Inhibition of genotoxic stress to improve T cell engineering

Abstract

Provided herein, among others, is a method and composition for manipulating T cells.The method comprises contacting T cells with nucleic acid and one or more inhibitors of cyclic GMP-AMP synthase (cGAS)-stimulator of interferon genes (STING) pathway.Provided herein is intended to increase cell survival rate, proliferation and gene editing efficiency, thereby enabling the total number of manipulated T cells to increase.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Application No. 63 / 377,251, filed September 27, 2022, which is incorporated herein by reference in its entirety for all purposes.

[0002] Electronic Sequence Listing Reference The contents of the electronic sequence listing (048893-565001WO_Sequence_Listing_ST26.xml; size: 1,720 bytes; created on September 26, 2023) are incorporated herein by reference in their entirety. [Background technology]

[0003] Gene editing allows for the manipulation of various cultured cell lines and primary cells, including T cells. Editing the cell genome allows for the generation of T cells specific to disease targets, such as cancer cell antigens recognized by edited T cell receptors, enabling the production of personalized cancer therapeutics. However, methods for manipulating T cells often reduce the recovery rate of the final cell product. For example, the toxic effects of T cell manipulation methods can result in reduced cell viability and proliferation rates. Furthermore, previous methods for manipulating T cells often have low gene editing efficiency, further contributing to low overall numbers of engineered T cells.

[0004] Disclosed herein, among other things, are solutions to these and other problems in the art. Summary of the Invention

[0005] In one aspect, a method of engineering a T cell is provided, comprising contacting the T cell with a nucleic acid and one or more cyclic GMP-AMP synthase (cGAS)-stimulator of interferon genes (STING) pathway inhibitors.

[0006] In one aspect, a method of increasing cell viability of a population of engineered T cells is provided, comprising contacting the population of T cells with one or more cyclic GMP-AMP synthase (cGAS)-stimulator of interferon genes (STING) pathway inhibitors and a nucleic acid, thereby forming a population of engineered T cells. In various embodiments, the population of engineered T cells has increased cell viability compared to a population of engineered T cells in which the population of T cells has not been contacted with one or more cGAS-STING pathway inhibitors.

[0007]

[0006] In one aspect, a method of increasing gene editing efficiency in a population of T cells is provided, comprising contacting the population of T cells with one or more cyclic GMP-AMP synthase (cGAS)-stimulator of interferon genes (STING) pathway inhibitors and a nucleic acid, thereby forming a population of engineered T cells. In various embodiments, the population of T cells has increased gene editing efficiency compared to a population of T cells not contacted with the one or more cGAS-STING pathway inhibitors.

[0008] In one aspect, a method for increasing the expansion of a population of engineered T cells is provided, comprising: i) contacting a population of T cells with one or more cyclic GMP-AMP synthase (cGAS)-stimulator of interferon genes (STING) pathway inhibitors and a nucleic acid, thereby forming a population of engineered T cells; and ii) expanding the population of engineered T cells, thereby forming an expanded population of engineered T cells. In various embodiments, the one or more cGAS-STING pathway inhibitors increase the population of expanded engineered T cells compared to a population of engineered T cells in which the population of T cells of step i) has not been contacted with the one or more cGAS-STING pathway inhibitors.

[0009] In one aspect, provided are engineered T cells produced by the methods provided herein, including embodiments thereof.

[0010] In one aspect, a population of engineered T cells is provided that is generated by contacting a population of T cells with a nucleic acid and one or more cyclic GMP-AMP synthase (cGAS)-stimulator of interferon genes (STING) pathway inhibitors.

[0011] In one aspect, a composition is provided that includes a population of T cells, a nucleic acid, and one or more cyclic GMP-AMP synthase (cGAS)-stimulator of interferon genes (STING) pathway inhibitors.

[0012] In one aspect, provided is a pharmaceutical composition comprising the engineered T cells provided herein, including embodiments thereof.

[0013] In one aspect, provided is a method of treating a disease in a subject in need thereof, the method comprising administering a therapeutically effective amount of an engineered T cell provided herein, including embodiments thereof, or a pharmaceutical composition provided herein comprising an embodiment thereof. [Brief explanation of the drawings]

[0014] [Figures 1A-1F]Plasmid DNA-mediated activation of the cGAS-STING pathway negatively affects T cell viability and proliferation after electroporation. (Figure 1A) Schematic of the exogenous T cell receptor DNA template used for CRISPR / Cas9-mediated homologous recombination repair at the TCR-α locus. The DNA template containing the TCR-α variant chain and TCR-β chain is designed to be inserted into the TCR-α(TRAC) locus while the endogenous TCR-α(VJ) and TCR-β are disrupted. (Figure 1B) Schematic of the 15-day workflow of the T cell activation, manipulation, and cell culture process. The entire process is carried out in chemically defined media. (Figure 1C-1E) Analysis of T cell viability (Figure 1C), T cell proliferation (Figure 1D), and knock-in / out efficiency (Figure 1E) during the 15-day T cell manipulation and culture process. T cell viability and cell proliferation were measured using a NucleoCounter NC-200. Knock-in / out efficiency was measured by flow cytometry and identified by MHC-peptide dextramer staining using flow cytometry (Figure 1F). Western blot analysis of DNA genotoxicity pathway activation (cGAS-STING-TBK1-IRF3) during and after the TCR manipulation process in the presence of different transfection components (RNP, DNA) used for gene editing as indicated.

[0015] [Figures 2A-2C] T cell viability, proliferation, and targeting data for different donors 48 hours after transfection. T cell viability (Figure 2A), T cell proliferation (Figure 2B), and knock-in / out efficiency (Figure 2C) were measured using different transfection process components (DNA, RNP) as indicated. T cell viability data and cell proliferation were measured using a NucleoCounter NC-200. Knock-in / out efficiency was measured by flow cytometry and identified by MHC-peptide dextramer staining in flow cytometry.

[0016] [Figure 3A-3B]Western blot analysis of AIM2 DNA inflammasome and Toll-like receptor (TLR) pathway activation during and after the TCR engineering process in the presence of different transfection components (RNP, DNA) as indicated. (Figure 3A) Cell lysates collected during the T cell engineering process were analyzed for activation of the Aim2-Cas1-IL-1B inflammasome pathway. (Figure 3B) Cell lysates collected during the T cell engineering process were analyzed for activation of the MyD88-TRAF6 TLR pathway.

[0017] [Figures 4A-4I] Individual donor T cell response data (3 donors) after inhibitor treatment. (Figures 4A, 4D, and 4G) Percent viability 48 hours after transfection was determined using a NucleoCounter NC-200 for donor 1, donor 2, and donor 3, respectively; (Figures 4B, 4E, and 4H) Final preparation (FDP) fold expansion after the 15-day T cell engineering process, calculated based on the seeded cell number on day 2, for donor 1, donor 2, and donor 3, respectively; (Figures 4C, 4F, and 4I) Total edited cell (TEC) numbers in the FDP for donor 1, donor 2, and donor 3, respectively. TECs were calculated based on a seeding density of 2 million cells per group after transfection. Numbers in each column are rounded up to the nearest integer. Total edited cell numbers were calculated by multiplying the fold expansion by the knock-in percentage.

[0018] [Figures 5A-5F]Individual donor T cell response data after inhibitor treatment (two additional donors in addition to the donors in Figure 4). (Figures 5A, 5D) Percent viability 48 hours after transfection was determined using a NucleoCounter NC-200 for donors 4 and 5, respectively; (Figures 5B, 5E) Final preparation (FDP) fold expansion after the 15-day T cell engineering process for donors 4 and 5, respectively, calculated based on the number of cells seeded on day 2; (Figures 5C, 5F) Total edited cell (TEC) numbers in the FDP for donors 4 and 5, respectively. TECs were calculated based on a cell seeding density of 2 million cells per group after transfection. Numbers in each column are rounded up to the nearest integer. Total edited cell numbers were calculated by multiplying the fold expansion by the knock-in percentage.

[0019] [Figures 6A-6C] Summary data for percent viability (Figure 6A), final cell product fold expansion (Figure 6B), and total edited cell (TEC) numbers (Figure 6C) 48 hours post-transfection from five independent donors. T cell viability data and fold expansion were measured using a NucleoCounter NC-200 machine. TECs were calculated by multiplying the fold expansion by the knock-in percentage. Data are presented as mean ± SEM (Figure 6A-6C). Compared to the control (CTR) group, ***P<0.001; **P<0.01; *P<0.05.

[0020] [Figure 7A-7C] Final T cell product cell editing ratio. (Figure 7A-7C) Knock-in and knock-out data from three representative donors, respectively, measured by flow cytometry. Knock-in ratios were identified by MHC-peptide dextramers and TCR double-positive staining in flow cytometry. Knock-out ratios are representative of MHC-peptide dextramers and TCR double-negatives. Control (CTR) represents the group without inhibitor treatment.

[0021] [Figures 8A-8F]BX795 pretreatment attenuates the T cell cGAS-STING pathway. (Figure 8A) Western blot analysis of TBK1 and IRF3 expression and phosphorylation with or without BX795 pretreatment (CTR group). N, 30 min, and 60 min represent immediately before transfection, 30 min after transfection, and 60 min after transfection, respectively. (Figures 8B-8E) Percent viability, fold expansion, knock-in / out efficiency, and total edited cell counts obtained from the same donor as in (Figure 8A). Viability data (Figure 8B) were collected 48 h after transfection using a NucleoCounter NC-200. Values ​​for fold expansion (Figure 8C), knock-in / out efficiency (Figure 8D), and total edited cells (Figure 8E) represent data obtained from the final day of culture (day 15). (Figure 8F) Comparison of cytokine expression (using qPCR) between groups with and without inhibitor treatment. Samples used for qPCR analysis were collected 6 h after transfection and analyzed in triplicate.

[0022] [Figures 9A-9D] Inhibitor pretreatment improves knockout and knockin ratios in the final T cell product. (Figure 9A) Total knockout percentage in the BX795 pretreatment group compared to the control group. (Figure 9B) Analysis of the percentage difference in knockout values ​​between the control and inhibitor pretreatment groups, set at 1. (Figure 9C) Knockin ratio in the BX795 pretreatment group compared to the control group. (Figure 9D) Analysis of the percentage difference in knockin between the BX795 pretreatment group and the control group, set at 1. Samples were collected on day 15, and ratios were measured by flow cytometry. Knockin / out ratios were determined by MHC-peptide dextramer staining in flow cytometry. ***P<0.001; **P<0.01; *P<0.05 compared to the control group.

[0023] [Figures 10A-10F]Pretreatment of T cell cultures with BX795 prior to transfection did not affect the final T cell product phenotype. The T cell phenotypic data shown in Figures 10A-10F were collected from six independent donors. The inhibitor type and dosage were used as indicated. Cell samples were collected on day 15, and phenotypic ratios were measured by flow cytometry. T stem cell memory (TSCM: CD45RA+CD45RO-CD95+CD27+), T central memory (TCM: CD45RA+CD45RO+CD95+CD27+), T cell effector memory (TEM: CD45RA+CD45RO+CD95+CD27-), and effector T cell (TE: CD45RA+CD45RO-CD95+CD27-) phenotypes were measured as indicated. All six donors were treated identically with respect to inhibitor pretreatment, electroporation process, and culture conditions and methods.

[0024] [Figures 11A-11F]T cell cultures pretreated with the BX795 inhibitor before transfection showed similar functional potential to untreated controls. (Figure 11A) Comparison of activation marker CD137 expression between the inhibitor pretreatment group and the control group. Cytokine expression: IFNγ (Figure 11B), TNF-α (Figure 11C), and granzyme B (Figure 11D) comparison between the inhibitor pretreatment group and the control group. Cell samples were collected on the final day (day 15). Cells were then co-cultured with the target peptide (WT1) at the indicated concentrations for 24 hours before flow cytometry analysis. (Figure 11E) Comparison of proliferation analysis between the inhibitor pretreatment group and the control group. Cell samples were collected on the final day (day 15). Cells were then labeled with CFSE dye and co-cultured with the target peptide (WT1) at the indicated concentrations for 72 hours before flow cytometry analysis. (Figure 11F) Cell killing analysis to compare T cell function between the inhibitor pretreatment group and the control group. On the final day (day 15), cell samples were collected and then co-cultured with T2 target cells pre-labeled with the target peptide at the indicated ratios for 20 hours. Target cell apoptosis levels were measured by calculating the Annexin V and 7-aminoactinomycin D (7-AAD) double-positive population using flow cytometry, and corrected for the target cell-only population.

[0025] [Figures 12A-12B] The inhibitor-pretreated and control groups had comparable T cell depletion profiles. The median fluorescence intensity (MFI) of Tim3 (Figure 12A) and PD-1 (Figure 12B) was analyzed by flow cytometry. Cell samples were collected on the final day of the process (day 15). Cells were then co-cultured with the target peptide (WT1) at the indicated concentrations for 24 hours before flow cytometry analysis.

[0026] [Figures 13A-13C]T cell cultures pretreated with the BX795 inhibitor before transfection showed similar functional potential to untreated control groups in three different donors. More donors (donors 1–3) were analyzed for T cell function, as shown in Figures 11A–11F. Cytokine expression of activation markers CD137, IFNγ, TNF-α, and granzyme B, as well as perforin expression, were measured for the inhibitor pretreatment and control groups. Cell samples were collected on the final day of the process (day 15). Cells were then co-cultured with the target peptide (WT1) at the indicated concentrations for 24 hours, followed by flow cytometry analysis. For proliferation analysis comparisons between the inhibitor pretreatment and control groups, samples were collected on day 15. Cells were then labeled with CFSE dye and co-cultured with the target peptide (WT1) at the indicated concentrations for 72 hours before flow cytometry analysis. For cell killing analysis, T cell function was compared between the inhibitor pretreatment and control groups. Cell samples were collected on day 15 and cocultured with T2 target cells pre-labeled with target peptides at the indicated ratios for 20 hours. Target cell apoptosis levels were measured by calculating the Annexin V and 7-aminoactinomycin D (7-AAD) double-positive population using flow cytometry, and corrected for the target cell-only population.

[0027] [Figures 14A-14C]Plasmid DNA transfection efficiency depends on the electroporation (pulse code) program used. The inhibitor BX795 enhances cell editing efficiency (knock-in) throughout the 15-day culture process. (Figure 14A) Comparison of the percentage of GFP-positive CD8+ T cells among different electroporation pulse codes: EH115 (strong), EW113 (medium), and EW100 (weak). DNA uptake was analyzed using 1 μg / ml of GFP plasmid according to the vendor's instructions. The percentage of GFP-positive CD8+ T cells was measured by flow cytometry 24 hours after electroporation. EH115 provides the most stringent conditions of the three pulse codes, while EW100 is recommended by the vendor as the basic pulse code with the mildest transfection conditions. (Figure 14B-C) To date, T cell knock-in and knock-out data from the untreated control group (Figure 14B) and the BX795 (2.5uM) pre-treated group (Figure 14C) measured by flow cytometry. Transfection was performed by day 2, and measurements began on day 4 (48 hours post-transfection). Knock-in ratios were identified by double-positive MHC-peptide dextramers and TCR staining by flow cytometry. Knock-outs are representative of the MHC-peptide dextramers and TCR double-negative population. DETAILED DESCRIPTION OF THE INVENTION

[0028] While various embodiments and aspects of the present invention have been shown and described herein, it will be apparent to those skilled in the art that such embodiments and aspects are provided by way of example only. Numerous variations, changes, and substitutions will now occur to those skilled in the art without departing from the invention. It should be understood that various alternatives to the embodiments described herein may be used in practicing the invention.

[0029] Any section headings used herein are for organizational purposes only and should not be construed as limiting the subject matter described. All documents or portions of documents cited in this application, including but not limited to patents, patent applications, papers, books, manuals, and articles, are expressly incorporated herein by reference in their entirety for any purpose.

[0030] The abbreviations used herein have their common meaning within the chemical and biological arts. The chemical structures and formulas depicted herein are constructed according to the standard rules of chemical valency known in the chemical arts.

[0031] Unless otherwise defined, technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art. In implementing the present invention, any methods, devices, and materials similar or equivalent to those described herein can be used. The following definitions are provided to facilitate understanding of certain terms frequently used herein and are not intended to limit the scope of the present disclosure.

[0032] "Nucleic acid" refers to nucleotides (e.g., deoxyribonucleotides or ribonucleotides) and polymers thereof, or their complements, in any form, whether single-stranded, double-stranded, or multiple-stranded; or nucleosides (e.g., deoxyribonucleosides or ribonucleosides). Examples of polynucleotides contemplated herein include single-stranded and double-stranded DNA, single-stranded and double-stranded RNA, and hybrid molecules that contain a mixture of single-stranded and double-stranded DNA and RNA. Examples of nucleic acids, e.g., polynucleotides contemplated herein include any type of RNA, such as mRNA, siRNA, miRNA, and guide RNA, and any type of DNA, such as genomic DNA, plasmid DNA, minicircle DNA, linear DNA, and any fragment thereof.

[0033] As used herein, the term "gene editing reagent" refers to components necessary for gene editing tools and may include enzymes, riboproteins, solutions, cofactors, etc. For example, gene editing reagents include zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), meganucleases, and one or more components necessary for clustered regularly interspaced short palindromic repeats system (CRISPR / Cas) gene editing.

[0034] As used herein, "zinc finger protein" (ZFP) refers to a chimeric protein containing a nuclease domain and a zinc-stabilized nucleic acid (e.g., DNA) binding domain. Each DNA-binding domain is typically referred to as a "finger," such that a zinc finger protein or polypeptide has at least one finger, more typically two, or three, or even four or five, up to at least six or more fingers. Each finger typically binds to 2-4 base pairs of DNA. Each finger may contain a zinc-chelating DNA-binding region of approximately 30 amino acids (e.g., U.S. Patent Application Publication No. 2012 / 0329067, the disclosure of which is incorporated herein by reference).

[0035] As used herein, "transcription activator-like effector" (TALE) refers to a protein composed of more than one TAL repeat and capable of sequence-specifically binding to nucleic acids. TALEs represent a class of DNA-binding proteins secreted by plant pathogenic bacteria such as Xanthomonas and Ralstonia species via their type III secretion systems upon infection of plant cells. Natural TALEs have been shown to specifically bind to plant promoter sequences, thereby regulating gene expression and activating effector-specific host genes to promote bacterial growth (Romer, P., et al., Science 318:645-648 (2007); Boch, J., et al., Annu. Rev. Phytopathol. 48:419-436 (2010); Kay, S., et al., Science 318:648-651 (2007); Kay, S., et al., Curr. Opin. Microbiol. 12:37-43 (2009)). The modular structure of TALs allows for the combination of DNA-binding domains with effector molecules such as nucleases. In particular, TALE nucleases enable the development of new genome engineering tools.

[0036] Natural TALEs are generally characterized by a central repeat domain and a carboxyl-terminal nuclear localization signal sequence (NLS) and transcription activation domain (AD). The central repeat domain typically consists of a variable number of 1.5-33.5 amino acid repeats, usually 33-35 residues long, except for the generally shorter carboxyl-terminal repeats called half-repeats. The repeats are mostly identical but differ in specific hypervariable residues. The DNA recognition specificity of TALEs is mediated by hypervariable residues, so-called repeat variable dimers (RVDs), typically located at positions 12 and 13 of each repeat, and each RVD targets a specific nucleotide in a given DNA sequence. Therefore, the sequential order of repeats in TAL proteins tends to correlate with the defined linear order of nucleotides in a given DNA sequence. The underlying RVD codes of several naturally occurring TALEs have been identified, allowing prediction of the sequence of consecutive repeats required for binding to a given DNA sequence (Boch, J., et al., Science 326:1509-1512 (2009); Moscou, MJ, et al., Science 326:1501 (2009)). Furthermore, TAL effectors generated with new repeat combinations have been shown to bind to target sequences predicted by this code. Target DNA sequences have generally been shown to begin with a 5' thymine base, which is recognized by TAL proteins.

[0037] Terms such as "RNA-guided DNA nuclease" or "RNA-guided DNA endonuclease" refer in their ordinary and customary sense to an enzyme that cleaves phosphodiester bonds within a DNA polynucleotide strand, where recognition of the phosphodiester bond is facilitated by a separate RNA sequence (e.g., a single guide RNA).

[0038] The term "class II CRISPR endonuclease" refers to an endonuclease that has endonuclease activity similar to Cas9 and is involved in the class II CRISPR system. An exemplary class II CRISPR system is the type II CRISPR locus from Streptococcus pyogenes SF370, which contains a cluster of four genes, Cas9, Cas1, Cas2, and Csn1, as well as two non-coding RNA elements, tracrRNA, and a characteristic array of repetitive sequences (direct repeats) spaced by short stretches of non-repetitive sequences (spacers, each approximately 30 bp). The Cpf1 enzyme belongs to a putative type V CRISPR-Cas system. Both type II and type V systems are included in class II of the CRISPR-Cas system. The C2c1 ("class 2 candidate 1") enzyme is a type VB enzyme of class II. The C2c2 ("class 2 candidate 2") enzyme is a type VI-A enzyme of class II. C2c3 ("Class 2 Candidate 3") enzymes are Class II VC enzymes. Non-limiting exemplary CRISPR-associated proteins include Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Csy1, Csy2, Csy3, Cse1, Cse2, Csc1, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmr1, Cmr3, Cmr4, Cmr5, These include Cmr6, Csb1, Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, Csx1, Csx15, Csf1, Csf2, Csf3, Csf4, Cpf1, C2c1, C2c3, Cas12a, Cas12b, Cas12c, Cas12d, Cas12e, Cas13a, Cas13b, and Cas13.

[0039]

[0013] As referred to herein, "CRISPR-associated protein 9," "Cas9," "Csn1," or "Cas9 protein" includes either recombinant or naturally occurring forms of the Cas9 endonuclease or variants or homologs thereof that maintain Cas9 endonuclease enzymatic activity (e.g., within at least 50%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% of the activity compared to Cas9). In embodiments, the variant or homolog has at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity over the entire sequence or a portion of the sequence (e.g., a portion of 50, 100, 150, or 200 contiguous amino acids) compared to a naturally occurring Cas9 protein. In embodiments, the Cas9 protein is substantially identical to the protein identified by UniProt reference number Q99ZW2, or a variant or homolog having substantial identity thereto. In embodiments, the Cas9 protein has at least 75% sequence identity with the amino acid sequence of the protein identified by UniProt reference number Q99ZW2. In embodiments, the Cas9 protein has at least 80% sequence identity with the amino acid sequence of the protein identified by UniProt reference number Q99ZW2. In embodiments, the Cas9 protein has at least 85% sequence identity with the amino acid sequence of the protein identified by UniProt reference number Q99ZW2. In embodiments, the Cas9 protein has at least 90% sequence identity with the amino acid sequence of the protein identified by UniProt reference number Q99ZW2. In embodiments, the Cas9 protein has at least 95% sequence identity with the amino acid sequence of the protein identified by UniProt reference number Q99ZW2.

[0040] As referred to herein, the "CRISPR-associated endonuclease Cas12a," "Cas12a," "Cas12," or "Cas12 protein" includes either recombinant or naturally occurring forms of the Cas12 endonuclease or variants or homologs thereof that maintain Cas12 endonuclease enzymatic activity (e.g., within at least 50%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% of the activity compared to Cas12). In various embodiments, the variants or homologs have at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity over the entire sequence or a portion of the sequence (e.g., a portion of 50, 100, 150, or 200 contiguous amino acids) compared to a naturally occurring Cas12 protein. In embodiments, the Cas12 protein is substantially identical to the protein identified by UniProt reference number A0Q7Q2, or a variant or homolog having substantial identity thereto.

[0041] "Cfp1" or "Cfp1 protein" as referred to herein includes either recombinant or naturally occurring forms of Cfp1 (CxxC finger protein 1) endonuclease or variants or homologs thereof that maintain Cfp1 endonuclease enzymatic activity (e.g., within at least 50%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% of the activity compared to Cfp1). In some embodiments, a variant or homolog has at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity over the entire sequence or a portion of the sequence (e.g., a portion of 50, 100, 150, or 200 contiguous amino acids) compared to a naturally occurring Cfp1 protein. In various embodiments, the Cfp1 protein is substantially identical to the protein identified by UniProt reference number Q9P0U4 or a variant or homolog having substantial identity thereto.

[0042]

[0013] The terms "RNA-guided RNA nuclease" or "RNA-guided RNAase" and the like refer in their ordinary and customary sense to an RNA-guided RNA nuclease that targets a specific phosphodiester bond within an RNA polynucleotide, where recognition of the phosphodiester bond is facilitated by a separate polynucleotide sequence (e.g., an RNA sequence (e.g., single guide RNA (sgRNA), guide RNA (gRNA)). Typically, the RNA-guided RNase targets single-stranded RNA. In embodiments, the RNA-guided RNase is Cas13 (e.g., Cas13a, Cas13b).

[0043] As referred to herein, "Cas13a" or "Cas13a protein" includes either recombinant or naturally occurring forms of Cas13a (CRISPR-associated endoribonuclease Cas13a) endonuclease, also known as CRISPR-associated endoribonuclease C2c2, C2c2, or variants or homologs thereof that maintain Cas13a endonuclease enzymatic activity (e.g., within the range of at least 50%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% activity compared to Cas13a). In some embodiments, variants or homologs have at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity over the entire sequence or a portion of the sequence (e.g., a portion of 50, 100, 150, or 200 contiguous amino acids) compared to a naturally occurring Cas13a protein. In embodiments, the Cas13a protein is substantially identical to the protein identified by UniProt reference number C7NBY4 or a variant or homolog having substantial identity thereto.

[0044] As referred to herein, "Cas13b" or "Cas13b protein" includes either recombinant or naturally occurring forms of Cas13b (CRISPR-associated RNA-guided ribonuclease Cas13b) endonuclease, or variants or homologs thereof that maintain Cas13b nuclease enzymatic activity (e.g., within the range of at least 50%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% activity compared to Cas13b). In some embodiments, the variant or homolog has at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity over the entire sequence or a portion of the sequence (e.g., a portion of 50, 100, 150, or 200 contiguous amino acids) compared to a naturally occurring Cas13b protein. In embodiments, the Cas13b protein is substantially identical to the protein identified by UniProt reference number A0A8G0P913 or a variant or homolog having substantial identity thereto.

[0045] In some embodiments, the gene editing reagent comprises Cas-CLOVER. In some embodiments, Cas-CLOVER comprises a Cro 051 nuclease domain fused to a catalytically dead Cas9. See, e.g., U.S. Patent Application Publication No. 2021 / 0107993 and Madison et al., Molecular Therapy Nucleic Acids, Vol. 29, pp. 979-995, September 13, 2022, each of which is incorporated herein by reference in its entirety. In some embodiments, the gene editing reagent comprises a nickase, e.g., nCas9 (nickase-Cas9). A nickase is an engineered Cas protein that can introduce single-strand breaks with the same specificity as regular CRISPR / Cas nucleases. See, e.g., International Publication No. WO 2014093694, incorporated herein by reference in its entirety.

[0046] The terms "guide RNA" and "gRNA" are used interchangeably and refer to a polynucleotide sequence comprising a crRNA sequence and, optionally, a tracrRNA sequence. In embodiments, a gRNA comprises a crRNA sequence and a tracrRNA sequence (e.g., a "single guide RNA" or "sgRNA"). In embodiments, a gRNA does not comprise a tracrRNA sequence. A crRNA sequence comprises a guide sequence (i.e., a "guide" or "spacer") and a tracr mate sequence (i.e., a direct repeat). The term "guide sequence" refers to a sequence that specifies a target site. Generally, a tracr mate sequence includes any sequence that has sufficient complementarity with a tracrRNA sequence to promote one or more of the following: (1) excision of the guide sequence adjacent to the tracr mate sequence in cells containing the corresponding tracr sequence; and (2) formation of a complex (e.g., a CRISPR complex) at a target sequence, where the complex (e.g., a CRISPR complex) comprises the tracr mate sequence hybridized to the tracr sequence.

[0047] In various embodiments, the gRNA is a single-stranded ribonucleic acid. In various aspects, the gRNA is about 10 to about 200 nucleic acid residues in length. In various aspects, the gRNA is about 50 to about 150 nucleic acid residues in length. In various aspects, the gRNA is about 80 to about 140 nucleic acid residues in length. In various aspects, the gRNA is about 90 to about 130 nucleic acid residues in length. In various aspects, the gRNA is about 100 to about 120 nucleic acid residues in length.

[0048] Generally, a guide sequence is any polynucleotide sequence that has sufficient complementarity with a target polynucleotide sequence to hybridize with the target sequence and direct sequence-specific binding of a CRISPR complex to the target sequence. In some embodiments, the degree of complementarity between a guide sequence and its corresponding target sequence is about 50%, 60%, 75%, 80%, 85%, 90%, 95%, 97.5%, 99% or more when optimally aligned using a suitable alignment algorithm. Optimal alignment can be determined using any suitable algorithm for aligning sequences, non-limiting examples of which include the Smith-Waterman algorithm, the Needleman-Wunsch algorithm, algorithms based on the Burrows-Wheeler transformation (e.g., Burrows Wheeler Aligner), ClustalW, Clustal X, BLAT, Novoalign (Novocraft Technologies), ELAND (Illumina, San Diego, Calif.), SOAP (available at soap.genomics.org.cn), and Maq (available at maq.sourceforge.net). In embodiments, the guide sequence is about 5, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 75, or more nucleotides in length. In various embodiments, guide sequence is less than about 75, 50, 45, 40, 35, 30, 25, 20, 15, 12 nucleotides in length or less.The ability of guide sequence to direct the sequence-specific binding of CRISPR complex to target sequence can be evaluated by any suitable assay.For example, the components of CRISPR system sufficient to form the CRISPR complex that comprises the guide sequence to be tested can be provided to the host cell that has corresponding target sequence, for example, by transfection with the vector that encodes the components of CRISPR sequence, and then evaluate the preferential cleavage within the target sequence, for example, by the Surveyor assay described herein.Similarly, cleavage of a target polynucleotide sequence can be assessed in vitro by providing components of a CRISPR complex that include the target sequence, a test guide sequence, and a control guide sequence that is different from the test guide sequence, and comparing the binding or cleavage rate at the target sequence between the test and control guide sequence reactions. Other assays are possible and will occur to those skilled in the art.

[0049] As used herein, the term "donor DNA" refers to single-stranded or double-stranded DNA that can be inserted into the genome of a cell (e.g., a T cell) using genetic modification methods (e.g., CRISPR). For example, the donor DNA may have homologous arms that are homologous to a region of a gene into which the donor DNA is inserted. For example, the donor DNA may form a complex with a Cas protein. In some cases, the cell may be transfected with gene editing reagents and the donor DNA. In various embodiments, the donor DNA is part of a plasmid, vector, or expression vector that facilitates delivery of the donor DNA into the cell. In various embodiments, the donor DNA is part of a circular DNA. In various embodiments, the donor DNA is part of a linear DNA. In various embodiments, the donor DNA may include one or more modifications.

[0050] The nucleic acid (such as donor DNA) used in the methods herein may be modified.For example, nucleic acid may contain known synthetic, natural and unnatural nucleotide analogs or modified backbone residues or bonds, which have the same binding properties as reference nucleic acid and are metabolized in the same manner as reference nucleotide.Examples of such analogs include, but are not limited to, phosphodiester derivatives, including, for example, phosphoramidate, phosphorodiamidate, phosphorothioate (also known as phosphorothioate, which has a double bond sulfur replacing the oxygen in phosphate), phosphorodithioate, phosphonocarboxylic acid, phosphonocarboxylate, phosphonoacetic acid, phosphonoformic acid, methylphosphonate, boronphosphonate, or O-methylphosphoramidite linkage (see Eckstein, OLIGONUCLEOTIDES AND ANALOGUES: A PRACTICAL APPROACH, Oxford University Press), and modifications to nucleotide bases such as 5-methylcytidine or pseudouridine; and peptide nucleic acid backbone and linkage. Other analog nucleic acids include those with normal backbones; non-ionic backbones, modified sugars, and non-ribose backbones (e.g., phosphorodiamidate morpholino oligos or locked nucleic acids (LNAs) known in the art), including those described in U.S. Patent Nos. 5,235,033 and 5,034,506 and Chapters 6 and 7, ASC Symposium Series 580, CARBOHYDRATE MODIFICATIONS IN ANTISENSE RESEARCH, Sanghui & Cook, eds. Nucleic acids containing one or more carbocyclic sugars are also included within one definition of nucleic acid. Mixtures of naturally occurring nucleic acids and analogs can be made; alternatively, mixtures of different nucleic acid analogs, as well as mixtures of naturally occurring nucleic acids and analogs, can be made. In various embodiments, the internucleotide linkages in DNA are phosphodiester, phosphodiester derivatives, or a combination of both.

[0051] As used herein, "TANK-binding kinase 1 protein" or "TBK1" includes either recombinant or naturally occurring forms of TANK-binding kinase 1 (TBK1) (also known as serine / threonine-protein kinase TBK1), NF-kappa-B-activating kinase, T2K, or variants or homologs thereof that maintain TANK-binding kinase 1 (TBK1) activity (e.g., in the range of at least 50%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% activity compared to TBK1). In various embodiments, the variant or homolog has at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity over the entire sequence or a portion of the sequence (e.g., a portion of 50, 100, 150, or 200 contiguous amino acids) compared to a naturally occurring TBK1 protein. In embodiments, the TBK1 protein is substantially identical to the protein identified by UniProt reference number Q9UHD2 or a variant or homolog having substantial identity thereto.

[0052] As used herein, "cyclic GMP-AMP synthase protein" or "cGAS" includes either recombinant or naturally occurring forms of cyclic GMP-AMP synthase protein (cGAS) or variants or homologs thereof that maintain cGAS activity (e.g., in the range of at least 50%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% activity compared to cGAS). In embodiments, the variant or homolog has at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity over the entire sequence or a portion of the sequence (e.g., a portion of 50, 100, 150, or 200 contiguous amino acids) compared to a naturally occurring cGAS protein. In embodiments, the cGAS protein is substantially identical to the protein identified by UniProt reference number Q8N884, or a variant or homolog having substantial identity thereto.

[0053] As used herein, "stimulator of interferon genes" or "STING" includes either recombinant or naturally occurring forms of stimulator of interferon genes (STING) (also called endoplasmic reticulum interferon stimulator), a mediator of IRF3 activation, transmembrane protein 173, or variants or homologs thereof that maintain STING activity (e.g., within the range of at least 50%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% activity compared to STING). In embodiments, the variant or homolog has at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity over the entire sequence or a portion of the sequence (e.g., a portion of 50, 100, 150, or 200 contiguous amino acids) compared to a naturally occurring STING protein. In embodiments, the STING protein is substantially identical to the protein identified by UniProt reference number Q86WV6 or a variant or homologue having substantial identity thereto.

[0054] As used herein, "interferon regulatory factor 3 protein" or "IRF3" includes either recombinant or naturally occurring forms of interferon regulatory factor 3 (IRF3) or variants or homologs thereof that maintain IRF3 activity (e.g., in the range of at least 50%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% activity compared to IRF3). In embodiments, the variant or homolog has at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity over the entire sequence or a portion of the sequence (e.g., a portion of 50, 100, 150, or 200 contiguous amino acids) compared to a naturally occurring IRF3 protein. In embodiments, the IRF3 protein is substantially identical to the protein identified by UniProt reference number Q14653, or a variant or homolog having substantial identity thereto.

[0055] The term "gene" refers to a segment of DNA involved in producing a protein. It includes regions preceding and following the coding region (leader and trailer) as well as intervening sequences (introns) between individual coding segments (exons). The leader, trailer, and introns contain regulatory elements required during transcription and translation of a gene. Furthermore, a "protein gene product" is a protein expressed from a particular gene.

[0056] The terms "plasmid," "vector," or "expression vector" refer to a nucleic acid molecule that encodes a gene and / or regulatory elements necessary for expression of a gene. In embodiments, the plasmid, vector, or expression vector is a circular nucleic acid. In embodiments, the plasmid, vector, or expression vector is not a linear nucleic acid. In embodiments, the plasmid, vector, or expression vector is a linear nucleic acid.

[0057] As used herein, the term "nanoplasmid" is used to refer to a circular nucleic acid that contains at least a nucleic acid sequence(s) of interest, a mini origin of replication (e.g., R6K), and a selectable marker (e.g., a small RNA selectable marker, RNA-OUT). In embodiments, the nanoplasmid contains less than 500 bp of prokaryotic DNA.

[0058] As used herein, the term "minicircle" generally refers to a circular nucleic acid of about 200 bases to about 5 kilobases in length. In various embodiments, the minicircle is about 2 kilobases to about 5 kilobases in length. In various embodiments, the minicircle does not contain prokaryotic DNA. Thus, in various embodiments, the minicircle contains at least the nucleic acid sequence(s) of interest and the elements necessary for expression of the nucleic acid sequence.

[0059] As used herein, terms such as "T cell engineering" or "T cell genetic engineering" refer to a type of genetic modification in which DNA is inserted, deleted, modified, or replaced at one or more specified locations within the genome of a T cell. Unlike earlier genetic engineering techniques that randomly insert genetic material into the host genome, T cell engineering targets genetic modifications at site-specific locations. Gene editing reagents can be used to engineer T cells, for example, to generate double-stranded breaks at specific points within a gene or genome where DNA is inserted. Gene editing reagents can include, for example, clustered regularly interspaced short palindromic repeats (CRISPR / Cas), ZFNs, or TALENs. Thus, an "engineered T cell" is a T cell in which DNA has been inserted, deleted, modified, or replaced at one or more specific locations within the T cell genome.

[0060] The term "recombinant," when used with reference to, for example, a virus, cell, nucleic acid, protein, or vector, indicates that the cell (e.g., T cell), nucleic acid, protein, or vector has been modified by the introduction of a heterologous nucleic acid or protein or the alteration of a native nucleic acid or protein, or that the cell is derived from a cell so modified. In some cases, recombinant cells express genes that are not found in the native (non-recombinant) form of the cell, or express native genes that are otherwise aberrantly expressed, under-expressed, or not expressed at all. Transgenic cells are those that express heterologous genes or coding sequences, typically as a result of recombinant methods.

[0061] The term "heterologous," when used with reference to portions of a nucleic acid, indicates that the nucleic acid comprises two or more subsequences that are not found in the same relationship to each other in nature. For example, a nucleic acid can be recombinantly produced, with two or more sequences from unrelated genes arranged to create a new functional nucleic acid, e.g., a promoter from one source and a coding region from another source. Similarly, a heterologous protein indicates that the protein comprises two or more subsequences that are not found in the same relationship to each other in nature (e.g., a fusion protein).

[0062] The term "exogenous" refers to a molecule or substance (e.g., a compound, nucleic acid, or protein) that originates from outside a given cell or organism. For example, an "exogenous promoter" as referred to herein is a promoter that does not originate from the cell or organism that expresses it. Conversely, the terms "endogenous" or "endogenous promoter" refer to a molecule or substance that is native to or originates from within a given cell or organism.

[0063] The term "isolated," when applied to a nucleic acid or protein, indicates that the nucleic acid or protein is essentially free from other cellular components with which it is naturally associated. It can be, for example, in a homogeneous state, either dry or in an aqueous solution. Purity and homogeneity are typically determined using analytical chemistry techniques such as polyacrylamide gel electrophoresis or high performance liquid chromatography. A nucleic acid that is the predominant species present in a preparation is substantially purified.

[0064] As used herein, the terms "electroporation," "electropermeabilization," and "electrotransfer" are used according to their simple ordinary meaning to refer to a technique in which an electric field is applied to cells to increase the permeability of the cell membrane, allowing chemicals, drugs, proteins, or nucleic acids, or combinations thereof, to be introduced into the cells.

[0065] The terms "transfection," "transduction," "transfecting," or "transducing" can be used interchangeably and are defined as the process of introducing a nucleic acid molecule or protein into a cell. Nucleic acids are introduced into cells using non-viral or viral-based methods. The nucleic acid molecule can be a genetic sequence encoding an entire protein or a functional portion thereof. Non-viral methods of transfection include any suitable transfection method that does not use viral DNA or viral particles as a delivery system for introducing nucleic acid molecules into cells. Exemplary non-viral transfection methods include calcium phosphate transfection, liposome transfection, nucleofection, sonoporation, heat shock transfection, magnetofection, and electroporation. In some embodiments, nucleic acid molecules are introduced into cells using electroporation according to standard procedures well known in the art. For viral-based transfection methods, any useful viral vector (e.g., adenoviral vector) can be used in the methods described herein. Examples of viral vectors include, but are not limited to, retroviral vectors, adenoviral vectors, lentiviral vectors, and adeno-associated viral vectors. In some embodiments, nucleic acid molecules are introduced into cells using adenovirus vectors according to standard procedures well known in the art. The term "transfection" or "transduction" also refers to the introduction of proteins into cells from the external environment. In various embodiments, protein transduction or transfection relies on the attachment of peptides or proteins that can cross the cell membrane to the target protein. See, for example, Ford et al. (2001) Gene Therapy 8:1-4 and Prochiantz (2007) Nat. Methods 4:119-20.

[0066] "Transduce" or "transduction" are used according to their plain ordinary meaning to refer to the process by which one or more foreign nucleic acids (i.e., DNA not naturally found in the cell) are introduced into a cell. Transduction can be accomplished by introducing a virus or viral vector (e.g., an adenoviral vector) into the cell.

[0067] The term "expression" or "expressed," as used herein with respect to a gene, refers to the transcription and / or translation product of that gene (e.g., TCR-alpha, TCR-beta, etc.). The expression level of a DNA molecule in a cell can be determined based on either the amount of corresponding mRNA present in the cell or the amount of protein encoded by that DNA produced by the cell. The expression level of a nucleic acid molecule can be detected by standard methods, including PCR or Northern blotting, well known in the art. See Sambrook et al., 1989 Molecular Cloning: A Laboratory Manual, 18.1-18.88.

[0068] "Contacting" is used according to its plain and ordinary meaning and refers to a process that allows at least two different species (e.g., compounds, including biomolecules or cells) to come into sufficient proximity for reaction, interaction, or physical contact. The two species can be, for example, a cGAS-STING pathway inhibitor provided herein and a T cell. In embodiments, contacting includes, for example, physically contacting a cGAS-STING pathway inhibitor described herein with a T cell. In embodiments, contacting can result in delivery of a compound to the cell. For example, contacting can result in delivery of a cGAS-STING inhibitor to the cell. In embodiments, contacting can result in delivery of a nucleic acid to the cell. In embodiments, "contacting" or "contacted" includes culturing a species, for example, a T cell, in the presence of a cGAS-STING pathway inhibitor.

[0069] A "control" or "standard control" refers to a sample, measurement, or value that serves as a reference, typically a known reference, for comparison with a test sample, measurement, or value. For example, a standard control can be engineered T cells generated without contacting the T cells with one or more cGAS-STING inhibitors provided herein, including embodiments thereof. In various embodiments, a standard control can be a population of engineered T cells generated without contacting a population of T cells with one or more cGAS-STING inhibitors provided herein, including embodiments thereof. Thus, a standard control can be engineered T cells generated by contacting T cells with a nucleic acid that does not contain one or more cGAS-STING inhibitors. A standard control can be a population of engineered T cells generated by contacting a population of T cells with a nucleic acid that does not contain one or more cGAS-STING inhibitors. Controls are also useful for determining the significance of data. For example, if the value of a given parameter varies widely in the control, the variation in the test sample is not considered significant. One of skill in the art will recognize that standard controls can be designed for assessment of any number of parameters (e.g., cell viability, cell proliferation, total edited cell number, gene editing efficiency, etc.).

[0070] Those skilled in the art will understand which standard control is most appropriate in a given situation, and can analyze data based on comparison with standard control value.Standard control is also useful for determining the significance (e.g., statistical significance) of data.For example, if the value of a given parameter varies greatly in standard control, the variation of test sample is not considered significant.

[0071] "T cells" or "T lymphocytes," as used herein, are a type of lymphocyte (a subtype of white blood cell) that plays a central role in cell-mediated immunity. They can be distinguished from other lymphocytes, such as B cells and natural killer cells, by the presence of T cell receptors on the cell surface. T cells include, for example, natural killer T (NKT) cells, cytotoxic T lymphocytes (CTLs), regulatory T (Treg) cells, and helper T cells. Different types of T cells can be distinguished by the use of T cell detection agents.

[0072] As defined herein, the terms "inhibition," "inhibit," "inhibiting," and the like, with respect to cell proliferation (e.g., cancer cell proliferation), mean to adversely affect a cell (e.g., reduce proliferation) or kill a cell. In some embodiments, inhibition refers to the reduction of a disease or disease symptom (e.g., cancer, cancer cell proliferation). In various embodiments, an "inhibitor" is a compound or protein that inhibits a receptor or another protein, for example, by binding, partially or completely blocking, reducing, preventing, delaying, inactivating, desensitizing, or downregulating activity (e.g., receptor activity or protein activity).

[0073] The term "disease" or "condition" refers to a state or condition of a patient or subject that can be treated with the compounds or methods provided herein. The disease may be cancer. In some further examples, "cancer" refers to human cancer. In various embodiments, the cancer is lymphoma, melanoma, or leukemia.

[0074] The term "associated with" or "associated with" in the context of a substance or activity or function of a substance associated with a disease (e.g., cancer) means that the substance or activity or function of the substance causes (in whole or in part) or the symptoms of the disease are caused (in whole or in part) by the substance or activity or function of the substance.

[0075] A "patient" or "subject in need thereof" refers to an organism suffering from or prone to a disease (e.g., cancer, etc.) or condition that can be treated by administration of a composition or pharmaceutical composition provided herein. Non-limiting examples include humans, other mammals, cows, rats, mice, dogs, monkeys, goats, sheep, cattle, deer, and other non-mammals. In some embodiments, the patient is a human.

[0076] As used herein, the term "cancer" refers to all types of cancer, neoplasms, or malignant tumors found in mammals (e.g., humans), including leukemia, lymphoma, carcinoma, and sarcoma. Exemplary cancers that can be treated with the compounds or methods provided herein include brain cancer, glioma, glioblastoma, neuroblastoma, prostate cancer, colorectal cancer, pancreatic cancer, medulloblastoma, melanoma, cervical cancer, stomach cancer, ovarian cancer, lung cancer, head cancer, Hodgkin's disease, and non-Hodgkin's lymphoma. Exemplary cancers that can be treated with the compounds or methods provided herein include cancer of the thyroid, endocrine system, brain, breast, cervix, colon, head and neck, liver, kidney, lung, ovary, pancreas, rectum, stomach, and uterus. Further examples include thyroid cancer, bile duct cancer, pancreatic adenocarcinoma, cutaneous melanoma, colon adenocarcinoma, rectal adenocarcinoma, gastric adenocarcinoma, esophageal carcinoma, head and neck squamous cell carcinoma, breast invasive carcinoma, lung adenocarcinoma, lung squamous cell carcinoma, non-small cell lung carcinoma, mesothelioma, multiple myeloma, neuroblastoma, glioma, glioblastoma multiforme, ovarian cancer, rhabdomyosarcoma, primary thrombocytosis, primary macroglobulinemia, primary brain tumor, malignant pancreatic insulinoma, malignant carcinoid, bladder cancer, premalignant skin lesions, testicular cancer, thyroid cancer, neuroblastoma, esophageal cancer, genitourinary cancer, malignant hypercalcemia, endometrial cancer, adrenocortical carcinoma, neoplasms of the endocrine or exocrine pancreas, medullary thyroid carcinoma, medullary thyroid carcinoma, melanoma, colorectal cancer, papillary thyroid carcinoma, hepatocellular carcinoma, or prostate cancer.

[0077] As used herein, "treating" or "treatment" of a symptom, disease, or disorder, or symptoms associated with a symptom, disease, or disorder, refers to an approach to obtaining beneficial or desired results, including clinical results. Beneficial or desired clinical results can include, but are not limited to, alleviation or amelioration of one or more symptoms or symptoms, reduction in the severity of the symptom, disorder, or disease, stabilization of the symptom, disorder, or disease state, prevention of the onset of the symptom, disorder, or disease, prevention of the spread of the symptom, disorder, or disease, delaying or slowing the progression of the symptom, disorder, or disease, delaying or slowing the onset of the symptom, disorder, or disease, improvement or remission of the symptom, disorder, or disease state, and remission, whether partial or total. "Treating" can also mean extending the survival of a subject beyond that expected in the absence of treatment. "Treating" can also mean inhibiting the progression of the symptom, disorder, or disease, temporarily slowing the progression of the symptom, disorder, or disease, but in some cases includes permanently halting the progression of the symptom, disorder, or disease.

[0078] The terms "dose" and "dosage" are used interchangeably herein. Dose refers to the amount of active ingredient given to an individual at each administration. Dosage varies depending on several factors, including the usual dose range for a given treatment, frequency of administration; size and tolerance of the individual; severity of symptoms; risk of side effects; and route of administration. Those skilled in the art will recognize that dosage may be modified depending on the above factors or based on the progress of treatment. The term "dosage form" refers to the specific format of a medicament or pharmaceutical composition and depends on the route of administration. For example, the dosage form may be a liquid form for injection, for example.

[0079] As used herein, a "therapeutically effective dose or amount" refers to a dose that produces the effect for which it is administered (e.g., treatment of a disease). The exact dose and formulation will depend on the purpose of the treatment and will be ascertainable by one of ordinary skill in the art using known techniques. For example, for a given parameter, a therapeutically effective amount will exhibit an increase or decrease of at least 5%, 10%, 15%, 20%, 25%, 40%, 50%, 60%, 75%, 80%, 90%, or at least 100%. Therapeutic efficacy can also be expressed as a "-fold" increase or decrease. For example, a therapeutically effective amount may have at least a 1.2-fold, 1.5-fold, 2-fold, 5-fold, or greater effect compared to a standard control. A therapeutically effective dose or amount may ameliorate one or more symptoms of a disease.

[0080] As used herein, the term "administration" is used according to its plain and ordinary meaning and includes any administration appropriate for cell therapy. Parenteral administration includes, for example, intravenous administration, intramuscular administration, intraarterial administration, intradermal administration, subcutaneous administration, intraperitoneal administration, intraperitoneal administration, and intracranial administration. In various embodiments, administration is intravenous administration.

[0081] As used herein, the term "signal transduction pathway" refers to a series of interactions between cellular and, optionally, extracellular components (e.g., proteins, nucleic acids, small molecules, ions, lipids) that transmit a change in one component to one or more other components, which may transmit changes to additional components, which may then be propagated to other signal transduction pathway components. In various embodiments, the signal transduction pathway is the cyclic GMP-AMP synthase (cGAS)-stimulator of interferon genes (STING) pathway. A signal transduction pathway can be activated, for example, by the presence of a compound (e.g., a circular nucleic acid) within a cell. For example, a signal transduction pathway can be activated by the presence of a circular nucleic acid (e.g., a plasmid) within the cytoplasm of a cell. In some cases, a signal transduction pathway can be activated by one or more conditions to which the cell is subjected (e.g., transduction, transfection, or electroporation of a nucleic acid into the cell).

[0082] methodProvided herein, among other things, are methods for engineering T cells, including contacting T cells with one or more cyclic GMP-AMP synthase (cGAS)-stimulator of interferon genes (STING) pathway inhibitors. The methods provided herein enable the formation of engineered T cells while inhibiting or reducing cytotoxicity associated with previously known methods of engineering T cells. For example, the methods provided herein are believed to be effective in reducing or inhibiting toxicity associated with the presence of DNA (e.g., plasmid DNA) in the cytoplasm of cells. For example, the methods provided herein are effective in reducing or inhibiting the cGAS-STING pathway, thereby reducing or inhibiting cell death. As used herein, "cyclic GMP-AMP synthase-stimulator of interferon genes pathway inhibitor" or "cGAS-STING pathway inhibitor" refers to a compound that inhibits or downregulates the activity or production of any one of the components of the cGAS-STING axis. For example, a cGAS-STING pathway inhibitor may inhibit or downregulate the activity or production of any one of the components of the cGAS pathway. In various embodiments, the cGAS-STING pathway inhibitor inhibits the binding of cGAS to double-stranded DNA (e.g., plasmid DNA). In various embodiments, the cGAS-STING pathway inhibitor inhibits the activity of cGAMP. In another example, the cGAS-STING pathway inhibitor may inhibit or downregulate the activity or production of any one of the components of the STING pathway. In various embodiments, the cGAS-STING pathway inhibitor may inhibit STING oligomerization. In another example, the cGAS-STING pathway inhibitor may inhibit or downregulate the activity or production of TANK-binding kinase 1 (TBK1). In various embodiments, the cGAS-STING pathway inhibitor inhibits TBK1 phosphorylation or STING phosphorylation. In various embodiments, the cGAS-STING pathway inhibitor inhibits the phosphorylation of interferon regulatory factor 3 (IRF-3). Thus, in various embodiments, the cGAS-STING pathway inhibitor comprises a kinase inhibitor (e.g., BX795, Aml, MRT, etc.). In various embodiments, the cGAS-STING pathway inhibitor is a kinase inhibitor.In one aspect, a method of engineering a T cell is provided, comprising contacting the T cell with a nucleic acid and one or more cyclic GMP-AMP synthase (cGAS)-stimulator of interferon genes (STING) pathway inhibitors.

[0083] In various embodiments, the nucleic acid is about 20 bases to about 30,000 bases in length. In various embodiments, the nucleic acid is about 1,000 bases to about 30,000 bases in length. In various embodiments, the nucleic acid is about 2,000 bases to about 30,000 bases in length. In various embodiments, the nucleic acid is about 3,000 bases to about 30,000 bases in length. In various embodiments, the nucleic acid is about 4,000 bases to about 30,000 bases in length. In various embodiments, the nucleic acid is about 5,000 bases to about 30,000 bases in length. In various embodiments, the nucleic acid is about 6,000 bases to about 30,000 bases in length. In various embodiments, the nucleic acid is about 7,000 bases to about 30,000 bases in length. In various embodiments, the nucleic acid is about 8,000 bases to about 30,000 bases in length. In various embodiments, the nucleic acid is about 9,000 bases to about 30,000 bases in length. In various embodiments, the nucleic acid is about 10,000 to about 30,000 bases in length. In various embodiments, the nucleic acid is about 11,000 to about 30,000 bases in length. In various embodiments, the nucleic acid is about 12,000 to about 30,000 bases in length. In various embodiments, the nucleic acid is about 13,000 to about 30,000 bases in length. In various embodiments, the nucleic acid is about 14,000 to about 30,000 bases in length. In various embodiments, the nucleic acid is about 15,000 to about 30,000 bases in length. In various embodiments, the nucleic acid is about 16,000 to about 30,000 bases in length. In various embodiments, the nucleic acid is about 17,000 to about 30,000 bases in length. In various embodiments, the nucleic acid is about 18,000 to about 30,000 bases in length. In various embodiments, the nucleic acid is about 19,000 to about 30,000 bases in length. In various embodiments, the nucleic acid is about 20,000 to about 30,000 bases in length. In various embodiments, the nucleic acid is about 21,000 to about 30,000 bases in length. In various embodiments, the nucleic acid is about 22,000 to about 30,000 bases in length. In various embodiments, the nucleic acid is about 23,000 to about 30,000 bases in length. In various embodiments, the nucleic acid is about 24,000 to about 30,000 bases in length. In various embodiments, the nucleic acid is about 25,000 to about 30,000 bases in length. In various embodiments, the nucleic acid is about 26,000 to about 30,000 bases in length. In various embodiments, the nucleic acid is about 27,000 to about 30,000 bases in length. In various embodiments, the nucleic acid is from about 28,000 bases to about 30,000 bases in length.In various embodiments, the nucleic acid is from about 29,000 bases to about 30,000 bases in length.

[0084] In various embodiments, the nucleic acid is about 20 bases to about 29,000 bases in length. In various embodiments, the nucleic acid is about 20 bases to about 28,000 bases in length. In various embodiments, the nucleic acid is about 20 bases to about 27,000 bases in length. In various embodiments, the nucleic acid is about 20 bases to about 26,000 bases in length. In various embodiments, the nucleic acid is about 20 bases to about 25,000 bases in length. In various embodiments, the nucleic acid is about 20 bases to about 24,000 bases in length. In various embodiments, the nucleic acid is about 20 bases to about 23,000 bases in length. In various embodiments, the nucleic acid is about 20 bases to about 22,000 bases in length. In various embodiments, the nucleic acid is about 20 bases to about 21,000 bases in length. In various embodiments, the nucleic acid is about 20 bases to about 20,000 bases in length. In various embodiments, the nucleic acid is about 20 bases to about 19,000 bases in length. In various embodiments, the nucleic acid is about 20 bases to about 18,000 bases in length. In various embodiments, the nucleic acid is about 20 bases to about 17,000 bases in length. In various embodiments, the nucleic acid is about 20 bases to about 16,000 bases in length. In various embodiments, the nucleic acid is about 20 bases to about 15,000 bases in length. In various embodiments, the nucleic acid is about 20 bases to about 14,000 bases in length. In various embodiments, the nucleic acid is about 20 bases to about 13,000 bases in length. In various embodiments, the nucleic acid is about 20 bases to about 12,000 bases in length. In various embodiments, the nucleic acid is about 20 bases to about 11,000 bases in length. In various embodiments, the nucleic acid is about 20 bases to about 10,000 bases in length. In various embodiments, the nucleic acid is about 20 bases to about 9,000 bases in length. In various embodiments, the nucleic acid is about 20 bases to about 8,000 bases in length. In various embodiments, the nucleic acid is about 20 bases to about 7000 bases in length. In various embodiments, the nucleic acid is about 20 bases to about 6000 bases in length. In various embodiments, the nucleic acid is about 20 bases to about 5000 bases in length. In various embodiments, the nucleic acid is about 20 bases to about 4000 bases in length. In various embodiments, the nucleic acid is about 20 bases to about 3000 bases in length. In various embodiments, the nucleic acid is about 20 bases to about 2000 bases in length. In various embodiments, the nucleic acid is about 20 bases to about 1000 bases in length.In embodiments, the nucleic acid is about 20, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10000, 11000, 12000, 13000, 14000, 15000, 16000, 17000, 18000, 19000, 20000, 21000, 22000, 23000, 24000, 25000, 26000, 27000, 28000, 29000, or 30000 in length.

[0085] In various embodiments, the nucleic acid is about 20 bases to about 5000 bases in length. In various embodiments, the nucleic acid is about 50 bases to about 5000 bases in length. In various embodiments, the nucleic acid is about 100 bases to about 5000 bases in length. In various embodiments, the nucleic acid is about 150 bases to about 5000 bases in length. In various embodiments, the nucleic acid is about 200 bases to about 5000 bases in length. In various embodiments, the nucleic acid is about 250 bases to about 5000 bases in length. In various embodiments, the nucleic acid is about 300 bases to about 5000 bases in length. In various embodiments, the nucleic acid is about 350 bases to about 5000 bases in length. In various embodiments, the nucleic acid is about 400 bases to about 5000 bases in length. In various embodiments, the nucleic acid is about 450 bases to about 5000 bases in length. In various embodiments, the nucleic acid is about 500 bases to about 5000 bases in length. In various embodiments, the nucleic acid is about 550 bases to about 5000 bases in length. In various embodiments, the nucleic acid is about 600 bases to about 5000 bases in length. In various embodiments, the nucleic acid is about 650 bases to about 5000 bases in length. In various embodiments, the nucleic acid is about 700 bases to about 5000 bases in length. In various embodiments, the nucleic acid is about 750 bases to about 5000 bases in length. In various embodiments, the nucleic acid is about 800 bases to about 5000 bases in length. In various embodiments, the nucleic acid is about 850 bases to about 5000 bases in length. In various embodiments, the nucleic acid is about 900 bases to about 5000 bases in length. In various embodiments, the nucleic acid is about 950 bases to about 5000 bases in length. In various embodiments, the nucleic acid is about 1000 bases to about 5000 bases in length. In various embodiments, the nucleic acid is about 1050 bases to about 5000 bases in length. In various embodiments, the nucleic acid is about 1100 bases to about 5000 bases in length. In various embodiments, the nucleic acid is about 1150 bases to about 5000 bases in length. In various embodiments, the nucleic acid is about 1200 bases to about 5000 bases in length. In various embodiments, the nucleic acid is about 1250 bases to about 5000 bases in length. In various embodiments, the nucleic acid is about 1300 bases to about 5000 bases in length. In various embodiments, the nucleic acid is about 1350 bases to about 5000 bases in length. In various embodiments, the nucleic acid is about 1400 bases to about 5000 bases in length. In various embodiments, the nucleic acid is about 1450 bases to about 5000 bases in length. In various embodiments, the nucleic acid is about 1500 bases to about 5000 bases in length.In various embodiments, the nucleic acid is about 1550 bases to about 5000 bases in length. In various embodiments, the nucleic acid is about 1600 bases to about 5000 bases in length. In various embodiments, the nucleic acid is about 1650 bases to about 5000 bases in length. In various embodiments, the nucleic acid is about 1700 bases to about 5000 bases in length. In various embodiments, the nucleic acid is about 1750 bases to about 5000 bases in length. In various embodiments, the nucleic acid is about 1800 bases to about 5000 bases in length. In various embodiments, the nucleic acid is about 1850 bases to about 5000 bases in length. In various embodiments, the nucleic acid is about 1900 bases to about 5000 bases in length. In various embodiments, the nucleic acid is about 1950 bases to about 5000 bases in length. In various embodiments, the nucleic acid is about 2000 bases to about 5000 bases in length. In various embodiments, the nucleic acid is about 2050 bases to about 5000 bases in length. In various embodiments, the nucleic acid is about 2100 bases to about 5000 bases in length. In various embodiments, the nucleic acid is about 2150 bases to about 5000 bases in length. In various embodiments, the nucleic acid is about 2200 bases to about 5000 bases in length. In various embodiments, the nucleic acid is about 2250 bases to about 5000 bases in length. In various embodiments, the nucleic acid is about 2300 bases to about 5000 bases in length. In various embodiments, the nucleic acid is about 2350 bases to about 5000 bases in length. In various embodiments, the nucleic acid is about 2400 bases to about 5000 bases in length. In various embodiments, the nucleic acid is about 2450 bases to about 5000 bases in length. In various embodiments, the nucleic acid is about 2500 bases to about 5000 bases in length. In various embodiments, the nucleic acid is about 2550 bases to about 5000 bases in length. In various embodiments, the nucleic acid is about 2600 bases to about 5000 bases in length. In various embodiments, the nucleic acid is about 2650 bases to about 5000 bases in length. In various embodiments, the nucleic acid is about 2700 bases to about 5000 bases in length. In various embodiments, the nucleic acid is about 2750 bases to about 5000 bases in length. In various embodiments, the nucleic acid is about 2800 bases to about 5000 bases in length. In various embodiments, the nucleic acid is about 2850 bases to about 5000 bases in length. In various embodiments, the nucleic acid is about 2900 bases to about 5000 bases in length. In various embodiments, the nucleic acid is about 2950 bases to about 5000 bases in length. In various embodiments, the nucleic acid is about 3000 bases to about 5000 bases in length. In various embodiments, the nucleic acid is from about 3050 bases to about 5000 bases in length.In various embodiments, the nucleic acid is about 3100 bases to about 5000 bases in length. In various embodiments, the nucleic acid is about 3150 bases to about 5000 bases in length. In various embodiments, the nucleic acid is about 3200 bases to about 5000 bases in length. In various embodiments, the nucleic acid is about 3250 bases to about 5000 bases in length. In various embodiments, the nucleic acid is about 3300 bases to about 5000 bases in length. In various embodiments, the nucleic acid is about 3350 bases to about 5000 bases in length. In various embodiments, the nucleic acid is about 3400 bases to about 5000 bases in length. In various embodiments, the nucleic acid is about 3450 bases to about 5000 bases in length. In various embodiments, the nucleic acid is about 3500 bases to about 5000 bases in length. In various embodiments, the nucleic acid is about 3550 bases to about 5000 bases in length. In various embodiments, the nucleic acid is about 3600 to about 5000 bases in length. In various embodiments, the nucleic acid is about 3650 to about 5000 bases in length. In various embodiments, the nucleic acid is about 3700 to about 5000 bases in length. In various embodiments, the nucleic acid is about 3750 to about 5000 bases in length. In various embodiments, the nucleic acid is about 3800 to about 5000 bases in length. In various embodiments, the nucleic acid is about 3850 to about 5000 bases in length. In various embodiments, the nucleic acid is about 3900 to about 5000 bases in length. In various embodiments, the nucleic acid is about 3950 to about 5000 bases in length. In various embodiments, the nucleic acid is about 4000 to about 5000 bases in length. In various embodiments, the nucleic acid is about 4050 to about 5000 bases in length. In various embodiments, the nucleic acid is about 4100 bases to about 5000 bases in length. In various embodiments, the nucleic acid is about 4150 bases to about 5000 bases in length. In various embodiments, the nucleic acid is about 4200 bases to about 5000 bases in length. In various embodiments, the nucleic acid is about 4250 bases to about 5000 bases in length. In various embodiments, the nucleic acid is about 4300 bases to about 5000 bases in length. In various embodiments, the nucleic acid is about 4350 bases to about 5000 bases in length. In various embodiments, the nucleic acid is about 4400 bases to about 5000 bases in length. In various embodiments, the nucleic acid is about 4450 bases to about 5000 bases in length. In various embodiments, the nucleic acid is about 4500 bases to about 5000 bases in length. In various embodiments, the nucleic acid is about 4550 bases to about 5000 bases in length. In various embodiments, the nucleic acid is from about 4600 bases to about 5000 bases in length.In various embodiments, the nucleic acid is about 4650 bases to about 5000 bases in length. In various embodiments, the nucleic acid is about 4700 bases to about 5000 bases in length. In various embodiments, the nucleic acid is about 4750 bases to about 5000 bases in length. In various embodiments, the nucleic acid is about 4800 bases to about 5000 bases in length. In various embodiments, the nucleic acid is about 4850 bases to about 5000 bases in length. In various embodiments, the nucleic acid is about 4900 bases to about 5000 bases in length. In various embodiments, the nucleic acid is about 4950 bases to about 5000 bases in length.

[0086] In various embodiments, the nucleic acid is about 20 bases to about 4950 bases in length. In various embodiments, the nucleic acid is about 20 bases to about 4900 bases in length. In various embodiments, the nucleic acid is about 20 bases to about 4850 bases in length. In various embodiments, the nucleic acid is about 20 bases to about 4800 bases in length. In various embodiments, the nucleic acid is about 20 bases to about 4750 bases in length. In various embodiments, the nucleic acid is about 20 bases to about 4700 bases in length. In various embodiments, the nucleic acid is about 20 bases to about 4650 bases in length. In various embodiments, the nucleic acid is about 20 bases to about 4600 bases in length. In various embodiments, the nucleic acid is about 20 bases to about 4550 bases in length. In various embodiments, the nucleic acid is about 20 bases to about 4500 bases in length. In various embodiments, the nucleic acid is about 20 bases to about 4450 bases in length. In various embodiments, the nucleic acid is about 20 bases to about 4400 bases in length. In various embodiments, the nucleic acid is about 20 bases to about 4350 bases in length. In various embodiments, the nucleic acid is about 20 bases to about 4300 bases in length. In various embodiments, the nucleic acid is about 20 bases to about 4250 bases in length. In various embodiments, the nucleic acid is about 20 bases to about 4200 bases in length. In various embodiments, the nucleic acid is about 20 bases to about 4150 bases in length. In various embodiments, the nucleic acid is about 20 bases to about 4100 bases in length. In various embodiments, the nucleic acid is about 20 bases to about 4050 bases in length. In various embodiments, the nucleic acid is about 20 bases to about 4000 bases in length. In various embodiments, the nucleic acid is about 20 bases to about 3950 bases in length. In various embodiments, the nucleic acid is about 20 bases to about 3900 bases in length. In various embodiments, the nucleic acid is about 20 bases to about 3850 bases in length. In various embodiments, the nucleic acid is about 20 bases to about 3800 bases in length. In various embodiments, the nucleic acid is about 20 bases to about 3750 bases in length. In various embodiments, the nucleic acid is about 20 bases to about 3700 bases in length. In various embodiments, the nucleic acid is about 20 bases to about 3650 bases in length. In various embodiments, the nucleic acid is about 20 bases to about 3600 bases in length. In various embodiments, the nucleic acid is about 20 bases to about 3550 bases in length. In various embodiments, the nucleic acid is about 20 bases to about 3500 bases in length. In various embodiments, the nucleic acid is about 20 bases to about 3450 bases in length. In various embodiments, the nucleic acid is about 20 bases to about 3400 bases in length. In various embodiments, the nucleic acid is about 20 bases to about 3350 bases in length.In various embodiments, the nucleic acid is about 20 bases to about 3300 bases in length. In various embodiments, the nucleic acid is about 20 bases to about 3250 bases in length. In various embodiments, the nucleic acid is about 20 bases to about 3200 bases in length. In various embodiments, the nucleic acid is about 20 bases to about 3150 bases in length. In various embodiments, the nucleic acid is about 20 bases to about 3100 bases in length. In various embodiments, the nucleic acid is about 20 bases to about 3050 bases in length. In various embodiments, the nucleic acid is about 20 bases to about 3000 bases in length. In various embodiments, the nucleic acid is about 20 bases to about 2950 bases in length. In various embodiments, the nucleic acid is about 20 bases to about 2900 bases in length. In various embodiments, the nucleic acid is about 20 bases to about 2850 bases in length. In various embodiments, the nucleic acid is about 20 bases to about 2800 bases in length. In various embodiments, the nucleic acid is about 20 bases to about 2750 bases in length. In various embodiments, the nucleic acid is about 20 bases to about 2700 bases in length. In various embodiments, the nucleic acid is about 20 bases to about 2650 bases in length. In various embodiments, the nucleic acid is about 20 bases to about 2600 bases in length. In various embodiments, the nucleic acid is about 20 bases to about 2550 bases in length. In various embodiments, the nucleic acid is about 20 bases to about 2500 bases in length. In various embodiments, the nucleic acid is about 20 bases to about 2450 bases in length. In various embodiments, the nucleic acid is about 20 bases to about 2400 bases in length. In various embodiments, the nucleic acid is about 20 bases to about 2350 bases in length. In various embodiments, the nucleic acid is about 20 bases to about 2300 bases in length. In various embodiments, the nucleic acid is about 20 bases to about 2250 bases in length. In various embodiments, the nucleic acid is about 20 bases to about 2200 bases in length. In various embodiments, the nucleic acid is about 20 bases to about 2150 bases in length. In various embodiments, the nucleic acid is about 20 bases to about 2100 bases in length. In various embodiments, the nucleic acid is about 20 bases to about 2050 bases in length. In various embodiments, the nucleic acid is about 20 bases to about 2000 bases in length. In various embodiments, the nucleic acid is about 20 bases to about 1950 bases in length. In various embodiments, the nucleic acid is about 20 bases to about 1900 bases in length. In various embodiments, the nucleic acid is about 20 bases to about 1850 bases in length. In various embodiments, the nucleic acid is about 20 bases to about 1800 bases in length. In various embodiments, the nucleic acid is about 20 bases to about 1750 bases in length. In various embodiments, the nucleic acid is about 20 bases to about 1700 bases in length.In various embodiments, the nucleic acid is about 20 bases to about 1650 bases in length. In various embodiments, the nucleic acid is about 20 bases to about 1600 bases in length. In various embodiments, the nucleic acid is about 20 bases to about 1550 bases in length. In various embodiments, the nucleic acid is about 20 bases to about 1500 bases in length. In various embodiments, the nucleic acid is about 20 bases to about 1450 bases in length. In various embodiments, the nucleic acid is about 20 bases to about 1400 bases in length. In various embodiments, the nucleic acid is about 20 bases to about 1350 bases in length. In various embodiments, the nucleic acid is about 20 bases to about 1300 bases in length. In various embodiments, the nucleic acid is about 20 bases to about 1250 bases in length. In various embodiments, the nucleic acid is about 20 bases to about 1200 bases in length. In various embodiments, the nucleic acid is about 20 bases to about 1150 bases in length. In various embodiments, the nucleic acid is about 20 bases to about 1100 bases in length. In various embodiments, the nucleic acid is about 20 bases to about 1050 bases in length. In various embodiments, the nucleic acid is about 20 bases to about 1000 bases in length. In various embodiments, the nucleic acid is about 20 bases to about 950 bases in length. In various embodiments, the nucleic acid is about 20 bases to about 900 bases in length. In various embodiments, the nucleic acid is about 20 bases to about 850 bases in length. In various embodiments, the nucleic acid is about 20 bases to about 800 bases in length. In various embodiments, the nucleic acid is about 20 bases to about 750 bases in length. In various embodiments, the nucleic acid is about 20 bases to about 700 bases in length. In various embodiments, the nucleic acid is about 20 bases to about 650 bases in length. In various embodiments, the nucleic acid is about 20 bases to about 600 bases in length. In various embodiments, the nucleic acid is about 20 bases to about 550 bases in length. In various embodiments, the nucleic acid is about 20 bases to about 500 bases in length. In various embodiments, the nucleic acid is about 20 bases to about 450 bases in length. In various embodiments, the nucleic acid is about 20 bases to about 400 bases in length. In various embodiments, the nucleic acid is about 20 bases to about 350 bases in length. In various embodiments, the nucleic acid is about 20 bases to about 300 bases in length. In various embodiments, the nucleic acid is about 20 bases to about 250 bases in length. In various embodiments, the nucleic acid is about 20 bases to about 200 bases in length. In various embodiments, the nucleic acid is about 20 bases to about 150 bases in length. In various embodiments, the nucleic acid is about 20 bases to about 100 bases in length. In various embodiments, the nucleic acid is about 20 bases to about 50 bases in length.In embodiments, the nucleic acid is at or near 20, 50, 100, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1050, 1100, 1200, 1250, 1300, 1350, 1400, 1450, 1500, 1550, 1600, 1650, 1700, 1750, 1800, 1850, 1900, 1950, 2000, 2050, 2100, 2200, 2250, 2300, 2350, 2400, 2450, 2500, 2550, 2600, 2650, 2700, 2750, 2800, 2850, 2900, 3000, 3050, 3100, 3200, 3250, 3300, 3350, 3400, 3450, 3500, 3550, 3600, 3650, 3700, 3750, 3800, 3850, 3900, 4000, 4100, 4200, 4300, 4400, 4500, 4600, 4700, 4800, 4900, 5000, 5100, 5200, 0, 2650, 7200, 2750, 2800, 2850, 2900, 2950, ​​3000, 3050, 3100, 3200, 3250, 3300, 3350, 3400, 3450, 3500, 3550, 3600, 3650, 3700, 3750, 3800, 3850, 3900, 3950, 4000, 4050, 4100, 4200, 4250, 4300, 4350, 4400, 4450, 4500, 4550, 4600, 4650, 4700, 4750, 4800, 4850, 4900, 4950, or 5000 bases in length.

[0087] In various embodiments, the nucleic acid is about 20 bases to about 1000 bases in length. In various embodiments, the nucleic acid is about 100 bases to about 1000 bases in length. In various embodiments, the nucleic acid is about 200 bases to about 1000 bases in length. In various embodiments, the nucleic acid is about 300 bases to about 1000 bases in length. In various embodiments, the nucleic acid is about 400 bases to about 1000 bases in length. In various embodiments, the nucleic acid is about 500 bases to about 1000 bases in length. In various embodiments, the nucleic acid is about 600 bases to about 1000 bases in length. In various embodiments, the nucleic acid is about 700 bases to about 1000 bases in length. In various embodiments, the nucleic acid is about 800 bases to about 1000 bases in length. In various embodiments, the nucleic acid is about 900 bases to about 1000 bases in length.

[0088] In various embodiments, the nucleic acid is about 20 bases to about 900 bases in length. In various embodiments, the nucleic acid is about 20 bases to about 800 bases in length. In various embodiments, the nucleic acid is about 20 bases to about 700 bases in length. In various embodiments, the nucleic acid is about 20 bases to about 600 bases in length. In various embodiments, the nucleic acid is about 20 bases to about 500 bases in length. In various embodiments, the nucleic acid is about 20 bases to about 400 bases in length. In various embodiments, the nucleic acid is about 20 bases to about 300 bases in length. In various embodiments, the nucleic acid is about 20 bases to about 200 bases in length. In various embodiments, the nucleic acid is about 20 bases to about 100 bases in length. In various embodiments, the nucleic acid is about 20, 100, 200, 300, 400, 500, 600, 700, 800, 900, or 1000 bases in length.

[0089] For the methods provided herein, in embodiments, the nucleic acid comprises donor DNA, which is optionally inserted into the genome of the T cell, thereby producing an engineered T cell. Thus, in embodiments, the nucleic acid comprises donor DNA. In embodiments, the nucleic acid is double-stranded circular DNA (e.g., a plasmid). The donor DNA can be delivered to the cell using various methods, including transfection, electroporation, and viral transduction. Thus, in embodiments, the nucleic acid is a plasmid, nanoplasmid, minicircle, linear plasmid, or viral vector comprising donor DNA. In embodiments, the nucleic acid is a plasmid comprising donor DNA. In embodiments, the nucleic acid is a nanoplasmid comprising donor DNA. In embodiments, the nucleic acid is a minicircle comprising donor DNA. In embodiments, the nucleic acid is a viral vector comprising donor DNA. In embodiments, the DNA is not linear DNA (e.g., double-stranded linear DNA). In embodiments, the DNA is circular DNA (e.g., double-stranded circular DNA). In embodiments, the DNA is linear DNA. In embodiments, the DNA is single-stranded DNA.

[0090] The methods provided herein are useful for generating T cell receptor (TCR)-engineered T cells. For example, the methods can be used to replace (e.g., knock out) endogenous TCRs in T cells with tumor-specific antigen-associated TCRs (e.g., knock-in). Thus, in various embodiments, the donor DNA encodes exogenous T cell receptor (TCR)-alpha or a fragment thereof, exogenous TCR-beta or a fragment thereof, or a combination thereof. In various embodiments, the donor DNA encodes exogenous T cell receptor (TCR)-alpha or a fragment thereof. In various embodiments, the donor DNA encodes exogenous T cell receptor (TCR)-beta or a fragment thereof.

[0091] In various embodiments, the donor DNA is about 5 to about 1000 bases in length. In various embodiments, the donor DNA is about 100 to about 1000 bases in length. In various embodiments, the donor DNA is about 200 to about 1000 bases in length. In various embodiments, the donor DNA is about 300 to about 1000 bases in length. In various embodiments, the donor DNA is about 400 to about 1000 bases in length. In various embodiments, the donor DNA is about 500 to about 1000 bases in length. In various embodiments, the donor DNA is about 600 to about 1000 bases in length. In various embodiments, the donor DNA is about 700 to about 1000 bases in length. In various embodiments, the donor DNA is about 800 to about 1000 bases in length. In various embodiments, the donor DNA is about 900 to about 1000 bases in length.

[0092] In various embodiments, the donor DNA is about 5 to about 900 bases in length. In various embodiments, the donor DNA is about 5 to about 800 bases in length. In various embodiments, the donor DNA is about 5 to about 700 bases in length. In various embodiments, the donor DNA is about 5 to about 600 bases in length. In various embodiments, the donor DNA is about 5 to about 500 bases in length. In various embodiments, the donor DNA is about 5 to about 400 bases in length. In various embodiments, the donor DNA is about 5 to about 300 bases in length. In various embodiments, the donor DNA is about 5 to about 200 bases in length. In various embodiments, the donor DNA is about 5 to about 100 bases in length. In various embodiments, the donor DNA is about 5, 100, 200, 300, 400, 500, 600, 700, 800, 900, or 1000 bases in length.

[0093] In various embodiments, the donor DNA is about 50 to about 600 bases in length. In various embodiments, the donor DNA is about 100 to about 600 bases in length. In various embodiments, the donor DNA is about 150 to about 600 bases in length. In various embodiments, the donor DNA is about 200 to about 600 bases in length. In various embodiments, the donor DNA is about 250 to about 600 bases in length. In various embodiments, the donor DNA is about 300 to about 600 bases in length. In various embodiments, the donor DNA is about 350 to about 600 bases in length. In various embodiments, the donor DNA is about 400 to about 600 bases in length. In various embodiments, the donor DNA is about 450 to about 600 bases in length. In various embodiments, the donor DNA is about 500 to about 600 bases in length. In various embodiments, the donor DNA is about 550 to about 600 bases in length.

[0094] In various embodiments, the donor DNA is about 50 to about 550 bases in length. In various embodiments, the donor DNA is about 50 to about 500 bases in length. In various embodiments, the donor DNA is about 50 to about 450 bases in length. In various embodiments, the donor DNA is about 50 to about 400 bases in length. In various embodiments, the donor DNA is about 50 to about 350 bases in length. In various embodiments, the donor DNA is about 50 to about 300 bases in length. In various embodiments, the donor DNA is about 50 to about 250 bases in length. In various embodiments, the donor DNA is about 50 to about 200 bases in length. In various embodiments, the donor DNA is about 50 to about 150 bases in length. In various embodiments, the donor DNA is about 150 to about 100 bases in length. In embodiments, the donor DNA is about 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, or 600 bases in length.

[0095] In various embodiments, the donor DNA is about 20 bases to about 5000 bases in length. In various embodiments, the donor DNA is about 250 bases to about 5000 bases in length. In various embodiments, the donor DNA is about 500 bases to about 5000 bases in length. In various embodiments, the donor DNA is about 750 bases to about 5000 bases in length. In various embodiments, the donor DNA is about 1000 bases to about 5000 bases in length. In various embodiments, the donor DNA is about 1250 bases to about 5000 bases in length. In various embodiments, the donor DNA is about 1500 bases to about 5000 bases in length. In various embodiments, the donor DNA is about 1750 bases to about 5000 bases in length. In various embodiments, the donor DNA is about 2000 bases to about 5000 bases in length. In various embodiments, the donor DNA is about 2250 bases to about 5000 bases in length. In various embodiments, the donor DNA is about 2500 bases to about 5000 bases in length. In various embodiments, the donor DNA is about 2750 bases to about 5000 bases in length. In various embodiments, the donor DNA is about 3000 bases to about 5000 bases in length. In various embodiments, the donor DNA is about 3250 bases to about 5000 bases in length. In various embodiments, the donor DNA is about 3500 bases to about 5000 bases in length. In various embodiments, the donor DNA is about 3750 bases to about 5000 bases in length. In various embodiments, the donor DNA is about 4000 bases to about 5000 bases in length. In various embodiments, the donor DNA is about 4250 bases to about 5000 bases in length. In various embodiments, the donor DNA is about 4500 bases to about 5000 bases in length. In various embodiments, the donor DNA is about 4750 bases to about 5000 bases in length.

[0096] In various embodiments, the donor DNA is about 20 bases to about 4750 bases in length. In various embodiments, the donor DNA is about 20 bases to about 4500 bases in length. In various embodiments, the donor DNA is about 20 bases to about 4250 bases in length. In various embodiments, the donor DNA is about 20 bases to about 4000 bases in length. In various embodiments, the donor DNA is about 20 bases to about 3750 bases in length. In various embodiments, the donor DNA is about 20 bases to about 3500 bases in length. In various embodiments, the donor DNA is about 20 bases to about 3250 bases in length. In various embodiments, the donor DNA is about 20 bases to about 3000 bases in length. In various embodiments, the donor DNA is about 20 bases to about 2750 bases in length. In various embodiments, the donor DNA is about 20 bases to about 2500 bases in length. In various embodiments, the donor DNA is about 20 bases to about 2250 bases in length. In various embodiments, the donor DNA is about 20 bases to about 2000 bases in length. In various embodiments, the donor DNA is about 20 bases to about 1750 bases in length. In various embodiments, the donor DNA is about 20 bases to about 1500 bases in length. In various embodiments, the donor DNA is about 20 bases to about 1250 bases in length. In various embodiments, the donor DNA is about 20 bases to about 1000 bases in length. In various embodiments, the donor DNA is about 20 bases to about 750 bases in length. In various embodiments, the donor DNA is about 20 bases to about 500 bases in length. In various embodiments, the donor DNA is about 20 bases to about 250 bases in length. In embodiments, the donor DNA is about 20, 250, 500, 750, 1000, 1250, 1500, 1750, 2000, 2250, 2500, 2750, 3000, 3250, 3500, 3750, 4000, 4250, 4500, 4750, or 5000 bases in length.

[0097] In some cases, delivery of nucleic acids to T cells can be facilitated by a delivery vehicle. The delivery vehicle can facilitate the interaction of nucleic acids with the T cell membrane, thereby allowing the nucleic acid to enter the T cell. In one example, the nucleic acid can be encapsulated in the delivery vehicle. In another example, the nucleic acid can be non-covalently associated with the delivery vehicle. Thus, in various embodiments, the nucleic acid is associated with the delivery vehicle. In various embodiments, the delivery vehicle is a lipid particle or nanoparticle. In various embodiments, the delivery vehicle is a lipid particle. In various embodiments, the delivery vehicle is a nanoparticle. In various embodiments, the delivery vehicle is a liposome or lipid nanoparticle.

[0098] For the methods provided herein, in embodiments, the T cells are primary T cells. "Primary T cells" is used according to its ordinary meaning in the biological arts to refer to T cells expanded directly from T cells extracted from a subject.

[0099] The methods provided herein, including embodiments thereof, may include contacting a T cell with a gene-editing reagent, thereby enabling editing of a target gene in the T cell. For example, the gene-editing reagent may promote knockout of an endogenous gene (e.g., an endogenous TCR) and knockin of a tumor antigen-specific TCR. Thus, in embodiments, the method further includes contacting the T cell with a gene-editing reagent. In embodiments, contacting the T cell with the gene-editing reagent includes contacting the T cell with a nucleic acid sequence encoding the gene-editing reagent. In embodiments, the T cell is contacted with the nucleic acid in the presence of a gene-editing agent or a nucleic acid sequence encoding the gene-editing reagent. In embodiments, the T cell is contacted with the nucleic acid in the presence of the gene-editing reagent. In embodiments, the T cell is contacted with the nucleic acid in the presence of the gene-editing reagent. In various embodiments, the T cell is contacted with the nucleic acid in the presence of a nucleic acid sequence encoding the gene-editing reagent.

[0100] In some embodiments, the gene editing reagent comprises an RNA-guided nuclease. In some embodiments, the RNA-guided nuclease is a CRISPR-Cas system. In some embodiments, the CRISPR-Cas system is selected from the group consisting of Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9, Cas10, Cas12, Cas13, nCas9, Cas-CLOVER, Csy1, Csy2, Csy3, Cse1, Cse2, Csc1, Csc2, In some embodiments, the CRISPR-Cas system comprises Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmr1, Cmr3, Cmr4, Cmr5, Cmr6, Csb1, Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, Csx1, Csx15, Csf1, Csf2, Csf3, or Csf4. In some embodiments, the CRISPR-Cas system comprises Cas1. In some embodiments, the CRISPR-Cas system comprises Cas1B. In some embodiments, the CRISPR-Cas system comprises Cas2. In some embodiments, the CRISPR-Cas system comprises Cas3. In some embodiments, the CRISPR-Cas system comprises Cas4. In some embodiments, the CRISPR-Cas system comprises Cas5. In embodiments, the CRISPR-Cas system comprises Cas6. In embodiments, the CRISPR-Cas system comprises Cas7. In embodiments, the CRISPR-Cas system comprises Cas8. In embodiments, the CRISPR-Cas system comprises Cas9. In embodiments, the CRISPR-Cas system comprises Cas10. In embodiments, the CRISPR-Cas system comprises Cas12. In embodiments, the CRISPR-Cas system comprises Cas13. In embodiments, the CRISPR-Cas system comprises nCas9. In embodiments, the CRISPR-Cas system comprises Cas-CLOVER. In embodiments, the CRISPR-Cas system comprises Csy1. In embodiments, the CRISPR-Cas system comprises Csy2. In embodiments, the CRISPR-Cas system comprises Csy3. In embodiments, the CRISPR-Cas system comprises Cse1.In embodiments, the CRISPR-Cas system comprises Cse2. In embodiments, the CRISPR-Cas system comprises Csc1. In embodiments, the CRISPR-Cas system comprises Csc2. In embodiments, the CRISPR-Cas system comprises Csm2. In embodiments, the CRISPR-Cas system comprises Csm3. In embodiments, the CRISPR-Cas system comprises Csm4. In embodiments, the CRISPR-Cas system comprises Csm5. In embodiments, the CRISPR-Cas system comprises Csm6. In embodiments, the CRISPR-Cas system comprises Cmr1. In embodiments, the CRISPR-Cas system comprises Cmr3. In embodiments, the CRISPR-Cas system comprises Cmr4. In embodiments, the CRISPR-Cas system comprises Cmr5. In embodiments, the CRISPR-Cas system comprises Cmr6. In embodiments, the CRISPR-Cas system comprises Csb1. In embodiments, the CRISPR-Cas system comprises Csb3. In embodiments, the CRISPR-Cas system comprises Csx17. In embodiments, the CRISPR-Cas system comprises Csx14. In embodiments, the CRISPR-Cas system comprises Csx10. In embodiments, the CRISPR-Cas system comprises Csx16. In embodiments, the CRISPR-Cas system comprises CsaX. In embodiments, the CRISPR-Cas system comprises Csx3. In embodiments, the CRISPR-Cas system comprises Csx1. In embodiments, the CRISPR-Cas system comprises Csx15. In embodiments, the CRISPR-Cas system comprises Csf1. In embodiments, the CRISPR-Cas system comprises Csf2. In embodiments, the CRISPR-Cas system comprises Csf3. In some embodiments, the CRISPR-Cas system comprises Csf4. In some embodiments, the gene editing reagent further comprises a guide RNA (gRNA).

[0101] In some embodiments, the gene editing reagent is MAD7, TALEN, or ZFN. In some embodiments, the gene editing reagent is MAD7. In some embodiments, the gene editing reagent is TALEN. In some embodiments, the gene editing reagent is ZFN. MAD7 is an engineered nuclease of the class 2 VA type CRISPR-Cas (Cas12a / Cpf1) family (refseq WP_055225123.1). See, for example, CRISPR J. April 2020;3(2):97-108, which is incorporated herein by reference in its entirety.

[0102] As described throughout this specification, including the figures and examples, donor DNA can be inserted into the TCR locus of a T cell, thereby forming an engineered T cell. Thus, for the methods provided herein, in embodiments, the donor DNA is inserted into an endogenous TCR locus. In embodiments, the endogenous TCR locus is an endogenous TCR-alpha locus, an endogenous TCR-beta locus, or a combination thereof. In embodiments, the endogenous TCR locus is an endogenous TCR-alpha locus. In embodiments, the endogenous TCR locus is an endogenous TCR-beta locus.

[0103] For the methods provided herein, gene editing reagents and nucleic acids can be delivered to T cells using various methods known in the art, including but not limited to electroporation and transfection methods. In various embodiments, contacting T cells with gene editing reagents comprises transfecting T cells with gene editing reagents. In various embodiments, contacting T cells with nucleic acids comprises transfecting T cells with nucleic acids.

[0104] As described above, the one or more cGAS-STING pathway inhibitors can be any compound that inhibits or downregulates the activity or production of any component of the cGAS pathway, the STING pathway, or the cGAS-STING axis, including components of TBK1. In various embodiments, the one or more cGAS-STING pathway inhibitors include a cGAS inhibitor. In various embodiments, the one or more cGAS-STING pathway inhibitors include a STING inhibitor. In various embodiments, the one or more cGAS-STING pathway inhibitors include a TANK-binding kinase 1 (TBK1) inhibitor. The cGAS, STING, or TBK1 inhibitor may include a kinase inhibitor. As used herein, "TANK-binding kinase 1 inhibitor" or "TBK1 inhibitor" refers to a compound that reduces or downregulates the activity or production of TANK-binding kinase 1. For example, a TBK1 inhibitor may inhibit the phosphorylation of TBK1 or a TBK1 target. In various embodiments, the TBK1 inhibitor is Amlexanox, BX795, or MRT67307.

[0105] In various embodiments, the one or more cGAS-STING pathway inhibitors are Amlexanox (Aml), MRT67307 (MRT), BX795, H151, ODN-A151 (ODN151), Ru.521, G140, or a combination thereof. In various embodiments, the one or more cGAS-STING pathway inhibitors comprise Amlexanox (Aml). In various embodiments, the one or more cGAS-STING pathway inhibitors comprise MRT. In various embodiments, the one or more cGAS-STING pathway inhibitors comprise BX795. In various embodiments, the one or more cGAS-STING pathway inhibitors comprise H151. In various embodiments, the one or more cGAS-STING pathway inhibitors comprise ODN151. In various embodiments, the one or more cGAS-STING pathway inhibitors comprise Ru.521. In various embodiments, the one or more cGAS-STING pathway inhibitors comprise G140. In various embodiments, the one or more cGAS-STING pathway inhibitors comprise a compound listed in Table 5.

[0106] As used herein, ODN-A151 (ODN151) refers to a nucleic acid sequence comprising 5'-TTAGGGTTAGGGTTAGGGTTAGGG-3 (SEQ ID NO: 1). In various embodiments, ODN-A151 is the nucleic acid sequence of SEQ ID NO: 1. In various embodiments, one or more internucleotide linkages of SEQ ID NO: 1 are via a phosphorothioate moiety (thiophosphate) moiety. A phosphorothioate moiety is a monothiophosphate (-P(O)3(S) 3- -) or dithiophosphate (-P(O)2(S)2 3- In embodiments, the phosphorothioate moiety can be a monothiophosphate (-P(O)3(S) 3- -).

[0107] In embodiments, the one or more cGAS-STING pathway inhibitors are Am1. In embodiments, the one or more cGAS-STING pathway inhibitors are MRT. In embodiments, the one or more cGAS-STING pathway inhibitors are BX795. In embodiments, the one or more cGAS-STING pathway inhibitors are H151. In embodiments, the one or more cGAS-STING pathway inhibitors are ODN151. In embodiments, the one or more cGAS-STING pathway inhibitors are Ru.521. In embodiments, the one or more cGAS-STING pathway inhibitors are G140.

[0108] In embodiments, the cGAS-STING pathway inhibitor is Am1. In embodiments, the cGAS-STING pathway inhibitor is MRT. In embodiments, the cGAS-STING pathway inhibitor is BX795. In embodiments, the cGAS-STING pathway inhibitor is H151. In embodiments, the cGAS-STING pathway inhibitor is ODN151. In embodiments, the cGAS-STING pathway inhibitor is Ru.521. In embodiments, the cGAS-STING pathway inhibitor is G140. In embodiments, the cGAS-STING inhibitor is Am1 and no other cGAS-STING pathway inhibitors. In embodiments, the cGAS-STING inhibitor is MRT and no other cGAS-STING pathway inhibitors. In embodiments, the cGAS-STING inhibitor is BX795 and no other cGAS-STING pathway inhibitors. In embodiments, the cGAS-STING inhibitor is H151 and no other cGAS-STING pathway inhibitors. In embodiments, the cGAS-STING inhibitor is ODN151 and no other cGAS-STING pathway inhibitors. In embodiments, the cGAS-STING inhibitor is Ru.521 and no other cGAS-STING pathway inhibitors. In embodiments, the cGAS-STING inhibitor is G140 and no other cGAS-STING pathway inhibitors.

[0109] In embodiments, the one or more cGAS-STING pathway inhibitors are selected from the following: Aml, BX795, ODN151, and MRT. In embodiments, the one or more cGAS-STING pathway inhibitors are ODN151. In embodiments, the one or more cGAS-STING pathway inhibitors are BX795. In embodiments, the one or more cGAS-STING pathway inhibitors comprise at least one cGAS-STING pathway inhibitor. In embodiments, the one or more cGAS-STING pathway inhibitors comprise a combination of cGAS-STING pathway inhibitors. In embodiments, the cGAS-STING pathway inhibitor does not comprise more than one cGAS-STING pathway inhibitor.

[0110] Applicants have discovered that treating T cells with one or more cGAS-STING pathway inhibitors before contacting the T cells with the nucleic acid or in the presence of the nucleic acid is effective in generating engineered T cells. In various embodiments, the T cells and the nucleic acid are contacted in the presence of one or more cGAS-STING pathway inhibitors. For example, the T cells can be transfected with the nucleic acid in the presence of one or more cGAS-STING pathway inhibitors. In another example, the T cells can be electroporated with the nucleic acid in the presence of one or more cGAS-STING pathway inhibitors. In various embodiments, the T cells are contacted sequentially with the nucleic acid and one or more cGAS-STING pathway inhibitors. In various embodiments, the T cells are contacted with one or more cGAS-STING pathway inhibitors before the nucleic acid. For example, one or more cGAS-STING pathway inhibitors can be added to a T cell culture before transfecting the T cells with the nucleic acid.

[0111] In various embodiments, T cells are contacted with one or more cGAS-STING pathway inhibitors for up to about 10 hours. In various embodiments, T cells are contacted with one or more cGAS-STING pathway inhibitors for about 1 hour to about 10 hours. In various embodiments, T cells are contacted with one or more cGAS-STING pathway inhibitors for about 2 hours to about 10 hours. In various embodiments, T cells are contacted with one or more cGAS-STING pathway inhibitors for about 3 hours to about 10 hours. In various embodiments, T cells are contacted with one or more cGAS-STING pathway inhibitors for about 4 hours to about 10 hours. In various embodiments, T cells are contacted with one or more cGAS-STING pathway inhibitors for about 5 hours to about 10 hours. In various embodiments, T cells are contacted with one or more cGAS-STING pathway inhibitors for about 6 hours to about 10 hours. In various embodiments, T cells are contacted with one or more cGAS-STING pathway inhibitors for about 7 hours to about 10 hours. In various embodiments, the T cells are contacted with one or more cGAS-STING pathway inhibitors for about 8 hours to about 10 hours. In various embodiments, the T cells are contacted with one or more cGAS-STING pathway inhibitors for about 9 hours to about 10 hours.

[0112] In various embodiments, T cells are contacted with one or more cGAS-STING pathway inhibitors for up to about 9 hours. In various embodiments, T cells are contacted with one or more cGAS-STING pathway inhibitors for about 1 hour to about 9 hours. In various embodiments, T cells are contacted with one or more cGAS-STING pathway inhibitors for about 2 hours to about 9 hours. In various embodiments, T cells are contacted with one or more cGAS-STING pathway inhibitors for about 2 hours to about 8 hours. In various embodiments, T cells are contacted with one or more cGAS-STING pathway inhibitors for about 2 hours to about 7 hours. In various embodiments, T cells are contacted with one or more cGAS-STING pathway inhibitors for about 2 hours to about 6 hours. In various embodiments, T cells are contacted with one or more cGAS-STING pathway inhibitors for about 2 hours to about 5 hours. In various embodiments, T cells are contacted with one or more cGAS-STING pathway inhibitors for about 2 hours to about 4 hours. In various embodiments, T cells are contacted with one or more cGAS-STING pathway inhibitors for about 2 hours to about 3 hours. In various embodiments, T cells are contacted with one or more cGAS-STING pathway inhibitors for about 2, 3, 4, 5, 6, 7, 8, 9, or 10 hours. In various embodiments, T cells are contacted with one or more cGAS-STING pathway inhibitors for about 6 hours. In various embodiments, T cells are contacted with one or more cGAS-STING pathway inhibitors for 6 hours.

[0113] In embodiments of the methods provided herein, T cells are contacted with one cGAS-STING pathway inhibitor. In embodiments, T cells are contacted with one cGAS-STING pathway inhibitor and no other cGAS-STING pathway inhibitor. In embodiments, the cGAS-STING pathway inhibitor is a cGAS-STING pathway inhibitor provided herein and does not include any other cGAS-STING pathway inhibitor.

[0114] Provided herein, inter alia, are methods for increasing the viability of engineered T cells, comprising contacting T cells with a nucleic acid and one or more cGAS-STING pathway inhibitors, thereby forming engineered T cells, wherein the engineered T cells have increased cell viability compared to engineered T cells formed without contacting the T cells with one or more cGAS-STING pathway inhibitors. The methods provided herein are useful for overcoming toxicity associated with T cell engineering, including toxicity associated with the delivery of nucleic acids to T cells or the presence of nucleic acids in the cytoplasm of T cells. Thus, the methods are intended to improve the viability of engineered T cells. "Cell viability" is used according to its ordinary meaning in the art and refers to the number or percentage of viable cells in a cell population. Cell viability can be assessed by measuring cell proliferation, cell membrane integrity, cell function, or metabolic activity. Cell viability can be measured by contacting cells with a nucleic acid-binding dye that enters only cells with compromised or damaged cell membranes. Cell viability can also be measured by contacting cells with a reagent that reacts with an enzyme in live cells or a reagent that detects the redox potential of the cells. For example, cell viability may be measured using a fluorescent detection assay, including assays using one or more of acridine orange, 4',6-diamidino-2-phenylindole (DAPI), propidium iodide (PI), or SYTOX Blue nucleic acid stain, etc. In various embodiments, cell viability may be measured by fluorescent microscopy or flow cytometry. Thus, in one aspect, a method of increasing cell viability of a population of engineered T cells is provided, comprising contacting the population of T cells with one or more cyclic GMP-AMP synthase (cGAS)-stimulator of interferon genes (STING) pathway inhibitors and a nucleic acid, thereby forming a population of engineered T cells, wherein the population of engineered T cells has increased cell viability compared to a population of engineered T cells not contacted with one or more cGAS-STING pathway inhibitors.

[0115] In some embodiments, the nucleic acid comprises donor DNA. In some embodiments, the nucleic acid is double-stranded circular DNA (e.g., a plasmid). In some embodiments, the nucleic acid is a plasmid, nanoplasmid, minicircle, or viral vector comprising donor DNA. In some embodiments, the donor DNA encodes exogenous T cell receptor (TCR)-beta or a fragment thereof, exogenous TCR-alpha or a fragment thereof, or a combination thereof. In some embodiments, the nucleic acid is associated with a delivery vehicle. In some embodiments, the delivery vehicle is a lipid particle or a nanoparticle.

[0116] For the methods provided herein, in embodiments, the T cells are primary T cells.

[0117]

[0013] In some embodiments, the method further comprises contacting the population of T cells with a gene editing reagent. In some embodiments, contacting the population of T cells with the gene editing reagent comprises contacting the population of T cells with a nucleic acid sequence encoding the gene editing reagent. In some embodiments, the T cells are contacted with the nucleic acid in the presence of a gene editing agent or a nucleic acid sequence encoding the gene editing reagent.

[0118] In embodiments, the donor DNA is inserted into an endogenous TCR locus, hi embodiments, the endogenous TCR locus is an endogenous TCR-alpha locus, an endogenous TCR-beta locus, or a combination thereof.

[0119] In various embodiments, contacting the T cell with a gene editing reagent comprises transfecting the T cell with the gene editing reagent.

[0120] In various embodiments, the one or more cGAS-STING pathway inhibitors comprise a cGAS inhibitor. In various embodiments, the one or more cGAS-STING pathway inhibitors comprise a STING inhibitor. In various embodiments, the one or more cGAS-STING pathway inhibitors comprise a TANK-binding kinase 1 (TBK1) inhibitor. In various embodiments, the one or more cGAS-STING pathway inhibitors are Amlexanox (Aml), MRT67307 (MRT), BX795, H151, ODN-A151 (ODN151), Ru.521, G140, or a combination thereof. In various embodiments, the one or more cGAS-STING pathway inhibitors comprise Amlexanox (Aml). In various embodiments, the one or more cGAS-STING pathway inhibitors comprise MRT. In various embodiments, the one or more cGAS-STING pathway inhibitors comprise BX795. In various embodiments, the one or more cGAS-STING pathway inhibitors comprise H151. In embodiments, the one or more cGAS-STING pathway inhibitors comprise ODN-A151. In embodiments, the one or more cGAS-STING pathway inhibitors comprise Ru.521. In embodiments, the one or more cGAS-STING pathway inhibitors comprise G140. In embodiments, the one or more cGAS-STING pathway inhibitors are Am1. In embodiments, the one or more cGAS-STING pathway inhibitors are MRT. In embodiments, the one or more cGAS-STING pathway inhibitors are BX795. In embodiments, the one or more cGAS-STING pathway inhibitors are H151. In embodiments, the one or more cGAS-STING pathway inhibitors are ODN-A151 (ODN151). In embodiments, the one or more cGAS-STING pathway inhibitors are Ru.521. In embodiments, the one or more cGAS-STING pathway inhibitors are G140.

[0121] In various embodiments, populations of T cells are independently contacted with one or more cGAS-STING pathway inhibitors at about 0.1 uM to about 50 uM. In various embodiments, populations of T cells are independently contacted with one or more cGAS-STING pathway inhibitors at about 1 uM to about 50 uM. In various embodiments, populations of T cells are independently contacted with one or more cGAS-STING pathway inhibitors at about 2 uM to about 50 uM. In various embodiments, populations of T cells are independently contacted with one or more cGAS-STING pathway inhibitors at about 3 uM to about 50 uM. In various embodiments, populations of T cells are independently contacted with one or more cGAS-STING pathway inhibitors at about 4 uM to about 50 uM. In various embodiments, populations of T cells are independently contacted with one or more cGAS-STING pathway inhibitors at about 5 uM to about 50 uM. In various embodiments, populations of T cells are independently contacted with one or more cGAS-STING pathway inhibitors at about 6 uM to about 50 uM. In various embodiments, populations of T cells are independently contacted with one or more cGAS-STING pathway inhibitors at about 7 uM to about 50 uM. In various embodiments, populations of T cells are independently contacted with one or more cGAS-STING pathway inhibitors at about 8 uM to about 50 uM. In various embodiments, populations of T cells are independently contacted with one or more cGAS-STING pathway inhibitors at about 9 uM to about 50 uM. In various embodiments, populations of T cells are independently contacted with one or more cGAS-STING pathway inhibitors at about 10 uM to about 50 uM. In various embodiments, populations of T cells are independently contacted with one or more cGAS-STING pathway inhibitors at about 11 uM to about 50 uM. In various embodiments, populations of T cells are independently contacted with one or more cGAS-STING pathway inhibitors at about 12 uM to about 50 uM. In various embodiments, populations of T cells are independently contacted with one or more cGAS-STING pathway inhibitors at about 13 uM to about 50 uM. In various embodiments, populations of T cells are independently contacted with one or more cGAS-STING pathway inhibitors at about 14 uM to about 50 uM. In various embodiments, populations of T cells are independently contacted with one or more cGAS-STING pathway inhibitors at about 15 uM to about 50 uM. In various embodiments, populations of T cells are independently contacted with one or more cGAS-STING pathway inhibitors at about 16 uM to about 50 uM.In various embodiments, populations of T cells are independently contacted with one or more cGAS-STING pathway inhibitors at about 17 uM to about 50 uM. In various embodiments, populations of T cells are independently contacted with one or more cGAS-STING pathway inhibitors at about 18 uM to about 50 uM. In various embodiments, populations of T cells are independently contacted with one or more cGAS-STING pathway inhibitors at about 19 uM to about 50 uM. In various embodiments, populations of T cells are independently contacted with one or more cGAS-STING pathway inhibitors at about 20 uM to about 50 uM. In various embodiments, populations of T cells are independently contacted with one or more cGAS-STING pathway inhibitors at about 21 uM to about 50 uM. In various embodiments, populations of T cells are independently contacted with one or more cGAS-STING pathway inhibitors at about 22 uM to about 50 uM. In various embodiments, populations of T cells are independently contacted with one or more cGAS-STING pathway inhibitors at about 23 uM to about 50 uM. In various embodiments, populations of T cells are independently contacted with one or more cGAS-STING pathway inhibitors at about 24 uM to about 50 uM. In various embodiments, populations of T cells are independently contacted with one or more cGAS-STING pathway inhibitors at about 25 uM to about 50 uM. In various embodiments, populations of T cells are independently contacted with one or more cGAS-STING pathway inhibitors at about 26 uM to about 50 uM. In various embodiments, populations of T cells are independently contacted with one or more cGAS-STING pathway inhibitors at about 27 uM to about 50 uM. In various embodiments, populations of T cells are independently contacted with one or more cGAS-STING pathway inhibitors at about 28 uM to about 50 uM. In various embodiments, the populations of T cells are independently contacted with one or more cGAS-STING pathway inhibitors at about 29 uM to about 50 uM. In various embodiments, the populations of T cells are independently contacted with one or more cGAS-STING pathway inhibitors at about 30 uM to about 50 uM. In various embodiments, the populations of T cells are independently contacted with one or more cGAS-STING pathway inhibitors at about 31 uM to about 50 uM. In various embodiments, the populations of T cells are independently contacted with one or more cGAS-STING pathway inhibitors at about 32 uM to about 50 uM. In various embodiments, the populations of T cells are independently contacted with one or more cGAS-STING pathway inhibitors at about 33 uM to about 50 uM.In various embodiments, populations of T cells are independently contacted with one or more cGAS-STING pathway inhibitors at about 34 uM to about 50 uM. In various embodiments, populations of T cells are independently contacted with one or more cGAS-STING pathway inhibitors at about 35 uM to about 50 uM. In various embodiments, populations of T cells are independently contacted with one or more cGAS-STING pathway inhibitors at about 36 uM to about 50 uM. In various embodiments, populations of T cells are independently contacted with one or more cGAS-STING pathway inhibitors at about 37 uM to about 50 uM. In various embodiments, populations of T cells are independently contacted with one or more cGAS-STING pathway inhibitors at about 38 uM to about 50 uM. In various embodiments, populations of T cells are independently contacted with one or more cGAS-STING pathway inhibitors at about 39 uM to about 50 uM. In various embodiments, populations of T cells are independently contacted with one or more cGAS-STING pathway inhibitors at about 40 uM to about 50 uM. In various embodiments, populations of T cells are independently contacted with one or more cGAS-STING pathway inhibitors at about 41 uM to about 50 uM. In various embodiments, populations of T cells are independently contacted with one or more cGAS-STING pathway inhibitors at about 42 uM to about 50 uM. In various embodiments, populations of T cells are independently contacted with one or more cGAS-STING pathway inhibitors at about 43 uM to about 50 uM. In various embodiments, populations of T cells are independently contacted with one or more cGAS-STING pathway inhibitors at about 44 uM to about 50 uM. In various embodiments, populations of T cells are independently contacted with one or more cGAS-STING pathway inhibitors at about 45 uM to about 50 uM. In various embodiments, populations of T cells are independently contacted with one or more cGAS-STING pathway inhibitors at about 46 uM to about 50 uM. In various embodiments, populations of T cells are independently contacted with one or more cGAS-STING pathway inhibitors at about 47 uM to about 50 uM. In various embodiments, populations of T cells are independently contacted with one or more cGAS-STING pathway inhibitors at about 48 uM to about 50 uM. In various embodiments, populations of T cells are independently contacted with one or more cGAS-STING pathway inhibitors at about 49 uM to about 50 uM.

[0122] In various embodiments, populations of T cells are independently contacted with one or more cGAS-STING pathway inhibitors at about 0.1 uM to about 49 uM. In various embodiments, populations of T cells are independently contacted with one or more cGAS-STING pathway inhibitors at about 0.1 uM to about 48 uM. In various embodiments, populations of T cells are independently contacted with one or more cGAS-STING pathway inhibitors at about 0.1 uM to about 47 uM. In various embodiments, populations of T cells are independently contacted with one or more cGAS-STING pathway inhibitors at about 0.1 uM to about 46 uM. In various embodiments, populations of T cells are independently contacted with one or more cGAS-STING pathway inhibitors at about 0.1 uM to about 45 uM. In various embodiments, populations of T cells are independently contacted with one or more cGAS-STING pathway inhibitors at about 0.1 uM to about 44 uM. In various embodiments, populations of T cells are independently contacted with one or more cGAS-STING pathway inhibitors at about 0.1 uM to about 43 uM. In various embodiments, populations of T cells are independently contacted with one or more cGAS-STING pathway inhibitors at about 0.1 uM to about 42 uM. In various embodiments, populations of T cells are independently contacted with one or more cGAS-STING pathway inhibitors at about 0.1 uM to about 41 uM. In various embodiments, populations of T cells are independently contacted with one or more cGAS-STING pathway inhibitors at about 0.1 uM to about 40 uM. In various embodiments, populations of T cells are independently contacted with one or more cGAS-STING pathway inhibitors at about 0.1 uM to about 39 uM. In various embodiments, populations of T cells are independently contacted with one or more cGAS-STING pathway inhibitors at about 0.1 uM to about 38 uM. In various embodiments, populations of T cells are independently contacted with one or more cGAS-STING pathway inhibitors at about 0.1 uM to about 37 uM. In various embodiments, populations of T cells are independently contacted with one or more cGAS-STING pathway inhibitors at about 0.1 uM to about 36 uM. In various embodiments, populations of T cells are independently contacted with one or more cGAS-STING pathway inhibitors at about 0.1 uM to about 35 uM. In various embodiments, populations of T cells are independently contacted with one or more cGAS-STING pathway inhibitors at about 0.1 uM to about 34 uM.In various embodiments, populations of T cells are independently contacted with one or more cGAS-STING pathway inhibitors at about 0.1 uM to about 33 uM. In various embodiments, populations of T cells are independently contacted with one or more cGAS-STING pathway inhibitors at about 0.1 uM to about 32 uM. In various embodiments, populations of T cells are independently contacted with one or more cGAS-STING pathway inhibitors at about 0.1 uM to about 31 uM. In various embodiments, populations of T cells are independently contacted with one or more cGAS-STING pathway inhibitors at about 0.1 uM to about 30 uM. In various embodiments, populations of T cells are independently contacted with one or more cGAS-STING pathway inhibitors at about 0.1 uM to about 29 uM. In various embodiments, populations of T cells are independently contacted with one or more cGAS-STING pathway inhibitors at about 0.1 uM to about 28 uM. In various embodiments, populations of T cells are independently contacted with one or more cGAS-STING pathway inhibitors at about 0.1 uM to about 27 uM. In various embodiments, populations of T cells are independently contacted with one or more cGAS-STING pathway inhibitors at about 0.1 uM to about 26 uM. In various embodiments, populations of T cells are independently contacted with one or more cGAS-STING pathway inhibitors at about 0.1 uM to about 25 uM. In various embodiments, populations of T cells are independently contacted with one or more cGAS-STING pathway inhibitors at about 0.1 uM to about 24 uM. In various embodiments, populations of T cells are independently contacted with one or more cGAS-STING pathway inhibitors at about 0.1 uM to about 23 uM. In various embodiments, populations of T cells are independently contacted with one or more cGAS-STING pathway inhibitors at about 0.1 uM to about 22 uM. In various embodiments, populations of T cells are independently contacted with one or more cGAS-STING pathway inhibitors at about 0.1 uM to about 21 uM. In various embodiments, populations of T cells are independently contacted with one or more cGAS-STING pathway inhibitors at about 0.1 uM to about 20 uM. In various embodiments, populations of T cells are independently contacted with one or more cGAS-STING pathway inhibitors at about 0.1 uM to about 19 uM. In various embodiments, populations of T cells are independently contacted with one or more cGAS-STING pathway inhibitors at about 0.1 uM to about 18 uM.In various embodiments, populations of T cells are independently contacted with one or more cGAS-STING pathway inhibitors at about 0.1 uM to about 17 uM. In various embodiments, populations of T cells are independently contacted with one or more cGAS-STING pathway inhibitors at about 0.1 uM to about 16 uM. In various embodiments, populations of T cells are independently contacted with one or more cGAS-STING pathway inhibitors at about 0.1 uM to about 15 uM. In various embodiments, populations of T cells are independently contacted with one or more cGAS-STING pathway inhibitors at about 0.1 uM to about 14 uM. In various embodiments, populations of T cells are independently contacted with one or more cGAS-STING pathway inhibitors at about 0.1 uM to about 13 uM. In various embodiments, populations of T cells are independently contacted with one or more cGAS-STING pathway inhibitors at about 0.1 uM to about 12 uM. In various embodiments, populations of T cells are independently contacted with one or more cGAS-STING pathway inhibitors at about 0.1 uM to about 11 uM. In various embodiments, populations of T cells are independently contacted with one or more cGAS-STING pathway inhibitors at about 0.1 uM to about 10 uM. In various embodiments, populations of T cells are independently contacted with one or more cGAS-STING pathway inhibitors at about 0.1 uM to about 9 uM. In various embodiments, populations of T cells are independently contacted with one or more cGAS-STING pathway inhibitors at about 0.1 uM to about 8 uM. In various embodiments, populations of T cells are independently contacted with one or more cGAS-STING pathway inhibitors at about 0.1 uM to about 7 uM. In various embodiments, populations of T cells are independently contacted with one or more cGAS-STING pathway inhibitors at about 0.1 uM to about 6 uM. In various embodiments, populations of T cells are independently contacted with one or more cGAS-STING pathway inhibitors at about 0.1 uM to about 5 uM. In various embodiments, populations of T cells are independently contacted with one or more cGAS-STING pathway inhibitors at about 0.1 uM to about 4 uM. In various embodiments, populations of T cells are independently contacted with one or more cGAS-STING pathway inhibitors at about 0.1 uM to about 3 uM. In various embodiments, populations of T cells are independently contacted with one or more cGAS-STING pathway inhibitors at about 0.1 uM to about 2 uM. In various embodiments, populations of T cells are independently contacted with one or more cGAS-STING pathway inhibitors at about 0.1 uM to about 1 uM.In various embodiments, the population of T cells is administered at a concentration of about 0.1 uM, 1 uM, 2 uM, 3 uM, 4 uM, 5 uM, 6 uM, 7 uM, 8 uM, 9 uM, 10 uM, 11 uM, 12 uM, 13 uM, 14 uM, 15 uM, 16 uM, 17 uM, 18 uM, 19 uM, 20 uM, 21 uM, 22 uM, 23 uM, 24 uM, 25 uM, 26 uM, 27 uM, and independently contacted with one or more cGAS-STING pathway inhibitors at 28uM, 29uM, 30uM, 31uM, 32uM, 33uM, 34uM, 35uM, 36uM, 37uM, 38uM, 39uM, 40uM, 41uM, 42uM, 43uM, 44uM, 45uM, 46uM, 47uM, 48uM, 49uM, or 50uM.

[0123] In embodiments, the population of T cells is contacted with the nucleic acid in the presence of one or more cGAS-STING pathway inhibitors. In embodiments, the population of T cells is contacted sequentially with the nucleic acid and one or more cGAS-STING pathway inhibitors. In embodiments, the population of T cells is contacted with the one or more cGAS-STING pathway inhibitors before the nucleic acid. In embodiments, the population of T cells is contacted with one cGAS-STING pathway inhibitor.

[0124] In various embodiments, the population of T cells is contacted with about 1 uM to about 10 uM BX795. In various embodiments, the population of T cells is contacted with about 1.5 uM to about 10 uM BX795. In various embodiments, the population of T cells is contacted with about 2 uM to about 10 uM BX795. In various embodiments, the population of T cells is contacted with about 2.5 uM to about 10 uM BX795. In various embodiments, the population of T cells is contacted with about 3 uM to about 10 uM BX795. In various embodiments, the population of T cells is contacted with about 3.5 uM to about 10 uM BX795. In various embodiments, the population of T cells is contacted with about 4 uM to about 10 uM BX795. In various embodiments, the population of T cells is contacted with about 4.5 uM to about 10 uM BX795. In various embodiments, the population of T cells is contacted with about 5 uM to about 10 uM BX795. In various embodiments, the population of T cells is contacted with about 5.5 uM to about 10 uM BX795. In various embodiments, the population of T cells is contacted with about 6 uM to about 10 uM BX795. In various embodiments, the population of T cells is contacted with about 6.5 uM to about 10 uM BX795. In various embodiments, the population of T cells is contacted with about 7 uM to about 10 uM BX795. In various embodiments, the population of T cells is contacted with about 7.5 uM to about 10 uM BX795. In various embodiments, the population of T cells is contacted with about 8 uM to about 10 uM BX795. In various embodiments, the population of T cells is contacted with about 8.5 uM to about 10 uM BX795. In various embodiments, the population of T cells is contacted with about 9 uM to about 10 uM BX795. In various embodiments, the population of T cells is contacted with about 9.5 uM to about 10 uM BX795.

[0125] In various embodiments, the population of T cells is contacted with about 1 uM to about 9.5 uM BX795. In various embodiments, the population of T cells is contacted with about 1 uM to about 9 uM BX795. In various embodiments, the population of T cells is contacted with about 1 uM to about 8.5 uM BX795. In various embodiments, the population of T cells is contacted with about 1 uM to about 8 uM BX795. In various embodiments, the population of T cells is contacted with about 1 uM to about 7.5 uM BX795. In various embodiments, the population of T cells is contacted with about 1 uM to about 7 uM BX795. In various embodiments, the population of T cells is contacted with about 1 uM to about 6.5 uM BX795. In various embodiments, the population of T cells is contacted with about 1 uM to about 6 uM BX795. In various embodiments, the population of T cells is contacted with about 1 uM to about 5.5 uM BX795. In various embodiments, the population of T cells is contacted with about 1 uM to about 5 uM BX795. In various embodiments, the population of T cells is contacted with about 1 uM to about 4.5 uM BX795. In various embodiments, the population of T cells is contacted with about 1 uM to about 4 uM BX795. In various embodiments, the population of T cells is contacted with about 1 uM to about 3.5 uM BX795. In various embodiments, the population of T cells is contacted with about 1 uM to about 3 uM BX795. In various embodiments, the population of T cells is contacted with about 1 uM to about 2.5 uM BX795. In various embodiments, the population of T cells is contacted with about 1 uM to about 2 uM BX795. In various embodiments, the population of T cells is contacted with about 1 uM to about 1.5 uM BX795. In various embodiments, the population of T cells is contacted with about 1 uM, 1.5 uM, 2 uM, 2.5 uM, 3 uM, 3.5 uM, 4 uM, 4.5 uM, 5 uM, 5.5 uM, 6 uM, 6.5 uM, 7 uM, 7.5 uM, 8 uM, 8.5 uM, 9 uM, 9.5 uM, or 10 uM BX795. In various embodiments, the population of T cells is contacted with about 2.5 uM BX795. In various embodiments, the population of T cells is contacted with about 5 uM BX795.

[0126] In various embodiments, the population of T cells is contacted with about 0.1 uM to about 8 uM of ODN151. In various embodiments, the population of T cells is contacted with about 0.5 uM to about 8 uM of ODN151. In various embodiments, the population of T cells is contacted with about 1 uM to about 8 uM of ODN151. In various embodiments, the population of T cells is contacted with about 1.5 uM to about 8 uM of ODN151. In various embodiments, the population of T cells is contacted with about 2 uM to about 8 uM of ODN151. In various embodiments, the population of T cells is contacted with about 2.5 uM to about 8 uM of ODN151. In various embodiments, the population of T cells is contacted with about 3 uM to about 8 uM of ODN151. In various embodiments, the population of T cells is contacted with about 3.5 uM to about 8 uM of ODN151. In various embodiments, the population of T cells is contacted with about 4 uM to about 8 uM of ODN151. In various embodiments, the population of T cells is contacted with about 4.5 uM to about 8 uM of ODN151. In various embodiments, the population of T cells is contacted with about 5 uM to about 8 uM of ODN151. In various embodiments, the population of T cells is contacted with about 5.5 uM to about 8 uM of ODN151. In various embodiments, the population of T cells is contacted with about 6 uM to about 8 uM of ODN151. In various embodiments, the population of T cells is contacted with about 6.5 uM to about 8 uM of ODN151. In various embodiments, the population of T cells is contacted with about 7 uM to about 8 uM of ODN151. In various embodiments, the population of T cells is contacted with about 7.5 uM to about 8 uM of ODN151.

[0127] In various embodiments, the population of T cells is contacted with about 0.1 uM to about 7.5 uM ODN151. In various embodiments, the population of T cells is contacted with about 0.1 uM to about 7 uM ODN151. In various embodiments, the population of T cells is contacted with about 0.1 uM to about 6.5 uM ODN151. In various embodiments, the population of T cells is contacted with about 0.1 uM to about 6 uM ODN151. In various embodiments, the population of T cells is contacted with about 0.1 uM to about 5.5 uM ODN151. In various embodiments, the population of T cells is contacted with about 0.1 uM to about 5 uM ODN151. In various embodiments, the population of T cells is contacted with about 0.1 uM to about 4.5 uM ODN151. In various embodiments, the population of T cells is contacted with about 0.1 uM to about 4 uM of ODN151. In various embodiments, the population of T cells is contacted with about 0.1 uM to about 3.5 uM of ODN151. In various embodiments, the population of T cells is contacted with about 0.1 uM to about 3 uM of ODN151. In various embodiments, the population of T cells is contacted with about 0.1 uM to about 2.5 uM of ODN151. In various embodiments, the population of T cells is contacted with about 0.1 uM to about 2 uM of ODN151. In various embodiments, the population of T cells is contacted with about 0.1 uM to about 1.5 uM of ODN151. In various embodiments, the population of T cells is contacted with about 0.1 uM to about 1 uM of ODN151. In various embodiments, the population of T cells is contacted with about 0.1 uM to about 0.5 uM of ODN 151. In various embodiments, the population of T cells is contacted with about 0.1 uM, 0.5 uM, 1 uM, 1.5 uM, 2 uM, 2.5 uM, 3 uM, 3.5 uM, 4 uM, 4.5 uM, 5 uM, 5.5 uM, 6 uM, 6.5 uM, 7 uM, 7.5 uM, 8 uM, 8.5 uM, 9 uM, 9.5 uM, or 10 uM of ODN 151.

[0128] As described above, the methods provided herein, including embodiments thereof, are effective to increase cell viability of engineered T cells compared to engineered T cells generated without treatment with one or more cGAS-STING pathway inhibitors. For example, the population of engineered T cells provided herein has increased cell viability compared to a population of engineered T cells generated without contacting the T cells with one or more cGAS-STING pathway inhibitors. In various embodiments, the cell viability of the population of engineered T cells is increased by at least about 0.5-fold to at least about 5-fold compared to a population of engineered T cells not contacted with one or more cGAS-STING pathway inhibitors. In various embodiments, the cell viability of the population of engineered T cells is increased by at least about 1-fold to at least about 5-fold compared to a population of engineered T cells not contacted with one or more cGAS-STING pathway inhibitors. In various embodiments, the cell viability of the population of engineered T cells is increased by at least about 1.5-fold to at least about 5-fold compared to a population of engineered T cells not contacted with one or more cGAS-STING pathway inhibitors. In various embodiments, the cell viability of the population of engineered T cells is increased by at least about 2-fold to at least about 5-fold compared to a population of engineered T cells not contacted with one or more cGAS-STING pathway inhibitors. In various embodiments, the cell viability of the population of engineered T cells is increased by at least about 2.5-fold to at least about 5-fold compared to a population of engineered T cells not contacted with one or more cGAS-STING pathway inhibitors. In various embodiments, the cell viability of the population of engineered T cells is increased by at least about 3-fold to at least about 5-fold compared to a population of engineered T cells not contacted with one or more cGAS-STING pathway inhibitors. In various embodiments, the cell viability of the population of engineered T cells is increased by at least about 3.5-fold to at least about 5-fold compared to a population of engineered T cells not contacted with one or more cGAS-STING pathway inhibitors. In various embodiments, the cell viability of the population of engineered T cells is increased by at least about 4-fold to at least about 5-fold compared to a population of engineered T cells not contacted with one or more cGAS-STING pathway inhibitors.

[0129] In various embodiments, the cell viability of the population of engineered T cells is increased by at least about 0.5-fold to at least about 4.5-fold compared to a population of engineered T cells not contacted with one or more cGAS-STING pathway inhibitors. In various embodiments, the cell viability of the population of engineered T cells is increased by at least about 0.5-fold to at least about 4-fold compared to a population of engineered T cells not contacted with one or more cGAS-STING pathway inhibitors. In various embodiments, the cell viability of the population of engineered T cells is increased by at least about 0.5-fold to at least about 3.5-fold compared to a population of engineered T cells not contacted with one or more cGAS-STING pathway inhibitors. In various embodiments, the cell viability of the population of engineered T cells is increased by at least about 0.5-fold to at least about 3-fold compared to a population of engineered T cells not contacted with one or more cGAS-STING pathway inhibitors. In various embodiments, the cell viability of the population of engineered T cells is increased by at least about 0.5-fold to at least about 2.5-fold compared to a population of engineered T cells not contacted with one or more cGAS-STING pathway inhibitors. In various embodiments, the cell viability of the population of engineered T cells is increased by at least about 0.5-fold to at least about 2-fold compared to a population of engineered T cells not contacted with one or more cGAS-STING pathway inhibitors. In various embodiments, the cell viability of the population of engineered T cells is increased by at least about 0.5-fold to at least about 1.5-fold compared to a population of engineered T cells not contacted with one or more cGAS-STING pathway inhibitors. In various embodiments, the cell viability of the population of engineered T cells is increased by at least about 0.5-fold to at least about 1-fold compared to a population of engineered T cells not contacted with one or more cGAS-STING pathway inhibitors. In various embodiments, the cell viability of the population of engineered T cells is increased by about 0.5-fold, 1-fold, 1.5-fold, 2-fold, 2.5-fold, 3-fold, 3.5-fold, 4-fold, 4.5-fold, or 5-fold compared to a population of engineered T cells not contacted with one or more cGAS-STING pathway inhibitors. In various embodiments, cell viability is increased by about 2-fold compared to a population of engineered T cells that have not been contacted with one or more cGAS-STING pathway inhibitors.In embodiments, cell viability is increased by 2-fold compared to a population of engineered T cells that have not been contacted with one or more cGAS-STING pathway inhibitors.

[0130] In various embodiments, the cell viability of the population of engineered T cells is increased by about 30% to about 95% compared to a population of engineered T cells not contacted with one or more cGAS-STING pathway inhibitors. In various embodiments, the cell viability of the population of engineered T cells is increased by about 35% to about 95% compared to a population of engineered T cells not contacted with one or more cGAS-STING pathway inhibitors. In various embodiments, the cell viability of the population of engineered T cells is increased by about 40% to about 95% compared to a population of engineered T cells not contacted with one or more cGAS-STING pathway inhibitors. In various embodiments, the cell viability of the population of engineered T cells is increased by about 45% to about 95% compared to a population of engineered T cells not contacted with one or more cGAS-STING pathway inhibitors. In various embodiments, the cell viability of the population of engineered T cells is increased by about 50% to about 95% compared to a population of engineered T cells not contacted with one or more cGAS-STING pathway inhibitors. In various embodiments, the cell viability of the population of engineered T cells is increased by about 55% to about 95% compared to a population of engineered T cells not contacted with one or more cGAS-STING pathway inhibitors. In various embodiments, the cell viability of the population of engineered T cells is increased by about 60% to about 95% compared to a population of engineered T cells not contacted with one or more cGAS-STING pathway inhibitors. In various embodiments, the cell viability of the population of engineered T cells is increased by about 65% to about 95% compared to a population of engineered T cells not contacted with one or more cGAS-STING pathway inhibitors. In various embodiments, the cell viability of the population of engineered T cells is increased by about 70% to about 95% compared to a population of engineered T cells not contacted with one or more cGAS-STING pathway inhibitors. In various embodiments, the cell viability of the population of engineered T cells is increased by about 75% to about 95% compared to a population of engineered T cells not contacted with one or more cGAS-STING pathway inhibitors. In various embodiments, the cell viability of the population of engineered T cells is increased by about 80% to about 95% compared to a population of engineered T cells that has not been contacted with one or more cGAS-STING pathway inhibitors.In various embodiments, the cell viability of the population of engineered T cells is increased by about 85% to about 95% compared to a population of engineered T cells not contacted with one or more cGAS-STING pathway inhibitors. In various embodiments, the cell viability of the population of engineered T cells is increased by about 90% to about 95% compared to a population of engineered T cells not contacted with one or more cGAS-STING pathway inhibitors.

[0131] In various embodiments, the cell viability of the population of engineered T cells is increased by about 35% to about 90% compared to a population of engineered T cells not contacted with one or more cGAS-STING pathway inhibitors. In various embodiments, the cell viability of the population of engineered T cells is increased by about 35% to about 85% compared to a population of engineered T cells not contacted with one or more cGAS-STING pathway inhibitors. In various embodiments, the cell viability of the population of engineered T cells is increased by about 35% to about 80% compared to a population of engineered T cells not contacted with one or more cGAS-STING pathway inhibitors. In various embodiments, the cell viability of the population of engineered T cells is increased by about 35% to about 75% compared to a population of engineered T cells not contacted with one or more cGAS-STING pathway inhibitors. In various embodiments, the cell viability of the population of engineered T cells is increased by about 35% to about 70% compared to a population of engineered T cells not contacted with one or more cGAS-STING pathway inhibitors. In various embodiments, the cell viability of the population of engineered T cells is increased by about 35% to about 65% compared to a population of engineered T cells not contacted with one or more cGAS-STING pathway inhibitors. In various embodiments, the cell viability of the population of engineered T cells is increased by about 35% to about 60% compared to a population of engineered T cells not contacted with one or more cGAS-STING pathway inhibitors. In various embodiments, the cell viability of the population of engineered T cells is increased by about 35% to about 55% compared to a population of engineered T cells not contacted with one or more cGAS-STING pathway inhibitors. In various embodiments, the cell viability of the population of engineered T cells is increased by about 35% to about 50% compared to a population of engineered T cells not contacted with one or more cGAS-STING pathway inhibitors. In various embodiments, the cell viability of the population of engineered T cells is increased by about 35% to about 45% compared to a population of engineered T cells not contacted with one or more cGAS-STING pathway inhibitors. In various embodiments, the cell viability of the population of engineered T cells is increased by about 35% to about 40% compared to a population of engineered T cells not contacted with one or more cGAS-STING pathway inhibitors.In various embodiments, the cell viability of the population of engineered T cells is increased by about 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% compared to a population of engineered T cells not contacted with one or more cGAS-STING pathway inhibitors.

[0132] The methods provided herein are effective in improving gene editing efficiency in T cells, thereby increasing the total number of engineered T cells. For example, the methods can increase the efficiency of gene knockout and / or gene knockin, thereby improving the gene editing efficiency of T cells. The methods can thereby improve the yield of engineered T cells generated from a population of T cells. For example, the methods provided herein can generate an increased population of engineered T cells from a population of T cells compared to a population of engineered T cells in which the T cells have not been contacted with one or more cGAS-STING pathway inhibitors. Thus, in one aspect, a method is provided for increasing gene editing efficiency in a population of T cells, comprising contacting a population of T cells with one or more cyclic GMP-AMP synthase (cGAS)-stimulator of interferon genes (STING) pathway inhibitors and a nucleic acid, thereby forming a population of engineered T cells, wherein the population of T cells has increased gene editing efficiency compared to a population of T cells that have not been contacted with one or more cGAS-STING pathway inhibitors.

[0133] In some embodiments, the nucleic acid comprises donor DNA. In some embodiments, the nucleic acid is a plasmid, nanoplasmid, minicircle, or viral vector comprising donor DNA. In some embodiments, the donor DNA encodes exogenous T cell receptor (TCR)-beta or a fragment thereof, exogenous TCR-alpha or a fragment thereof, or a combination thereof. In some embodiments, the nucleic acid is associated with a delivery vehicle. In some embodiments, the delivery vehicle is a lipid particle or a nanoparticle.

[0134] In various embodiments, the population of T cells comprises primary T cells.

[0135] In some embodiments, the method further comprises contacting the population of T cells with a gene editing reagent. In some embodiments, contacting the population of T cells with the gene editing reagent comprises contacting the population of T cells with a nucleic acid sequence encoding the gene editing reagent. In some embodiments, the population of T cells is contacted with the nucleic acid in the presence of a gene editing agent or a nucleic acid sequence encoding the gene editing reagent. In some embodiments, the gene editing reagent comprises an RNA-guided nuclease. In some embodiments, the RNA-guided nuclease is a CRISPR-Cas system.

[0136] In embodiments, the donor DNA is inserted into an endogenous TCR locus, hi embodiments, the endogenous TCR locus is an endogenous TCR-alpha locus, an endogenous TCR-beta locus, or a combination thereof.

[0137] In embodiments, contacting the population of T cells with a gene editing reagent comprises transfecting the population of T cells with the gene editing reagent. In embodiments, contacting the population of T cells with a nucleic acid comprises transfecting the population of T cells with the nucleic acid.

[0138] In various embodiments, the one or more cGAS-STING pathway inhibitors comprise a cGAS inhibitor. In various embodiments, the one or more cGAS-STING pathway inhibitors comprise a STING inhibitor. In various embodiments, the one or more cGAS-STING pathway inhibitors comprise a TANK-binding kinase 1 (TBK1) inhibitor. In various embodiments, the one or more cGAS-STING pathway inhibitors are Amlexanox (Aml), MRT67307 (MRT), BX795, H151, ODN-A151 (ODN151), Ru.521, G140, or a combination thereof. In various embodiments, the one or more cGAS-STING pathway inhibitors comprise Amlexanox (Aml). In various embodiments, the one or more cGAS-STING pathway inhibitors comprise MRT. In various embodiments, the one or more cGAS-STING pathway inhibitors comprise BX795. In various embodiments, the one or more cGAS-STING pathway inhibitors comprise H151. In embodiments, the one or more cGAS-STING pathway inhibitors comprise ODN151. In embodiments, the one or more cGAS-STING pathway inhibitors comprise Ru.521. In embodiments, the one or more cGAS-STING pathway inhibitors comprise G140.

[0139] In embodiments, the one or more cGAS-STING pathway inhibitors are Am1. In embodiments, the one or more cGAS-STING pathway inhibitors are MRT. In embodiments, the one or more cGAS-STING pathway inhibitors are BX795. In embodiments, the one or more cGAS-STING pathway inhibitors are H151. In embodiments, the one or more cGAS-STING pathway inhibitors are ODN151. In embodiments, the one or more cGAS-STING pathway inhibitors are Ru.521. In embodiments, the one or more cGAS-STING pathway inhibitors are G140. In embodiments, the cGAS-STING pathway inhibitor is Am1 and does not include other cGAS-STING pathway inhibitors. In embodiments, the cGAS-STING pathway inhibitor is MRT and does not include other cGAS-STING pathway inhibitors. In embodiments, the cGAS-STING pathway inhibitor is BX795 and does not include other cGAS-STING pathway inhibitors. In embodiments, the cGAS-STING pathway inhibitor is H151 and does not include other cGAS-STING pathway inhibitors. In embodiments, the cGAS-STING pathway inhibitor is ODN151 and does not include other cGAS-STING pathway inhibitors. In embodiments, the cGAS-STING pathway inhibitor is Ru.521 and does not include other cGAS-STING pathway inhibitors. In embodiments, the cGAS-STING pathway inhibitor is G140 and does not include other cGAS-STING pathway inhibitors.

[0140] In various embodiments, populations of T cells are independently contacted with one or more cGAS-STING pathway inhibitors at about 0.1 uM to about 50 uM. In various embodiments, populations of T cells are independently contacted with one or more cGAS-STING pathway inhibitors at about 2 uM to about 50 uM. In various embodiments, populations of T cells are independently contacted with one or more cGAS-STING pathway inhibitors at about 4 uM to about 50 uM. In various embodiments, populations of T cells are independently contacted with one or more cGAS-STING pathway inhibitors at about 6 uM to about 50 uM. In various embodiments, populations of T cells are independently contacted with one or more cGAS-STING pathway inhibitors at about 8 uM to about 50 uM. In various embodiments, populations of T cells are independently contacted with one or more cGAS-STING pathway inhibitors at about 10 uM to about 50 uM. In various embodiments, populations of T cells are independently contacted with one or more cGAS-STING pathway inhibitors at about 12 uM to about 50 uM. In various embodiments, populations of T cells are independently contacted with one or more cGAS-STING pathway inhibitors at about 14 uM to about 50 uM. In various embodiments, populations of T cells are independently contacted with one or more cGAS-STING pathway inhibitors at about 16 uM to about 50 uM. In various embodiments, populations of T cells are independently contacted with one or more cGAS-STING pathway inhibitors at about 18 uM to about 50 uM. In various embodiments, populations of T cells are independently contacted with one or more cGAS-STING pathway inhibitors at about 20 uM to about 50 uM. In various embodiments, populations of T cells are independently contacted with one or more cGAS-STING pathway inhibitors at about 22 uM to about 50 uM. In various embodiments, the populations of T cells are independently contacted with one or more cGAS-STING pathway inhibitors at about 24 uM to about 50 uM. In various embodiments, the populations of T cells are independently contacted with one or more cGAS-STING pathway inhibitors at about 26 uM to about 50 uM. In various embodiments, the populations of T cells are independently contacted with one or more cGAS-STING pathway inhibitors at about 28 uM to about 50 uM. In various embodiments, the populations of T cells are independently contacted with one or more cGAS-STING pathway inhibitors at about 30 uM to about 50 uM. In various embodiments, the populations of T cells are independently contacted with one or more cGAS-STING pathway inhibitors at about 32 uM to about 50 uM.In various embodiments, populations of T cells are independently contacted with one or more cGAS-STING pathway inhibitors at about 34 uM to about 50 uM. In various embodiments, populations of T cells are independently contacted with one or more cGAS-STING pathway inhibitors at about 36 uM to about 50 uM. In various embodiments, populations of T cells are independently contacted with one or more cGAS-STING pathway inhibitors at about 38 uM to about 50 uM. In various embodiments, populations of T cells are independently contacted with one or more cGAS-STING pathway inhibitors at about 40 uM to about 50 uM. In various embodiments, populations of T cells are independently contacted with one or more cGAS-STING pathway inhibitors at about 42 uM to about 50 uM. In various embodiments, populations of T cells are independently contacted with one or more cGAS-STING pathway inhibitors at about 44 uM to about 50 uM. In various embodiments, the populations of T cells are independently contacted with one or more cGAS-STING pathway inhibitors at about 46 uM to about 50 uM. In various embodiments, the populations of T cells are independently contacted with one or more cGAS-STING pathway inhibitors at about 48 uM to about 50 uM.

[0141] In various embodiments, populations of T cells are independently contacted with one or more cGAS-STING pathway inhibitors at about 0.1 uM to about 48 uM. In various embodiments, populations of T cells are independently contacted with one or more cGAS-STING pathway inhibitors at about 0.1 uM to about 46 uM. In various embodiments, populations of T cells are independently contacted with one or more cGAS-STING pathway inhibitors at about 0.1 uM to about 44 uM. In various embodiments, populations of T cells are independently contacted with one or more cGAS-STING pathway inhibitors at about 0.1 uM to about 42 uM. In various embodiments, populations of T cells are independently contacted with one or more cGAS-STING pathway inhibitors at about 0.1 uM to about 40 uM. In various embodiments, populations of T cells are independently contacted with one or more cGAS-STING pathway inhibitors at about 0.1 uM to about 38 uM. In various embodiments, populations of T cells are independently contacted with one or more cGAS-STING pathway inhibitors at about 0.1 uM to about 36 uM. In various embodiments, populations of T cells are independently contacted with one or more cGAS-STING pathway inhibitors at about 0.1 uM to about 34 uM. In various embodiments, populations of T cells are independently contacted with one or more cGAS-STING pathway inhibitors at about 0.1 uM to about 32 uM. In various embodiments, populations of T cells are independently contacted with one or more cGAS-STING pathway inhibitors at about 0.1 uM to about 30 uM. In various embodiments, populations of T cells are independently contacted with one or more cGAS-STING pathway inhibitors at about 0.1 uM to about 28 uM. In various embodiments, populations of T cells are independently contacted with one or more cGAS-STING pathway inhibitors at about 0.1 uM to about 26 uM. In various embodiments, populations of T cells are independently contacted with one or more cGAS-STING pathway inhibitors at about 0.1 uM to about 24 uM. In various embodiments, populations of T cells are independently contacted with one or more cGAS-STING pathway inhibitors at about 0.1 uM to about 22 uM. In various embodiments, populations of T cells are independently contacted with one or more cGAS-STING pathway inhibitors at about 0.1 uM to about 20 uM. In various embodiments, populations of T cells are independently contacted with one or more cGAS-STING pathway inhibitors at about 0.1 uM to about 18 uM.In various embodiments, populations of T cells are independently contacted with one or more cGAS-STING pathway inhibitors at about 0.1 uM to about 16 uM. In various embodiments, populations of T cells are independently contacted with one or more cGAS-STING pathway inhibitors at about 0.1 uM to about 14 uM. In various embodiments, populations of T cells are independently contacted with one or more cGAS-STING pathway inhibitors at about 0.1 uM to about 12 uM. In various embodiments, populations of T cells are independently contacted with one or more cGAS-STING pathway inhibitors at about 0.1 uM to about 10 uM. In various embodiments, populations of T cells are independently contacted with one or more cGAS-STING pathway inhibitors at about 0.1 uM to about 8 uM. In various embodiments, populations of T cells are independently contacted with one or more cGAS-STING pathway inhibitors at about 0.1 uM to about 6 uM. In various embodiments, populations of T cells are independently contacted with one or more cGAS-STING pathway inhibitors at about 0.1 uM to about 4 uM. In various embodiments, populations of T cells are independently contacted with one or more cGAS-STING pathway inhibitors at about 0.1 uM to about 2 uM. In various embodiments, populations of T cells are independently contacted with one or more cGAS-STING pathway inhibitors at about 0.1 uM, 2 uM, 4 uM, 6 uM, 8 uM, 10 uM, 12 uM, 14 uM, 16 uM, 18 uM, 20 uM, 22 uM, 24 uM, 26 uM, 28 uM, 30 uM, 32 uM, 34 uM, 36 uM, 38 uM, 40 uM, 42 uM, 44 uM, 46 uM, 48 uM, or 50 uM.

[0142] In embodiments, the population of T cells is contacted with the nucleic acid in the presence of one or more cGAS-STING pathway inhibitors. In embodiments, the population of T cells is contacted sequentially with the nucleic acid and one or more cGAS-STING pathway inhibitors. In some embodiments, the population of T cells is contacted with one or more cGAS-STING pathway inhibitors prior to the nucleic acid. In some embodiments, the population of T cells is contacted with a cGAS-STING pathway inhibitor provided herein and not with other cGAS-STING pathway inhibitors.

[0143] In various embodiments, the population of T cells is contacted with about 2 uM to about 8 uM BX795. In various embodiments, the population of T cells is contacted with about 2.5 uM to about 8 uM BX795. In various embodiments, the population of T cells is contacted with about 3 uM to about 8 uM BX795. In various embodiments, the population of T cells is contacted with about 3.5 uM to about 8 uM BX795. In various embodiments, the population of T cells is contacted with about 4 uM to about 8 uM BX795. In various embodiments, the population of T cells is contacted with about 4.5 uM to about 8 uM BX795. In various embodiments, the population of T cells is contacted with about 5 uM to about 8 uM BX795. In various embodiments, the population of T cells is contacted with about 5.5 uM to about 8 uM BX795. In various embodiments, the population of T cells is contacted with about 6 uM to about 8 uM BX795. In various embodiments, the population of T cells is contacted with about 6.5 uM to about 8 uM BX795. In various embodiments, the population of T cells is contacted with about 7 uM to about 8 uM BX795. In various embodiments, the population of T cells is contacted with about 7.5 uM to about 8 uM BX795.

[0144] In various embodiments, the population of T cells is contacted with about 2 uM to about 7.5 uM BX795. In various embodiments, the population of T cells is contacted with about 2 uM to about 7 uM BX795. In various embodiments, the population of T cells is contacted with about 2 uM to about 6.5 uM BX795. In various embodiments, the population of T cells is contacted with about 2 uM to about 6 uM BX795. In various embodiments, the population of T cells is contacted with about 2 uM to about 5.5 uM BX795. In various embodiments, the population of T cells is contacted with about 2 uM to about 5 uM BX795. In various embodiments, the population of T cells is contacted with about 2 uM to about 4.5 uM BX795. In various embodiments, the population of T cells is contacted with about 2 uM to about 4 uM BX795. In various embodiments, the population of T cells is contacted with about 2 uM to about 3.5 uM BX795. In various embodiments, the population of T cells is contacted with about 2 uM to about 3 uM BX795. In various embodiments, the population of T cells is contacted with about 2 uM to about 2.5 uM BX795. In various embodiments, the population of T cells is contacted with about 2 uM, 2.5 uM, 3 uM, 3.5 uM, 4 uM, 4.5 uM, 5 uM, 5.5 uM, 6 uM, 6.5 uM, 7 uM, 7.5 uM, or 8 uM BX795.

[0145] In various embodiments, the population of T cells is contacted with about 0.1 uM to about 5 uM of ODNA151. In various embodiments, the population of T cells is contacted with about 0.25 uM to about 5 uM of ODNA151. In various embodiments, the population of T cells is contacted with about 0.5 uM to about 5 uM of ODNA151. In various embodiments, the population of T cells is contacted with about 0.75 uM to about 5 uM of ODNA151. In various embodiments, the population of T cells is contacted with about 1 uM to about 5 uM of ODNA151. In various embodiments, the population of T cells is contacted with about 1.25 uM to about 5 uM of ODNA151. In various embodiments, the population of T cells is contacted with about 1.5 uM to about 5 uM of ODNA151. In various embodiments, the population of T cells is contacted with about 1.75 uM to about 5 uM of ODNA151. In various embodiments, the population of T cells is contacted with about 2 uM to about 5 uM of ODNA151. In various embodiments, the population of T cells is contacted with about 2.25 uM to about 5 uM of ODNA151. In various embodiments, the population of T cells is contacted with about 2.5 uM to about 5 uM of ODNA151. In various embodiments, the population of T cells is contacted with about 2.75 uM to about 5 uM of ODNA151. In various embodiments, the population of T cells is contacted with about 3 uM to about 5 uM of ODNA151. In various embodiments, the population of T cells is contacted with about 3.25 uM to about 5 uM of ODNA151. In various embodiments, the population of T cells is contacted with about 3.5 uM to about 5 uM of ODNA151. In various embodiments, the population of T cells is contacted with about 3.75 uM to about 5 uM of ODNA151. In various embodiments, the population of T cells is contacted with about 4 uM to about 5 uM of ODNA151. In various embodiments, the population of T cells is contacted with about 4.25 uM to about 5 uM of ODNA151. In various embodiments, the population of T cells is contacted with about 4.5 uM to about 5 uM of ODNA151. In various embodiments, the population of T cells is contacted with about 4.75 uM to about 5 uM of ODNA151.

[0146] In various embodiments, the population of T cells is contacted with about 0.1 uM to about 4.75 uM of ODNA151. In various embodiments, the population of T cells is contacted with about 0.1 uM to about 4.5 uM of ODNA151. In various embodiments, the population of T cells is contacted with about 0.1 uM to about 4.25 uM of ODNA151. In various embodiments, the population of T cells is contacted with about 0.1 uM to about 4 uM of ODNA151. In various embodiments, the population of T cells is contacted with about 0.1 uM to about 3.75 uM of ODNA151. In various embodiments, the population of T cells is contacted with about 0.1 uM to about 3.5 uM of ODNA151. In various embodiments, the population of T cells is contacted with about 0.1 uM to about 3.25 uM of ODNA151. In various embodiments, the population of T cells is contacted with about 0.1 uM to about 3 uM of ODNA151. In various embodiments, the population of T cells is contacted with about 0.1 uM to about 2.75 uM of ODNA151. In various embodiments, the population of T cells is contacted with about 0.1 uM to about 2.5 uM of ODNA151. In various embodiments, the population of T cells is contacted with about 0.1 uM to about 2.25 uM of ODNA151. In various embodiments, the population of T cells is contacted with about 0.1 uM to about 2 uM of ODNA151. In various embodiments, the population of T cells is contacted with about 0.1 uM to about 1.75 uM of ODNA151. In various embodiments, the population of T cells is contacted with about 0.1 uM to about 1.5 uM of ODNA151. In various embodiments, the population of T cells is contacted with about 0.1 uM to about 1.25 uM of ODNA151. In various embodiments, the population of T cells is contacted with about 0.1 uM to about 1 uM of ODNA151. In various embodiments, the population of T cells is contacted with about 0.1 uM to about 0.75 uM of ODNA151. In various embodiments, the population of T cells is contacted with about 0.1 uM to about 0.5 uM of ODNA151. In various embodiments, the population of T cells is contacted with about 0.1 uM to about 0.25 uM of ODNA151. In various embodiments, the population of T cells is contacted with about 0.1 uM, 0.25 uM, 0.5 uM, 0.75 uM, 1 uM, 1.25 uM, 1.5 uM, 1.75 uM, 2 uM, 2.25 uM, 2.5 uM, 2.75 uM, 3 uM, 3.25 uM, 3.5 uM, 3.75 uM, 4 uM, 4.25 uM, 4.5 uM, 4.75 uM, or 5 uM of ODNA151.

[0147] In various embodiments, the population of T cells is contacted with about 10 uM to about 50 uM Aml. In various embodiments, the population of T cells is contacted with about 12 uM to about 50 uM Aml. In various embodiments, the population of T cells is contacted with about 14 uM to about 50 uM Aml. In various embodiments, the population of T cells is contacted with about 16 uM to about 50 uM Aml. In various embodiments, the population of T cells is contacted with about 18 uM to about 50 uM Aml. In various embodiments, the population of T cells is contacted with about 20 uM to about 50 uM Aml. In various embodiments, the population of T cells is contacted with about 22 uM to about 50 uM Aml. In various embodiments, the population of T cells is contacted with about 24 uM to about 50 uM Aml. In various embodiments, the population of T cells is contacted with about 26 uM to about 50 uM Aml. In various embodiments, the population of T cells is contacted with about 28 uM to about 50 uM Aml. In various embodiments, the population of T cells is contacted with about 30 uM to about 50 uM Aml. In various embodiments, the population of T cells is contacted with about 32 uM to about 50 uM Aml. In various embodiments, the population of T cells is contacted with about 34 uM to about 50 uM Aml. In various embodiments, the population of T cells is contacted with about 36 uM to about 50 uM Aml. In various embodiments, the population of T cells is contacted with about 38 uM to about 50 uM Aml. In various embodiments, the population of T cells is contacted with about 40 uM to about 50 uM Aml. In various embodiments, the population of T cells is contacted with about 42 uM to about 50 uM Aml. In various embodiments, the population of T cells is contacted with about 44 uM to about 50 uM Aml. In various embodiments, the population of T cells is contacted with about 46 uM to about 50 uM Aml. In various embodiments, the population of T cells is contacted with about 48 uM to about 50 uM Aml.

[0148] In various embodiments, the population of T cells is contacted with about 10 uM to about 48 uM Aml. In various embodiments, the population of T cells is contacted with about 10 uM to about 46 uM Aml. In various embodiments, the population of T cells is contacted with about 10 uM to about 44 uM Aml. In various embodiments, the population of T cells is contacted with about 10 uM to about 42 uM Aml. In various embodiments, the population of T cells is contacted with about 10 uM to about 40 uM Aml. In various embodiments, the population of T cells is contacted with about 10 uM to about 38 uM Aml. In various embodiments, the population of T cells is contacted with about 10 uM to about 36 uM Aml. In various embodiments, the population of T cells is contacted with about 10 uM to about 34 uM Aml. In various embodiments, the population of T cells is contacted with about 10 uM to about 32 uM Aml. In various embodiments, the population of T cells is contacted with about 10 uM to about 30 uM Aml. In various embodiments, the population of T cells is contacted with about 10 uM to about 28 uM Aml. In various embodiments, the population of T cells is contacted with about 10 uM to about 26 uM Aml. In various embodiments, the population of T cells is contacted with about 10 uM to about 24 uM Aml. In various embodiments, the population of T cells is contacted with about 10 uM to about 22 uM Aml. In various embodiments, the population of T cells is contacted with about 10 uM to about 20 uM Aml. In various embodiments, the population of T cells is contacted with about 10 uM to about 18 uM Aml. In various embodiments, the population of T cells is contacted with about 10 uM to about 16 uM Aml. In various embodiments, the population of T cells is contacted with about 10 uM to about 14 uM Aml. In various embodiments, the population of T cells is contacted with about 10 uM to about 12 uM of Aml, hi various embodiments, the population of T cells is contacted with about 10 uM, 12 uM, 14 uM, 16 uM, 18 uM, 20 uM, 22 uM, 24 uM, 26 uM, 28 uM, 30 uM, 32 uM, 34 uM, 36 uM, 38 uM, 40 uM, 42 uM, 44 uM, 46 uM, 48 uM, or 50 uM of Aml.

[0149] In various embodiments, the population of T cells is contacted with about 1 uM to about 10 uM MRT. In various embodiments, the population of T cells is contacted with about 1.5 uM to about 10 uM MRT. In various embodiments, the population of T cells is contacted with about 2 uM to about 10 uM MRT. In various embodiments, the population of T cells is contacted with about 2.5 uM to about 10 uM MRT. In various embodiments, the population of T cells is contacted with about 3 uM to about 10 uM MRT. In various embodiments, the population of T cells is contacted with about 3.5 uM to about 10 uM MRT. In various embodiments, the population of T cells is contacted with about 4 uM to about 10 uM MRT. In various embodiments, the population of T cells is contacted with about 4.5 uM to about 10 uM MRT. In various embodiments, the population of T cells is contacted with about 5 uM to about 10 uM MRT. In various embodiments, the population of T cells is contacted with about 5.5 uM to about 10 uM MRT. In various embodiments, the population of T cells is contacted with about 6 uM to about 10 uM MRT. In various embodiments, the population of T cells is contacted with about 6.5 uM to about 10 uM MRT. In various embodiments, the population of T cells is contacted with about 7 uM to about 10 uM MRT. In various embodiments, the population of T cells is contacted with about 7.5 uM to about 10 uM MRT. In various embodiments, the population of T cells is contacted with about 8 uM to about 10 uM MRT. In various embodiments, the population of T cells is contacted with about 8.5 uM to about 10 uM MRT. In various embodiments, the population of T cells is contacted with about 9 uM to about 10 uM MRT. In various embodiments, the population of T cells is contacted with about 9.5 uM to about 10 uM MRT.

[0150] In various embodiments, the population of T cells is contacted with about 1 uM to about 9.5 uM MRT. In various embodiments, the population of T cells is contacted with about 1 uM to about 9 uM MRT. In various embodiments, the population of T cells is contacted with about 1 uM to about 8.5 uM MRT. In various embodiments, the population of T cells is contacted with about 1 uM to about 8 uM MRT. In various embodiments, the population of T cells is contacted with about 1 uM to about 7.5 uM MRT. In various embodiments, the population of T cells is contacted with about 1 uM to about 7 uM MRT. In various embodiments, the population of T cells is contacted with about 1 uM to about 6.5 uM MRT. In various embodiments, the population of T cells is contacted with about 1 uM to about 6 uM MRT. In various embodiments, the population of T cells is contacted with about 1 uM to about 5.5 uM MRT. In various embodiments, the population of T cells is contacted with about 1 uM to about 5 uM MRT. In various embodiments, the population of T cells is contacted with about 1 uM to about 4.5 uM MRT. In various embodiments, the population of T cells is contacted with about 1 uM to about 4 uM MRT. In various embodiments, the population of T cells is contacted with about 1 uM to about 3.5 uM MRT. In various embodiments, the population of T cells is contacted with about 1 uM to about 3 uM MRT. In various embodiments, the population of T cells is contacted with about 1 uM to about 2.5 uM MRT. In various embodiments, the population of T cells is contacted with about 1 uM to about 2 uM MRT. In various embodiments, the population of T cells is contacted with about 1 uM to about 1.5 uM MRT. In embodiments, the population of T cells is contacted with about 1 uM, 1.5 uM, 2 uM, 2.5 uM, 3 uM, 3.5 uM, 4 uM, 4.5 uM, 5 uM, 5.5 uM, 6 uM, 6.5 uM, 7 uM, 7.5 uM, 8 uM, 8.5 uM, 9 uM, 9.5 uM, or 10 uM MRT.

[0151] In various embodiments, the gene editing efficiency of the population of T cells is increased by at least about 0.5-fold to at least about 5-fold compared to a population of T cells not contacted with one or more cGAS-STING pathway inhibitors. In various embodiments, the gene editing efficiency of the population of T cells is increased by at least about 1-fold to at least about 5-fold compared to a population of T cells not contacted with one or more cGAS-STING pathway inhibitors. In various embodiments, the gene editing efficiency of the population of T cells is increased by at least about 1.5-fold to at least about 5-fold compared to a population of T cells not contacted with one or more cGAS-STING pathway inhibitors. In various embodiments, the gene editing efficiency of the population of T cells is increased by at least about 2-fold to at least about 5-fold compared to a population of T cells not contacted with one or more cGAS-STING pathway inhibitors. In various embodiments, the gene editing efficiency of the population of T cells is increased by at least about 2.5-fold to at least about 5-fold compared to a population of T cells not contacted with one or more cGAS-STING pathway inhibitors. In various embodiments, the gene editing efficiency of the population of T cells is increased by at least about 3-fold to at least about 5-fold compared to a population of T cells not contacted with one or more cGAS-STING pathway inhibitors. In various embodiments, the gene editing efficiency of the population of T cells is increased by at least about 3.5-fold to at least about 4-fold compared to a population of T cells not contacted with one or more cGAS-STING pathway inhibitors. In various embodiments, the gene editing efficiency of the population of T cells is increased by at least about 4-fold to at least about 5-fold compared to a population of T cells not contacted with one or more cGAS-STING pathway inhibitors. In various embodiments, the gene editing efficiency of the population of T cells is increased by at least about 4.5-fold to at least about 5-fold compared to a population of T cells not contacted with one or more cGAS-STING pathway inhibitors.

[0152] In various embodiments, the gene editing efficiency of the population of T cells is increased by at least about 0.5-fold to at least about 4.5-fold compared to a population of T cells not contacted with one or more cGAS-STING pathway inhibitors. In various embodiments, the gene editing efficiency of the population of T cells is increased by at least about 0.5-fold to at least about 4-fold compared to a population of T cells not contacted with one or more cGAS-STING pathway inhibitors. In various embodiments, the gene editing efficiency of the population of T cells is increased by at least about 0.5-fold to at least about 3.5-fold compared to a population of T cells not contacted with one or more cGAS-STING pathway inhibitors. In various embodiments, the gene editing efficiency of the population of T cells is increased by at least about 0.5-fold to at least about 3-fold compared to a population of T cells not contacted with one or more cGAS-STING pathway inhibitors. In various embodiments, the gene editing efficiency of the population of T cells is increased by at least about 0.5-fold to at least about 2.5-fold compared to a population of T cells not contacted with one or more cGAS-STING pathway inhibitors. In various embodiments, the gene editing efficiency of the population of T cells is increased by at least about 0.5-fold to at least about 2-fold compared to a population of T cells not contacted with one or more cGAS-STING pathway inhibitors. In various embodiments, the gene editing efficiency of the population of T cells is increased by at least about 0.5-fold to at least about 1.5-fold compared to a population of T cells not contacted with one or more cGAS-STING pathway inhibitors. In various embodiments, the gene editing efficiency of the population of T cells is increased by at least about 0.5-fold to at least about 1-fold compared to a population of T cells not contacted with one or more cGAS-STING pathway inhibitors. In various embodiments, the gene editing efficiency of the population of T cells is increased by at least about 0.5-fold, 1-fold, 1.5-fold, 2-fold, 2.5-fold, 3-fold, 3.5-fold, 4-fold, 4.5-fold, or 5-fold compared to a population of T cells not contacted with one or more cGAS-STING pathway inhibitors.

[0153] In various embodiments, the gene editing efficiency of the population of T cells is increased by about 2-fold to about 3-fold compared to a population of T cells not contacted with one or more cGAS-STING pathway inhibitors. In various embodiments, the gene editing efficiency of the population of T cells is increased by about 2.2-fold to about 3-fold compared to a population of T cells not contacted with one or more cGAS-STING pathway inhibitors. In various embodiments, the gene editing efficiency of the population of T cells is increased by about 2.4-fold to about 3-fold compared to a population of T cells not contacted with one or more cGAS-STING pathway inhibitors. In various embodiments, the gene editing efficiency of the population of T cells is increased by about 2.6-fold to about 3-fold compared to a population of T cells not contacted with one or more cGAS-STING pathway inhibitors. In various embodiments, the gene editing efficiency of the population of T cells is increased by about 2.8-fold to about 3-fold compared to a population of T cells not contacted with one or more cGAS-STING pathway inhibitors.

[0154] In various embodiments, the gene editing efficiency of the population of T cells is increased by about 2-fold to about 2.8-fold compared to a population of T cells not contacted with one or more cGAS-STING pathway inhibitors. In various embodiments, the gene editing efficiency of the population of T cells is increased by about 2-fold to about 2.6-fold compared to a population of T cells not contacted with one or more cGAS-STING pathway inhibitors. In various embodiments, the gene editing efficiency of the population of T cells is increased by about 2-fold to about 2.4-fold compared to a population of T cells not contacted with one or more cGAS-STING pathway inhibitors. In various embodiments, the gene editing efficiency of the population of T cells is increased by about 2-fold to about 2.2-fold compared to a population of T cells not contacted with one or more cGAS-STING pathway inhibitors. In various embodiments, the gene editing efficiency of the population of T cells is increased by about 2-fold, 2.2-fold, 2.4-fold, 2.6-fold, 2.8-fold, or 3-fold compared to a population of T cells not contacted with one or more cGAS-STING pathway inhibitors.

[0155] In various embodiments, the gene editing efficiency of the population of T cells is increased by about 60% to about 99% compared to a population of T cells not contacted with one or more cGAS-STING pathway inhibitors. In various embodiments, the gene editing efficiency of the population of T cells is increased by about 63% to about 99% compared to a population of T cells not contacted with one or more cGAS-STING pathway inhibitors. In various embodiments, the gene editing efficiency of the population of T cells is increased by about 66% to about 99% compared to a population of T cells not contacted with one or more cGAS-STING pathway inhibitors. In various embodiments, the gene editing efficiency of the population of T cells is increased by about 69% to about 99% compared to a population of T cells not contacted with one or more cGAS-STING pathway inhibitors. In various embodiments, the gene editing efficiency of the population of T cells is increased by about 72% to about 99% compared to a population of T cells not contacted with one or more cGAS-STING pathway inhibitors. In various embodiments, the gene editing efficiency of the population of T cells is increased by about 75% to about 99% compared to a population of T cells not contacted with one or more cGAS-STING pathway inhibitors. In various embodiments, the gene editing efficiency of the population of T cells is increased by about 78% to about 99% compared to a population of T cells not contacted with one or more cGAS-STING pathway inhibitors. In various embodiments, the gene editing efficiency of the population of T cells is increased by about 81% to about 99% compared to a population of T cells not contacted with one or more cGAS-STING pathway inhibitors. In various embodiments, the gene editing efficiency of the population of T cells is increased by about 84% to about 99% compared to a population of T cells not contacted with one or more cGAS-STING pathway inhibitors. In various embodiments, the knockout efficiency of the population of T cells is increased by about 87% to about 99% compared to a population of T cells not contacted with one or more cGAS-STING pathway inhibitors. In various embodiments, the gene editing efficiency of the population of T cells is increased by about 90% to about 99% compared to a population of T cells not contacted with one or more cGAS-STING pathway inhibitors. In various embodiments, the gene editing efficiency of the population of T cells is increased by about 93% to about 99% compared to a population of T cells not contacted with one or more cGAS-STING pathway inhibitors. In various embodiments, the gene editing efficiency of the population of T cells is increased by about 96% to about 99% compared to a population of T cells not contacted with one or more cGAS-STING pathway inhibitors.

[0156] In various embodiments, the gene editing efficiency of the population of T cells is increased by about 60% to about 96% compared to a population of T cells not contacted with one or more cGAS-STING pathway inhibitors. In various embodiments, the gene editing efficiency of the population of T cells is increased by about 60% to about 93% compared to a population of T cells not contacted with one or more cGAS-STING pathway inhibitors. In various embodiments, the gene editing efficiency of the population of T cells is increased by about 60% to about 90% compared to a population of T cells not contacted with one or more cGAS-STING pathway inhibitors. In various embodiments, the gene editing efficiency of the population of T cells is increased by about 60% to about 87% compared to a population of T cells not contacted with one or more cGAS-STING pathway inhibitors. In various embodiments, the gene editing efficiency of the population of T cells is increased by about 60% to about 84% compared to a population of T cells not contacted with one or more cGAS-STING pathway inhibitors. In various embodiments, the gene editing efficiency of the population of T cells is increased by about 60% to about 81% compared to a population of T cells not contacted with one or more cGAS-STING pathway inhibitors. In various embodiments, the gene editing efficiency of the population of T cells is increased by about 60% to about 78% compared to a population of T cells not contacted with one or more cGAS-STING pathway inhibitors. In various embodiments, the gene editing efficiency of the population of T cells is increased by about 60% to about 75% compared to a population of T cells not contacted with one or more cGAS-STING pathway inhibitors. In various embodiments, the gene editing efficiency of the population of T cells is increased by about 60% to about 72% compared to a population of T cells not contacted with one or more cGAS-STING pathway inhibitors. In various embodiments, the gene editing efficiency of the population of T cells is increased by about 60% to about 69% compared to a population of T cells not contacted with one or more cGAS-STING pathway inhibitors. In various embodiments, the gene editing efficiency of the population of T cells is increased by about 60% to about 66% compared to a population of T cells not contacted with one or more cGAS-STING pathway inhibitors. In various embodiments, the gene editing efficiency of the population of T cells is increased by about 60% to about 63% compared to a population of T cells not contacted with one or more cGAS-STING pathway inhibitors.In various embodiments, the gene editing efficiency of the population of T cells is increased by about 60%, 63%, 66%, 69%, 72%, 75%, 78%, 81%, 84%, 87%, 90%, 93%, 96%, or 99% compared to a population of T cells not contacted with the one or more cGAS-STING pathway inhibitors.

[0157] In various embodiments, the knockout efficiency of the engineered population of T cells is between about 20% and about 100%. In various embodiments, the knockout efficiency of the engineered population of T cells is between about 30% and about 100%. In various embodiments, the knockout efficiency of the engineered population of T cells is between about 40% and about 100%. In various embodiments, the knockout efficiency of the engineered population of T cells is between about 50% and about 100%. In various embodiments, the knockout efficiency of the engineered population of T cells is between about 60% and about 100%. In various embodiments, the knockout efficiency of the engineered population of T cells is between about 70% and about 100%. In various embodiments, the knockout efficiency of the engineered population of T cells is between about 70% and about 99%. In various embodiments, the knockout efficiency of the engineered population of T cells is between about 72% and about 99%. In various embodiments, the knockout efficiency of the engineered population of T cells is between about 74% and about 99%. In various embodiments, the knockout efficiency of the engineered population of T cells is between about 76% and about 99%. In various embodiments, the knockout efficiency of the engineered population of T cells is between about 78% and about 99%. In various embodiments, the knockout efficiency of the engineered population of T cells is between about 80% and about 99%. In various embodiments, the knockout efficiency of the engineered population of T cells is between about 82% and about 99%. In various embodiments, the knockout efficiency of the engineered population of T cells is between about 84% and about 99%. In various embodiments, the knockout efficiency of the engineered population of T cells is between about 86% and about 99%. In various embodiments, the knockout efficiency of the engineered population of T cells is between about 88% and about 99%. In various embodiments, the knockout efficiency of the engineered population of T cells is between about 90% and about 99%. In various embodiments, the knockout efficiency of the engineered population of T cells is between about 92% and about 99%. In various embodiments, the knockout efficiency of the engineered population of T cells is between about 94% and about 99%. In various embodiments, the knockout efficiency of the engineered population of T cells is between about 96% and about 99%. In various embodiments, the knockout efficiency of the engineered population of T cells is between about 98% and about 99%.

[0158] In various embodiments, the knockout efficiency of the engineered population of T cells is between about 50% and about 98%. In various embodiments, the knockout efficiency of the engineered population of T cells is between about 60% and about 98%. In various embodiments, the knockout efficiency of the engineered population of T cells is between about 70% and about 98%. In various embodiments, the knockout efficiency of the engineered population of T cells is between about 70% and about 96%. In various embodiments, the knockout efficiency of the engineered population of T cells is between about 70% and about 94%. In various embodiments, the knockout efficiency of the engineered population of T cells is between about 70% and about 92%. In various embodiments, the knockout efficiency of the engineered population of T cells is between about 70% and about 90%. In various embodiments, the knockout efficiency of the engineered population of T cells is between about 70% and about 88%. In various embodiments, the knockout efficiency of the engineered population of T cells is between about 70% and about 86%. In various embodiments, the knockout efficiency of the engineered population of T cells is between about 70% and about 84%. In various embodiments, the knockout efficiency of the engineered population of T cells is between about 70% and about 82%. In various embodiments, the knockout efficiency of the engineered population of T cells is between about 70% and about 80%. In various embodiments, the knockout efficiency of the engineered population of T cells is between about 70% and about 78%. In various embodiments, the knockout efficiency of the engineered population of T cells is between about 70% and about 76%. In various embodiments, the knockout efficiency of the engineered population of T cells is between about 70% and about 74%. In various embodiments, the knockout efficiency of the engineered population of T cells is between about 70% and about 72%. In embodiments, the knockout efficiency of the engineered population of T cells is about 20%, 30%, 40%, 50%, 55%, 60%, 65%, 70%, 72%, 74%, 76%, 78%, 80%, 82%, 84%, 86%, 88%, 90%, 92%, 94%, 96%, 98%, 99%, or about 100%. For the methods provided herein, in embodiments, the knockout efficiency of the population of T cells is about 90%. In embodiments, the knockout efficiency of the population of T cells is 90%.

[0159] In various embodiments, the knock-in efficiency for the population of T cells is about 20% to about 100%. In various embodiments, the knock-in efficiency for the population of T cells is about 20% to about 99%. In various embodiments, the knock-in efficiency for the population of T cells is about 25% to about 99%. In various embodiments, the knock-in efficiency for the population of T cells is about 30% to about 99%. In various embodiments, the knock-in efficiency for the population of T cells is about 35% to about 99%. In various embodiments, the knock-in efficiency for the population of T cells is about 40% to about 99%. In various embodiments, the knock-in efficiency for the population of T cells is about 45% to about 99%. In various embodiments, the knock-in efficiency for the population of T cells is about 50% to about 99%. In various embodiments, the knock-in efficiency for the population of T cells is about 55% to about 99%. In various embodiments, the knock-in efficiency for the population of T cells is about 60% to about 99%. In various embodiments, the knock-in efficiency for the population of T cells is about 65% to about 99%. In various embodiments, the knock-in efficiency for the population of T cells is about 70% to about 99%. In various embodiments, the knock-in efficiency for the population of T cells is about 75% to about 99%. In various embodiments, the knock-in efficiency for the population of T cells is about 80% to about 99%. In various embodiments, the knock-in efficiency for the population of T cells is about 85% to about 99%. In various embodiments, the knock-in efficiency for the population of T cells is about 90% to about 99%. In various embodiments, the knock-in efficiency for the population of T cells is about 95% to about 99%.

[0160] In various embodiments, the knock-in efficiency for the population of T cells is about 20% to about 95%. In various embodiments, the knock-in efficiency for the population of T cells is about 20% to about 90%. In various embodiments, the knock-in efficiency for the population of T cells is about 20% to about 85%. In various embodiments, the knock-in efficiency for the population of T cells is about 20% to about 80%. In various embodiments, the knock-in efficiency for the population of T cells is about 20% to about 75%. In various embodiments, the knock-in efficiency for the population of T cells is about 20% to about 70%. In various embodiments, the knock-in efficiency for the population of T cells is about 20% to about 65%. In various embodiments, the knock-in efficiency for the population of T cells is about 20% to about 60%. In various embodiments, the knock-in efficiency for the population of T cells is about 20% to about 55%. In various embodiments, the knock-in efficiency for the population of T cells is about 20% to about 50%. In various embodiments, the knock-in efficiency for the population of T cells is about 20% to about 45%. In various embodiments, the knock-in efficiency for the population of T cells is about 20% to about 40%. In various embodiments, the knock-in efficiency for the population of T cells is about 20% to about 35%. In various embodiments, the knock-in efficiency for the population of T cells is about 20% to about 30%. In various embodiments, the knock-in efficiency for the population of T cells is about 20% to about 25%. In various embodiments, the knock-in efficiency for the population of T cells is about 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100%. In various embodiments, the knock-in efficiency is about 60%. In various embodiments, the knock-in efficiency is 60%.

[0161] Provided herein, inter alia, are methods for increasing the proliferation of engineered T cells. The methods provided herein are believed to be effective in improving the proliferation of engineered T cells, thereby increasing the total number of engineered T cells in culture. The proliferation of engineered T cells is improved compared to engineered T cells generated without contact with one or more cGAS-STING pathway inhibitors. Thus, in one aspect, a method is provided for increasing the proliferation of a population of engineered T cells, comprising: i) contacting a population of T cells with one or more cyclic GMP-AMP synthase (cGAS)-stimulator of interferon genes (STING) pathway inhibitors and a nucleic acid, thereby forming a population of engineered T cells; and ii) expanding the population of engineered T cells, thereby forming a population of expanded engineered T cells, wherein the one or more cGAS-STING pathway inhibitors increase the population of expanded engineered T cells in step i) compared to a population of engineered T cells not contacted with one or more cGAS-STING pathway inhibitors.

[0162] In some embodiments, the nucleic acid comprises donor DNA. In some embodiments, the nucleic acid is a plasmid, nanoplasmid, minicircle, or viral vector comprising donor DNA. In some embodiments, the donor DNA encodes exogenous T cell receptor (TCR)-beta or a fragment thereof, exogenous TCR-alpha or a fragment thereof, or a combination thereof. In some embodiments, the nucleic acid is associated with a delivery vehicle. In some embodiments, the delivery vehicle is a lipid particle or a nanoparticle.

[0163] In various embodiments, the population of T cells comprises primary T cells.

[0164] In some embodiments, the T cell is contacted with a nucleic acid sequence encoding the gene editing reagent. In some embodiments, the T cell is contacted with the nucleic acid in the presence of a gene editing agent or a nucleic acid sequence encoding the gene editing reagent.

[0165] In embodiments, the donor DNA is inserted into an endogenous TCR locus, hi embodiments, the endogenous TCR locus is an endogenous TCR-alpha locus, an endogenous TCR-beta locus, or a combination thereof.

[0166] In embodiments, contacting the T cell with a gene editing reagent comprises transfecting the T cell with the gene editing reagent. In embodiments, contacting the T cell with a nucleic acid comprises transfecting the T cell with the nucleic acid.

[0167] In various embodiments, the one or more cGAS-STING pathway inhibitors comprise a cGAS inhibitor. In various embodiments, the one or more cGAS-STING pathway inhibitors comprise a TANK-binding kinase 1 (TBK1) inhibitor. In various embodiments, the one or more cGAS-STING pathway inhibitors are Amlexanox (Aml), MRT67307 (MRT), BX795, H151, ODN-A151 (ODN151), Ru.521, G140, or a combination thereof. In various embodiments, the one or more cGAS-STING pathway inhibitors comprise Amlexanox (Aml). In various embodiments, the one or more cGAS-STING pathway inhibitors comprise MRT. In various embodiments, the one or more cGAS-STING pathway inhibitors comprise BX795. In various embodiments, the one or more cGAS-STING pathway inhibitors comprise H151. In various embodiments, the one or more cGAS-STING pathway inhibitors comprise ODN-A151. In embodiments, the one or more cGAS-STING pathway inhibitors comprise ODN-151. In embodiments, the one or more cGAS-STING pathway inhibitors comprise Ru.521. In embodiments, the one or more cGAS-STING pathway inhibitors comprise G140.

[0168] In embodiments, the cGAS-STING pathway inhibitor is Am1. In embodiments, the cGAS-STING pathway inhibitor is MRT. In embodiments, the cGAS-STING pathway inhibitor is BX795. In embodiments, the cGAS-STING pathway inhibitor is H151. In embodiments, the cGAS-STING pathway inhibitor is ODN-A151. In embodiments, the cGAS-STING pathway inhibitor is ODN151. In embodiments, the cGAS-STING pathway inhibitor is Ru.521. In embodiments, the cGAS-STING pathway inhibitor is G140. In embodiments, the cGAS-STING pathway inhibitor is Am1 and no other cGAS-STING pathway inhibitors are present. In embodiments, the cGAS-STING pathway inhibitor is MRT and no other cGAS-STING pathway inhibitors are present. In embodiments, the cGAS-STING pathway inhibitor is BX795 and no other cGAS-STING pathway inhibitors. In embodiments, the cGAS-STING pathway inhibitor is H151 and no other cGAS-STING pathway inhibitors. In embodiments, the cGAS-STING pathway inhibitor is ODN-A151 and no other cGAS-STING pathway inhibitors. In embodiments, the cGAS-STING pathway inhibitor is ODN151 and no other cGAS-STING pathway inhibitors. In embodiments, the cGAS-STING pathway inhibitor is Ru.521 and no other cGAS-STING pathway inhibitors. In embodiments, the cGAS-STING pathway inhibitor is G140 and no other cGAS-STING pathway inhibitors.

[0169] In various embodiments, populations of T cells are independently contacted with one or more cGAS-STING pathway inhibitors at about 1 uM to about 50 uM. In various embodiments, populations of T cells are independently contacted with one or more cGAS-STING pathway inhibitors at about 2 uM to about 50 uM. In various embodiments, populations of T cells are independently contacted with one or more cGAS-STING pathway inhibitors at about 4 uM to about 50 uM. In various embodiments, populations of T cells are independently contacted with one or more cGAS-STING pathway inhibitors at about 6 uM to about 50 uM. In various embodiments, populations of T cells are independently contacted with one or more cGAS-STING pathway inhibitors at about 8 uM to about 50 uM. In various embodiments, populations of T cells are independently contacted with one or more cGAS-STING pathway inhibitors at about 10 uM to about 50 uM. In various embodiments, populations of T cells are independently contacted with one or more cGAS-STING pathway inhibitors at about 12 uM to about 50 uM. In various embodiments, populations of T cells are independently contacted with one or more cGAS-STING pathway inhibitors at about 14 uM to about 50 uM. In various embodiments, populations of T cells are independently contacted with one or more cGAS-STING pathway inhibitors at about 16 uM to about 50 uM. In various embodiments, populations of T cells are independently contacted with one or more cGAS-STING pathway inhibitors at about 18 uM to about 50 uM. In various embodiments, populations of T cells are independently contacted with one or more cGAS-STING pathway inhibitors at about 20 uM to about 50 uM. In various embodiments, populations of T cells are independently contacted with one or more cGAS-STING pathway inhibitors at about 22 uM to about 50 uM. In various embodiments, the populations of T cells are independently contacted with one or more cGAS-STING pathway inhibitors at about 24 uM to about 50 uM. In various embodiments, the populations of T cells are independently contacted with one or more cGAS-STING pathway inhibitors at about 26 uM to about 50 uM. In various embodiments, the populations of T cells are independently contacted with one or more cGAS-STING pathway inhibitors at about 28 uM to about 50 uM. In various embodiments, the populations of T cells are independently contacted with one or more cGAS-STING pathway inhibitors at about 30 uM to about 50 uM. In various embodiments, the populations of T cells are independently contacted with one or more cGAS-STING pathway inhibitors at about 32 uM to about 50 uM.In various embodiments, populations of T cells are independently contacted with one or more cGAS-STING pathway inhibitors at about 34 uM to about 50 uM. In various embodiments, populations of T cells are independently contacted with one or more cGAS-STING pathway inhibitors at about 36 uM to about 50 uM. In various embodiments, populations of T cells are independently contacted with one or more cGAS-STING pathway inhibitors at about 38 uM to about 50 uM. In various embodiments, populations of T cells are independently contacted with one or more cGAS-STING pathway inhibitors at about 40 uM to about 50 uM. In various embodiments, populations of T cells are independently contacted with one or more cGAS-STING pathway inhibitors at about 42 uM to about 50 uM. In various embodiments, populations of T cells are independently contacted with one or more cGAS-STING pathway inhibitors at about 44 uM to about 50 uM. In various embodiments, the populations of T cells are independently contacted with one or more cGAS-STING pathway inhibitors at about 46 uM to about 50 uM. In various embodiments, the populations of T cells are independently contacted with one or more cGAS-STING pathway inhibitors at about 48 uM to about 50 uM.

[0170] In various embodiments, populations of T cells are independently contacted with one or more cGAS-STING pathway inhibitors at about 1 uM to about 48 uM. In various embodiments, populations of T cells are independently contacted with one or more cGAS-STING pathway inhibitors at about 1 uM to about 46 uM. In various embodiments, populations of T cells are independently contacted with one or more cGAS-STING pathway inhibitors at about 1 uM to about 44 uM. In various embodiments, populations of T cells are independently contacted with one or more cGAS-STING pathway inhibitors at about 1 uM to about 42 uM. In various embodiments, populations of T cells are independently contacted with one or more cGAS-STING pathway inhibitors at about 1 uM to about 40 uM. In various embodiments, populations of T cells are independently contacted with one or more cGAS-STING pathway inhibitors at about 1 uM to about 38 uM. In various embodiments, populations of T cells are independently contacted with one or more cGAS-STING pathway inhibitors at about 1 uM to about 36 uM. In various embodiments, populations of T cells are independently contacted with one or more cGAS-STING pathway inhibitors at about 1 uM to about 32 uM. In various embodiments, populations of T cells are independently contacted with one or more cGAS-STING pathway inhibitors at about 1 uM to about 30 uM. In various embodiments, populations of T cells are independently contacted with one or more cGAS-STING pathway inhibitors at about 1 uM to about 28 uM. In various embodiments, populations of T cells are independently contacted with one or more cGAS-STING pathway inhibitors at about 1 uM to about 26 uM. In various embodiments, populations of T cells are independently contacted with one or more cGAS-STING pathway inhibitors at about 1 uM to about 24 uM. In various embodiments, the populations of T cells are independently contacted with one or more cGAS-STING pathway inhibitors at about 1 uM to about 22 uM. In various embodiments, the populations of T cells are independently contacted with one or more cGAS-STING pathway inhibitors at about 1 uM to about 20 uM. In various embodiments, the populations of T cells are independently contacted with one or more cGAS-STING pathway inhibitors at about 1 uM to about 18 uM. In various embodiments, the populations of T cells are independently contacted with one or more cGAS-STING pathway inhibitors at about 1 uM to about 16 uM. In various embodiments, the populations of T cells are independently contacted with one or more cGAS-STING pathway inhibitors at about 1 uM to about 14 uM.In various embodiments, the populations of T cells are independently contacted with one or more cGAS-STING pathway inhibitors at about 1 uM to about 12 uM. In various embodiments, the populations of T cells are independently contacted with one or more cGAS-STING pathway inhibitors at about 1 uM to about 10 uM. In various embodiments, the populations of T cells are independently contacted with one or more cGAS-STING pathway inhibitors at about 1 uM to about 8 uM. In various embodiments, the populations of T cells are independently contacted with one or more cGAS-STING pathway inhibitors at about 1 uM to about 6 uM. In various embodiments, the populations of T cells are independently contacted with one or more cGAS-STING pathway inhibitors at about 1 uM to about 4 uM. In various embodiments, the populations of T cells are independently contacted with one or more cGAS-STING pathway inhibitors at about 1 uM to about 2 uM. In various embodiments, the populations of T cells are independently contacted with about 1 uM, 2 uM, 4 uM, 6 uM, 8 uM, 10 uM, 12 uM, 14 uM, 16 uM, 18 uM, 20 uM, 22 uM, 24 uM, 26 uM, 28 uM, 30 uM, 32 uM, 34 uM, 36 uM, 38 uM, 40 uM, 42 uM, 44 uM, 46 uM, 48 uM, or 50 uM of one or more cGAS-STING pathway inhibitors.

[0171] In embodiments, the population of T cells is contacted with the nucleic acid in the presence of one or more cGAS-STING pathway inhibitors. In embodiments, the population of T cells is contacted sequentially with the nucleic acid and the one or more cGAS-STING pathway inhibitors. In embodiments, the population of T cells is contacted with the one or more cGAS-STING pathway inhibitors prior to the nucleic acid.

[0172] In embodiments, the T cells are contacted with one cGAS-STING pathway inhibitor. In embodiments, the T cells are contacted with one cGAS-STING pathway inhibitor provided herein and no other cGAS-STING pathway inhibitors.

[0173] In various embodiments, the population of T cells is contacted with about 1 uM to about 10 uM BX795. In various embodiments, the population of T cells is contacted with about 0.5 uM to about 10 uM BX795. In various embodiments, the population of T cells is contacted with about 2 uM to about 10 uM BX795. In various embodiments, the population of T cells is contacted with about 2.5 uM to about 10 uM BX795. In various embodiments, the population of T cells is contacted with about 3 uM to about 10 uM BX795. In various embodiments, the population of T cells is contacted with about 3.5 uM to about 10 uM BX795. In various embodiments, the population of T cells is contacted with about 4 uM to about 10 uM BX795. In various embodiments, the population of T cells is contacted with about 4.5 uM to about 10 uM BX795. In various embodiments, the population of T cells is contacted with about 5 uM to about 10 uM BX795. In various embodiments, the population of T cells is contacted with about 5.5 uM to about 10 uM BX795. In various embodiments, the population of T cells is contacted with about 6 uM to about 10 uM BX795. In various embodiments, the population of T cells is contacted with about 6.5 uM to about 10 uM BX795. In various embodiments, the population of T cells is contacted with about 7 uM to about 10 uM BX795. In various embodiments, the population of T cells is contacted with about 7.5 uM to about 10 uM BX795. In various embodiments, the population of T cells is contacted with about 8 uM to about 10 uM BX795. In various embodiments, the population of T cells is contacted with about 8.5 uM to about 10 uM BX795. In various embodiments, the population of T cells is contacted with about 8 uM to about 10 uM BX795. In various embodiments, the population of T cells is contacted with about 8.5 uM to about 10 uM BX795. In various embodiments, the population of T cells is contacted with about 9 uM to about 10 uM BX795. In various embodiments, the population of T cells is contacted with about 9.5 uM to about 10 uM BX795.

[0174] In various embodiments, the population of T cells is contacted with about 1 uM to about 9.5 uM BX795. In various embodiments, the population of T cells is contacted with about 0.5 uM to about 9 uM BX795. In various embodiments, the population of T cells is contacted with about 1 uM to about 8.5 uM BX795. In various embodiments, the population of T cells is contacted with about 0.5 uM to about 8 uM BX795. In various embodiments, the population of T cells is contacted with about 1 uM to about 7.5 uM BX795. In various embodiments, the population of T cells is contacted with about 0.5 uM to about 7 uM BX795. In various embodiments, the population of T cells is contacted with about 1 uM to about 6.5 uM BX795. In various embodiments, the population of T cells is contacted with about 0.5 uM to about 6 uM BX795. In various embodiments, the population of T cells is contacted with about 1 uM to about 5.5 uM BX795. In various embodiments, the population of T cells is contacted with about 0.5 uM to about 5 uM BX795. In various embodiments, the population of T cells is contacted with about 1 uM to about 4.5 uM BX795. In various embodiments, the population of T cells is contacted with about 0.5 uM to about 4 uM BX795. In various embodiments, the population of T cells is contacted with about 1 uM to about 3.5 uM BX795. In various embodiments, the population of T cells is contacted with about 0.5 uM to about 3 uM BX795. In various embodiments, the population of T cells is contacted with about 1 uM to about 2.5 uM BX795. In various embodiments, the population of T cells is contacted with about 0.5 uM to about 2 uM BX795. In various embodiments, the population of T cells is contacted with about 1 uM to about 1.5 uM BX795. In various embodiments, the population of T cells is contacted with about 0.5 uM to about 1 uM BX795. In various embodiments, the population of T cells is contacted with about 1 uM, 1.5 uM, 2 uM, 2.5 uM, 3 uM, 3.5 uM, 4 uM, 4.5 uM, 5 uM, 5.5 uM, 6 uM, 6.5 uM, 7 uM, 7.5 uM, 8 uM, 8.5 uM, 9 uM, 9.5 uM, or 10 uM BX795.

[0175] In various embodiments, the population of T cells is contacted with about 0.1 uM to about 8 uM of ODNA151. In various embodiments, the population of T cells is contacted with about 0.5 uM to about 8 uM of ODNA151. In various embodiments, the population of T cells is contacted with about 1 uM to about 8 uM of ODNA151. In various embodiments, the population of T cells is contacted with about 1.5 uM to about 8 uM of ODNA151. In various embodiments, the population of T cells is contacted with about 2 uM to about 8 uM of ODNA151. In various embodiments, the population of T cells is contacted with about 2.5 uM to about 8 uM of ODNA151. In various embodiments, the population of T cells is contacted with about 3 uM to about 8 uM of ODNA151. In various embodiments, the population of T cells is contacted with about 3.5 uM to about 8 uM of ODNA151. In various embodiments, the population of T cells is contacted with about 4 uM to about 8 uM of ODNA151. In various embodiments, the population of T cells is contacted with about 4.5 uM to about 8 uM of ODNA151. In various embodiments, the population of T cells is contacted with about 5 uM to about 8 uM of ODNA151. In various embodiments, the population of T cells is contacted with about 5.5 uM to about 8 uM of ODNA151. In various embodiments, the population of T cells is contacted with about 6 uM to about 8 uM of ODNA151. In various embodiments, the population of T cells is contacted with about 6.5 uM to about 8 uM of ODNA151. In various embodiments, the population of T cells is contacted with about 7 uM to about 8 uM of ODNA151. In various embodiments, the population of T cells is contacted with about 7.5 uM to about 8 uM of ODNA151.

[0176] In various embodiments, the population of T cells is contacted with about 0.1 uM to about 7.5 uM of ODNA151. In various embodiments, the population of T cells is contacted with about 0.1 uM to about 7 uM of ODNA151. In various embodiments, the population of T cells is contacted with about 0.1 uM to about 7.5 uM of ODNA151. In various embodiments, the population of T cells is contacted with about 0.1 uM to about 7 uM of ODNA151. In various embodiments, the population of T cells is contacted with about 0.1 uM to about 6.5 uM of ODNA151. In various embodiments, the population of T cells is contacted with about 0.1 uM to about 6 uM of ODNA151. In various embodiments, the population of T cells is contacted with about 0.1 uM to about 5.5 uM of ODNA151. In various embodiments, the population of T cells is contacted with about 0.1 uM to about 5 uM of ODNA151. In various embodiments, the population of T cells is contacted with about 0.1 uM to about 4.5 uM of ODNA151. In various embodiments, the population of T cells is contacted with about 0.1 uM to about 4 uM of ODNA151. In various embodiments, the population of T cells is contacted with about 0.1 uM to about 3.5 uM of ODNA151. In various embodiments, the population of T cells is contacted with about 0.1 uM to about 3 uM of ODNA151. In various embodiments, the population of T cells is contacted with about 0.1 uM to about 2.5 uM of ODNA151. In various embodiments, the population of T cells is contacted with about 0.1 uM to about 2 uM of ODNA151. In various embodiments, the population of T cells is contacted with about 0.1 uM to about 1.5 uM of ODNA151. In various embodiments, the population of T cells is contacted with about 0.1 uM to about 1 uM of ODNA151. In various embodiments, the population of T cells is contacted with about 0.1 uM to about 0.5 uM of ODNA151. In various embodiments, the population of T cells is contacted with about 0.1 uM, 0.5 uM, 1 uM, 1.5 uM, 2 uM, 2.5 uM, 3 uM, 3.5 uM, 4 uM, 4.5 uM, 5 uM, 5.5 uM, 6 uM, 6.5 uM, 7 uM, 7.5 uM, or 8 uM of ODNA151.

[0177] In various embodiments, the population of T cells is contacted with about 5 uM to about 50 uM of AML. In various embodiments, the population of T cells is contacted with about 15 uM to about 50 uM of AML. In various embodiments, the population of T cells is contacted with about 20 uM to about 50 uM of AML. In various embodiments, the population of T cells is contacted with about 25 uM to about 50 uM of AML. In various embodiments, the population of T cells is contacted with about 30 uM to about 50 uM of AML. In various embodiments, the population of T cells is contacted with about 35 uM to about 50 uM of AML. In various embodiments, the population of T cells is contacted with about 40 uM to about 50 uM of AML. In various embodiments, the population of T cells is contacted with about 45 uM to about 50 uM of AML.

[0178] In various embodiments, the population of T cells is contacted with about 5 uM to about 45 uM AML. In various embodiments, the population of T cells is contacted with about 5 uM to about 40 uM AML. In various embodiments, the population of T cells is contacted with about 5 uM to about 35 uM AML. In various embodiments, the population of T cells is contacted with about 5 uM to about 30 uM AML. In various embodiments, the population of T cells is contacted with about 5 uM to about 25 uM AML. In various embodiments, the population of T cells is contacted with about 5 uM to about 20 uM AML. In various embodiments, the population of T cells is contacted with about 5 uM to about 15 uM AML. In various embodiments, the population of T cells is contacted with about 5 uM to about 10 uM AML. In embodiments, the population of T cells is contacted with about 5 uM, 10 uM, 15 uM, 20 uM, 25 uM, 30 uM, 35 uM, 40 uM, 45 uM, or 50 uM of AML.

[0179] In various embodiments, the population of T cells is contacted with about 1 uM to about 25 uM MRT. In various embodiments, the population of T cells is contacted with about 2 uM to about 25 uM MRT. In various embodiments, the population of T cells is contacted with about 3 uM to about 25 uM MRT. In various embodiments, the population of T cells is contacted with about 4 uM to about 25 uM MRT. In various embodiments, the population of T cells is contacted with about 5 uM to about 25 uM MRT. In various embodiments, the population of T cells is contacted with about 6 uM to about 25 uM MRT. In various embodiments, the population of T cells is contacted with about 7 uM to about 25 uM MRT. In various embodiments, the population of T cells is contacted with about 8 uM to about 25 uM MRT. In various embodiments, the population of T cells is contacted with about 9 uM to about 25 uM MRT. In various embodiments, the population of T cells is contacted with about 10 uM to about 25 uM MRT. In various embodiments, the population of T cells is contacted with about 11 uM to about 25 uM MRT. In various embodiments, the population of T cells is contacted with about 12 uM to about 25 uM MRT. In various embodiments, the population of T cells is contacted with about 13 uM to about 25 uM MRT. In various embodiments, the population of T cells is contacted with about 14 uM to about 25 uM MRT. In various embodiments, the population of T cells is contacted with about 15 uM to about 25 uM MRT. In various embodiments, the population of T cells is contacted with about 16 uM to about 25 uM MRT. In various embodiments, the population of T cells is contacted with about 17 uM to about 25 uM MRT. In various embodiments, the population of T cells is contacted with about 18 uM to about 25 uM MRT. In various embodiments, the population of T cells is contacted with about 19 uM to about 25 uM MRT. In various embodiments, the population of T cells is contacted with about 20 uM to about 25 uM MRT. In various embodiments, the population of T cells is contacted with about 21 uM to about 25 uM MRT. In various embodiments, the population of T cells is contacted with about 22 uM to about 25 uM MRT. In various embodiments, the population of T cells is contacted with about 23 uM to about 25 uM MRT. In various embodiments, the population of T cells is contacted with about 24 uM to about 25 uM MRT.

[0180] In various embodiments, the population of T cells is contacted with about 1 uM to about 24 uM MRT. In various embodiments, the population of T cells is contacted with about 1 uM to about 23 uM MRT. In various embodiments, the population of T cells is contacted with about 1 uM to about 22 uM MRT. In various embodiments, the population of T cells is contacted with about 1 uM to about 21 uM MRT. In various embodiments, the population of T cells is contacted with about 1 uM to about 20 uM MRT. In various embodiments, the population of T cells is contacted with about 1 uM to about 19 uM MRT. In various embodiments, the population of T cells is contacted with about 1 uM to about 18 uM MRT. In various embodiments, the population of T cells is contacted with about 1 uM to about 17 uM MRT. In various embodiments, the population of T cells is contacted with about 1 uM to about 16 uM MRT. In various embodiments, the population of T cells is contacted with about 1 uM to about 15 uM MRT. In various embodiments, the population of T cells is contacted with about 1 uM to about 14 uM MRT. In various embodiments, the population of T cells is contacted with about 1 uM to about 13 uM MRT. In various embodiments, the population of T cells is contacted with about 1 uM to about 12 uM MRT. In various embodiments, the population of T cells is contacted with about 1 uM to about 11 uM MRT. In various embodiments, the population of T cells is contacted with about 1 uM to about 10 uM MRT. In various embodiments, the population of T cells is contacted with about 1 uM to about 9 uM MRT. In various embodiments, the population of T cells is contacted with about 1 uM to about 8 uM MRT. In various embodiments, the population of T cells is contacted with about 1 uM to about 7 uM MRT. In various embodiments, the population of T cells is contacted with about 1 uM to about 6 uM MRT. In various embodiments, the population of T cells is contacted with about 1 uM to about 5 uM MRT. In various embodiments, the population of T cells is contacted with about 1 uM to about 4 uM MRT. In various embodiments, the population of T cells is contacted with about 1 uM to about 3 uM MRT. In various embodiments, the population of T cells is contacted with about 1 uM to about 2 uM MRT. In embodiments, the population of T cells is contacted with about 1 uM, 2 uM, 3 uM, 4 uM, 5 uM, 6 uM, 7 uM, 8 uM, 9 uM, 10 uM, 11 uM, 12 uM, 13 uM, 14 uM, 15 uM, 16 uM, 17 uM, 18 uM, 19 uM, 20 uM, 21 uM, 22 uM, 23 uM, 24 uM, or 25 uM of MRT.

[0181] In various embodiments, the expanded population of engineered T cells is increased by at least about 0.5-fold to at least about 5-fold compared to the population of engineered T cells where the population of T cells in step i) is not contacted with one or more cGAS-STING pathway inhibitors. In various embodiments, the expanded population of engineered T cells is increased by at least about 1-fold to at least about 5-fold compared to the population of engineered T cells where the population of T cells in step i) is not contacted with one or more cGAS-STING pathway inhibitors. In various embodiments, the expanded population of engineered T cells is increased by at least about 1.5-fold to at least about 5-fold compared to the population of engineered T cells where the population of T cells in step i) is not contacted with one or more cGAS-STING pathway inhibitors. In various embodiments, the expanded population of engineered T cells is increased by at least about 2-fold to at least about 5-fold compared to the population of engineered T cells where the population of T cells in step i) is not contacted with one or more cGAS-STING pathway inhibitors. In various embodiments, the expanded population of engineered T cells is increased by at least about 2.5-fold to at least about 5-fold compared to the population of engineered T cells where the population of T cells in step i) is not contacted with one or more cGAS-STING pathway inhibitors. In various embodiments, the expanded population of engineered T cells is increased by at least about 3-fold to at least about 5-fold compared to the population of engineered T cells where the population of T cells in step i) is not contacted with one or more cGAS-STING pathway inhibitors. In various embodiments, the expanded population of engineered T cells is increased by at least about 3.5-fold to at least about 5-fold compared to the population of engineered T cells where the population of T cells in step i) is not contacted with one or more cGAS-STING pathway inhibitors. In various embodiments, the expanded population of engineered T cells is increased by at least about 4-fold to at least about 5-fold compared to the population of engineered T cells where the population of T cells in step i) is not contacted with one or more cGAS-STING pathway inhibitors. In various embodiments, the population of expanded engineered T cells is increased by at least about 4.5-fold to at least about 5-fold compared to a population of engineered T cells in which the population of T cells in step i) has not been contacted with one or more cGAS-STING pathway inhibitors.

[0182] In various embodiments, the expanded population of engineered T cells is increased by at least about 0.5-fold to at least about 4.5-fold compared to the population of engineered T cells where the population of T cells in step i) is not contacted with one or more cGAS-STING pathway inhibitors. In various embodiments, the expanded population of engineered T cells is increased by at least about 0.5-fold to at least about 4-fold compared to the population of engineered T cells where the population of T cells in step i) is not contacted with one or more cGAS-STING pathway inhibitors. In various embodiments, the expanded population of engineered T cells is increased by at least about 0.5-fold to at least about 3.5-fold compared to the population of engineered T cells where the population of T cells in step i) is not contacted with one or more cGAS-STING pathway inhibitors. In various embodiments, the expanded population of engineered T cells is increased by at least about 0.5-fold to at least about 3-fold compared to the population of engineered T cells where the population of T cells in step i) is not contacted with one or more cGAS-STING pathway inhibitors. In various embodiments, the expanded population of engineered T cells is increased by at least about 0.5-fold to at least about 2.5-fold compared to the population of engineered T cells where the population of T cells in step i) is not contacted with one or more cGAS-STING pathway inhibitors. In various embodiments, the expanded population of engineered T cells is increased by at least about 0.5-fold to at least about 2-fold compared to the population of engineered T cells where the population of T cells in step i) is not contacted with one or more cGAS-STING pathway inhibitors. In various embodiments, the expanded population of engineered T cells is increased by at least about 0.5-fold to at least about 1.5-fold compared to the population of engineered T cells where the population of T cells in step i) is not contacted with one or more cGAS-STING pathway inhibitors. In various embodiments, the expanded population of engineered T cells is increased by at least about 0.5-fold to at least about 1-fold compared to the population of engineered T cells where the population of T cells in step i) is not contacted with one or more cGAS-STING pathway inhibitors. In various embodiments, the expanded population of engineered T cells is increased by at least about 0.5-fold, 1-fold, 2-fold, 2.5-fold, 3-fold, 3.5-fold, 4-fold, 4.5-fold, or 5-fold compared to a population of engineered T cells in which the population of T cells in step i) has not been contacted with the one or more cGAS-STING pathway inhibitors.

[0183] For the methods provided herein, in embodiments, the population of expanded engineered T cells is increased by about 2-fold to about 3-fold compared to a population of engineered T cells in which the population of T cells in step i) has not been contacted with one or more cGAS-STING pathway inhibitors.

[0184] In various embodiments, the population of engineered T cells expands by at least about 0.5-fold to at least about 100-fold compared to the population of engineered T cells where the population of T cells in step i) is not contacted with one or more cGAS-STING pathway inhibitors. In various embodiments, the population of engineered T cells expands by at least about 5-fold to at least about 100-fold compared to the population of engineered T cells where the population of T cells in step i) is not contacted with one or more cGAS-STING pathway inhibitors. In various embodiments, the population of engineered T cells expands by at least about 10-fold to at least about 100-fold compared to the population of engineered T cells where the population of T cells in step i) is not contacted with one or more cGAS-STING pathway inhibitors. In various embodiments, the population of engineered T cells expands by at least about 15-fold to at least about 100-fold compared to the population of engineered T cells where the population of T cells in step i) is not contacted with one or more cGAS-STING pathway inhibitors. In various embodiments, the population of engineered T cells expands by at least about 20-fold to at least about 100-fold compared to the population of engineered T cells where the population of T cells in step i) is not contacted with one or more cGAS-STING pathway inhibitors. In various embodiments, the population of engineered T cells expands by at least about 25-fold to at least about 100-fold compared to the population of engineered T cells where the population of T cells in step i) is not contacted with one or more cGAS-STING pathway inhibitors. In various embodiments, the population of engineered T cells expands by at least about 30-fold to at least about 100-fold compared to the population of engineered T cells where the population of T cells in step i) is not contacted with one or more cGAS-STING pathway inhibitors. In various embodiments, the population of engineered T cells expands by at least about 35-fold to at least about 100-fold compared to the population of engineered T cells where the population of T cells in step i) is not contacted with one or more cGAS-STING pathway inhibitors. In various embodiments, the population of engineered T cells expands by at least about 40-fold to at least about 100-fold compared to a population of engineered T cells in which the population of T cells in step i) has not been contacted with one or more cGAS-STING pathway inhibitors.In various embodiments, the population of engineered T cells expands by at least about 45-fold to at least about 100-fold compared to the population of engineered T cells where the population of T cells in step i) is not contacted with one or more cGAS-STING pathway inhibitors. In various embodiments, the population of engineered T cells expands by at least about 50-fold to at least about 100-fold compared to the population of engineered T cells where the population of T cells in step i) is not contacted with one or more cGAS-STING pathway inhibitors. In various embodiments, the population of engineered T cells expands by at least about 55-fold to at least about 100-fold compared to the population of engineered T cells where the population of T cells in step i) is not contacted with one or more cGAS-STING pathway inhibitors. In various embodiments, the population of engineered T cells expands by at least about 60-fold to at least about 100-fold compared to the population of engineered T cells where the population of T cells in step i) is not contacted with one or more cGAS-STING pathway inhibitors. In various embodiments, the population of engineered T cells expands by at least about 65-fold to at least about 100-fold compared to the population of engineered T cells where the population of T cells in step i) is not contacted with one or more cGAS-STING pathway inhibitors. In various embodiments, the population of engineered T cells expands by at least about 70-fold to at least about 100-fold compared to the population of engineered T cells where the population of T cells in step i) is not contacted with one or more cGAS-STING pathway inhibitors. In various embodiments, the population of engineered T cells expands by at least about 75-fold to at least about 100-fold compared to the population of engineered T cells where the population of T cells in step i) is not contacted with one or more cGAS-STING pathway inhibitors. In various embodiments, the population of engineered T cells expands by at least about 80-fold to at least about 100-fold compared to the population of engineered T cells where the population of T cells in step i) is not contacted with one or more cGAS-STING pathway inhibitors. In various embodiments, the population of engineered T cells expands by at least about 85-fold to at least about 100-fold compared to a population of engineered T cells in which the population of T cells in step i) has not been contacted with the one or more cGAS-STING pathway inhibitors.In embodiments, the population of engineered T cells expands by at least about 90-fold to at least about 100-fold compared to the population of engineered T cells where the population of T cells in step i) is not contacted with one or more cGAS-STING pathway inhibitors. In embodiments, the population of engineered T cells expands by at least about 95-fold to at least about 100-fold compared to the population of engineered T cells where the population of T cells in step i) is not contacted with one or more cGAS-STING pathway inhibitors.

[0185] In various embodiments, the population of engineered T cells expands by at least about 0.5-fold to at least about 95-fold compared to the population of engineered T cells where the population of T cells in step i) is not contacted with one or more cGAS-STING pathway inhibitors. In various embodiments, the population of engineered T cells expands by at least about 0.5-fold to at least about 90-fold compared to the population of engineered T cells where the population of T cells in step i) is not contacted with one or more cGAS-STING pathway inhibitors. In various embodiments, the population of engineered T cells expands by at least about 0.5-fold to at least about 85-fold compared to the population of engineered T cells where the population of T cells in step i) is not contacted with one or more cGAS-STING pathway inhibitors. In various embodiments, the population of engineered T cells expands by at least about 0.5-fold to at least about 80-fold compared to the population of engineered T cells where the population of T cells in step i) is not contacted with one or more cGAS-STING pathway inhibitors. In various embodiments, the population of engineered T cells expands by at least about 0.5-fold to at least about 75-fold compared to the population of engineered T cells where the population of T cells in step i) is not contacted with one or more cGAS-STING pathway inhibitors. In various embodiments, the population of engineered T cells expands by at least about 0.5-fold to at least about 70-fold compared to the population of engineered T cells where the population of T cells in step i) is not contacted with one or more cGAS-STING pathway inhibitors. In various embodiments, the population of engineered T cells expands by at least about 0.5-fold to at least about 65-fold compared to the population of engineered T cells where the population of T cells in step i) is not contacted with one or more cGAS-STING pathway inhibitors. In various embodiments, the population of engineered T cells expands by at least about 0.5-fold to at least about 60-fold compared to the population of engineered T cells where the population of T cells in step i) is not contacted with one or more cGAS-STING pathway inhibitors. In various embodiments, the population of engineered T cells expands by at least about 0.5-fold to at least about 55-fold compared to a population of engineered T cells in which the population of T cells in step i) has not been contacted with the one or more cGAS-STING pathway inhibitors.In various embodiments, the population of engineered T cells expands by at least about 0.5-fold to at least about 50-fold compared to the population of engineered T cells where the population of T cells in step i) is not contacted with one or more cGAS-STING pathway inhibitors. In various embodiments, the population of engineered T cells expands by at least about 0.5-fold to at least about 45-fold compared to the population of engineered T cells where the population of T cells in step i) is not contacted with one or more cGAS-STING pathway inhibitors. In various embodiments, the population of engineered T cells expands by at least about 0.5-fold to at least about 40-fold compared to the population of engineered T cells where the population of T cells in step i) is not contacted with one or more cGAS-STING pathway inhibitors. In various embodiments, the population of engineered T cells expands by at least about 0.5-fold to at least about 35-fold compared to the population of engineered T cells where the population of T cells in step i) is not contacted with one or more cGAS-STING pathway inhibitors. In various embodiments, the population of engineered T cells expands by at least about 0.5-fold to at least about 30-fold compared to the population of engineered T cells where the population of T cells in step i) is not contacted with one or more cGAS-STING pathway inhibitors. In various embodiments, the population of engineered T cells expands by at least about 0.5-fold to at least about 25-fold compared to the population of engineered T cells where the population of T cells in step i) is not contacted with one or more cGAS-STING pathway inhibitors. In various embodiments, the population of engineered T cells expands by at least about 0.5-fold to at least about 20-fold compared to the population of engineered T cells where the population of T cells in step i) is not contacted with one or more cGAS-STING pathway inhibitors. In various embodiments, the population of engineered T cells expands by at least about 0.5-fold to at least about 15-fold compared to the population of engineered T cells where the population of T cells in step i) is not contacted with one or more cGAS-STING pathway inhibitors. In various embodiments, the population of engineered T cells expands by at least about 0.5-fold to at least about 10-fold compared to a population of engineered T cells in which the population of T cells in step i) has not been contacted with the one or more cGAS-STING pathway inhibitors.In various embodiments, the population of engineered T cells expands by at least about 0.5-fold to at least about 5-fold compared to the population of engineered T cells where the population of T cells in step i) is not contacted with one or more cGAS-STING pathway inhibitors. In various embodiments, the population of engineered T cells expands by at least about 0.5-fold, 5-fold, 10-fold, 15-fold, 20-fold, 25-fold, 30-fold, 35-fold, 40-fold, 45-fold, 50-fold, 55-fold, 60-fold, 65-fold, 70-fold, 75-fold, 80-fold, 85-fold, 90-fold, 95-fold, or 100-fold compared to the population of engineered T cells where the population of T cells in step i) is not contacted with one or more cGAS-STING pathway inhibitors.

[0186] Engineered T cell compositions Provided herein, inter alia, are compositions comprising engineered T cells produced by the methods provided herein, including embodiments thereof. In one aspect, provided are engineered T cells produced by the methods provided herein, including embodiments thereof.

[0187] Provided herein, among other things, are compositions comprising a population of engineered T cells produced by the methods provided herein, including embodiments thereof. The population of engineered T cells may have increased survival and / or proliferation compared to a population of engineered T cells produced by a method in which the population of T cells is not contacted with a cGAS-STING pathway inhibitor prior to generating the population of engineered T cells. Thus, in one aspect, a population of engineered T cells is provided that is produced by contacting a population of T cells with a nucleic acid and one or more cyclic GMP-AMP synthase (cGAS)-stimulator of interferon genes (STING) pathway inhibitors.

[0188] In some embodiments, the nucleic acid comprises donor DNA. In some embodiments, the nucleic acid is a plasmid, nanoplasmid, minicircle, or viral vector comprising donor DNA. In some embodiments, the donor DNA encodes exogenous T cell receptor (TCR)-alpha or a fragment thereof, exogenous TCR-beta or a fragment thereof, or a combination thereof. In some embodiments, the nucleic acid is associated with a delivery vehicle. In some embodiments, the delivery vehicle is a lipid particle or a nanoparticle.

[0189] In various embodiments, the population of T cells comprises primary T cells.

[0190] In some embodiments, the population of engineered T cells provided herein is further contacted with a gene editing reagent. In some embodiments, the donor DNA is inserted into endogenous TCR locus. In some embodiments, the endogenous TCR locus is endogenous TCR-alpha locus, endogenous TCR-beta locus, or a combination thereof.

[0191] In embodiments, contacting the T cell with a gene editing reagent comprises transfecting the T cell with the gene editing reagent. In embodiments, contacting the T cell with a nucleic acid comprises transfecting the T cell with the nucleic acid.

[0192] In embodiments, the one or more cGAS-STING pathway inhibitors comprise a cGAS inhibitor. In embodiments, the one or more cGAS-STING pathway inhibitors comprise a STING inhibitor. In embodiments, the one or more cGAS-STING pathway inhibitors comprise a TANK-binding kinase 1 (TBK1) inhibitor.

[0193] In embodiments, the one or more cGAS-STING pathway inhibitors are Amlexanox (AmI), MRT67307 (MRT), BX795, H151, ODN-A151 (ODN151), Ru.521, G140, or a combination thereof. In embodiments, the one or more cGAS-STING pathway inhibitors are selected from the following: AmI, BX795, ODN151, and MRT. In embodiments, the one or more cGAS-STING pathway inhibitors are ODN151. In embodiments, the one or more cGAS-STING pathway inhibitors are BX795.

[0194] In embodiments, the population of T cells is contacted with the nucleic acid in the presence of one or more cGAS-STING pathway inhibitors. In embodiments, the population of T cells is contacted sequentially with the nucleic acid and the one or more cGAS-STING pathway inhibitors. In embodiments, the population of T cells is contacted with the one or more cGAS-STING pathway inhibitors prior to the nucleic acid.

[0195] In various embodiments, the population of T cells is contacted with one or more cGAS-STING pathway inhibitors for up to about 10 hours, for example, from about 2 hours to about 10 hours. In various embodiments, the population of T cells is contacted with one or more cGAS-STING pathway inhibitors for about 3 hours to about 10 hours. In various embodiments, the population of T cells is contacted with one or more cGAS-STING pathway inhibitors for about 4 hours to about 10 hours. In various embodiments, the population of T cells is contacted with one or more cGAS-STING pathway inhibitors for about 5 hours to about 10 hours. In various embodiments, the population of T cells is contacted with one or more cGAS-STING pathway inhibitors for about 6 hours to about 10 hours. In various embodiments, the population of T cells is contacted with one or more cGAS-STING pathway inhibitors for about 7 hours to about 10 hours. In various embodiments, the population of T cells is contacted with one or more cGAS-STING pathway inhibitors for about 8 hours to about 10 hours. In various embodiments, the population of T cells is contacted with one or more cGAS-STING pathway inhibitors for about 9 hours to about 10 hours.

[0196] In various embodiments, the population of T cells is contacted with one or more cGAS-STING pathway inhibitors for up to about 9 hours, for example, from about 2 hours to about 9 hours. In various embodiments, the population of T cells is contacted with one or more cGAS-STING pathway inhibitors for about 2 hours to about 8 hours. In various embodiments, the population of T cells is contacted with one or more cGAS-STING pathway inhibitors for about 2 hours to about 7 hours. In various embodiments, the population of T cells is contacted with one or more cGAS-STING pathway inhibitors for about 2 hours to about 6 hours. In various embodiments, the population of T cells is contacted with one or more cGAS-STING pathway inhibitors for about 2 hours to about 5 hours. In various embodiments, the population of T cells is contacted with one or more cGAS-STING pathway inhibitors for about 2 hours to about 4 hours. In various embodiments, the population of T cells is contacted with one or more cGAS-STING pathway inhibitors for about 2 hours to about 3 hours. In embodiments, the population of T cells is contacted with the one or more cGAS-STING pathway inhibitors for about 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, or 10 hours. In embodiments, the T cells are contacted with the one or more cGAS-STING pathway inhibitors for about 6 hours. In embodiments, the T cells are contacted with the one or more cGAS-STING pathway inhibitors for 6 hours.

[0197] In embodiments, the population of T cells is contacted with one cGAS-STING pathway inhibitor. In embodiments, the population of T cells is contacted with one cGAS-STING pathway inhibitor and no other cGAS-STING pathway inhibitor.

[0198] T cell composition Provided herein are compositions useful for generating a population of engineered T cells, comprising a population of T cells and one or more cGAS-STING inhibitors. The applicant has demonstrated that one or more cGAS-STING inhibitors increase gene editing efficiency in T cells. The applicant has further demonstrated that the compositions provided herein, including embodiments thereof, result in a population of engineered T cells with increased cell viability and proliferation compared to compositions that do not include one or more cGAS-STING inhibitors. Thus, in one aspect, a composition is provided that includes a population of T cells, a nucleic acid, and one or more cyclic GMP-AMP synthase (cGAS)-stimulator of interferon genes (STING) pathway inhibitors.

[0199] In some embodiments, the nucleic acid comprises donor DNA. In some embodiments, the nucleic acid is a plasmid, nanoplasmid, minicircle, or viral vector comprising donor DNA. In some embodiments, the donor DNA encodes exogenous T cell receptor (TCR)-alpha or a fragment thereof, exogenous TCR-beta or a fragment thereof, or a combination thereof. In some embodiments, the nucleic acid is associated with a delivery vehicle. In some embodiments, the delivery vehicle is a lipid particle or a nanoparticle.

[0200] In various embodiments, the population of T cells comprises primary T cells.

[0201] In various embodiments, the composition further comprises a gene editing reagent.

[0202] In embodiments, the one or more cGAS-STING pathway inhibitors comprise a cGAS inhibitor. In embodiments, the one or more cGAS-STING pathway inhibitors comprise a STING inhibitor. In embodiments, the one or more cGAS-STING pathway inhibitors comprise a TANK-binding kinase 1 (TBK1) inhibitor.

[0203] In various embodiments, the one or more cGAS-STING pathway inhibitors are Amlexanox (Aml), MRT67307 (MRT), BX795, H151, ODN-A151 (ODN151), Ru.521, G140, or a combination thereof. In various embodiments, the one or more cGAS-STING pathway inhibitors comprise Amlexanox (Aml). In various embodiments, the one or more cGAS-STING pathway inhibitors comprise MRT. In various embodiments, the one or more cGAS-STING pathway inhibitors comprise BX795. In various embodiments, the one or more cGAS-STING pathway inhibitors comprise H151. In various embodiments, the one or more cGAS-STING pathway inhibitors comprise ODN151. In various embodiments, the one or more cGAS-STING pathway inhibitors comprise Ru.521. In various embodiments, the one or more cGAS-STING pathway inhibitors comprise G140.

[0204] In embodiments, the cGAS-STING pathway inhibitor is Am1. In embodiments, the cGAS-STING pathway inhibitor is MRT. In embodiments, the cGAS-STING pathway inhibitor is BX795. In embodiments, the cGAS-STING pathway inhibitor is H151. In embodiments, the cGAS-STING pathway inhibitor is ODN151. In embodiments, the cGAS-STING pathway inhibitor is Ru.521. In embodiments, the cGAS-STING pathway inhibitor is G140. In embodiments, the cGAS-STING pathway inhibitor is Am1 and no other cGAS-STING pathway inhibitors. In embodiments, the cGAS-STING pathway inhibitor is MRT and no other cGAS-STING pathway inhibitors. In embodiments, the cGAS-STING pathway inhibitor is BX795 and no other cGAS-STING pathway inhibitors. In embodiments, the cGAS-STING pathway inhibitor is H151 and no other cGAS-STING pathway inhibitors. In embodiments, the cGAS-STING pathway inhibitor is ODN151 and no other cGAS-STING pathway inhibitors. In embodiments, the cGAS-STING pathway inhibitor is Ru.521 and no other cGAS-STING pathway inhibitors. In embodiments, the cGAS-STING pathway inhibitor is G140 and no other cGAS-STING pathway inhibitors.

[0205] In embodiments, the one or more cGAS-STING pathway inhibitors are selected from the following: Aml, BX795, ODN151, and MRT. In embodiments, the one or more cGAS-STING pathway inhibitors are ODN151. In embodiments, the one or more cGAS-STING pathway inhibitors are BX795.

[0206] Pharmaceutical Composition The compositions provided herein, including T cell compositions and engineered T cell compositions, are believed to be effective in treating diseases (e.g., cancer). For example, the engineered T cells provided herein may contain an exogenous T cell receptor specific to a cancer cell antigen. Thus, in one aspect, a pharmaceutical composition is provided comprising the engineered T cells provided herein, including embodiments thereof. In various embodiments, the pharmaceutical composition further comprises a pharmaceutically acceptable excipient (e.g., saline).

[0207] Treatment method It is contemplated that the engineered T cells provided herein, including embodiments thereof, are specific for disease-specific antigens (e.g., cancer cell antigens), thereby enabling effective targeting of cancer cells. The engineered T cells may, for example, comprise one or more exogenous T cell receptors engineered to be specific for an individual's cancer cells, enabling individualized and specific targeting of cancer cells. Thus, in one aspect, a method of treating a disease in a subject in need thereof is provided, comprising administering a therapeutically effective amount of an engineered T cell provided herein, including embodiments thereof, or a pharmaceutical composition provided herein comprising an embodiment thereof. In various embodiments, the method comprises administering a therapeutically effective amount of an engineered T cell provided herein, including embodiments thereof. In various embodiments, the method comprises administering a therapeutically effective amount of a pharmaceutical composition provided herein, including embodiments thereof.

[0208] For the methods provided herein, in embodiments, the engineered T cells may be generated from a subject. For example, T cells may be extracted from a subject and contacted ex vivo with nucleic acid (e.g., donor nucleic acid) and one or more cGAS-STING inhibitors, thereby generating engineered T cells that are administered to the original subject. Thus, in embodiments, the engineered T cells are autologous T cells. In embodiments, the engineered T cells may be generated from T cells that are not taken from a subject. For example, the engineered T cells may be generated from a healthy subject (e.g., a subject without cancer). Thus, in embodiments, the engineered T cells are allogeneic T cells.

[0209] For the methods provided herein, in embodiments, the disease is cancer. In embodiments, the cancer is melanoma, lymphoma, or leukemia. In embodiments, the cancer is melanoma. In embodiments, the cancer is lymphoma. In embodiments, the cancer is leukemia.

[0210] It will be understood that the examples and embodiments described herein are for illustrative purposes only, and that various modifications or variations in light thereof will be suggested to those skilled in the art and are to be included within the spirit and scope of this application and the appended claims. All publications, patents, and patent applications cited herein are hereby incorporated by reference in their entirety for all purposes. [Example]

[0211] Example 1: Introduction to an exemplary study Over the past decade, a better understanding of tumor immunotherapy combined with the development of human cell engineering techniques has made adoptive cell therapy (ACT) a viable treatment option. ACT is a highly personalized cancer treatment approach that relies on engineering cancer-targeting lymphocytes ex vivo and then administering these cancer-killing T cells in vivo back to the original patient. Unlike other forms of cancer immunotherapy, which rely on the generation of anti-tumor lymphocytes in the patient's body, which lack specificity and efficiency under most circumstances, ACT offers multiple advantages, including high potency, specificity, large numbers of lymphocytes, and sustained circulation time. Today, three forms of ACT have been developed and are in clinical use: tumor-infiltrating lymphocytes (TILs), chimeric antigen receptor T cells (CAR-Ts), and T cell receptor-engineered T cells (TCR-Ts). TILs and CAR-Ts have shown promise in patients with melanoma, lymphoma, and leukemia, achieving promising clinical results and improving both the quality and longevity of patients' lives. However, serious side effects, such as severe cytokine release syndrome (CRS), cardiovascular toxicity, and unexpected neurological complications, remain unaddressed. Furthermore, TIL and CAR-T approaches have met with limited success in solid tumor treatment. To overcome these challenges, TCR-engineered T cells have been utilized for the past 20 years, and numerous clinical and preclinical studies have linked their impact on mediating tumor lysis and eradication in various tumor types. For example, in early 2022, Immunocore Inc.'s Kimmtrak, the first TCR-engineered T cell therapeutic approach for melanoma treatment, was approved by the FDA as a treatment option.

[0212] The T cell receptor (TCR) consists of an alpha (α) chain and a beta (β) chain associated with the CD3 complex on the surface of T cells. TCRs bind non-covalently to peptide histocompatibility complex class I (MHC-I) molecules on the surface of tumor cells with high specificity due to their ability to distinguish between MHC-I molecules loaded with non-self or mutated self-peptides and MHC-I molecules loaded with self-peptides. However, in certain cases, tumor cell evasion of immune surveillance, reduced surface expression of MHC-I molecules, or low levels of mutated self-peptides on the cell surface can suppress T cell-mediated tumor cell killing. TCR engineering approaches aim to solve this problem by knocking in tumor-specific antigen-associated TCRs while simultaneously knocking out endogenous TCRs. Nowadays, whole genome or exome sequencing technologies are used to screen for somatic point mutations in tumor tissues and identify unique peptide antigens, called neoantigens, with the potential for MHC-I presentation. A proprietary algorithm is then used to engineer TCRs capable of recognizing the MHC-I / neo-antigen complex with the goal of improving the specificity of T cell-mediated tumor cell killing. By further optimizing the TCR subtype and unique characteristics, TCR-engineered T cells may offer a unique therapeutic opportunity that could benefit many patients suffering from diseases such as synovial cell sarcoma, melanoma, and myeloma.

[0213] Many approaches have been developed to improve the efficiency of T cell engineering. For example, viral vectors have been successfully used for gene editing purposes, but the risk of integrating viral sequences into the human cell genome and the complications that such an approach can bring cannot be ignored. The use of adeno-associated viruses as an advanced version of viral editing technology appears to have improved safety concerns, but there are limitations regarding the gene size and site-specific delivery of target transgenes. Because CAR-T engineering does not have strict requirements regarding the genome insertion site, viral-based gene editing approaches are widely used in CAR-T approaches. However, more precise genome editing techniques that enable site-specific insertion of transgene(s) are highly desired and have not yet been fully discovered, and the technical needs for site-specific insertion of the desired transgene(s) have not yet been implemented.

[0214] On the other hand, non-viral gene editing can enable convenient, safe, and efficient reprogramming of cultured and primary cells. Compared to the traditional recombinant viral vectors mentioned above, for example, using electroporation to deliver genome editing reagents such as CRISPR-associated protein 9 (Cas9), guide RNA (gRNA), and DNA templates to T cells can save time, reduce costs, lower safety risks, and enable highly specific genome editing / insertion with the ability to integrate much larger DNA sequences. Through the homology-directed repair (HDR) mechanism, the DNA template can be integrated into the precise site(s) nicked by the CRISPR-Cas ribonucleoprotein (RNP consisting of the Cas9 protein and gRNA) within the T cell genome. This approach has been utilized by Roth et al. to target human T cells, NK cells, and induced pluripotent stem cells (iPSCs); however, its use in clinical settings has not become widespread because the editing efficiency and level of off-target activity remain to be determined. Furthermore, the functional consequences of the electroporation process on cultured T cells have not been fully investigated. Previous studies have shown that up to 80% of electroporated cells are negatively affected after electroporation, exhibiting decreased viability, reduced growth, and altered gene expression. Although several correlations between cytotoxicity and DNA format (ssDNA, dsDNA, cDNA), length, and delivery method have been implicated, the underlying causes of post-electroporation cytotoxicity observed in T cell cultures have not been fully characterized. Therefore, there is an urgent need to mitigate the cytotoxicity of electroporation during the TCR manipulation process to enhance gene editing efficiency and cell growth conditions.

[0215] In this study, we describe an effective, safe, and convenient approach to dramatically improve electroporation-mediated T cell manipulation efficiency, proliferation, and therefore total edited cell (TEC) number levels during TCR manipulation. Using the WT1 peptide-associated TCR as a DNA template, we generated a clinically relevant model system in which we tested the effects of various components of the electroporation-based DNA delivery method. We investigated the underlying molecular mechanisms of cytotoxicity during the T cell manipulation process and identified double-stranded DNA as the most potent inducer of cellular DNA genotoxicity in cultured T cells. We then screened several small molecule inhibitors of the DNA genotoxicity pathway and analyzed their effects on T cell growth, viability, and TEC number to improve TCR manipulation efficiency. Furthermore, the identified top DNA genotoxicity inhibitors did not adversely affect the phenotype and function of the final formulation (FDP), such as its tumor cell-killing ability. The aforementioned DNA genotoxicity mitigation approach has been shown to be effective in lymphocyte samples from different healthy donors, implying potential for widespread application in clinical studies.

[0216] Example 2: Inhibition of genomic stress dramatically improves T cell engineering efficiency result

[0217] DNA plasmids activate the cGAS-STING pathway, which negatively affects T cell viability and proliferation after electroporation

[0218] To achieve high gene editing efficiency with low toxicity in the TCR engineering process, we co-electroporated human primary CD8+ T cells with CRISPR-Cas9 ribonucleoprotein (RNP) and nanoplasmid DNA templates, since circular DNA has been reported to be less toxic than linear DNA [2,3]. A 1572-bp WT1 peptide-specific TCR template was targeted to TRAC exon 1 (Figure 1A) by homologous recombination repair. An overview of the optimized 15-day process, including T cell activation, electroporation, expansion, and harvesting, is shown in Figure 1B. To address the underlying causes of reduced viability and low growth rates after electroporation, we electroporated T cells with different components of the CRISPR / Cas9 gene targeting reagent (RNP, DNA template, etc.). An EH115 pulse code was used with a 100-µl cuvette in a Lonza Nucleofector device according to the manufacturer's protocol. During this process, we measured viability, T cell growth rate, and knock-in (KI) / knock-out (KO) efficiency. Our data showed that the presence of plasmid DNA (DNA and DNA + RNP only) during the electroporation process correlated with significantly reduced viability and growth in T cells (Figures 1C, 1D, 2A-2C). No dramatic effects on viability and growth were observed in the RNP-only or control (no DNA, no RNP) groups, as they were comparable to the non-transfected (no TFX) group. Compared to the DNA-only group, the RNP + DNA group showed slower recovery and reduced growth / proliferation rates. These findings suggest that neither the electroporation process nor RNP was the primary reason for the reduced viability and growth of T cell cultures, but that DNA was identified as a major contributor to the reduced T cell growth and viability during the TCR engineering process. Notably, knock-in / knock-out (KI / KO) data demonstrated that the most optimized electroporation program can reach upwards of 85-90% KO and 50% KI / KO efficiencies (Figure 1E) without nonspecific KI / KO populations from the RNP-only, no RNP, or no DNA arms.

[0219] Based on previous reports in monocytes [4,5], we hypothesized that genotoxic stress after transfection would cause decreased viability and reduced culture proliferation due to the presence of plasmid DNA in T cells. To confirm this, we subjected cell samples from days 2 to 15 to Western blot analysis. Indeed, both the DNA-only and DNA + RNP groups activated the cGAS-STING pathway, as evidenced by the corresponding phosphorylation of STING, TBK1, and IRF3 (Figure 1F). The RNP-only group, despite achieving a high knockout percentage (Figure 1E), did not activate the cGAS-STING pathway (Figure 1F). To rule out other potential pathways that may affect culture growth or proliferation in the presence of single-stranded DNA (ssDNA) or double-stranded DNA (dsDNA), we examined the activation of the inflammasome-related pathway (Aim2 / IL-1B) and the DNA endosome activation pathway (TLR9 / MyD88) in T cells after transfection. Our findings showed that none of these pathways were activated (Figure 3A,B), outlining the differences between virus-based and nanoparticle-based DNA delivery approaches [6,7,8].

[0220] Pretreatment with BX795, AML and MRT increases T cell viability, proliferation and total edited cell numbers during the TCR engineering process

[0221] Because plasmid DNA appears to activate the cGAS-STING pathway, resulting in reduced viability and growth inhibition in T cells after electroporation, we investigated whether blocking activation of the cGAS-STING pathway could alleviate the negative effects of plasmid DNA on culture viability and growth. Six inhibitors targeting cGAS (G140, Ru521), STING (H151), and TBK1 (Amlexanox, BX795, MRT67307) were tested (Table 1). To avoid potential contamination and nonspecific immune responses introduced by exposure to human serum, serum-free, chemically defined culture medium was used in this study. Because adding inhibitors after transfection negatively affected culture growth, T cells were treated with these inhibitors only before transfection at the indicated concentrations (Table 1) and for the specified time periods. Pretreatment of T cells with several of these inhibitors at optimized concentrations significantly improved culture viability 48 hours after transfection (Figure 4A, 4D, 4G, 5A, 5D). On average, culture viability improved approximately two-fold compared to the untreated control group (CTR) when using the inhibitors BX795 (5 μM), Aml (25 μM), and MRT (2.5 μM) (Figure 6A). Furthermore, the final T cell product collected on day 15 showed higher cell proliferation (Figure 4B, 4E, 4H, 5B, 5E) compared to the control group, where the average proliferation was only 6.6-fold. Cells pretreated with BX795 (5 μM), Aml (25 μM), and MRT (5 μM) reached 20.3-, 14.5-, and 17.2-fold proliferation, respectively. On average, treatment of T cells with the aforementioned inhibitors (at the given concentrations) improved culture proliferation by approximately 2- to 3-fold, which was consistent across all donors ( Figure 6B ).

[0222] Because inhibition of the cGAS-STING pathway only mitigates plasmid DNA-mediated genotoxicity, no impact on gene editing efficiency was expected. Therefore, the knock-in / knock-out (KO+KI) rates for TCR targeting were nearly equivalent across all donors (Figure 7). The only exception was a donor with poor cell health, where treatment of cultured cells with a cGAS-STING pathway inhibitor improved the KO+KI rate due to better survival and therefore proliferation of cells with a KO+KI phenotype (Figure 7C). This is further discussed in the following section.

[0223] The total number of edited cells (TECs) in the final preparation is perhaps the most important metric for adoptive cell transfer (ACT), as it is expected to translate into clinical therapeutic impact, among other indications, and is therefore considered one of the most important criteria for cell therapy purposes. By multiplying the total cell number of the final cell product on day 15 by the percentage of knock-in, we obtain TECs, which represent the output of our T cell engineering and culture. As shown for the control condition (CTR), with a seeding density of 2 million cells and no inhibitor treatment, the TEC number averaged approximately 5.24 million (Figure 6C). However, when T cells were pretreated with the indicated inhibitors, a significant increase in TEC output was observed in all donors (Figure 4C, Figure 4F, Figure 4I, Figure 5C, Figure 5F), which was consistent with the cell proliferation data. Pretreatment with BX795 (2.5uM), Aml (25uM), and MRT (5uM) increased TEC numbers from an average of 5.24 million to 21.1 million, 14.7 million, and 17.4 million, respectively, contributing to an average increase of 303.4%, 267.0%, and 180% in TECs compared to the control group (Figure 6C).

[0224] The use of statistical analysis in autologous T cell engineering studies is challenging due to the large donor-to-donor variability in T cell editing efficiency and proliferation rates. However, interestingly, for T cells pretreated with medium or high concentrations (2.5 μM and 5 μM) of the inhibitor BX795, TEC analysis revealed a statistically significant improvement compared to the control group (Figure 6C), resulting in a more than three-fold improvement in TEC.

[0225] BX795 pretreatment improves cell viability and proliferation by inhibiting IRF3 phosphorylation.

[0226] Based on the data, BX795 (2.5 μM and 5 μM) was most effective in improving culture viability and growth and was therefore used for further study. BX795 is a kinase inhibitor that targets TBK1, a kinase that functions downstream of the cGAS-STING pathway [9-11]. Western blot analysis revealed that pretreatment with BX795 successfully attenuated the phosphorylation of IRF3, a downstream target of TBK1, at both medium and high concentrations after transfection (Figure 8A). As a result, downstream cytokine expression, such as type 1 interferons (IFNa, IFNB) and proinflammatory cytokines (IL-6), was reduced upon pretreatment with BX795 (Figure 8F), indicating successful inhibition of the cGAS-STING pathway during the TCR manipulation process. However, TBK1 phosphorylation itself was not attenuated after BX795 pretreatment but was actually enhanced, consistent with a previous report that showed a two-fold increase in TBK1 phosphorylation upon BX795 treatment [9]. As previously shown (Figures 4A-I and 5A-F), BX795 treatment resulted in higher % survival (Figure 8B), increased T cell proliferation (Figure 8C), equal or better KI / KO ratios (Figure 8E), and improved TEC numbers.

[0227] BX795 inhibitor treatment improves knockout and knockin ratios in the final T cell product.

[0228] Pretreatment of cultured T cells with the BX795 inhibitor is not expected to adversely affect Cas9 protein-mediated targeted cleavage of the gene of interest, nor should it affect the homologous recombination repair process. However, data from the final cell product show that T cells pretreated with BX795 had a significantly higher total knockout percentage than the control group (Figure 9A). BX795-pretreated T cells had statistically significantly higher editing values, reaching knockout efficiencies of 86.7% (BX795-M) and 83.5% (BX795-H) compared to the control group, which had a knockout efficiency of approximately 70% (Figure 9B). Notably, BX795 pretreatment of T cells also resulted in a higher knock-in percentage in the final cell product, increasing the knock-in ratio from 49.5% (CTR) to 61% (BX795-M) (Figure 9C), which is statistically significant compared to the non-inhibitor-treated control group (Figure 9D). It is unlikely that pretreatment of T cells with the BX795 inhibitor could enhance Cas9 protein-mediated cleavage of the target gene(s) or improve the plasmid DNA repair process based on homologous recombination. The observed improvement in knock-in and knock-out ratios in the final T cell product (Figures 4C, 4F, 4I, 5C, 5F, 6C) is likely due to the survival and growth of edited T cells, as observed trends of improved TEC with increased fold expansion and higher viability in cultures pretreated with BX795 (Figure 6C). T cells with a successful KO+KI ratio have a lower chance of survival and proliferation due to metabolic shifts and higher cellular stresses, and pretreatment of T cells with BX795 can partially alleviate some of these cellular stresses.

[0229] BX795 inhibitor treatment does not affect memory T cell phenotype.

[0230] Because memory T cells have the potential for proliferation and long-term survival once reintroduced into patients, the number of memory T cells in the final T cell product is expected to strongly correlate with clinical efficacy during T cell therapy [14,15]. The balance of glycolysis versus fatty acid oxidation metabolism in T cells, as well as growth-related signaling pathways (such as the Jak-STAT pathway), can influence T cell phenotype

[16] . With our optimized T cell activation and culture method, over 95% of cells possess either the central memory T cell (TCM) or stem cell memory T cell (TSCM) phenotype in the final cell product (Figures 10A-F). A high percentage of memory T cell phenotype potentially promotes higher proliferation and enhances the tumor-killing ability of T cells. Pretreatment of T cell cultures with the BX795 inhibitor did not adversely affect T cell phenotype compared to untreated controls for different donors (Figures 10A-F). The higher growth rate observed in the inhibitor-treated group may potentially alter cell metabolism, cytokine concentrations, and metabolite levels in the culture medium, leading to changes in T cell phenotype, making this a very important criterion for inhibitor selection / application. Fortunately, BX795 treatment did not affect T cell phenotype.

[0231] CD8+ T cells from cultures treated with the BX795 inhibitor exhibit activation, proliferation, and target cell killing comparable to untreated controls.

[0232] The cGAS-STING pathway is important in detecting and neutralizing genotoxic stress. It is a major component of the innate immune system, capable of triggering inflammation in response to cytoplasmic DNA detection [17,18]. By blocking the cGAS-STING pathway, we observed significant improvements in culture viability, cell proliferation rate, and therefore total edited cell numbers. To ensure that treating cells with BX795 did not adversely affect the function of the T cell product, we used cells from four independent donors and followed the activation, transfection, and culture protocols (described below). T cells harvested from inhibitor-treated and control groups were pulsed for 24 hours with the indicated concentrations of a specific peptide (WT1) that binds to the engineered TCR. Flow cytometry data showed that BX795-pretreated T cells had activation profiles comparable to untreated control cells in terms of CD137 (Figure 11A), IFNg (Figure 11B), TNFa (Figure 11C), and granzyme B expression (Figure 11D). The data show that pretreatment of T cells with the indicated concentrations of the BX795 inhibitor did not affect T cell activation or cytokine expression. Another major concern is exhaustion in engineered T cell cultures. To measure the level of T cell exhaustion, we assessed the expression of exhaustion markers such as Tim3 and PD-1. No significant differences in the levels of T cell exhaustion markers were observed at medium or low peptide concentrations in the inhibitor-treated or non-treated groups compared to the negative control (Figures 12A and 12B). As expected, T cells pretreated with the inhibitor had similar levels of Tim3 and PD-1 expression as the non-treated control (Figures 12A and 12B).

[0233] We next examined T cell proliferation after treatment with BX795. The final T cell products obtained from BX795-pretreated or -untreated groups were labeled with CFSE far-red and co-cultured with WT1 peptide at the indicated concentrations for 72 hours. When analyzed by flow cytometry, T cell proliferation in response to antigen-specific stimulation was comparable between BX795-treated and -untreated samples (Figure 11E). T cells treated with higher concentrations of WT1 peptide achieved a high (approximately 60%) proliferation rate, suggesting robust, antigen-specific proliferation of the final T cell product. Finally, we compared T cells isolated from BX795-pretreated and -untreated control cultures in a target cell killing assay. Target cells T2 (T) were labeled with CFSE far-red and then pulsed with 20 μM WT1 peptide before co-incubating with our final T cell product (E) in a ratio-dependent manner. Our data confirmed that T cells pretreated with the inhibitor had comparable target cell killing capabilities to untreated controls at all E:T ratios (Figure 11F). Notably, compared with CTLs obtained from conventional vaccine strategies, our T cells achieved high killing efficiency at much lower E:T ratios [19,20], outlining the potential benefits of our TCR-engineered T cells for antigen-specific tumor cell killing. Collectively, our data demonstrate the safety and feasibility of using the BX795 cGAS-STING pathway inhibitor for the T cell engineering process and outline the potentially broad impact of such inhibitors in the field of adoptive T cell therapy. The use of the cGAS-STING inhibitor specifically improved T cell engineering efficiency, cell proliferation rate, and therefore total edited cell number. By improving T cell viability and growth after electroporation, we were able to obtain 2-3 times higher TECs with activation, proliferation, and target cell killing capabilities comparable to those of controls. Notably, we demonstrated that this phenomenon was not donor-specific or restricted to a particular antigen, as similar favorable results were observed for T cells obtained from all donors in this study (Figures 4A-I, 5A-F, 13A-C).

[0234] Consideration

[0235] In this study, we introduced a facile, efficient, and manufacturing-friendly solution to improve T cell viability and growth during the TCR engineering process. Pretreatment of T cells with a TBK1 inhibitor resulted in higher T cell proliferation and improved TCR editing efficiency without affecting the expected phenotype(s) of the final T cell preparation (FDP). We successfully performed TCR engineering by electroporation of RNPs and long DNA sequences into T cells, while simultaneously improving editing efficiency, electroporation scale, and cell proliferation rate. Our data reveal a direct correlation between the electroporation program (pulse code) used and T cell viability and proliferation rate. We hypothesized that this phenomenon is related to the amount of DNA introduced into T cells during the electroporation process, where stronger pulse codes (e.g., EH115) result in the delivery of more plasmid DNA constructs to T cells compared with milder pulse codes (e.g., EW100). We verified this by using a GFP plasmid construct during T cell transfection (Figure 14A). This also explains why the use of stronger pulse codes results in reduced viability, as our data suggest that plasmid DNA is the primary cause of cell death and growth reduction after electroporation. When using a TBK1 inhibitor, we observed better T cell proliferation with multiple pulse codes (EW113, EH115, etc.). Furthermore, the BX795 inhibitor performed better in the EH115 group, likely due to the introduction of more plasmid DNA, which causes higher levels of genotoxicity.

[0236] To our knowledge, this is the first report demonstrating that the TBK1 inhibitor BX795 enhances TCR editing efficacy in a non-viral cell editing approach. Because BX795 has not been reported to directly affect homologous recombination DNA repair (HDR) or DNA internalization processes, the improved knock-in rate mediated by BX795 pretreatment is unlikely to be due to enhanced dsDNA repair. Although some literature suggests a potential link between the cGAS pathway and HDR function, a clear mechanism has not been delineated. We hypothesize that BX795 enhances the survival of edited T cells both during electroporation and subsequent post-transfection culture by reducing genotoxicity. This assertion is partially supported by time-gradient TCR-dextramer flow cytometry analysis (Figures 14B and 14C), which shows that BX795 treatment increases exogenous TCR expression both initially (days 4 and 5) and throughout the culture process (days 6–15) compared to the control group, demonstrating a higher increase curve.

[0237] The mechanism of genotoxic stress is complex. Although our Western blot and qPCR data show a significant reduction in cGAS / STING pathway activation after BX795 treatment (Figure 8A), we believe that BX795 treatment does not completely resolve plasmid DNA-mediated genotoxic stress. The levels of type 1 interferons, such as TNFα and TNFβ, did not show a significant decrease upon BX795 treatment, likely due to the removal of the BX795 inhibitor before transfection. This issue can be resolved by exposing T cells to a TBK1 inhibitor both before and after electroporation, or by extending the treatment time.

[0238] T cell culture expansion after transfection can be significantly enhanced by CD3 / CD28 stimulation or exposure to TCR-specific peptide antigens presented on MHC-I molecules. However, this unintentionally results in T cell exhaustion and a high percentage of effector T cells in the FDP. Previous studies in CAR-T research have shown that memory phenotype CD8+ T cells (TCMs, TSCMs, etc.) maintain better circulation time, proliferation capacity, and target tumor cell killing ability when reinfused into patients. In this study, we used IL-7 / IL-15 cytokines to promote the generation of memory CD8+ T cells, resulting in over 95% memory TCMs or TSCMs in the FDP (Figure 10A-F). However, this negatively impacted T cell growth rates, achieving an average T cell expansion of only 5.24-fold. Pretreatment of these T cell cultures with a TBK1 inhibitor prior to electroporation significantly improved the proliferation rate of the T cell cultures by up to 20.3-fold (Figures 4A-I, 5A-F, 6A-C) without any detectable adverse effect on T cell phenotype.

[0239] Indeed, nonviral genome targeting approaches have the potential to reduce costs, improve safety, and shorten the timeline for developing next-generation cell-based immunotherapies. Advances in DNA synthesis and next-generation sequencing technologies enable the synthesis of gRNA and DNA plasmid templates within a few days. However, to use electroporation as the most efficient nonviral gene delivery / editing approach, various burdens must be overcome, such as its harsh effects on cultured cells and plasmid DNA-mediated genotoxic stress. In this regard, many parameters, such as electroporation equipment, buffers, and electroporation programs, need to be tested and optimized to find a balance between knock-in efficiency, cell proliferation, and overall T cell phenotype. Our study introduces a new approach to effectively address the genotoxic stress response in T cells derived from different donors in a simple and cost-effective manner. Treating T cell cultures with a cGAS inhibitor before transfection can improve cell viability, promote cell growth, and increase editing efficiency. Our process is scalable, cost-effective, and applicable not only to T cell engineering but also to any nonviral cell engineering process.

[0240] method

[0241] RNA ribonucleoproteins and DNA plasmids

[0242] Single guide RNA sequences for TRAC and TRBC were obtained as described by Oh, Senger et al. and ordered from Synthego (Menlo Park, CA, USA). SpyFi Cas9 protein was purchased from Aldevron (Fargo, ND, USA) and used at a final concentration of 0.05 mg / ml for electroporation. For a 100 μl electroporation procedure, 2.5 μg of Cas9 protein was pre-complexed with a 3 molar excess of sgRNA for each knockout site and then mixed prior to delivery. Nanoplasmid carrying the WT1 TCR sequence was ordered from Nature Technologies (Lincoln, NE, USA) and used at a working concentration of 150 μg / ml for electroporation.

[0243] Cell isolation and activation

[0244] Peripheral blood mononuclear cells (PBMCs) were isolated from human cryopreserved leukoplakates using Ficoll gradient centrifugation (400 rpm, 25 min). All samples were obtained from healthy donors genotyped for the HLA A*02:01 MHC class I complex. CD8+ T cells were then isolated using a Miltenyi AutoMACS cell separator according to the manufacturer's protocol. The isolated CD8+ T cells were then activated with Miltenyi T Cell TransACT Reagent (1:100) (Cat. No. 130-111-160), 25 ng / mL IL-7 (Cat. No. 130-095-367), and 50 ng / mL IL-15 (Cat. No. 130-095-760) in Fuji Prime-XV medium (Irvine Scientific Cat. No. 91154) for 48 hours.

[0245] Electroporation

[0246] Cells were electroporated using the 4D-Nucleofector System (Lonza) according to the manufacturer's protocol. After activation and inhibitor treatment, 10 × 10 610 million (or 10 million) CD8+ T cells were washed and resuspended in Lonza P3 Primary Cell Nucleofector Solution (Cat. No. V4XP-3024), then mixed with premixed RNP and DNA plasmids and transferred to a Lonza 100 µL cuvette. After electroporation, 400 µL of FUJI Prime-XV medium was added to the cuvette and incubated for 15 minutes before the cells were seeded into culture flasks. The pulse code used in this manuscript is EH115 for all conditions.

[0247] cell culture

[0248] All inhibitors were purchased from Invivogen. Unless otherwise stated, cells were cultured in Fuji Prime-XV medium (complete medium) containing 25 ng / mL IL-7 and 50 ng / mL IL-15. After electroporation, T cells were plated at 1–2 million cells / cm in 24-well gREX (Wilsonwolf, catalog: 80192M). 2 The cells were carefully transferred at a seeding density of 1000 x g. 8 ml of complete medium was added per well, and 50% of the medium was replaced after 6 days. The cells were incubated at 37°C and 5% CO2. A NucleoCounter NC-200 automated cell counter equipped with a Via2-Cassette was used in this study for cell counting and viability analysis.

[0249] Flow cytometry

[0250] All surface and intracellular staining antibodies were purchased from BioLegend and BD Biosciences as listed in Table 2. Dextramer antibodies for knock-in analysis were purchased from Immudex (catalog: WB3469-PE, WT1) and used according to the manufacturer's protocol. Surface, CFSE, 7-AAD / Annexin V, and intracellular staining were performed as previously described

[17] . Samples were analyzed using a BD FACSLyric flow cytometer (BD Biosciences) and FlowJo v10 software. For proliferation and cell death assays, data were normalized by negative or target cell-only controls.

[0251] Western blot

[0252] Antibodies for Western blotting were purchased from Cell Signaling and are listed in Table 3. T cell pellets were collected, washed, and stored as frozen pellets at -80°C until use. Cell pellet processing, Western blot experiments, and data analysis were performed as previously described (Tang, JBC, 2020).

[0253] qPCR

[0254] The RNeasy Mini Kit (catalog number 74106) was purchased from Qiagen. The TaqMan RNA-to-CT 1-Step Kit (catalog number 4392938) and TaqMan primer-probe assay using the reporter dye FAM and MGB quencher were purchased from Thermo Fisher (assay information is listed in Table 4). After RNA isolation, 2 ng of RNA was mixed with the TaqMan primer-probe mix, TaqMan RNA-to-CT 1-Step Master Mix, and RT enzyme mix in a 10 μL qPCR system according to the manufacturer's protocol. Reaction readouts were measured using a QuantStudio 6 Flex machine, and data were analyzed using QuantStudio Real-Time PCR Software v1.2. The PCR cycling conditions were 50°C for 30 minutes, 95°C for 10 minutes, and 40 cycles of 95°C for 15 seconds and 60°C for 1 minute. All data points were collected in triplicate using RNA18S as an internal control (sample ID: #Hs03928990_g1).

[0255] Data Analysis and Statistics

[0256] Western blot data were analyzed with Image Lab 6.1 (Bio-Rad). Flow cytometry data were analyzed using FlowJo software v10 (BD Biosciences). Data from different assays were sorted in Excel (Microsoft). Graphs were generated and presented with Graphpad Prism 10. For all statistical analyses, data were presented as mean ± SEM.

[0257] Table 1. Inhibitor treatment concentrations used

[0258] Two days after activation, CD8+ T cells were washed and resuspended in FUJI Prime-XV complete medium containing the indicated concentrations of inhibitors for 6 hours. After treatment, cells were washed and resuspended in Lonza P3 buffer before electroporation. After transfection, T cells were cultured in FUJI Prime-XV complete medium without inhibitors. [Table 1]

[0259] [Table 2]

[0260] [Table 3]

[0261] [Table 4]

[0262] [Table 5] References

[0263] [1]Roth T.et.al,Reprogramming human T cell function and specificity with non-viral genome targeting.Nature.559,405-409(2018)

[0264] [2]Cornu,TI,Mussolino,C.&Cathomen,T.Refining strategies to translate genome editing to the clinic.Nat.Med.23,415-423(2017).

[0265] [3]Hornung,V.&Latz,E.Intracellular DNA recognition.Nat.Rev.Immunol.10,123-130(2010).

[0266] [4]Luecke,S.et.al,cGAS is activated by DNA in a length-dependent manner.EMBO Rep.18,1707-1715(2017)

[0267] [5]Hansen,K.et.al,Listeria monocytogenes induces IFNβ expression through an IFI16-,cGAS-and STING-dependent pathway.EMBO J.33,1654-1666(2014)

[0268] [6]Rathiam V.et.al,The AIM2 inflammasome is essential for host defense against cytosolic bacteria and DNA viruses.Nature Immunology.11,pages395-402(2010)

[0269] [7]Ferrer J.et.al,Dual Toll-Like Receptor Targeting Liposomal Spherical Nucleic Acids.Bioconjugate Chem.30,944-951(2019)

[0270] [8]Radovic-Moreno A.et.al,Immunomodulatory spherical nucleic acids.Proc.Natl.Acad.Sci.U.S.A.,112,3892-3897(2015)

[0271] [9]Clark K.et.al,Use of the Pharmacological Inhibitor BX795 to Study the Regulation and Physiological Roles of TBK1 and IKB Kinase.Mechanism of Signal Trans.284,14136-14146(2009)

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[10] Feldman R.et.al,Novel Small Molecule Inhibitors of 3-Phosphoinositide-dependent Kinase-1.Mechanism of Signal Trans.280,19867-19874

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[11] Steinhagen F.et.al,Suppressive oligodeoxynucleotides containing TTAGGG motifs inhibit cGAS activation in human monocytes.Eur.J.Immunol.48,605-61(2018)

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[12] Eichholz K.et.al,Immune-Complexed adenovirus induce AIM2-mediated pyroptosis in human dendritic cells.PLOS Pathogens.https: / / doi.org / 10.1371 / journal.ppat.1005871.(2016)

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[13] Kaminski J.et al,Synthetic oligodeoxynucleotides containing suppressive TTAGGG motifs inhibit AIM2 inflammasome activation.J Immunol.191,3876-83(2013)

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[17] Zhang X.et.al,Structures ...

Claims

1. 1. A method of manipulating a T cell, comprising contacting the T cell with a nucleic acid and one or more cyclic GMP-AMP synthase (cGAS)-stimulator of interferon genes (STING) pathway inhibitors.

2. The method of claim 1 , wherein the nucleic acid comprises donor DNA.

3. The method of claim 1 or 2, wherein the nucleic acid is a double-stranded circular nucleic acid.

4. The method of claim 2 or 3, wherein the nucleic acid is a plasmid, nanoplasmid, minicircle or viral vector containing the donor DNA.

5. 5. The method of any one of claims 2 to 4, wherein the donor DNA encodes an exogenous T cell receptor (TCR)-alpha or a fragment thereof, an exogenous TCR-beta or a fragment thereof, or a combination thereof.

6. The method of any one of claims 1 to 5, wherein the nucleic acid is associated with a delivery vehicle.

7. 7. The method of claim 6, wherein the delivery vehicle is a lipid particle or a nanoparticle.

8. The method of any one of claims 1 to 7, wherein the T cells are primary T cells.

9. 9. The method of any one of claims 1-8, further comprising contacting the T cells with a gene editing reagent.

10. 10. The method of Claim 9, wherein contacting the T cell with the gene editing reagent comprises contacting the T cell with a nucleic acid sequence encoding the gene editing reagent.

11. 11. The method of Claim 10, wherein the T cell is contacted with the nucleic acid in the presence of the nucleic acid sequence encoding a gene editing agent or the gene editing reagent.

12. 12. The method of any one of claims 9 to 11, wherein the gene editing reagent comprises an RNA-guided nuclease.

13. The method of claim 12, wherein the RNA-guided nuclease is a CRISPR-Cas system.

14. The CRISPR-Cas system includes Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9, Cas10, Cas12, Cas13, nCas9, Cas-CLOVER, Csy1, Csy2, Csy3, Cse1, Cse2, Csc1, Csc2, Csa5, Csn2, 10. The method of claim 9, comprising Csm2, Csm3, Csm4, Csm5, Csm6, Cmr1, Cmr3, Cmr4, Cmr5, Cmr6, Csb1, Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, Csx1, Csx15, Csf1, Csf2, Csf3, or Csf4.

15. The method of claim 14, wherein the CRISPR-Cas system comprises Cas9, nCas9, or Cas-CLOVER.

16. 16. The method of claim 15, wherein the CRISPR-Cas system comprises Cas9.

17. 16. The method of claim 15, wherein the CRISPR-Cas system comprises nCas9.

18. 16. The method of claim 15, wherein the CRISPR-Cas system comprises Cas-CLOVER.

19. 19. The method of any one of claims 12 to 18, wherein the gene editing reagent further comprises a guide RNA.

20. 12. The method of any one of claims 9 to 11, wherein the gene editing reagent comprises MAD7, a TALEN, or a ZFN.

21. 21. The method of Claim 20, wherein the gene editing reagent comprises MAD7.

22. 21. The method of claim 20, wherein the gene editing reagent comprises a TALEN.

23. 21. The method of Claim 20, wherein the gene editing reagent comprises a ZFN.

24. 24. The method of any one of claims 2 to 23, wherein the donor DNA is inserted into an endogenous TCR locus.

25. 25. The method of claim 24, wherein the endogenous TCR locus is an endogenous TCR-alpha locus, an endogenous TCR-beta locus, or a combination thereof.

26. 26. The method of any one of claims 9-25, wherein contacting the T cell with the gene editing reagent comprises transfecting the T cell with the gene editing reagent.

27. 27. The method of any one of claims 1 to 26, wherein contacting the T cell with the nucleic acid comprises transfecting the T cell with the nucleic acid.

28. The method of any one of claims 1 to 27, wherein the one or more cGAS-STING pathway inhibitors comprises a cGAS inhibitor.

29. The method of any one of claims 1 to 28, wherein the one or more cGAS-STING pathway inhibitors comprise a STING inhibitor.

30. 30. The method of any one of claims 1 to 29, wherein the one or more cGAS-STING pathway inhibitors comprise a TANK-binding kinase 1 (TBK1) inhibitor.

31. 31. The method of any one of claims 1 to 30, wherein the one or more cGAS-STING pathway inhibitors are Amlexanox (Aml), MRT67307 (MRT), BX795, H151, ODN-A151 (ODN151), Ru.521, G140, or a combination thereof.

32. 32. The method of claim 31, wherein the one or more cGAS-STING pathway inhibitors are selected from Am1, BX795, ODN151, and MRT.

33. 32. The method of claim 31, wherein the one or more cGAS-STING pathway inhibitors is ODN151.

34. 32. The method of claim 31, wherein the one or more cGAS-STING pathway inhibitors is BX795.

35. 35. The method of any one of claims 1 to 34, wherein the T cell and the nucleic acid are contacted in the presence of one or more cGAS-STING pathway inhibitors.

36. 35. The method of any one of claims 1 to 34, wherein the T cells are contacted sequentially with the nucleic acid and the one or more cGAS-STING pathway inhibitors.

37. 37. The method of claim 36, wherein the T cells are contacted with the one or more cGAS-STING pathway inhibitors prior to the nucleic acid.

38. 38. The method of any one of claims 1-37, wherein the T cells are contacted with the one or more cGAS-STING pathway inhibitors for about 2 hours to about 10 hours.

39. 39. The method of claim 38, wherein the T cells are contacted with the one or more cGAS-STING pathway inhibitors for about 6 hours.

40. 40. The method of any one of claims 1 to 39, wherein the T cells are contacted with one cGAS-STING pathway inhibitor.

41. 1. A method of increasing cell viability of a population of engineered T cells, comprising contacting a population of T cells with one or more cyclic GMP-AMP synthase (cGAS)-stimulator of interferon genes (STING) pathway inhibitors and a nucleic acid, thereby forming the population of engineered T cells, wherein the population of engineered T cells has increased cell viability compared to a population of engineered T cells in which the population of T cells has not been contacted with one or more cGAS-STING pathway inhibitors.

42. 42. The method of claim 41 , wherein the nucleic acid comprises donor DNA.

43. 43. The method of claim 41 or 42, wherein the nucleic acid is a double-stranded circular nucleic acid.

44. 44. The method of claim 42 or 43, wherein the nucleic acid is a plasmid, nanoplasmid, minicircle, or viral vector comprising the donor DNA.

45. 45. The method of any one of claims 42 to 44, wherein the donor DNA encodes an exogenous T cell receptor (TCR)-beta or a fragment thereof, an exogenous TCR-alpha or a fragment thereof, or a combination thereof.

46. 46. ​​The method of any one of claims 41 to 45, wherein the nucleic acid is associated with a delivery vehicle.

47. 47. The method of claim 46, wherein the delivery vehicle is a lipid particle or a nanoparticle.

48. 48. The method of any one of claims 41 to 47, wherein the T cells are primary T cells.

49. 49. The method of any one of claims 41-48, further comprising contacting the population of T cells with a gene editing reagent.

50. 50. The method of Claim 49, wherein contacting the population of T cells with the gene editing reagent comprises contacting the population of T cells with a nucleic acid sequence encoding the gene editing reagent.

51. 51. The method of Claim 50, wherein the T cell is contacted with the nucleic acid in the presence of the nucleic acid sequence encoding a gene editing agent or the gene editing reagent.

52. 52. The method of any one of claims 49-51, wherein the gene editing reagent comprises an RNA-guided nuclease.

53. 53. The method of claim 52, wherein the RNA-guided nuclease is a CRISPR-Cas system.

54. The CRISPR-Cas system is selected from the group consisting of Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9, Cas10, Cas12, Cas13, nCas9, Cas-CLOVER, Csy1, Csy2, Csy3, Cse1, Cse2, Csc1, Csc2, Csa5, and Csn2. , Csm2, Csm3, Csm4, Csm5, Csm6, Cmr1, Cmr3, Cmr4, Cmr5, Cmr6, Csb1, Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, Csx1, Csx15, Csf1, Csf2, Csf3, or Csf4.

55. 55. The method of claim 54, wherein the CRISPR-Cas system comprises Cas9, nCas9, or Cas-CLOVER.

56. 56. The method of claim 55, wherein the CRISPR-Cas system comprises Cas9.

57. 56. The method of claim 55, wherein the CRISPR-Cas system comprises nCas9.

58. 56. The method of claim 55, wherein the CRISPR-Cas system comprises Cas-CLOVER.

59. 59. The method of any one of claims 51-58, wherein the gene editing reagent further comprises a guide RNA.

60. 52. The method of any one of claims 49-51, wherein the gene editing reagent comprises MAD7, a TALEN, or a ZFN.

61. 61. The method of Claim 60, wherein the gene editing reagent comprises MAD7.

62. 61. The method of Claim 60, wherein the gene editing reagent comprises a TALEN.

63. 61. The method of Claim 60, wherein the gene editing reagent comprises a ZFN.

64. 64. The method of any one of claims 42 to 63, wherein the donor DNA is inserted into an endogenous TCR locus.

65. 65. The method of claim 64, wherein the endogenous TCR locus is an endogenous TCR-alpha locus, an endogenous TCR-beta locus, or a combination thereof.

66. 66. The method of any one of claims 49-65, wherein contacting the T cell with the gene editing reagent comprises transfecting the T cell with the gene editing reagent.

67. 67. The method of any one of claims 41-66, wherein contacting the T cell with the nucleic acid comprises transfecting the T cell with the nucleic acid.

68. The method of any one of claims 41 to 51, wherein the one or more cGAS-STING pathway inhibitors comprise a cGAS inhibitor.

69. The method of any one of claims 41 to 68, wherein the one or more cGAS-STING pathway inhibitors comprise a STING inhibitor.

70. 70. The method of any one of claims 41 to 69, wherein the one or more cGAS-STING pathway inhibitors comprise a TANK-binding kinase 1 (TBK1) inhibitor.

71. 71. The method of any one of claims 41-70, wherein the one or more cGAS-STING pathway inhibitors are Amlexanox (Aml), MRT67307 (MRT), BX795, H151, ODN-A151 (ODN151), Ru.521, G140, or a combination thereof.

72. 72. The method of any one of claims 41-71, wherein the populations of T cells are independently contacted with about 0.1 uM to about 50 uM of the one or more cGAS-STING pathway inhibitors.

73. 73. The method of any one of claims 41-72, wherein the population of T cells is contacted with the nucleic acid in the presence of one or more cGAS-STING pathway inhibitors.

74. 74. The method of any one of claims 41-73, wherein the population of T cells is contacted sequentially with the nucleic acid and the one or more cGAS-STING pathway inhibitors.

75. 75. The method of claim 74, wherein the population of T cells is contacted with the one or more cGAS-STING pathway inhibitors prior to the nucleic acid.

76. The method of any one of claims 41 to 75, wherein the population of T cells is contacted with one cGAS-STING pathway inhibitor.

77. 77. The method of claim 76, wherein the population of T cells is contacted with about 1 uM to about 10 uM BX795.

78. 77. The method of claim 76, wherein the population of T cells is contacted with about 0.1 uM to about 8 uM ODN151.

79. 79. The method of any one of claims 41-78, wherein the cell viability of the population of engineered T cells is increased by at least about 0.5-fold to at least about 5-fold compared to a population of engineered T cells not contacted with one or more cGAS-STING pathway inhibitors.

80. 80. The method of claim 79, wherein the cell viability is increased by about 2-fold.

81. 81. The method of any one of claims 41-80, wherein the cell viability of the engineered population of T cells is increased by about 30% to about 95%.

82. 1. A method of increasing gene editing efficiency in a population of T cells, comprising contacting the population of T cells with one or more cyclic GMP-AMP synthase (cGAS)-stimulator of interferon genes (STING) pathway inhibitors and a nucleic acid, thereby forming a population of engineered T cells, wherein the population of T cells has increased gene editing efficiency compared to a population of T cells not contacted with the one or more cGAS-STING pathway inhibitors.

83. 83. The method of claim 82, wherein the nucleic acid comprises donor DNA.

84. 84. The method of claim 82 or 83, wherein the nucleic acid is a double-stranded circular nucleic acid.

85. 85. The method of claim 83 or 84, wherein the nucleic acid is a plasmid, nanoplasmid, minicircle, or viral vector comprising the donor DNA.

86. 86. The method of any one of claims 83-85, wherein the donor DNA encodes exogenous T cell receptor (TCR)-beta or a fragment thereof, exogenous TCR-alpha or a fragment thereof, or a combination thereof.

87. 87. The method of any one of claims 82 to 86, wherein the nucleic acid is associated with a delivery vehicle.

88. 88. The method of claim 87, wherein the delivery vehicle is a lipid particle or a nanoparticle.

89. 87. The method of any one of claims 82 to 86, wherein the population of T cells comprises primary T cells.

90. 90. The method of any one of claims 82-89, further comprising contacting the population of T cells with a gene editing reagent.

91. 91. The method of Claim 90, wherein contacting the T cells with the gene editing reagent comprises contacting the population of T cells with a nucleic acid sequence encoding the gene editing reagent.

92. 92. The method of Claim 91, wherein the population of T cells is contacted with the nucleic acid in the presence of a gene editing agent or the nucleic acid sequence encoding the gene editing reagent.

93. 93. The method of any one of claims 90-92, wherein the gene editing reagent comprises an RNA-guided nuclease.

94. 94. The method of claim 93, wherein the RNA-guided nuclease is a CRISPR-Cas system.

95. The CRISPR-Cas system is selected from the group consisting of Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9, Cas10, Cas12, Cas13, nCas9, Cas-CLOVER, Csy1, Csy2, Csy3, Cse1, Cse2, Csc1, Csc2, Csa5, and Csn2. , Csm2, Csm3, Csm4, Csm5, Csm6, Cmr1, Cmr3, Cmr4, Cmr5, Cmr6, Csb1, Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, Csx1, Csx15, Csf1, Csf2, Csf3, or Csf4.

96. 96. The method of claim 95, wherein the CRISPR-Cas system comprises Cas9, nCas9, or Cas-CLOVER.

97. 97. The method of claim 96, wherein the CRISPR-Cas system comprises Cas9.

98. 97. The method of claim 96, wherein the CRISPR-Cas system comprises nCas9.

99. 97. The method of claim 96, wherein the CRISPR-Cas system comprises Cas-CLOVER.

100. 100. The method of any one of claims 90-99, wherein the gene editing reagent further comprises a guide RNA.

101. 93. The method of any one of claims 90-92, wherein the gene editing reagent comprises MAD7, a TALEN, or a ZFN.

102. 102. The method of Claim 101, wherein the gene editing reagent comprises MAD7.

103. 102. The method of Claim 101, wherein the gene editing reagent comprises a TALEN.

104. 102. The method of Claim 101, wherein the gene editing reagent comprises a ZFN.

105. 105. The method of any one of claims 83 to 104, wherein the donor DNA is inserted into an endogenous TCR locus.

106. 106. The method of claim 105, wherein the endogenous TCR locus is an endogenous TCR-alpha locus, an endogenous TCR-beta locus, or a combination thereof.

107. 107. The method of any one of claims 90-106, wherein contacting the population of T cells with the gene editing reagent comprises transfecting the population of T cells with the gene editing reagent.

108. 108. The method of any one of claims 82-107, wherein contacting the population of T cells with the nucleic acid comprises transfecting the population of T cells with the nucleic acid.

109. The method of any one of claims 82 to 92, wherein the one or more cGAS-STING pathway inhibitors comprises a cGAS inhibitor.

110. The method of any one of claims 82 to 109, wherein the one or more cGAS-STING pathway inhibitors comprise a STING inhibitor.

111. The method of any one of claims 82 to 110, wherein the one or more cGAS-STING pathway inhibitors comprise a TANK-binding kinase 1 (TBK1) inhibitor.

112. 112. The method of any one of claims 82-111, wherein the one or more cGAS-STING pathway inhibitors are Amlexanox (Aml), MRT67307 (MRT), BX795, H151, ODN-A151 (ODN151), Ru.521, G140, or a combination thereof.

113. The method of any one of claims 82-112, wherein the populations of T cells are independently contacted with about 0.1 uM to about 50 uM of the one or more cGAS-STING pathway inhibitors.

114. The method of any one of claims 82-113, wherein the population of T cells is contacted with the nucleic acid in the presence of one or more cGAS-STING pathway inhibitors.

115. The method of any one of claims 82-113, wherein the population of T cells is contacted sequentially with the nucleic acid and the one or more cGAS-STING pathway inhibitors.

116. 116. The method of claim 115, wherein the population of T cells is contacted with the one or more cGAS-STING pathway inhibitors prior to the nucleic acid.

117. The method of any one of claims 82 to 116, wherein the population of T cells is contacted with one cGAS-STING pathway inhibitor.

118. 118. The method of claim 117, wherein the population of T cells is contacted with about 2 uM to about 8 uM BX795.

119. 118. The method of claim 117, wherein the population of T cells is contacted with about 0.1 uM to about 5 uM ODNA151.

120. 118. The method of claim 117, wherein the population of T cells is contacted with about 10 uM to about 50 uM Aml.

121. 118. The method of claim 117, wherein the population of T cells is contacted with about 1 uM to about 10 uM MRT.

122. 122. The method of any one of claims 82-121, wherein the gene editing efficiency of the engineered population of T cells is increased by at least about 0.5 fold to at least about 5 fold compared to a population of engineered T cells in which the population of T cells is not contacted with one or more cGAS-STING pathway inhibitors.

123. 123. The method of Claim 122, wherein the gene editing efficiency of the engineered population of T cells is increased by about 2 to about 3 fold.

124. 122. The method of any one of claims 82-121, wherein the gene editing efficiency of the engineered population of T cells is between about 60% and about 99%.

125. 122. The method of any one of claims 82-121, wherein the knockout efficiency of the engineered population of T cells is between about 10% and about 100%.

126. 110. The method of claim 109, wherein the knockout efficiency of the engineered population of T cells is between about 30% and about 100%.

127. 110. The method of claim 109, wherein the knockout efficiency of the engineered population of T cells is between about 50% and about 100%.

128. 110. The method of claim 109, wherein the knockout efficiency of the engineered population of T cells is between about 60% and about 100%.

129. 110. The method of claim 109, wherein the knockout efficiency of the engineered population of T cells is between about 60% and about 99%.

130. 110. The method of claim 109, wherein the knockout efficiency of the engineered population of T cells is between about 60% and about 95%.

131. 126. The method of Claim 125, wherein the knockout efficiency is selected from about 50%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95% and about 99%.

132. 113. The method of any one of claims 82-112, wherein the knock-in efficiency of the engineered population of T cells is between about 20% and about 99%.

133. 114. The method of Claim 113, wherein said population of engineered T cells has a knock-in efficiency of about 30% to about 99%.

134. 114. The method of Claim 113, wherein said population of engineered T cells has a knock-in efficiency of between about 40% and about 99%.

135. 114. The method of Claim 113, wherein said population of engineered T cells has a knock-in efficiency of about 50% to about 99%.

136. 133. The method of Claim 132, wherein the knock-in efficiency is selected from about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80% and about 90%.

137. 1. A method for increasing the expansion of a population of engineered T cells, comprising: i) contacting a population of T cells with one or more cyclic GMP-AMP synthase (cGAS)-stimulator of interferon genes (STING) pathway inhibitors and nucleic acids, thereby forming said engineered population of T cells; and ii) expanding the population of engineered T cells, thereby forming an expanded population of engineered T cells; wherein the one or more cGAS-STING pathway inhibitors increase the expanded population of engineered T cells compared to a population of engineered T cells in step i) that has not been contacted with one or more cGAS-STING pathway inhibitors.

138. 138. The method of claim 137, wherein the nucleic acid comprises donor DNA.

139. 139. The method of claim 137 or 138, wherein the nucleic acid is a double-stranded circular nucleic acid.

140. 140. The method of claim 138 or 139, wherein the nucleic acid is a plasmid, nanoplasmid, minicircle or viral vector containing donor DNA.

141. 141. The method of any one of claims 138-140, wherein the donor DNA encodes exogenous T cell receptor (TCR)-beta or a fragment thereof, exogenous TCR-alpha or a fragment thereof, or a combination thereof.

142. 142. The method of any one of claims 137 to 141, wherein the nucleic acid is associated with a delivery vehicle.

143. 143. The method of claim 142, wherein the delivery vehicle is a lipid particle or a nanoparticle.

144. 144. The method of any one of claims 137 to 143, wherein the T cells are primary T cells.

145. 145. The method of any one of claims 137-144, wherein step i) further comprises contacting the T cells with a gene editing reagent.

146. 146. The method of Claim 145, wherein contacting the T cell with the gene editing reagent comprises contacting the T cell with a nucleic acid sequence encoding the gene editing reagent.

147. 147. The method of Claim 146, wherein the T cell is contacted with the nucleic acid in the presence of the nucleic acid sequence encoding a gene editing agent or the gene editing reagent.

148. 148. The method of any one of claims 145-147, wherein the gene editing reagent comprises an RNA-guided nuclease.

149. The method of claim 148, wherein the RNA-guided nuclease is a CRISPR-Cas system.

150. The CRISPR-Cas system is selected from the group consisting of Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9, Cas10, Cas12, Cas13, nCas9, Cas-CLOVER, Csy1, Csy2, Csy3, Cse1, Cse2, Csc1, Csc2, Csa5, Csn2, 150. The method of claim 149, comprising Csm2, Csm3, Csm4, Csm5, Csm6, Cmr1, Cmr3, Cmr4, Cmr5, Cmr6, Csb1, Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, Csx1, Csx15, Csf1, Csf2, Csf3, or Csf4.

151. 151. The method of Claim 150, wherein the CRISPR-Cas system comprises Cas9, nCas9, or Cas-CLOVER.

152. 152. The method of claim 151, wherein the CRISPR-Cas system comprises Cas9.

153. 152. The method of claim 151, wherein the CRISPR-Cas system comprises nCas9.

154. 152. The method of claim 151, wherein the CRISPR-Cas system comprises Cas-CLOVER.

155. 155. The method of any one of Claims 148-154, wherein the gene editing reagent further comprises a guide RNA.

156. 148. The method of any one of claims 145-147, wherein the gene editing reagent comprises MAD7, a TALEN, or a ZFN.

157. The method of Claim 156, wherein the gene editing reagent comprises MAD7.

158. The method of Claim 156, wherein the gene editing reagent comprises a TALEN.

159. The method of Claim 156, wherein the gene editing reagent comprises a ZFN.

160. 160. The method of any one of claims 138-159, wherein the donor DNA is inserted into an endogenous TCR locus.

161. 161. The method of claim 160, wherein the endogenous TCR locus is an endogenous TCR-alpha locus, an endogenous TCR-beta locus, or a combination thereof.

162. 162. The method of any one of claims 145-161, wherein contacting the T cell with the gene editing reagent comprises transfecting the T cell with the gene editing reagent.

163. 163. The method of any one of claims 137-162, wherein contacting the T cell with the nucleic acid comprises transfecting the T cell with the nucleic acid.

164. The method of any one of claims 137 to 163, wherein the one or more cGAS-STING pathway inhibitors comprises a cGAS inhibitor.

165. The method of any one of claims 137 to 164, wherein the one or more cGAS-STING pathway inhibitors comprise a STING inhibitor.

166. The method of any one of claims 137 to 165, wherein the one or more cGAS-STING pathway inhibitors comprise a TANK-binding kinase 1 (TBK1) inhibitor.

167. 167. The method of any one of claims 137-166, wherein the one or more cGAS-STING pathway inhibitors are Amlexanox (Aml), MRT67307 (MRT), BX795, H151, ODN-A151 (ODN151), Ru.521, G140, or a combination thereof.

168. 168. The method of any one of claims 137-167, wherein the populations of T cells are independently contacted with about 1 uM to about 50 uM of the one or more cGAS-STING pathway inhibitors.

169. 169. The method of any one of claims 137-168, wherein the population of T cells is contacted with the nucleic acid in the presence of one or more cGAS-STING pathway inhibitors.

170. 169. The method of any one of claims 137-168, wherein the population of T cells is contacted sequentially with the nucleic acid and the one or more cGAS-STING pathway inhibitors.

171. 171. The method of claim 170, wherein the population of T cells is contacted with the one or more cGAS-STING pathway inhibitors prior to the nucleic acid.

172. The method of any one of claims 137 to 171, wherein the T cells are contacted with one cGAS-STING pathway inhibitor.

173. 173. The method of claim 172, wherein the population of T cells is contacted with about 1 uM to about 10 uM BX795.

174. 173. The method of claim 172, wherein the population of T cells is contacted with about 0.1 uM to about 8 uM ODNA151.

175. 118. The method of claim 117, wherein the population of T cells is contacted with about 5 uM to about 50 uM AML.

176. 118. The method of claim 117, wherein the population of T cells is contacted with about 1 uM to about 25 uM MRT.

177. 177. The method of any one of claims 137-176, wherein the expanded population of engineered T cells is increased by at least about 0.5-fold to at least about 5-fold compared to a population of engineered T cells in which the population of T cells in step i) has not been contacted with one or more cGAS-STING pathway inhibitors.

178. 178. The method of claim 177, wherein the expanded engineered T cell population is increased by about 2 to about 3 fold.

179. 177. The method of any one of claims 137-176, wherein the population of engineered T cells expands by at least about 0.5 fold to at least about 100 fold.

180. 180. The method of claim 179, wherein the engineered T cells expand by about 20 fold.

181. 181. An engineered T cell produced by the method of any one of claims 1 to 180.

182. A population of engineered T cells produced by contacting a population of T cells with a nucleic acid and one or more cyclic GMP-AMP synthase (cGAS)-stimulator of interferon genes (STING) pathway inhibitors.

183. 183. The population of engineered T cells of claim 182, wherein said nucleic acid comprises donor DNA.

184. 184. The population of engineered T cells of claim 182 or 183, wherein the nucleic acid is a double-stranded circular nucleic acid.

185. 185. The population of engineered T cells of claim 183 or 184, wherein the nucleic acid is a plasmid, nanoplasmid, minicircle or viral vector comprising donor DNA.

186. 186. The population of engineered T cells of any one of claims 183-185, wherein the donor DNA encodes exogenous T cell receptor (TCR)-alpha or a fragment thereof, exogenous TCR-beta or a fragment thereof, or a combination thereof.

187. 187. The population of engineered T cells of any one of claims 182-186, wherein the nucleic acid is associated with a delivery vehicle.

188. 188. The population of engineered T cells of claim 187, wherein the delivery vehicle is a lipid particle or a nanoparticle.

189. 189. The population of engineered T cells of any one of claims 182-188, wherein the T cells are primary T cells.

190. 190. The population of engineered T cells of any one of claims 182-189, wherein the population of T cells is further contacted with a gene editing reagent.

191. 191. The population of engineered T cells of any one of claims 183-190, wherein the donor DNA is inserted into an endogenous TCR locus.

192. 192. The population of engineered T cells of claim 191, wherein the endogenous TCR locus is an endogenous TCR-alpha locus, an endogenous TCR-beta locus, or a combination thereof.

193. 193. The population of engineered T cells of any one of Claims 190-192, wherein contacting the T cells with the gene editing reagent comprises transfecting the T cells with the gene editing reagent.

194. 194. The population of engineered T cells of any one of claims 182-193, wherein contacting said T cells with said nucleic acid comprises transfecting said T cells with said nucleic acid.

195. 195. The population of engineered T cells of any one of claims 182-194, wherein the one or more cGAS-STING pathway inhibitors comprise a cGAS inhibitor.

196. 196. The population of engineered T cells of any one of claims 182-195, wherein the one or more cGAS-STING pathway inhibitors comprise a STING inhibitor.

197. 197. The population of engineered T cells of any one of claims 182-196, wherein the one or more cGAS-STING pathway inhibitors comprise a TANK-binding kinase 1 (TBK1) inhibitor.

198. 198. The population of engineered T cells of any one of claims 182-197, wherein the one or more cGAS-STING pathway inhibitors are Amlexanox (Aml), MRT67307 (MRT), BX795, H151, ODN-A151 (ODN151), Ru.521, G140, or a combination thereof.

199. 200. The population of engineered T cells of claim 198, wherein the one or more cGAS-STING pathway inhibitors are selected from Am1, BX795, ODN151, and MRT.

200. 200. The population of engineered T cells of claim 198, wherein said one or more cGAS-STING pathway inhibitors is ODN151.

201. 200. The population of engineered T cells of claim 198, wherein said one or more cGAS-STING pathway inhibitors is BX795.

202. 202. The population of engineered T cells of any one of claims 182-201, wherein the T cells and the nucleic acid are contacted in the presence of one or more cGAS-STING pathway inhibitors.

203. 202. The population of engineered T cells of any one of claims 182-201, wherein the T cells are contacted sequentially with the nucleic acid and the one or more cGAS-STING pathway inhibitors.

204. 204. The population of engineered T cells of claim 203, wherein the T cells are contacted with the one or more cGAS-STING pathway inhibitors prior to the nucleic acid.

205. 205. The population of engineered T cells of any one of claims 182-204, wherein the T cells are contacted with the one or more cGAS-STING pathway inhibitors for about 2 hours to about 10 hours.

206. 206. The population of engineered T cells of claim 205, wherein the T cells are contacted with the one or more cGAS-STING pathway inhibitors for about 6 hours.

207. 207. The population of engineered T cells of any one of claims 182-206, wherein the T cells are contacted with one cGAS-STING pathway inhibitor.

208. A composition comprising a population of T cells, a nucleic acid, and one or more cyclic GMP-AMP synthase (cGAS)-stimulator of interferon genes (STING) pathway inhibitors.

209. The composition of claim 208, wherein the nucleic acid comprises donor DNA.

210. The composition of claim 208 or 209, wherein the nucleic acid is a plasmid, nanoplasmid, minicircle or viral vector containing donor DNA.

211. The composition of claim 209 or 210, wherein the donor DNA encodes exogenous T cell receptor (TCR)-alpha or a fragment thereof, exogenous TCR-beta or a fragment thereof, or a combination thereof.

212. The composition of any one of claims 208 to 211, wherein the nucleic acid is associated with a delivery vehicle.

213. The composition of claim 212, wherein the delivery vehicle is a lipid particle or a nanoparticle.

214. The composition of any one of claims 208 to 213, wherein the population of T cells comprises primary T cells.

215. The composition of any one of claims 208-214, further comprising a gene editing reagent.

216. The composition of any one of claims 208-215, wherein the one or more cGAS-STING pathway inhibitors comprises a cGAS inhibitor.

217. The composition of any one of claims 208-216, wherein the one or more cGAS-STING pathway inhibitors comprise a STING inhibitor.

218. The composition of any one of claims 208-217, wherein the one or more cGAS-STING pathway inhibitors comprise a TANK-binding kinase 1 (TBK1) inhibitor.

219. The composition of any one of claims 208-218, wherein the one or more cGAS-STING pathway inhibitors are Amlexanox (Aml), MRT67307 (MRT), BX795, H151, ODN-A151 (ODN151), Ru.521, G140, or a combination thereof.

220. The composition of claim 219, wherein the one or more cGAS-STING pathway inhibitors are selected from Am1, BX795, ODN151, and MRT.

221. The composition of claim 219, wherein the one or more cGAS-STING pathway inhibitors is ODN151.

222. The composition of claim 219, wherein the one or more cGAS-STING pathway inhibitors is BX795.

223. 182. A pharmaceutical composition comprising the engineered T cell of claim 181.

224. 224. A method of treating a disease in a subject in need thereof comprising administering a therapeutically effective amount of the engineered T cell of claim 181 or the pharmaceutical composition of claim 223.

225. 225. The method of claim 224, wherein the engineered T cells are autologous T cells.

226. 225. The method of claim 224, wherein the engineered T cells are allogeneic T cells.

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