target nucleic acid cleavage. In such embodiments, the RGN may be fused with a heterologous protein that is capable of modifying the target nucleic acid in a non-cleavage based manner. For example, in some embodiments, a deactivated RGN protein is fused to transcription activator or transcription repressor domains (e.g., the Kruppel associated box (KRAB or SKD); the Mad mSIN3 interaction domain (SID or SID4X); the ERF repressor domain (ERD); the MAX- interacting protein 1 (MXI1); methyl-CpG binding protein 2 (MECP2); etc.). In some such cases, the deactivated RGN fusion protein is targeted by the gRNA to a specific location (i.e., sequence) in the target nucleic acid and exerts locus-specific regulation such as blocking RNA polymerase binding to a promoter (which selectively inhibits transcription activator function), and/or modifying the local chromatin status (e.g., when a fusion sequence is used that modifies the target DNA or modifies a polypeptide associated with the target DNA). In some cases, the changes are transient (e.g., transcription repression or activation). In some cases, the changes are inheritable (e.g., when epigenetic modifications are made to the target DNA or to proteins associated with the target DNA, e.g., nucleosomal histones). [00169] In some embodiments, the RGN nickase mutant. Nickase mutants comprise only one catalytically active domain. The RGN nickase mutants retain DNA binding based on gRNA specificity, but are capable of cutting only one strand of DNA resulting in a single- strand break (e.g. a “nick”). [00170] In some of embodiments, the nickase comprises an inactive RuvC domain. RuvC domains have an RNase H fold structure (see, e.g., Nishimasu et al. (2014) Cell 156(5):935-949, which is incorporated by reference in its entirety). RuvC domains of RGNs are often split RuvC domains, comprising two or more non-adjacent regions within the linear amino acid sequence. A non-limiting example of a mutation within a RuvC domain that inactivates its nuclease activity is the D10A mutation that mutates the first aspartic acid residue in the split RuvC nuclease domain. [00171] In some embodiments, the nickase RGN of the fusion protein comprises a mutation (e.g., a H840A mutation, wherein amino acid numbering is based on the Streptococcus pyogenes Cas9 sequence which renders the RGN capable of cleaving only the target strand (the strand which comprises the PAM) of a nucleic acid duplex. In some of these embodiments, the nickase comprises an inactive HNH nuclease domain. The HNH nuclease domain of RGNs have a ββα-metal fold (see, e.g., Nishimasu et al.2014). The HNH nuclease domain of the Streptococcus pyogenes Cas9, for example, comprises amino acid residues 775- 908. A non-limiting example of a mutation within a HNH domain that inactivates its nuclease activity is the H840A mutation that mutates the first histidine of the HNH nuclease domain. In some embodiments, the H840A mutant nickases are useful for prime editing with a reverse transcriptase. [00172] In some embodiments, the RGN polypeptides described herein can be engineered to alter the PAM/PFS specificity of the RGN polypeptide. In some embodiments, a mutant RGN polypeptide has a PAM/PFS specificity that is different from the PAM/PFS specificity of the parental RGN polypeptide. For example, a naturally occurring RGN protein can be modified to alter the PAM/PFS sequence that the mutant RGN polypeptide recognizes to decrease off target sites, improve specificity, or eliminate a PAM/PFS recognition requirement. In some embodiments, a RGN protein can be modified to increase the length of the PAM/PFS recognition sequence. In some embodiments, the length of the PAM recognition sequence is at least 4, 5, 6, 7, 8, 9, 10 or 15 amino acids in length. RGN polypeptides that recognize different PAM/PFS sequences and/or have reduced off-target activity can be generated using directed evolution. Exemplary methods and systems that can be used for directed evolution of RGN polypeptides are described, e.g., in Esvelt et al. Nature 2011, 472(7344): 499-503. [00173] In other embodiments, the nuclease-dead RGNs or a RGN with only nickase activity can be targeted to particular genomic locations to modify the sequence of a target polynucleotide through fusion to a base-editing polypeptide, for example a deaminase polypeptide or active variant or fragment thereof that deaminates a nucleotide base, resulting in conversion from one nucleotide base to another. The base-editing polypeptide can be fused to the RGN at its N-terminal or C-terminal end. Additionally, the base-editing polypeptide may be fused to the RGN via a peptide linker. A non-limiting example of a deaminase polypeptide that is useful for such compositions and methods include cytidine deaminase or the adenosine deaminase base editor described in Gaudelli et al. (2017) Nature 551:464-471, U.S. Publ. Nos. 2017/0121693 and 2018/0073012, and International Publ. No. WO/2018/027078, each of which is herein incorporated by reference in its entirety. [00174] A “base-editing polypeptide” or a “base editor,” as referred to herein, is a fusion protein comprising an RGN domain that is capable of binding to a DNA target molecule and a deaminase domain that is capable of deaminating target cytidine or adenine residues in a DNA target molecule. In some embodiments, the RGN domain is a deactivated RGN or comprises a nickase activity. In some embodiments, the deaminase is a cytidine deaminase or an adenine deaminase. In some embodiments, the deaminase is an apolipoprotein B mRNA-editing complex 1 (APOBEC1) deaminase domain. In some embodiments, the base editor is present as part of a fusion protein or protein complex. In some embodiments, the fusion protein comprises a RGN and a base editor. In some embodiments, the fusion protein comprises an RGN and an evolved base editor. In some embodiments, the base editor is in a fusion protein described in any of WO2020139783A2, WO202215969A1, WO2022056254A2, WO2022204093A1, and PCT/IB2023/061192 filed November 6, 2023, each of which is incorporated herein by reference in its entirety. [00175] The term "deaminase" or "deaminase domain," as used herein, refers to a protein or enzyme that catalyzes a deamination reaction. In some embodiments, the deaminase or deaminase domain is a cytidine deaminase, catalyzing the hydrolytic deamination of cytidine or deoxycytidine to uridine or deoxyuridine, respectively. In some embodiments, the deaminase or deaminase domain is a cytidine deaminase domain, catalyzing the hydrolytic deamination of cytosine to uracil. In some embodiments, the deaminase or deaminase domain is an adenine deaminase, catalyzing the hydrolytic deamination of adenine or deoxyadenine to inosine or deoxyinosine, respectively. In some embodiments, the deaminase or deaminase domain is an adenine deaminase domain, catalyzing the hydrolytic deamination of adenine to inosine. In some embodiments, the deaminase or deaminase domain is a naturally-occurring deaminase from an organism, such as a human, chimpanzee, gorilla, monkey, cow, dog, rat, or mouse. In some embodiments, the deaminase or deaminase domain is a variant of a naturally- occurring deaminase from an organism, that does not occur in nature. For example, in some embodiments, the deaminase or deaminase domain is at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75% at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5%) identical to a naturally- occurring deaminase from an organism. [00176] In other embodiments, the nuclease-dead RGNs or a RGN with only nickase activity can be targeted to particular genomic locations to modify the sequence of a target polynucleotide through fusion to a prime editing polypeptide. In some embodiments, the prime- editing polypeptide is a reverse transcriptase. [00177] Prime editing is a versatile and precise genome editing method that directly writes new genetic information into a specified DNA site using a nucleic acid programmable DNA binding protein working in association with a polymerase (described in, e.g., US 11,447,770B1; WO2021072328; WO2021226558; WO2020156575; WO2021042047; US11193123; each incorporated by reference in its entirety herein). The prime editing system uses an RGN that is a nickase, and the system is programmed with a prime editing (PE) guide RNA (“PEgRNA”). The PEgRNA is a guide RNA that both specifies the target sequence and provides the template for polymerization of the replacement strand containing the edit by way of an extension engineered onto the guide RNA (e.g., at the 5' or 3' end, or at an internal portion of the guide RNA). The RGN nickase/prime editing polypeptide fusion is guided to the target sequence by the PEgRNA and nicks the target strand upstream of sequence to be edited and upstream of the PAM, creating a 3′ flap on the target strand. The pegRNA includes a primer binding site (PBS) that is complementary to the 3′ flap of the target strand. In some embodiments, a PBS is at least about 5, 6, 7, 8, 9, 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 certain embodiments, the pegRNA comprises a PBS that is at least 5 (e.g., at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 28, 19, or 20) nucleotides in length. In some embodiments, the pegRNA may comprise a PBS that is at least 8 nucleotides in length. Hybridrization of the PBS and 3′ flap of the target strand allows polymerization of the replacement strand containing the edit using the extension of the PEgRNA as template. The extension of the PEgRNA can be formed from RNA or DNA. In the case of an RNA extension, the polymerase of the prime editor can be an RNA-dependent DNA polymerase (such as a reverse transcriptase). In the case of a DNA extension, the polymerase of the prime editor may be a DNA-dependent DNA polymerase. [00178] The replacement strand containing the desired edit (e.g., a single nucleobase substitution) shares the same sequence as the target strand of the target sequence to be edited (with the exception that it includes the desired edit). Through DNA repair and/or replication machinery, the target strand of the target sequence is replaced by the newly synthesized replacement strand containing the desired edit. In some cases, prime editing may be thought of as a “search-and-replace” genome editing technology since the prime editors not only search and locate the desired target sequence to be edited, but at the same time, encode a replacement strand containing a desired edit which is installed in place of the corresponding target strand of the target sequence. Thus, in some embodiments, a guide RNA useful in the presently disclosed compositions and methods comprises an extension comprising an edit template for prime editing. In some embodiments, a prime editing polypeptide that can be fused to an RGN includes a DNA polymerase (e.g., an RNA-dependent DNA polymerase). In certain embodiments, the DNA polymerase is a reverse transcriptase. In certain embodiments, the RGN is a nickase. gRNAs [00179] The present disclosure includes guide RNAs (gRNAs) that direct an effector protein to a specific target nucleic acid sequence. A gRNA comprises a nucleic acid-targeting segment and protein-binding segment. The protein-binding segment of a gRNA comprises, in part, two complementary stretches of nucleotides that hybridize to one another to form a double stranded RNA duplex (dsRNA duplex), which facilitates binding to the effector protein. The nucleic acid-binding segment (or “nucleic acid-binding sequence”) of a gRNA comprises a nucleotide sequence that is complementary to and capable of binding to a specific target nucleic acid sequence. The nucleic acid-targeting segment of a gRNA can be modified (e.g., by genetic engineering) to hybridize to any desired sequence within a target nucleic acid sequence. [00180] The protein-binding segment of the gRNA interacts with an effector protein and the interaction of the gRNA molecule and effector protein results in binding to the target nucleic acid sequence and produces one or more modifications within or around the target nucleic acid sequence. The precise location of the target modification site is determined by both (i) base-pairing complementarity between the gRNA and the target nucleic acid sequence; and (ii) the location of a short motif, referred to as the protospacer adjacent motif (PAM), in the target DNA sequence (referred to as a protospacer flanking sequence (PFS) in target RNA sequences). The PAM/PFS sequence is required for Cas binding to the target nucleic acid sequence. A variety of PAM/PFS sequences are known in the art and are suitable for use with a particular Cas endonuclease (e.g., a Cas9 endonuclease) (See e.g., Nat Methods. 2013 Nov; 10(11): 1116–1121 and Sci Rep.2014; 4: 5405). In some embodiments, the PAM sequence is located within between 60 and 5 base pairs of the target modification site in a target DNA sequence. In some embodiments, the PAM sequence is located within 50 base pairs of the target modification site in a target DNA sequence. In some embodiments, the PAM sequence is located within 10 base pairs of the target modification site in a target DNA sequence. The DNA sequences that can be targeted by this method are limited only by the relative distance of the PAM sequence to the target modification site and the presence of a unique 20 base pair sequence to mediate sequence-specific, gRNA-mediated Cas binding. In some embodiments, the PFS sequence is located at the 3’ end of the target RNA sequence. In some embodiments, the target modification site is located at the 5’ terminus of the target locus. In some embodiments, the target modification site is located at the 3’ end of the target locus. In some embodiments, the target modification site is located within an intron or an exon of the target locus. [00181] In some embodiments, the present disclosure includes a polynucleotide encoding a gRNA. In some embodiments, a gRNA-encoding nucleic acid is comprised in an expression vector, e.g., a recombinant expression vector. In some embodiments, the present disclosure includes a polynucleotide encoding an effector protein. In some embodiments, the polynucleotide encoding an effector protein is comprised in an expression vector, e.g., a recombinant expression vector. [00182] In some embodiments, the protein-binding segment of a gRNA is comprised in one RNA molecule and the DNA-binding segment is comprised in another separate RNA molecule. Such embodiments are referred to herein as “double-molecule gRNAs” or “two- molecule gRNA” or “dual gRNAs.” In some embodiments, the gRNA is a single RNA molecule and is referred to herein as a “single-guide RNA” or an “sgRNA.” The term “guide RNA” or “gRNA” is inclusive, referring both to two-molecule guide RNAs and sgRNAs. In some embodiments, a gRNA comprises a single RNA molecule (sgRNA). [00183] The specificity of a gRNA for a target loci is mediated by the sequence of the nucleic acid-binding segment, which comprises about 20 nucleotides that are complementary to a target nucleic acid sequence within the target locus. In some embodiments, the corresponding target nucleic acid sequence is approximately 20 nucleotides in length. In some embodiments, the nucleic acid-binding segments of the gRNA sequences of the present disclosure are at least 90% complementary to a target nucleic acid sequence within a target locus. In some embodiments, the nucleic acid-binding segments of the gRNA sequences of the present disclosure are at least 95%, 96%, 97%, 98%, or 99% complementary to a target nucleic acid sequence within a target locus. In some embodiments, the nucleic acid-binding segments of the gRNA sequences of the present disclosure are 100% complementary to a target nucleic acid sequence within a target locus. [00184] In some embodiments, the target nucleic acid sequence is an RNA target sequence. In some embodiments, the target nucleic acid sequence is a DNA target sequence. [00185] In some embodiments, the nucleic acid-binding segments of the gRNA sequences described herein are designed to minimize off-target binding using algorithms known in the art (e.g., Cas-OFF finder) to identify target sequences that are unique to a particular target locus or target gene. [00186] In some embodiments, the gRNAs described herein can comprise one or more modified nucleosides or nucleotides which introduce stability toward nucleases. In such embodiments, these modified gRNAs may elicit a reduced innate immune as compared to a non-modified gRNA. The term “innate immune response” includes a cellular response to exogenous nucleic acids, including single stranded nucleic acids, generally of viral or bacterial origin, which involves the induction of cytokine expression and release, particularly the interferons, and cell death. [00187] In some embodiments, a software tool can be used to optimize the choice of gRNA within a user’s target sequence, e.g., to minimize total off-target activity across the genome. Off target activity may be other than cleavage. For example, for each possible gRNA choice using S. pyogenes Cas9, software tools can identify all potential off-target sequences (preceding either NAG or NGG PAMs) across the genome that contain up to a certain number (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) of mismatched base-pairs. The cleavage efficiency at each off-target sequence can be predicted, e.g., using an experimentally-derived weighting scheme. Each possible gRNA can then be ranked according to its total predicted off-target cleavage; the top-ranked gRNAs represent those that are likely to have the greatest on-target and the least off-target cleavage. Other functions, e.g., automated reagent design for gRNA vector construction, primer design for the on-target Surveyor assay, and primer design for high- throughput detection and quantification of off-target cleavage via next-generation sequencing, can also be included in the tool. IV. Methods of producing modified cells [00188] In some embodiments, the present disclosure provides methods for producing modified cells. In some embodiments, the methods comprise introducing a gene-regulating system into a population of immune effector cells wherein the gene-regulating system is capable of reducing expression and/or function of at least two endogenous target genes (e.g., ACAT1 and DNMT3A). In some embodiments, the immune effector cells are further modified to express a TCR or a CAR. In some embodiments, the immune effector cells are T-cells. [00189] The components of the gene-regulating systems described herein, e.g., a nucleic acid-, protein-, or nucleic acid/protein-based system can be introduced into target cells in a variety of forms using a variety of delivery methods and formulations. In some embodiments, a polynucleotide encoding one or more components of the system is delivered by a recombinant vector (e.g., a viral vector or plasmid). In some embodiments, where the system comprises more than a single component, a vector may comprise a plurality of polynucleotides, each encoding a component of the system. In some embodiments, where the system comprises more than a single component, a plurality of vectors may be used, wherein each vector comprises a polynucleotide encoding a particular component of the system. In some embodiments, a vector may also comprise a sequence encoding a signal peptide (e.g., for nuclear localization, nucleolar localization, mitochondrial localization), fused to the polynucleotide encoding the one or more components of the system. For example, a vector may comprise a nuclear localization sequence (e.g., from SV40) fused to the polynucleotide encoding the one or more components of the system. In some embodiments, the introduction of the gene-regulating system to the cell occurs in vitro. In some embodiments, the introduction of the gene-regulating system to the cell occurs in vivo. In some embodiments, the introduction of the gene-regulating system to the cell occurs ex vivo. [00190] In some embodiments, the recombinant vector comprising a polynucleotide encoding one or more components of a gene-regulating system described herein is a viral vector. Suitable viral vectors include, but are not limited to, viral vectors based on vaccinia virus; poliovirus; adenovirus (see, e.g., Li et al., Invest Opthalmol Vis Sci 35:25432549, 1994; Borras et al., Gene Ther 6:515 524, 1999; Li and Davidson, PNAS 92:7700 7704, 1995; Sakamoto et al., H Gene Ther 5:10881097, 1999; WO 94/12649, WO 93/03769; WO 93/19191 ; WO 94/28938; WO 95/11984 and WO 95/00655); adeno-associated virus (see, e.g., U.S. Patent No.7,078,387; Ali et al., Hum Gene Ther 9:8186, 1998, Flannery et al„ PNAS 94:6916 6921 , 1997; Bennett et al., Invest Opthalmol Vis Sci 38:28572863, 1997; Jomary et al., Gene Ther 4:683690, 1997, Rolling et al., Hum Gene Ther 10:641648, 1999; Ali et al., Hum Mol Genet 5:591594, 1996; Srivastava in WO 93/09239, Samulski et al., J. Vir. (1989) 63:3822- 3828; Mendelson et al„ Virol. (1988) 166:154-165; and Flotte et al., PNAS (1993) 90:10613- 10617); SV40; herpes simplex virus; human immunodeficiency virus (see, e.g., Miyoshi et al., PNAS 94:1031923, 1997; Takahashi et al., J Virol 73:78127816, 1999); a retroviral vector (e.g., Murine Leukemia Virus, spleen necrosis virus, and vectors derived from retroviruses such as Rous Sarcoma Virus, Harvey Sarcoma Virus, avian leukosis virus, a lentivirus, human immunodeficiency virus, myeloproliferative sarcoma virus, and mammary tumor virus); and the like. [00191] In some embodiments, the recombinant vector comprising a polynucleotide encoding one or more components of a gene-regulating system described herein is a plasmid. Numerous suitable plasmid expression vectors are known to those of skill in the art, and many are commercially available. The following vectors are provided by way of example; for eukaryotic host cells: pXT1, pSG5 (Stratagene), pSVK3, pBPV, pMSG, and pSVLSV40 (Pharmacia). However, any other plasmid vector may be used so long as it is compatible with the host cell. Depending on the cell type and gene-regulating system utilized, any of a number of suitable transcription and translation control elements, including constitutive and inducible promoters, transcription enhancer elements, transcription terminators, etc. may be used in the expression vector (see e.g., Bitter et al. (1987) Methods in Enzymology, 153:516-544). [00192] In some embodiments, a polynucleotide sequence encoding one or more components of a gene-regulating system described herein is operably linked to a control element, e.g., a transcriptional control element, such as a promoter. The transcriptional control element may be functional in either a eukaryotic cell (e.g., a mammalian cell) or a prokaryotic cell (e.g., bacterial or archaeal cell). In some embodiments, a polynucleotide sequence encoding one or more components of a gene-regulating system described herein is operably linked to multiple control elements that allow expression of the polynucleotide in both prokaryotic and eukaryotic cells. Depending on the cell type and gene-regulating system utilized, any of a number of suitable transcription and translation control elements, including constitutive and inducible promoters, transcription enhancer elements, transcription terminators, etc. may be used in the expression vector (see e.g., Bitter et al. (1987) Methods in Enzymology, 153:516-544). [00193] Non-limiting examples of suitable eukaryotic promoters (promoters functional in a eukaryotic cell) include those from cytomegalovirus (CMV) immediate early, herpes simplex virus (HSV) thymidine kinase, early and late SV40, long terminal repeats (LTRs) from retrovirus, and mouse metallothionein-l. Selection of the appropriate vector and promoter is well within the level of ordinary skill in the art. The expression vector may also contain a ribosome binding site for translation initiation and a transcription terminator. The expression vector may also include appropriate sequences for amplifying expression. The expression vector may also include nucleotide sequences encoding protein tags (e.g., 6xHis tag, hemagglutinin tag, green fluorescent protein, etc.) that are fused to the effector protein, thus resulting in a chimeric polypeptide. [00194] In some embodiments, a polynucleotide sequence encoding one or more components of a gene-regulating system described herein is operably linked to an inducible promoter. In some embodiments, a polynucleotide sequence encoding one or more components of a gene-regulating system described herein is operably linked to a constitutive promoter. [00195] Methods of introducing polynucleotides and recombinant vectors into a host cell are known in the art, and any known method can be used to introduce components of a gene- regulating system into a cell. Suitable methods include e.g., viral or bacteriophage infection, transfection, conjugation, protoplast fusion, lipofection, electroporation, calcium phosphate precipitation, polyethyleneimine (PEI)-mediated transfection, DEAE-dextran mediated transfection, liposome-mediated transfection, particle gun technology, calcium phosphate precipitation, direct micro injection, nanoparticle-mediated nucleic acid delivery (see, e.g., Panyam et al., Adv Drug Deliv Rev.2012 Sep 13. pii: S0169-409X(12)00283-9), microfluidics delivery methods (See e.g., International PCT Publication No. WO 2013/059343 herein incorporated by reference), lipid nanoparticles (LNPs) (See e.g. WO2022173531, WO2022150485, and WO2022150485 the contents of each of which are incorporate by reference) and the like. In some embodiments, delivery via electroporation comprises mixing the cells with the components of a gene-regulating system in a cartridge, chamber, or cuvette and applying one or more electrical impulses of defined duration and amplitude. In some embodiments, cells are mixed with components of a gene-regulating system in a vessel connected to a device (e.g., a pump) which feeds the mixture into a cartridge, chamber, or cuvette wherein one or more electrical impulses of defined duration and amplitude are applied, after which the cells are delivered to a second vessel. [00196] In some embodiments, one or more components of a gene-regulating system, or polynucleotide sequence encoding one or more components of a gene-regulating system described herein are introduced to a cell in a non-viral delivery vehicle, such as a transposon, a nanoparticle (e.g., a lipid nanoparticle), a liposome, an exosome, an attenuated bacterium, or a virus-like particle. In some embodiments, the vehicle is an attenuated bacterium (e.g., naturally or artificially engineered to be invasive but attenuated to prevent pathogenesis including Listeria monocytogenes, certain Salmonella strains, Bifidobacterium longum, and modified Escherichia coli), bacteria having nutritional and tissue-specific tropism to target specific cells, and bacteria having modified surface proteins to alter target cell specificity. In some embodiments, the vehicle is a genetically modified bacteriophage (e.g., engineered phages having large packaging capacity, less immunogenicity, containing mammalian plasmid maintenance sequences and having incorporated targeting ligands). In some embodiments, the vehicle is a mammalian virus-like particle. For example, modified viral particles can be generated (e.g., by purification of the “empty” particles followed by ex vivo assembly of the virus with the desired cargo). The vehicle can also be engineered to incorporate targeting ligands to alter target tissue specificity. In some embodiments, the vehicle is a biological liposome. For example, the biological liposome is a phospholipid-based particle derived from human cells (e.g., erythrocyte ghosts, which are red blood cells broken down into spherical structures derived from the subject and wherein tissue targeting can be achieved by attachment of various tissue or cell-specific ligands), secretory exosomes, or subjectiderived membrane- bound nanovescicles (30 -100 nm) of endocytic origin (e.g., can be produced from various cell types and can therefore be taken up by cells without the need for targeting ligands). [00197] In some embodiments, the methods of producing modified cells described herein comprise obtaining a population of cells from a sample. In some embodiments, a sample comprises a tissue sample, a fluid sample, a cell sample, a protein sample, or a DNA or RNA sample. In some embodiments, a tissue sample may be derived from any tissue type including, but not limited to skin, hair (including roots), bone marrow, bone, muscle, salivary gland, esophagus, stomach, small intestine (e.g., tissue from the duodenum, jejunum, or ileum), large intestine, liver, gallbladder, pancreas, lung, kidney, bladder, uterus, ovary, vagina, placenta, testes, thyroid, adrenal gland, cardiac tissue, thymus, spleen, lymph node, spinal cord, brain, eye, ear, tongue, cartilage, white adipose tissue, or brown adipose tissue. In some embodiments, a tissue sample may be derived from a cancerous, pre-cancerous, or non-cancerous tumor. In some embodiments, a fluid sample comprises buccal swabs, blood, plasma, oral mucous, vaginal mucous, peripheral blood, cord blood, saliva, semen, urine, ascites fluid, pleural fluid, spinal fluid, pulmonary lavage, tears, sweat, semen, seminal fluid, seminal plasma, prostatic fluid, pre-ejaculatory fluid (Cowper’s fluid), excreta, cerebrospinal fluid, lymph, cell culture media comprising one or more populations of cells, buffered solutions comprising one or more populations of cells, and the like. [00198] In some embodiments, the sample is processed to enrich or isolate a particular cell type, such as an immune effector cell, from the remainder of the sample. In certain embodiments, the sample is a peripheral blood sample which is then subject to leukapheresis to separate the red blood cells and platelets and to isolate lymphocytes. In some embodiments, the sample is a leukopak from which immune effector cells can be isolated or enriched. In some embodiments, the sample is a tumor sample that is further processed to isolate lymphocytes present in the tumor (i.e., to isolate tumor infiltrating lymphocytes). [00199] In some embodiments, the isolated cells are expanded in culture to produce an expanded population of cells. One or more activating or growth factors may be added to the culture system during the expansion process. For example, in some embodiments, one or more cytokines (such as IL-2, IL-15, and/or IL-7) can be added to the culture system to enhance or promote cell proliferation and expansion. In some embodiments, one or more activating antibodies, such as an anti-CD3 antibody, may be added to the culture system to enhance or promote cell proliferation and expansion. In some embodiments, the immune effector cells may be co-cultured with feeder cells during the expansion process. In some embodiments, the methods provided herein comprise one or more expansion phases. For example, in some embodiments, the immune effector cells can be expanded after isolation from a sample, allowed to rest, and then expanded again. In some embodiments, the cells can be expanded in one set of expansion conditions followed by a second round of expansion in a second, different, set of expansion conditions. Methods for ex vivo expansion of immune cells are known in the art, for example, as described in US Patent Application Publication Nos. 20180282694 and 20170152478 and US Patent Nos.8,383,099 and 8,034,334. [00200] At any point during the culture and expansion process, the gene-regulating systems described herein can be introduced to the cells to produce a population of modified cells. In some embodiments, the gene-regulating system is introduced to the population of cells immediately after enrichment from a sample. In some embodiments, the gene-regulating system is introduced to the population of cells before, during, or after the one or more expansion process. [00201] In some embodiments, the modified cells produced by the methods described herein may be used immediately. Alternatively, the cells may be frozen at liquid nitrogen temperatures and stored for long periods of time, being thawed and capable of being reused. In such cases, the cells will usually be frozen in 10% dimethylsulfoxide (DMSO), 50% serum, 40% buffered medium, or some other such solution as is commonly used in the art to preserve cells at such freezing temperatures, and thawed in a manner as commonly known in the art for thawing frozen cultured cells. In some embodiments, the modified cells may be cultured in vitro under various culture conditions. The cells may be expanded in culture, i.e. grown under conditions that promote their proliferation. Culture medium may be liquid or semi-solid, e.g. containing agar, methylcellulose, etc. The cell population may be suspended in an appropriate nutrient medium, such as AIM-V, Iscove’s modified DMEM or RPMI 1640, PRIMEXV, XVIVO15 etc, normally supplemented with fetal calf serum (about 5-10%), human AB serum, L-glutamine or GlutaMAX, a thiol, particularly 2-mercaptoethanol, and may contain antibiotics, e.g. penicillin and streptomycin. The culture may contain growth factors to which the regulatory T cells are responsive. Growth factors, as defined herein, are molecules capable of promoting survival, growth and/or differentiation of cells, either in culture or in the intact tissue, through specific effects on a transmembrane receptor. Growth factors include polypeptides and non- polypeptide factors. V. Compositions and Kits [00202] The term “composition” as used herein refers to a formulation of a modified cell described herein that is capable of being administered or delivered to a subject or cell. Typically, formulations include all physiologically acceptable compositions including derivatives and/or prodrugs, solvates, stereoisomers, racemates, or tautomers thereof with any physiologically acceptable carriers, diluents, and/or excipients. A “therapeutic composition” or “pharmaceutical composition” (used interchangeably herein) is a composition of a modified cell capable of being administered to a subject for the treatment of a particular disease or disorder. [00203] The phrase “pharmaceutically acceptable” is employed herein to refer to those compounds, materials, compositions, and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio. [00204] As used herein “pharmaceutically acceptable carrier, diluent or excipient” includes without limitation any adjuvant, carrier, excipient, glidant, sweetening agent, diluent, preservative, dye/colorant, flavor enhancer, surfactant, wetting agent, dispersing agent, suspending agent, stabilizer, isotonic agent, solvent, surfactant, and/or emulsifier which has been approved by the United States Food and Drug Administration as being acceptable for use in humans and/or domestic animals. Exemplary pharmaceutically acceptable carriers include, but are not limited to, to sugars, such as lactose, glucose and sucrose; starches, such as corn starch and potato starch; cellulose, and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; tragacanth; malt; gelatin; talc; cocoa butter, waxes, animal and vegetable fats, paraffins, silicones, bentonites, silicic acid, zinc oxide; oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; glycols, such as propylene glycol; polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol; esters, such as ethyl oleate and ethyl laurate; agar; buffering agents, such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen- free water; isotonic saline; Ringer’s solution; ethyl alcohol; phosphate buffer solutions; and any other compatible substances employed in pharmaceutical formulations. Except insofar as any conventional media and/or agent is incompatible with the agents of the present disclosure, its use in therapeutic compositions is contemplated. Supplementary active ingredients also can be incorporated into the compositions. [00205] “Pharmaceutically acceptable salt” includes both acid and base addition salts. Pharmaceutically-acceptable salts include the acid addition salts (formed with the free amino groups of the protein) and which are formed with inorganic acids such as, for example, hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid and the like, and organic acids such as, but not limited to, acetic acid, 2,2-dichloroacetic acid, adipic acid, alginic acid, ascorbic acid, aspartic acid, benzenesulfonic acid, benzoic acid, 4- acetamidobenzoic acid, camphoric acid, camphor-10-sulfonic acid, capric acid, caproic acid, caprylic acid, carbonic acid, cinnamic acid, citric acid, cyclamic acid, dodecylsulfuric acid, ethane-1,2-disulfonic acid, ethanesulfonic acid, 2-hydroxyethanesulfonic acid, formic acid, fumaric acid, galactaric acid, gentisic acid, glucoheptonic acid, gluconic acid, glucuronic acid, glutamic acid, glutaric acid, 2-oxo-glutaric acid, glycerophosphoric acid, glycolic acid, hippuric acid, isobutyric acid, lactic acid, lactobionic acid, lauric acid, maleic acid, malic acid, malonic acid, mandelic acid, methanesulfonic acid, mucic acid, naphthalene-1,5-disulfonic acid, naphthalene-2-sulfonic acid, 1-hydroxy-2-naphthoic acid, nicotinic acid, oleic acid, orotic acid, oxalic acid, palmitic acid, pamoic acid, propionic acid, pyroglutamic acid, pyruvic acid, salicylic acid, 4-aminosalicylic acid, sebacic acid, stearic acid, succinic acid, tartaric acid, thiocyanic acid, ptoluenesulfonic acid, trifluoroacetic acid, undecylenic acid, and the like. Salts formed with the free carboxyl groups can also be derived from inorganic bases such as, for example, sodium, potassium, lithium, ammonium, calcium, magnesium, iron, zinc, copper, manganese, aluminum salts and the like. Salts derived from organic bases include, but are not limited to, salts of primary, secondary, and tertiary amines, substituted amines including naturally occurring substituted amines, cyclic amines and basic ion exchange resins, such as ammonia, isopropylamine, trimethylamine, diethylamine, triethylamine, tripropylamine, diethanolamine, ethanolamine, deanol, 2-dimethylaminoethanol, 2-diethylaminoethanol, dicyclohexylamine, lysine, arginine, histidine, caffeine, procaine, hydrabamine, choline, betaine, benethamine, benzathine, ethylenediamine, glucosamine, methylglucamine, theobromine, triethanolamine, tromethamine, purines, piperazine, piperidine, N- ethylpiperidine, polyamine resins and the like. Particularly preferred organic bases are isopropylamine, diethylamine, ethanolamine, trimethylamine, dicyclohexylamine, choline and caffeine. [00206] Wetting agents, emulsifiers and lubricants, such as sodium lauryl sulfate and magnesium stearate, as well as coloring agents, release agents, coating agents, sweetening, flavoring and perfuming agents, preservatives and antioxidants can also be present in the compositions. [00207] Examples of pharmaceutically-acceptable antioxidants include: (1) water soluble antioxidants, such as ascorbic acid, cysteine hydrochloride, sodium bisulfate, sodium metabisulfite, sodium sulfite and the like; (2) oil-soluble antioxidants, such as ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propyl gallate, alpha-tocopherol, and the like; and (3) metal chelating agents, such as citric acid, ethylenediamine tetraacetic acid (EDTA), sorbitol, tartaric acid, phosphoric acid, and the like. [00208] Further guidance regarding formulations that are suitable for various types of administration can be found in Remington’s Pharmaceutical Sciences, Mace Publishing Company, Philadelphia, Pa., 17th ed. (1985). For a brief review of methods for drug delivery, see, Langer, Science 249:1527-1533 (1990). [00209] In some embodiments, the present disclosure provides kits for carrying out a method described herein. In some embodiments, a kit for modifying a cell can include: (a) one or more nucleic acid molecules (or polynucleotides encoding the same) capable of reducing the expression or modifying the function of a gene product encoded by at least two endogenous target genes (e.g., ACAT1 and DNMT3A); (b) one or more proteins (or polynucleotides encoding the same) capable of reducing the expression or modifying the function of a gene product encoded by at least two endogenous target genes (e.g., ACAT1 and DNMT3A); (c) one or more gRNAs (or polynucleotides encoding the same) capable of binding to a target DNA sequence in an endogenous gene; (d) one or more effector proteins (or polynucleotides encoding the same) capable of interacting with a gRNA and modifying a target DNA sequence in an endogenous gene; (e) one or more guide DNAs (gDNAs) (or polynucleotides encoding the same) capable of binding to a target DNA sequence in an endogenous gene; (f) one or more effector proteins (or polynucleotides encoding the same) capable of interacting with a gDNA and modifying a target DNA sequence in an endogenous gene; (g) one or more gRNAs (or polynucleotides encoding the same) capable of binding to a target mRNA sequence encoded by an endogenous gene; (h) one or more effector proteins (or polynucleotides encoding the same) capable of interacting with a gRNA and modifying a target mRNA sequence encoded by an endogenous gene; or (i) any combination of the above. [00210] In some embodiments, the kit for modifying a cell comprises one or more components of a gene-regulating system (or one or more polynucleotides encoding the one or more components) and a reagent for reconstituting and/or diluting the components. In some embodiments, a kit modifying a cell comprises one or more components of a gene-regulating system (or one or more polynucleotides encoding the one or more components) and further comprises one or more additional reagents, where such additional reagents can be selected from: a buffer for introducing the gene-regulating system into a cell; a wash buffer; a control reagent; a control expression vector or RNA polynucleotide; a reagent for in vitro production of the gene-regulating system from DNA, and the like. Components of a kit can be in separate containers or can be combined in a single container. [00211] In some embodiments, a kit for treating a disease or disorder can include: a modified immune described herein (e.g., a modified cell comprising reduced expression and/or function of ACAT1 and DNMT3A). [00212] In some embodiments, the kit for treating a disease or disorder comprises a modified cell described herein and a reagent for reconstituting and/or diluting the components. Components of a kit can be in separate containers or can be combined in a single container. [00213] In addition to above-mentioned components, in some embodiments a kit further comprises instructions for using the components of the kit to practice the methods of the present disclosure. The instructions for practicing the methods are generally recorded on a suitable recording medium. For example, the instructions may be printed on a substrate, such as paper or plastic, etc. As such, the instructions may be present in the kits as a package insert or in the labeling of the container of the kit or components thereof (i.e., associated with the packaging or sub-packaging). In other embodiments, the instructions are present as an electronic storage data file present on a suitable computer readable storage medium, e.g. CD-ROM, diskette, flash drive, etc. In yet other embodiments, the actual instructions are not present in the kit, but means for obtaining the instructions from a remote source, e.g. via the internet, are provided. An example of this embodiment is a kit that includes a web address where the instructions can be viewed and/or from which the instructions can be downloaded. As with the instructions, this means for obtaining the instructions is recorded on a suitable substrate. VI. Therapeutic Methods and Applications [00214] In some embodiments, the modified cells described herein may be used in a variety of therapeutic applications. For example, in some embodiments the modified cells described herein may be administered to a subject to treat a disease. [00215] In some embodiments, the subject may be a neonate, a juvenile, or an adult. Of particular interest are mammalian subjects. Mammalian species that may be treated with the present methods include canines and felines; equines; bovines; ovines; etc. and primates, particularly humans. Animal models, particularly small mammals (e.g. mice, rats, guinea pigs, hamsters, rabbits, etc.) may be used for experimental investigations. [00216] Administration of the modified cells described herein, populations thereof, and compositions thereof can occur by injection, irrigation, inhalation, consumption, electro- osmosis, hemodialysis, iontophoresis, and other methods known in the art. In some embodiments, administration route is local or systemic. In some embodiments administration route is intraarterial, intracranial, intradermal, intraduodenal, intrammamary, intrameningeal, intraperitoneal, intrathecal, intratumoral, intravenous, intravitreal, ophthalmic, parenteral, spinal, subcutaneous, ureteral, urethral, vaginal, or intrauterine. [00217] In some embodiments, the administration route is by infusion (e.g., continuous or bolus). Examples of methods for local administration, that is, delivery to the site of injury or disease, include through an Ommaya reservoir, e.g. for intrathecal delivery (See e.g., US Patent Nos. 5,222,982 and 5,385,582, incorporated herein by reference); by bolus injection, e.g. by a syringe, e.g. into a joint; by continuous infusion, e.g. by cannulation, such as with convection (See e.g., US Patent Application Publication No. 2007-0254842, incorporated herein by reference); or by implanting a device upon which the cells have been reversibly affixed (see e.g. US Patent Application Publication Nos. 2008-0081064 and 2009-0196903, incorporated herein by reference). In some embodiments, the administration route is by topical administration or direct injection. In some embodiments, the modified cells described herein may be provided to the subject alone or with a suitable substrate or matrix, e.g. to support their growth and/or organization in the tissue to which they are being transplanted. [00218] In some embodiments, at least 1 x 10³ cells are administered to a subject. In some embodiments, at least 5 x 10³ cells, 1 x 10⁴ cells, 5 x 10⁴ cells, 1 x 10⁵ cells, 5 x 10⁵ cells, 1 x 10⁶, 2 x 10⁶, 3 x 10⁶, 4 x 10⁶, 5 x 10⁶, 1 x 10⁷, 1 x 10⁸, 5 x 10⁸, 1 x 10⁹, 5 x 10⁹, 1 x 10¹⁰, 5 x 10¹⁰, 1 x 10¹¹, 5 x 10¹¹, 1 x 10¹², 5 x 10¹², or more cells are administered to a subject. In some embodiments, between about 1 x 10⁷ and about 1 x 10¹² cells are administered to a subject. In some embodiments, between about 1 x 10⁸ and about 1 x 10¹² cells are administered to a subject. In some embodiments, between about 1 x 10⁹ and about 1 x 10¹² cells are administered to a subject. In some embodiments, between about 1 x 10¹⁰ and about 1 x 10¹² cells are administered to a subject. In some embodiments, between about 1 x 10¹¹ and about 1 x 10¹² cells are administered to a subject. In some embodiments, between about 1 x 10⁷ and about 1 x 10¹¹ cells are administered to a subject. In some embodiments, between about 1 x 10⁷ and about 1 x 10¹⁰ cells are administered to a subject. In some embodiments, between about 1 x 10⁷ and about 1 x 10⁹ cells are administered to a subject. In some embodiments, between about 1 x 10⁷ and about 1 x 10⁸ cells are administered to a subject. The number of administrations of treatment to a subject may vary. In some embodiments, introducing the modified cells into the subject may be a one-time event. In some embodiments, such treatment may require an on- going series of repeated treatments. In some embodiments, multiple administrations of the modified cells may be required before an effect is observed. The exact protocols depend upon the disease or condition, the stage of the disease and parameters of the individual subject being treated. [00219] In some embodiments, the modified cells described herein are administered to a subject. In some embodiments, the modified cells described herein administered to a subject are autologous cells. The term “autologous” in this context refers to cells that have been derived from the same subject to which they are administered. For example, cells may be obtained from a subject, modified ex vivo according to the methods described herein, and then administered to the same subject in order to treat a disease. In such embodiments, the cells administered to the subject are autologous immune effector cells. In some embodiments, the modified cells, or compositions thereof, administered to a subject are allogenic immune effector cells. The term “allogenic” in this context refers to cells that have been derived from one subject and are administered to another subject. For example, cells may be obtained from a first subject, modified ex vivo according to the methods described herein and then administered to a second subject in order to treat a disease. In such embodiments, the cells administered to the subject are allogenic immune effector cells. [00220] In some embodiments, the modified cells described herein are administered to a subject in order to treat a disease. In some embodiments, treatment comprises delivering an effective amount of a population of cells (e.g., a population of modified cells) or composition thereof to a subject in need thereof. In some embodiments, treating refers to the treatment of a disease in a mammal, e.g., in a human, including (a) inhibiting the disease, i.e., arresting disease development or preventing disease progression; (b) relieving the disease, i.e., causing regression of the disease state or relieving one or more symptoms of the disease; and (c) curing the disease, i.e., remission of one or more disease symptoms. In some embodiments, treatment may refer to a short-term (e.g., temporary and/or acute) and/or a long-term (e.g., sustained) reduction in one or more disease symptoms. In some embodiments, treatment results in an improvement or remediation of the symptoms of the disease. The improvement is an observable or measurable improvement or may be an improvement in the general feeling of well-being of the subject. [00221] The effective amount of a modified cell administered to a particular subject will depend on a variety of factors, several of which will differ from patient to patient including the disorder being treated and the severity of the disorder; activity of the specific agent(s) employed; the age, body weight, general health, sex and diet of the patient; the timing of administration, route of administration; the duration of the treatment; drugs used in combination; the judgment of the prescribing physician; and like factors known in the medical arts. [00222] In some embodiments, the effective amount of a modified cell may be the number of cells required to result in at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, or more fold decrease in tumor mass or volume, decrease in the number of tumor cells, or decrease in the number of metastases. In some embodiments, the effective amount of a modified cell may be the number of cells required to achieve an increase in life expectancy, an increase in progression-free or disease-free survival, or amelioration of various physiological symptoms associated with the disease being treated. In some embodiments, an effective amount of modified cells will be at least 1 x 10³ cells, for example 5 x 10³ cells, 1 x 10⁴ cells, 5 x 10⁴ cells, 1 x 10⁵ cells, 5 x 10⁵ cells, 1 x 10⁶, 2 x 10⁶, 3 x 10⁶, 4 x 10⁶, 5 x 10⁶, 1 x 10⁷, 1 x 10⁸, 5 x 10⁸, 1 x 10⁹, 5 x 10⁹, 1 x 10¹⁰, 5 x 10¹⁰, 1 x 10¹¹, 5 x 10¹¹, 1 x 10¹², 5 x 10¹², or more cells. [00223] In some embodiments, the modified cells and gene-regulating systems described herein may be used in the treatment of a cell-proliferative disorder, such as a cancer. In some embodiments, the modified cells and gene-regulating systems described herein may be used to reduce the size of a tumor, kill tumor cells, prevent tumor cell proliferation, prevent growth of a tumor, eliminate a tumor from a patient, prevent relapse of a tumor, prevent tumor metastasis, induce remission in a patient, or any combination thereof. In some embodiments, the methods induce a complete response. In some embodiments, the methods induce a partial response. In some embodiments, the method results in remission of the cancer. Cancers that may be treated using the compositions and methods disclosed herein include cancers of the blood and solid tumors. For example, cancers that may be treated using the compositions and methods disclosed herein include, but are not limited to, adenoma, carcinoma, sarcoma, leukemia or lymphoma. In some embodiments, the cancer is chronic lymphocytic leukemia (CLL), B cell acute lymphocytic leukemia (B-ALL), acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), non-Hodgkin’s lymphoma (NHL), diffuse large cell lymphoma (DLCL), diffuse large B cell lymphoma (DLBCL), Hodgkin’s lymphoma, multiple myeloma, renal cell carcinoma (RCC), neuroblastoma, colorectal cancer, breast cancer, ovarian cancer, melanoma, sarcoma, prostate cancer, lung cancer, esophageal cancer, hepatocellular carcinoma, pancreatic cancer, astrocytoma, mesothelioma, head and neck cancer, and medulloblastoma, and liver cancer. EXAMPLES Example 1: Materials and Methods [00224] The experiments described herein utilize an RNA guided nuclease and base editing polypeptide system to modulate expression of one or more endogenous target genes in T cell populations. [00225] A nonlimiting example workflow for producing modified T cells is shown in FIG.1. Briefly, T cells are thawed and activated on D0. On Day 1 T cells are transduced with a lentiviral vector to introduce a transgenic T cell receptor (TCR) or Chimeric Antigen Receptor (CAR), enabling specific tumor antigen recognition. On Day 3 cells undergo an electroporation (EP) step to knock out specific genes of interest to improve T cell function (such as increased proliferation, increased infiltration into tumors, persistence of the immune cells in a subject, and/or increased resistance to immune cell exhaustion). T cells are then refed at D7 and harvested at D10, followed by cryopreservation. Cells are then thawed, and 2 days after thaw (Day 12) they are evaluated for functional readouts. [00226] For multiplex editing experiments, expression cassettes were produced and introduced into vectors for mammalian expression. The base editor was codon-optimized for human expression and operably fused at the 5' end to an SV40 nuclear localization sequence with a 32aa linker fused to the RNA guided nuclease and operably fused at the 3' end to USP2 and nucleoplasmin NLS sequences. Each expression cassette was under control of a CMV promoter. The construct was then subcloned into a proprietary vector from Trilink Biotechnologies for the purpose of mRNA synthesis (Trilink). The mRNA was synthesized with full substitutions of 5-Methoxyuridine, capped with CleanCap (Trilink), synthesized with an additional 120 Polyadenylated tail, and resuspended in 1mM Sodium Citrate, pH6.4 (Trilink). Purified mRNA was tested at 2ug per 1e6 cells per nucleofection for guide screening and optimization purposes. [00227] Guide RNA (gRNA)s were synthesized by Integrated DNA technologies. Guides were synthesized with phosphorothioated 2'-O-methyl modifications to the first and last 3bp of each guide. [00228] The components described above were introduced into primary human T Cells. [00229] Total protein was extracted from cell pellets with 1/3 RIPA (Abcam) with 2/3 1x PBS (ThermoFisher). Cell pellets were lysed and centrifuged at 1000 RPM for 5mins. The supernatant was collected and total protein concentration was determined via BCA protein assay (ThermoFisher) following manufacturer’s protocol. Protein was diluted to a 0.2mg/mL concentration with 0.1x sample buffer (ProteinSimple). Antibodies were diluted in the provided antibody diluent for antibodies against ACAT1 (ab168342, Abcam) or DNMT3a (ab188470, Abcam) were used at a 1:50 dilution with the appropriate secondary. Samples and plate were prepared following manufacturer’s instructions. [00230] Total genomic DNA was harvested using a genomic DNA isolation kit (Machery-Nagel) according to the manufacturer’s instructions. All PCR reactions were performed using 10 μL of 2X Master Mix Phusion High-Fidelity DNA polymerase (Thermo Scientific) in a 20 μL reaction including 0.5 μM of each primer. Large genomic regions encompassing each target gene were first amplified using PCR#1 primers, using a program of: 98°C., 1 min; 30 cycles of [98°C., 10 sec; 62°C., 15 sec; 72°C., 5 min]; 72°C., 5 min; 12°C., forever. One μL of this PCR reaction was then further amplified using primers specific for each guide (PCR#2 primers), using a program of: 98°C., 1 min; 35 cycles of [98°C., 10 sec; 67°C., 15 sec; 72°C., 30 sec]; 72°C., 5 min; 12°C., forever. Primers for PCR#2 include Nextera Read 1 and Read 2 Transposase Adapter overhang sequences for Illumina sequencing. [00231] Day 0: Human T cell thaw and Activation (Initiation): Three days prior to Amaxa nucleofection, positively selected (CD4/CD8 positive selection) enriched T cells were thawed, counted, seeded, and activated into a T150 flask containing complete CTS Optimizer T-cell Expansion SFM (Gibco) supplemented with OpTmizer T-Cell Expansion Supplement (2.6% v/v, Gibco), CTS Immune Cell SR (2.5% v/v, Gibco) 1X GlutaMAX Supplement (Gibco) and 1% Penicillin-Streptomycin (Gibco). Base media was also supplemented with Recombinant human IL-2 (300IU/mL, Miltenyi Biotec), human IL-7 (5 ng/mL, Miltenyi Biotec) and human IL-15 (5 ng/mL, Miltenyi Biotec). T Cells were activated with anti CD3/CD28 Dynabeads at a ratio of 1:1 bead/cell. Cells were initially seeded at 1x10^6 cells per ml and grown for 3 days. [00232] Day 1: Lentiviral transduction of T cells: Lentivirus was thawed and diluted with TCGM to obtain the desired multiplicity of infection (MOI) and added to the T cells. The Lentiviral transduction mix may contain a transduction enhancer. [00233] Day 3: Electroporation of T cells: T cells were harvested and counted the T cells. Electroporation mastermix was prepared by mixing the desired amount of mRNA encoding the RGN and guide RNA. T cells were resuspended in electroporation buffer (P3) at the desired cell density, and the electroporation master mix was added and the mixture was transferred to a nucleovette for electroporation with the selected nucleofector program. After electroporation, cells were immediately retrieved and transferred to conicals prefilled with TCGM and rested for 1 h at 37C in the CO2 incubator. After 1 h, cells were transferred to the appropriate vessel for expansion and placed at 37C in the CO2 incubator. [00234] Day 7: Refeeding modified T cells: Culture vessel(s) were removed from the incubator and refed by adding the appropriate amount of cytokines diluted in TCGM. [00235] Day 10: Harvest and cryopreservation of modified T cells: Contents of all vessels were harvested into correspondingly labeled conical tubes. Cells were centrifuged and resuspended in preferred media in volume appropriate for counting. After counting, cells were centrifuged cryopreservation. Cells were resuspended cells in appropriate volume of ice cold DPBS to rinse, centrifuged at room temperature, and resuspended in appropriate volume of cryopreserving media. Cryovials were transferred to ice until ready to transfer to CoolCell freezing containers. CoolCell freezing containers with cells were placed at -80C for at least 4 hours and transferred to appropriate locations for long-term storage. [00236] Day 12: Functional assessment of modified T cells: T cells were thawed and counted. T cells were resuspended in TCGM at 1E6 cells/mL and plated in appropriate vessel. T cells were rested for two days until harvest for functional characterization. Example 2: Genomic deletion of DNMT3A increases T cell persistence [00237] Experiments were performed to assess the effects of genomic deletion of DNMT3A in receptor engineered T cells. Modified, receptor engineered DNMT3A knockout (KO) T cells expressing the NY-ESO-1 TCR were prepared as described in Example 1 above using both spCas9 and a Life Edit nuclease (Life Edit Therapeutics, North Carolina) respectively. The spCas9 gRNA was designed according to Prinzing et al. (2021) Sci Transl Med. November 17; 13(620) for use with spCas9. [00238] As shown in FIG. 2A, the indel percentage of UTD T cells with DNMT3A genomic deletion (UTD DNMT3a KO) and engineered NY-ESO-1 TCR expressing T cells with DNMT3A genomic deletion (NY-ESO-1 TCR DNMT3a KO) were 80 and 85 percent using the spCas9 system, respectively. These levels of editing efficiency led to robust reduction in protein expression of DNMT3a, as shown in the Western blot in FIG.2B. Similar % deletion was observed using a Life Edit nuclease (Life Edit Therapeutics, North Carolina) (FIG.14B). DNMT3a protein expression levels in UTD T cells with DNMT3A genomic deletion (UTD DNMT3a KO) and engineered NY-ESO-1 TCR expressing T cells with DNMT3A genomic deletion (NY-ESO-1 TCR DNMT3a KO) were significantly reduced when compared to that of engineered NY-ESO-1 TCR expressing T cells that underwent the EP step however without DNMT3A genomic deletion (NY-ESO-1 TCR EP Only). [00239] As shown in FIG.3A, the vector copy number of lentivirus expressing the NY- ESO-1 TCR was comparable in engineered NY-ESO-1 TCR expressing T cells that underwent the EP step however without DNMT3A genomic deletion (NY-ESO-1 TCR EP Only) and in engineered NY-ESO-1 TCR expressing T cells with DNMT3A genomic deletion (NY-ESO-1 TCR DNMT3a KO). Tetramer staining for NY-ESO-1 TCR and analysis by flow cytometry were performed to further determine transduction efficiency. Cells were gated sequentially as live > lymphocytes > single cells > CD3+. As shown in the FACS plots in FIG. 3B, in both populations of the engineered NY-ESO-1 TCR expressing T cells with and without DNMT3A genomic deletion, around 50 percent of CD3+ cells had detectable cell surface expression of the NY-ESO-1 TCR. [00240] Population doubling level (PDL) was determined to assess whether deletion of DNMT3A impacts the in vitro expansion of T cell cultures. As shown in FIG.4, the PDL was comparable for UTD T cells with DNMT3A genomic deletion (UTD DNMT3a KO), engineered NY-ESO-1 TCR expressing T cells that underwent the EP step however without DNMT3A genomic deletion (NY-ESO-1 TCR EP Only) and engineered NY-ESO-1 TCR expressing T cells with DNMT3A genomic deletion (NY-ESO-1 TCR DNMT3a KO). [00241] To determine whether genomic deletion of DNMT3A changes the cytotoxic capacity of engineered T cells, each of the following cell populations were co-cultured with NY-ESO-1+ cells from the A375 melanoma and H1299.A2 non-small cell lung cancer cell lines: (1) UTD T cells with DNMT3A genomic deletion (UTD DNMT3a KO), (2) engineered NY-ESO-1 TCR expressing T cells that underwent the EP step however without DNMT3A genomic deletion (NY-ESO-1 TCR EP Only) and (3) engineered NY-ESO-1 TCR expressing T cells with DNMT3A genomic deletion (NY-ESO-1 TCR DNMT3a KO). Co-cultures were done at three effector to target ratios of 10:1, 5:1, and 2.5:1. Percent cytotoxicity was determined by measurement of impedance on the Xcelligence system after 6 hours. As shown in FIG. 5, UTD T cells with DNMT3A genomic deletion but not expressing the NY-ESO-1 TCR were ineffective in killing target cells, as expected, whereas engineered NY-ESO-1 TCR expressing T cells with and without DNMT3A genomic deletion achieved similar levels of cytotoxicity at each effector to target ratios tested. These results show that DNMT3A deletion does not negatively impact the cytotoxic function of receptor engineered T cells. Furthermore, as shown in FIG.6, the concentration of IFNγ detected in the supernatant of these co-cultures at the E:T ratio of 5:1 was comparable for engineered NY-ESO-1 TCR expressing T cells with and without DNMT3A genomic deletion. FIG.17A shows data generated with a different donor and using T cells generated according to a second set of culture conditions. Both populations of these cells produced similarly high levels of IFNγ whereas UTD T cells with DNMT3A genomic deletion but not expressing the NY-ESO-1 TCR released very little IFNγ. These results show that DNMT3A deletion does not negatively impact the level of IFNγ released by receptor engineered T cells in response to antigen stimulation. [00242] As described in the present application and commonly known in the field, prolonged antigen stimulation can lead to T cell exhaustion, whereby antigen-specific T cells exhibit decreased effector function and fail to persist in the body. To study the impact of DNMT3A deletion on T cell persistence in vitro, a restimulation assay was used, as shown in FIG. 7. Briefly, cells were thawed at D0, and following two days of rest, T cells were co- cultured with fluorescently labelled tumor cells at an effector:target (E:T) ratio of 2.5:1. New tumor cells were added to the wells at intervals of 2-3 days from each other. Cytotoxicity as well as T cell proliferation were evaluated via image analysis within the Incucyte system. [00243] After receptor engineered T cells were thawed and rested for 48 hours, they were co-cultured with target tumor cells at a 2.5:1 effector to target ratio in the wells of a 6- well plate. Every 2 to 3 days, the engineered T cells were harvested and counted to determine their proliferative potential and re-plated with a fresh population of tumor cells at the same E:T ratio. Each co-culture was considered one round of antigen stimulation and up to 7 rounds of antigen stimulations were performed over the course of 17 days after cells were thawed. As shown in FIG. 8A, whereas the proliferative potential (calculated as theoretical cell growth from increased factor of absolute cell counts between stimulations and multiplied this with previous stimulation cell count) of NY-ESO-1 TCR expressing T cells began to decline after 5 stimulations with A375 cells and after 4 stimulations with H1299.A2 cells, NY-ESO-1 TCR expressing T cells with DNMT3A genomic deletion continued to display robust growth up to 6 rounds of stimulations. As shown in FIG. 8B, NY-ESO-1 TCR expressing T cells with and without genomic DNMT3A deletion (TCR WT and TCR DNMT3a KO respectively) demonstrated increased in vitro persistence. FIG.17B shows data generated with five different donors, with T cells generated according to a second set of culture conditions. These results show that deletion of DNMT3A increases persistence of NY-ESO-1 TCR T cells in vitro. [00244] To determine how increased persistence of DNMT3A KO engineered T cells impact effector functionality in vivo, mice were individually inoculated with 5x10⁶ of NY- ESO-1+ H1299.A2 cells subcutaneously. Body weights for the mice and the tumor volume were determined and used to randomize animals into the following treatment groups (N=5/group) ten days post inoculation: 1. PBS (Vehicle), 2. control, electroporated UTD T cells (UTD EP Only) (1.52E7 cells/mouse), 3. UTD T cells with DNMT3A genomic deletion (UTD DNMT3a KO) (1.64E7 cells/mouse), 4. engineered NY-ESO-1 TCR expressing T cells with DNMT3A genomic deletion (NY-ESO-1 TCR DNMT3a KO) (1.64E7 cells/mouse, corresponding to 1E7 tetramer positive cells/mouse; or 3.27E6 cells/mouse, corresponding to 2E6 tetramer positive cells/mouse). [00245] Ten days after inoculation, vehicle control was administered or cells were adoptively transferred to the mice. As shown in FIG. 9, tumor volume in mice treated with vehicle control, control, electroporated T cells and T cells with DNMT3A genomic deletion but not expressing the NY-ESO-1 TCR continued to increase until tumors reached a maximum allowable volume of 2000mm³22 days post treatment and animals were sacrificed. Tumors in mice treated with engineered NY-ESO-1 TCR expressing T cells with and without DNMT3A deletion became undetectable 8 days after adoptive cell transfer. However, whereas tumors remained undetectable in mice treated with engineered, DNMT3A KO T cells up to 34 days post treatment, tumors in mice treated with cells where DNMT3A was not deleted began to grow after 29 days post treatment and were on average larger than their pre-treatment volume at 36 days post treatment. FIG.17C shows data generated with five different donors and using T cells generated according to a second set of culture conditions. These results show that DNMT3A deletion allowed engineered T cells to persist and to continue to kill tumor cells in vivo while their WT counterparts eventually failed to control tumor growth. Example 3: Genomic deletion of ACAT1 increases IFN-gamma cytokine secretion and T cell persistence. [00246] Experiments were performed to assess the effects of genomic deletion of ACAT1 in receptor engineered T cells. Modified, receptor engineered ACAT1 KO T cells expressing the NY-ESO-1 TCR were prepared as described in Example 1 above using a Life Edit nuclease (Life Edit Therapeutics, North Carolina) and corresponding gRNAs. [00247] As shown in the Western blot in FIG.10, ACAT1 protein expression levels in T cells where ACAT1 genomic deletion was carried out using different gRNAs (guide 1, guide 2, guide 3, and guide 4) were significantly reduced when compared to that of T cells electroporated in the absence of ACAT1 gRNA (EP control). [00248] As shown in FIG. 11A, the vector copy number of lentivirus expressing the NY-ESO-1 TCR was comparable in engineered NY-ESO-1 TCR expressing T cells that underwent the EP step however without ACAT1 genomic deletion (NY-ESO-1 TCR EP Only) and in engineered NY-ESO-1 TCR expressing T cells with ACAT1 genomic deletion (NY- ESO-1 TCR ACAT1 KO). Tetramer staining for NY-ESO-1 TCR and analysis by flow cytometry were performed to further determine transduction efficiency. Cells were gated sequentially as live > lymphocytes > single cells > CD3+. As shown in the FACS plots in FIG. 11B, in both populations of the engineered NY-ESO-1 TCR expressing T cells with and without ACAT1 genomic deletion, around 60 percent of CD3+ cells had detectable cell surface expression of the NY-ESO-1 TCR. [00249] Population doubling level (PDL) was determined to assess whether deletion of ACAT1 impacts the in vitro expansion of T cell cultures. As shown in FIG. 12, the PDL was comparable for control, electroporated T cells (UTD EP Only), engineered NY-ESO-1 TCR expressing T cells that underwent the EP step however without ACAT1 genomic deletion (NY- ESO-1 TCR EP Only) and engineered NY-ESO-1 TCR expressing T cells with ACAT1 genomic deletion (NY-ESO-1 TCR ACAT1 KO). The similar PDL between these populations shows that genomic deletion of ACAT1 does not hamper T cell expansion. [00250] To determine whether genomic deletion of ACAT1 changes the capacity of engineered T cells to secret IFNγ, each of the following cell populations were co-cultured with NY-ESO-1+ cells from the A375 melanoma and H1299.A2 non-small cell lung cancer cell lines and T2 cells previously loaded with the NY-ESO-1 peptide (positive control, peptide- pulsed T2 cells): (1) electroporated UTD T cells (UTD EP only), (2) UTD T cells with ACAT1 genomic deletion (UTD ACAT1 KO), (3) engineered NY-ESO-1 TCR expressing T cells that underwent the EP step however without ACAT1 genomic deletion (NY-ESO-1 TCR EP Only), and (4) engineered NY-ESO-1 TCR expressing T cells with ACAT1 genomic deletion (NY- ESO-1 TCR ACAT1 KO). Co-cultures were done at an effector to target ratio of 5:1. As shown in FIG. 13A, control, electroporated UTD T cells with and without ACAT1 genomic deletion but not expressing the NY-ESO-1 TCR released very little IFNγ, as expected. Importantly, the concentration of IFNγ detected in the supernatant of these co-cultures was significantly higher for engineered NY-ESO-1 TCR expressing T cells with ACAT1 genomic deletion, with fold increases in IFNγ concentration ranging from 2-5 times that of engineered NY-ESO-1 TCR expressing T cells that underwent the EP step however without ACAT1 genomic deletion. These results show that ACAT1 deletion increases the level of IFNγ released by receptor engineered T cells in response to antigen stimulation. FIG.18A shows data generated with five different donors and using T cells generated according to a second set of culture conditions. A similar experiment performed with engineered T cells expressing a CD19-targeted CAR (FIG. 13B). These experiments show a 1.2-5 fold increase in IFN γ for both the TCR and CAR. Further TCR and CAR expression were similar between wild type and knock out groups for the TCR and CAR (data not shown). [00251] FIG. 13A shows the concentration of IFN-gamma detected in the supernatant of co-cultures of electroporated UTD T cells (UTD EP only), UTD T cells with ACAT1 genomic deletion (UTD ACAT1 KO), of engineered NY-ESO-1 TCR expressing T cells that underwent the EP step however without ACAT1 genomic deletion (NY-ESO-1 TCR EP Only) and of engineered NY-ESO-1 TCR expressing T cells with ACAT1 genomic deletion (NY- ESO-1 TCR ACAT1 KO) individually with each of the following: NY-ESO-1+ A375 melanoma cell line, NY-ESO-1+ H1299.A2 non-small cell lung carcinoma cell line, and peptide-pulsed T2 cells. FIG. 13B shows the concentration of IFN-gamma detected in the supernatant of co-cultures of electroporated UTD T cells (UTD), engineered CD19-targeted CAR expressing T cells that underwent the EP step however without the ACAT1 genomic deletion (CAR WT), and of engineered CD19-targeted CAR expressing T cells with ACAT1 genomic deletion (CAR ACAT1 KO) individually with the K562 tumor cell line (“tumor negative”) and with the Daudi tumor cell line (“tumor positive”). In contrast with the single DNMT3a knockout shown in FIG. 6, this cell comprising a ACAT1 knockout (e.g. a DNMT3a/ACAT1 double KO) shows a significant increase in IFN-gamma production. [00252] To study the impact of ACAT1 deletion on T cell persistence in vitro, a restimulation assay using the Incucyte system was used, as shown in FIG.7 and described for DNMT3A in Example 2. As shown in FIG.18B, NY-ESO-1 TCR expressing T cells with and without genomic ACAT1 deletion (TCR – EP ONLY and TCR – ACAT1 KO respectively) demonstrated increased in vitro persistence following repeated stimulation with A375 melanoma (top) and H1299.A2 non-small cell lung cancer (bottom) cell lines. These results show that deletion of ACAT1 increases persistence of NY-ESO-1 TCR T cells in vitro. Example 4: Genomic deletion of DNMT3A and ACAT1. [00253] To determine how genomic deletion of both DNMT3A and ACAT1 changes the cytotoxic capacity of engineered T cells compared to genomic deletion of each of the genes individually, each of the following cell populations were co-cultured with target NY-ESO-1+ cells of the H1299.A2 non-small cell lung cancer cell line were engineered with Nuclight red in an Incucyte assay: (1) electroporated NY-ESO-1 TCR expressing T cells, (2) NY-ESO-1 TCR expressing T cells with DNMT3A genomic deletion, (3) NY-ESO-1 TCR expressing T cells with ACAT1 genomic deletion, and (4) NY-ESO-1 TCR expressing T cells with both DNMT3A and ACAT1 genomic deletions, at a E:T ratio of 2.5:1. Every 2 to 3 days, the tumor cells were re-challenged with 10e4/well regardless of the E:T ratio. The H1299.A2 cell count per well normalized to time zero was determined for each well of the co-culture assay every 2 hours for 12 and a half days. As shown in FIG. 14A, while cytotoxic activity was similar for all four populations of engineered T cells in the first 4 cycles of co-cultures, only the DNMT3A single KO, ACAT1 single KO, and DNMT3A/ACAT1 double KO engineered T cells maintained cytotoxicity against target H1299.A2 cells after 4 cycles. Importantly, the double KO engineered T cells were significantly more effective at killing target cells in the fifth and sixth rounds of co-cultures when compared to the single KO engineered T cells. These results demonstrate that genomic deletion of DNMT3A and ACAT1 confers increased cytotoxic functions to engineered T cells and that reduced expression of both these genes further enhances engineered T cell effector functions compared to reduced expression of just one of these two genes Example 5: Multiplex editing demonstrated in T cells [00254] The ability to knock-out multiple endogenous genes in a cell was demonstrated. Cells modified to delete DNMT3A, ACAT1, and TRAC in T cells were prepared as described in Example 1. Here, the cells were editing using a construct comprising a Life Edit nuclease and base editor (Life Edit Therapeutics, North Carolina) with corresponding gRNAs. As shown in FIG. 15A-15B, successful multiplex editing of ACAT1, DNMT3A, and TRAC was demonstrated (FIG.15A) and confirmed by Western Blot and Flow Cytometry (FIG.15B). [00255] Additional experiments were performed to demonstrate the ability to multiplex edit 5 genes, ACAT1, B2M, DNMT3a, TRAC, and TRBC1/2). For mRNA multiplex delivery, 4ug of base editor mRNA, 8ug of the sgRNA targeting ACAT1 and 4ug each of the sgRNAs targeting B2M, DNMT3a, TRAC and TRBC1/2 were co-transfected with 1x10^6 T Cells. After 96 hours of growth, cells were taken for total genomic DNA, flow cytometry analysis and protein expression analysis. As shown in FIG. 16B successful multiplex editing of ACAT1, B2M, DNMT3a, TRAC, and TRBC1/2 was demonstrated and high knockdown was confirmed by Western Blot and Flow Cytometry (FIG.16C).

Claims

CLAIMS 1. A modified cell comprising reduced expression of at least two endogenous genes relative to the expression of the at least two endogenous genes in a non-modified cell, wherein the at least two endogenous genes comprise ACAT1 and DNMT3A.

2. A modified cell comprising a knockout of at least two endogenous genes, wherein the at least two endogenous genes comprise ACAT1 and DNMT3A.

3. The modified cell of claim 1 or claim 2, further comprising reduced expression of one or more additional endogenous target genes selected from PDCD1, CD274, CTLA4, IDO, LAG3, HAVCR2, TIGIT, A2AR, A2BR, B7-H3, BTLA, KIR, NOX2, VISTA, SIGLEC7, B2M, TAP1, TAPBP, TRA, TRB, TRD, TRG, TRAC, CD52, MCJ, TRBC1, TRBC2, HLA-A, HLA-B, HLA-C, HLA-DR, HLA-DQ, HLA-DP, and CIITA.

4. The modified cell of any of claims 1-3, further comprising reduced expression of B2M, TRAC, TRBC1, and TRBC2.

5. The modified cell of any of claims 1-4, further comprising reduced expression of TRAC.

6. A modified cell comprising reduced expression of at least two endogenous genes relative to the expression of the at least two endogenous genes in a non-modified cell, wherein the at least two endogenous genes comprise MCJ and DNMT3A.

7. A modified cell comprising a knockout of at least two endogenous genes, wherein the at least two endogenous genes comprise MCJ and DNMT3A.

8. The modified cell of claim 6 or claim 7, further comprising reduced expression of one or more additional endogenous target genes selected from PDCD1, CD274, CTLA4, IDO, LAG3, HAVCR2, TIGIT, A2AR, A2BR, B7-H3, BTLA, KIR, NOX2, VISTA, SIGLEC7, B2M, TAP1, TAPBP, TRA, TRB, TRD, TRG, TRAC, CD52, ACAT1, TRBC1, TRBC2, HLA-A, HLA-B, HLA-C, HLA-DR, HLA-DQ, HLA-DP, and CIITA.

9. The modified cell of any of claims 6-8, further comprising reduced expression of B2M, TRAC, TRBC1, and TRBC2.

10. A modified cell comprising a knockout of the endogenous genes ACAT1 and DNMT3A.

11. A modified cell comprising a knockout of the endogenous genes ACAT1, DNMT3A, B2M, TRAC, TRBC1, and TRBC2.

12. A modified cell comprising a knockout of the endogenous genes MCJ and DNMT3A.

13. A modified cell comprising a knockout of the endogenous genes MCJ, DNMT3A, B2M, TRAC, TRBC1, and TRBC2.

14. The modified cell of any of claims 10-13, further comprising a knockout of one or more additional endogenous target genes selected from PDCD1, CD274, CTLA4, IDO, LAG3, HAVCR2, TIGIT, A2AR, A2BR, B7-H3, BTLA, KIR, NOX2, VISTA, SIGLEC7, B2M, TAP1, TAPBP, TRA, TRB, TRD, TRG, TRAC, CD52, ACAT1, TRBC1, TRBC2, HLA-A, HLA-B, HLA-C, HLA-DR, HLA-DQ, HLA-DP, and CIITA.

15. The modified cell of any one of claims 1-14, wherein the cell is an immune effector cell or a stem cell.

16. The modified cell of claim 15, wherein the immune effector cell is selected from a T cell, a natural killer (NK) cell, and an NKT cell.

17. The modified cell of claim 15, wherein the stem cell is an induced pluripotent stem cell (iPSC).

18. The modified cell of any one of claims 1-17, wherein the reduced expression of the at least two endogenous target genes or knocked-out expression of the at least two endogenous genes enhances an effector function and/or decreases exhaustion of the effector cell.

19. The modified cell of claim 18, wherein the effector function is selected from cell proliferation, cytokine production, cell viability, tumor infiltration, and cytotoxicity.

20. The modified cell of any of claims 1-19, further comprising expression of an exogenous polynucleotide or polypeptide.

21. The modified cell of claim 20, wherein the exogenous polynucleotide encodes an engineered immune receptor.

22. The modified cell of claim 20, wherein the exogenous polypeptide is an engineered immune receptor.

23. The modified cell of any of claims 1-20, further comprising an engineered immune receptor displayed on the cell surface.

24. The modified cell of claim 23, wherein the engineered immune receptor is a chimeric antigen receptor (CAR) comprising an antigen-binding domain, a transmembrane domain, and an intracellular signaling domain.

25. The modified cell of claim 23, wherein the engineered immune receptor is an engineered T cell receptor (TCR).

26. The modified cell of any one of claims 23-25, wherein the engineered immune receptor specifically binds to an antigen expressed on a target cell, wherein the antigen is a tumor-associated antigen.

27. The modified cell of any one of claims 1-26, wherein the expression of the at least two endogenous genes is reduced or knocked-out by a gene-regulating system capable of reducing or knocking-out the expression of the at least two endogenous target genes.

28. The modified cell of claim 27, wherein the gene-regulating system comprises (i) a nucleic acid molecule; (ii) an enzymatic protein; or (iii) a nucleic acid molecule and an enzymatic protein.

29. The modified cell of claim 28, wherein the gene-regulating system comprises a nucleic acid molecule selected from an siRNA, an shRNA, a microRNA (miR), an antagomiR, or an antisense RNA.

30. The modified cell of claim 28, wherein the gene-regulating system comprises an enzymatic protein, and wherein the enzymatic protein has been engineered to specifically bind to a target sequence in one or more of the endogenous genes.

31. The modified cell of claim 30, wherein the protein is a Transcription activator-like effector nuclease (TALEN), a zinc-finger nuclease, or a meganuclease.

32. The modified cell of claim 30, wherein the gene-regulating system comprises a nucleic acid molecule and an enzymatic protein, wherein the nucleic acid molecule is a guide RNA (gRNA) molecule, and the enzymatic protein is or comprises an RNA- guided nuclease.

33. The modified cell of claim 32, wherein the RNA-guided nuclease is a Cas protein, a Cas ortholog, spCas protein, or an IscB protein.

34. The modified cell of claim 33, wherein the Cas protein is a wild-type Cas protein comprising two enzymatically active domains, and capable of inducing double stranded DNA breaks.

35. The modified cell of claim 33, wherein the Cas protein is a Cas nickase mutant comprising one enzymatically active domain and capable of inducing single stranded DNA breaks.

36. The modified cell of claim 33, wherein the Cas protein is a deactivated Cas protein (dCas).

37. The modified cell of claim 32, wherein the RNA-guided nuclease comprises a nickase, wherein the nickase induces single stranded DNA breaks.

38. The modified cell of any one of claims 32 - 37, wherein the RNA-guided nuclease or Cas nickase or dCas is fused to a heterologous protein.

39. The modified cell of claim 38, wherein the heterologous protein is selected from a deaminase, a transposase, and a reverse transcriptase.

40. The modified cell of claim 39, wherein the deaminase is selected from a cytidine deaminase and an adenosine deaminase.

41. The modified cell of any one of claims 26 - 40, wherein the gene-regulating system is introduced to the cell by transfection, transduction, electroporation, or physical disruption of the cell membrane by a microfluidics device.

42. The modified cell of claim 41, wherein the gene-regulating system is introduced to the cell by transfection with a lipid nanoparticle.

43. The modified cell of claim 41, wherein the gene-regulating system is introduced to the cell by transduction with an adeno-associated virus vector.

44. The modified cell of claim 41, wherein the gene-regulating system is introduced as one or more polynucleotides encoding one or more components of the system, a protein, or a ribonucleoprotein (RNP) complex.

45. A composition comprising the modified cells of any one of claims 1-44.

46. The composition of claim 45, wherein the composition comprises at least 1 x 10⁴, 1 x 10⁵, or 1 x 10⁶ modified cells.

47. The composition of claim 45 or 46, suitable for administration to a subject in need thereof.

48. The composition of any one of claims 45– 47, comprising autologous cells derived from the subject in need thereof.

49. The composition of any one of claims 45– 47, comprising allogeneic cells derived from a donor subject.

50. A method of producing a modified cell comprising: a. introducing a gene-regulating system into the cell, wherein the gene-regulating system is capable of reducing expression of at least two endogenous target genes, comprising ACAT1 and/or DNMT3A; and b. culturing the cell such that the expression of the at least two endogenous target genes is reduced compared to cell that has not been modified.

51. A method of producing a modified cell comprising: a. introducing a gene-regulating system into the cell, wherein the gene-regulating system is capable of reducing expression of at least two endogenous target genes, comprising MCJ and/or DNMT3A; and b. culturing the cell such that the expression of the at least two endogenous target genes is reduced compared to cell that has not been modified.

52. The method of claim 50 or 51, further comprising: a. introducing a gene-regulating system into the cell, wherein the gene-regulating system is capable of reducing expression of B2M, TRAC, TRBC1, and TRBC2; and b. culturing the cell such that the expression of B2M, TRAC, TRBC1, and TRBC2, reduced compared to cell that has not been modified.

53. The method of any of claims 50-52, further comprising introducing a polynucleotide sequence encoding an engineered immune receptor selected from a CAR and a TCR.

54. The method of any one of claims 50-53, wherein the gene-regulating system and/or the polynucleotide encoding the engineered immune receptor are introduced to the cell by transfection, transduction, electroporation, or physical disruption of the cell membrane by a microfluidics device.

55. A method of treating a disease or disorder in a subject in need thereof comprising administering an effective amount of the modified cells of any one of claims 1–44, or the composition of any one of claims 45–49.

56. The method of claim 55, wherein the disease or disorder is a cancer.

57. The method of claim 56, wherein the cancer is selected from a leukemia, a lymphoma, or a solid tumor.

58. The method of any one of claims 55– 57, wherein the modified cells are autologous to the subject.

59. The method of any one of claims 55– 57, wherein the modified cells are allogenic to the subject.

60. A method of producing multiplexed edits in a modified cell comprising: a. introducing a gene-regulating system into a cell, wherein the gene-regulating system is capable of reducing expression of at least two endogenous target genes, comprising ACAT1 or MCJ and/or DNMT3A; and b. culturing the cell such that the expression of the at least two endogenous target genes is reduced compared to cell that has not been modified.

61. A method of producing multiplexed edits in a modified cell comprising: a. introducing a gene-regulating system into a cell, wherein the gene-regulating system is capable of reducing expression of at least two endogenous target genes, comprising ACAT1, DNMT3A, B2M, TRAC, TRBC1, and TRBC2; and b. culturing the cell such that the expression of the at least two endogenous target genes is reduced compared to cell that has not been modified.

62. A method of producing multiplexed edits in a modified cell comprising: a. Introducing a gene-regulating system into a cell, wherein the gene-regulating system is capable of reducing expression of at least two endogenous target genes, comprising MCJ, DNMT3A, B2M, TRAC, TRBC1, and TRBC2; and b. culturing the cell such that the expression of the at least two endogenous target genes is reduced compared to cell that has not been modified.

63. A modified cell comprising reduced expression of at least two endogenous genes relative to the expression of the at least two endogenous genes in a non-modified cell, wherein the at least two endogenous genes comprise ACAT1 and DNMT3A, wherein the modified cell exhibits increased cytokine production and decreased cell exhaustion compared to an unmodified cell.

64. A modified cell comprising a knockout of at least two endogenous genes, wherein the at least two endogenous genes comprise ACAT1 and DNMT3A, wherein the modified cell exhibits increased cytokine production and decreased cell exhaustion compared to an unmodified cell.

65. A composition for producing multiplexed edits in a cell, wherein the composition comprises a gene-regulating system, wherein the gene-regulating system comprises guide RNAs (gRNAs) targeting each of ACAT1, DNMT3a, TRAC, TRBC1/2, and B2M and an RNA-guided nuclease.

66. The composition of claim 65, wherein the RNA-guided nuclease is a Cas protein, a Cas ortholog, spCas protein, or an IscB protein.

67. The composition of claim 66, wherein the Cas protein is a wild-type Cas protein comprising two enzymatically active domains, and capable of inducing double stranded DNA breaks.

68. The composition of claim 66, wherein the Cas protein is a Cas nickase mutant comprising one enzymatically active domain and capable of inducing single stranded DNA breaks.

69. The composition of claim 66, wherein the Cas protein is a deactivated Cas protein (dCas).

70. The composition of claim 69, wherein the RNA-guided nuclease comprises a nickase, wherein the nickase induces single stranded DNA breaks.

71. The composition of any of claims 65-70, wherein the RNA-guided nuclease or Cas nickase or dCas is fused to a heterologous protein.

72. The composition of claim 71, wherein the heterologous protein is selected from a deaminase, a transposase, and a reverse transcriptase.

73. The composition of claim 72, wherein the deaminase is selected from a cytidine deaminase and an adenosine deaminase.

74. A modified cell produced by a) transfecting the composition of any one of claims 64-73 into the cell; and b) culturing the cell such that the expression of ACAT1, DNMT3a, TRAC, TRBC1/2, and B2M are reduced compared to a cell that has not been modified.

75. The modified cell of claim 74 that is further modified by introducing a polynucleotide sequence encoding a transgene.

76. The modified cell of claim 75, wherein the transgene expresses an engineered immune receptor.

77. The modified cell of claim 76, wherein the engineered immune receptor is a chimeric antigen receptor (CAR) comprising an antigen-binding domain, a transmembrane domain, and an intracellular signaling domain.

78. The modified cell of claim 76, wherein the engineered immune receptor is an engineered T cell receptor (TCR).

79. The modified cell of any one of claims 76-78, wherein the engineered immune receptor specifically binds to an antigen expressed on a target cell.

80. The modified cell of claim 79, wherein the antigen is a tumor-associated antigen.

81. The modified cell of any one of claims 74-80, wherein the cell is an immune effector cell or a stem cell.

82. The modified cell of claim 81, wherein the immune effector cell is selected from a T cell, a natural killer (NK) cell, and an NKT cell.

83. The modified cell of claim 81, wherein the stem cell is an induced pluripotent stem cell (iPSC).

84. A method of treating a disease or disorder in a subject in need thereof comprising administering an effective amount of the modified cells of any one of claims 74-83, or the composition of any one of claims 65-73.

85. The method of claim 84, wherein the disease or disorder is a cancer.

86. The method of claim 85, wherein the cancer is selected from a leukemia, a lymphoma, or a solid tumor.

87. The method of any one of claims 84-86, wherein the modified cells are autologous to the subject.

88. The method of any one of claims 84-86, wherein the modified cells are allogenic to the subject.