Inhibition of genotoxic stress to improve t cell engineering

Abstract

Provided herein, inter alia, are methods and compositions for engineering T cells. The method comprises contacting the T cell with a nucleic acid and one or more cyclic GMP-AMP synthetase (cGAS)-interferon gene stimulating factor (STING) pathway inhibitors. The methods provided herein contemplate to increase cell viability, expansion and gene editing efficiency, thereby allowing for an increase in the total number of engineered T cells.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority to U.S. Provisional Application No. 63 / 377,251, filed on September 27, 2022, the contents of which are hereby incorporated by reference in their entirety and for all purposes.

[0003] References to Electronic Sequence Listings

[0004] The contents of the electronic sequence listing (048893-565001WO_Sequence_Listing_ST26.xml; size: 1,720 bytes; and creation date: September 26, 2023) are hereby incorporated by reference in their entirety. Background Art

[0005] Gene editing allows the engineering of various cultured cell lines and primary cells, including T cells. Editing the cell genome enables the generation of T cells specific for disease targets (e.g., cancer cell antigens recognized by edited T cell receptors), allowing the generation of personalized cancer therapies. However, methods for engineering T cells often result in low recovery rates of the final cell products. For example, the toxic effects of T cell engineering methods may result in decreased cell viability and amplification rates. In addition, previous methods for engineering T cells often result in low gene editing efficiencies, which further lead to low total engineered T cell numbers.

[0006] Solutions to these and other problems in the art are disclosed herein, among other things. Summary of the invention

[0007] In one aspect, a method of engineering a T cell is provided, the method 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.

[0008] In one aspect, a method for increasing the cell viability of an engineered T cell population is provided, the method comprising contacting the T cell population with one or more cyclic GMP-AMP synthase (cGAS)-interferon gene stimulator (STING) pathway inhibitors and nucleic acids, thereby forming an engineered T cell population. In an embodiment, the engineered T cell population has increased cell viability relative to an engineered T cell population in which the T cell population is not contacted with one or more cGAS-STING pathway inhibitors.

[0009] In one aspect, a method for increasing gene editing efficiency in a T cell population is provided, the method comprising contacting a T cell population with one or more cyclic GMP-AMP synthase (cGAS)-interferon gene stimulator (STING) pathway inhibitors and nucleic acids to form an engineered T cell population. In an embodiment, the population of T cells has an increased gene editing efficiency relative to a T cell population not contacted with one or more cGAS-STING pathway inhibitors.

[0010] In one aspect, a method for increasing the expansion of an engineered T cell population is provided, the method comprising i) contacting a T cell population with one or more cyclic GMP-AMP synthase (cGAS)-interferon gene stimulator (STING) pathway inhibitors and a nucleic acid, thereby forming an engineered T cell population, and ii) expanding the engineered T cell population, thereby forming an expanded engineered T cell population. In an embodiment, one or more cGAS-STING pathway inhibitors increase the expanded engineered T cell population relative to an engineered T cell population in which the T cell population of step i) is not contacted with one or more cGAS-STING pathway inhibitors.

[0011] In one aspect, an engineered T cell prepared by the method provided herein (including the embodiments thereof) is provided.

[0012] In one aspect, an engineered T cell population is provided by contacting a T cell population with a nucleic acid and one or more cyclic GMP-AMP synthase (cGAS)-stimulator of interferon genes (STING) pathway inhibitors.

[0013] 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.

[0014] In one aspect, a pharmaceutical composition is provided, comprising the engineered T cells provided herein (including the embodiments thereof).

[0015] In one aspect, a method of treating a disease in a subject in need thereof is provided, the method comprising administering a therapeutically effective amount of an engineered T cell provided herein (including embodiments thereof) or a pharmaceutical composition provided herein (including embodiments thereof). BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figures 1A to 1F Plasmid DNA-mediated activation of the cGAS-STING pathway negatively affects T cell viability and expansion after electroporation. Figure 1A) Schematic diagram of an exogenous T cell receptor DNA template for CRISPR / Cas9-mediated homology-directed repair at the TCR-α locus. The DNA template including the TCR-α variant chain and the TCR-β chain is intended to be inserted into the TCR-α (TRAC) locus, while the endogenous TCR-α (VJ) and TCR-β have been disrupted; ( Figure 1B )Schematic diagram of the 15-day workflow of T cell activation, engineering, and cell culture. The entire process is performed in chemically defined medium; ( Figures 1C to 1E ) T cell viability during the 15-day T cell engineering and culture process ( Figure 1C ), T cell expansion ( Figure 1D ) and knock-in / knock-out efficiency ( Figure 1E ) analysis; T cell viability and cell expansion were measured by NucleoCounter NC-200. Knock-in / knock-out efficiency was measured by flow cytometry and identified by MHC-peptide dextramer staining using flow cytometry. ( Figure 1F ) Western blot analysis of DNA genotoxic pathway activation (cGAS-STING-TBK1-IRF3) during and after the TCR engineering process in the presence of different components of the transfection for gene editing (RNP, DNA) as shown.

[0017] Figures 2A to 2C . T cell viability, expansion and targeting data for different donors 48 hours after transfection. T cell viability using different transfection process components (DNA, RNP) as shown ( Figure 2A ), T cell proliferation rate ( Figure 2B ) and knock-in / knock-out efficiency ( Figure 2C ). T cell viability data and cell expansion rate were measured by NucleoCounter NC-200. Knock-in / knock-out efficiency was measured by flow cytometry and identified by MHC-peptide dextramer staining by flow cytometry.

[0018] Figures 3A to 3B .Western blot analysis of AIM2 DNA inflammasome and toll-like receptor (TLR) pathway activation in the presence of different transfection components (RNP, DNA) as indicated during and after the TCR engineering process. 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.

[0019] Figures 4A to 4I.Individual donor T cell response data after inhibitor treatment (three donors). Figure 4A , 4D 4G) The percentage of survival rate of donors 1, 2 and 3 48 hours after transfection was determined using NucleoCounter NC-200; ( Figure 4B , 4E , 4H) Calculated based on the number of seeded cells on day 2, corresponding to the expansion fold of the final drug product (FDP) after 15 days of T cell engineering process for donors 1, 2 and 3, respectively; ( Figure 4C , 4F , 4I) correspond to the total edited cell (TEC) number in FDP of donors 1, 2 and 3, respectively. TEC was calculated based on the seeding density of 2 million cells after transfection in each group. The numbers on each column were rounded to integers. The total edited cell number was calculated by multiplying the expansion fold by the knock-in percentage.

[0020] Figures 5A to 5F : Individual donor T cell response data after inhibitor treatment (there are two more donors in addition to the donor in Figure 4). Figure 5A , 5D ) The percentage of viability of donors 4 and 5 after 48 hours of transfection was determined using NucleoCounter NC-200; ( Figure 5B , 5E ) The final drug product (FDP) expansion fold of donors 4 and 5 after 15 days of T cell engineering process was calculated based on the number of seeded cells on day 2, ( Figure 5C , 5F ) Total edited cell (TEC) number in each FDP of donors 4 and 5. TEC was calculated based on the seeding density of 2 million cells after transfection in each group. Numbers on each column were rounded to integers. Total edited cell number was calculated by multiplying the expansion fold by the knock-in percentage.

[0021] Figures 6A to 6C : Percent viability 48 hours after transfection from five independent donors ( Fig. 6A ), the final cell product amplification factor ( Figure 6B ) and total number of editing cells (TEC) ( Figure 6C ). T cell viability data and expansion fold were measured by NucleoCounter NC-200 machine. TEC was calculated by multiplying the expansion fold by the knock-in percentage. Data are expressed as mean ± SEM ( Figures 6A to 6C ). ***P<0.001; **P<0.01; *P<0.05, compared with the control (CTR) group.

[0022] Figures 7A to 7C : The cell editing ratio of the final T cell product. ( Figures 7A to 7C ) Knock-in and knock-out data from 3 representative donors measured by flow cytometry. Knock-in rate was identified by flow cytometry by MHC-peptide dextramer and TCR double positive staining. Knockout represents MHC-peptide dextramer and TCR double negative. Control (CTR) represents the group without inhibitor treatment.

[0023] Figures 8A to 8F .BX795 pretreatment attenuates the cGAS-STING pathway in T cells. ( Fig. 8A ) Western blot analysis of TBK1 and IRF3 expression and phosphorylation with or without (CTR group) BX795 pretreatment. N, 30 min and 60 min indicate immediately before transfection, 30 min after transfection and 60 min after transfection, respectively. ( Figures 8B to 8E ) from and ( Fig. 8A ) obtained from the same donor in the same experiment. Viability data were collected 48 hours after transfection using NucleoCounter NC-200 ( Figure 8B ). Amplification factor ( Figure 8C ), knock-in / knock-out efficiency ( Fig.8D ) and total editing cells ( Fig. 8E ) values ​​represent data obtained from the last day of culture (day 15). Fig.8F ) Comparison of cytokine expression between groups with and without inhibitor treatment (using qPCR). Samples for qPCR analysis were collected 6 hours after transfection and analyzed in triplicate.

[0024] Figures 9A to 9D :Inhibitor pretreatment improves the knockout and knockin ratios of the final T cell products. ( Fig.9A ) Total knockout percentage in the BX795 pretreatment group compared to the control group. ( Fig. 9B ) Analysis of the difference in percent knockdown values ​​between control (set to 1) and inhibitor pre-treated groups. Fig. 9C )Knock-in ratio of BX795 pretreatment group compared with control group. ( Fig.9D ) Analysis of the difference in knock-in percentage between the BX795 pretreatment group and the control group (set to 1). Samples were collected on day 15 and the ratios were measured by flow cytometry. The knock-in / knock-out ratios were identified by flow cytometry by MHC-peptide dextramer staining. ***P<0.001; **P<0.01; *P<0.05, compared to the control group.

[0025] Figures 10A to 10F : Pretreatment of T cell cultures with BX795 before transfection had no effect on the phenotype of the final T cell product. Figures 10A to 10FT cell phenotype data as indicated. Inhibitor types and doses 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 cells (TE: CD45RA + CD45RO - CD95 + CD27 - All six donors were treated similarly with respect to inhibitor pretreatment, electroporation procedures, and culture conditions and methods.

[0026] Figures 11A to 11F T cell cultures pretreated with the BX795 inhibitor prior to transfection displayed functional potential similar to that of untreated controls. Fig.11A Comparison of activation marker CD137 expression between the inhibitor pretreatment group and the control group. Cytokine expression: IFNγ( Fig. 11B )、TNF-α( Fig. 11C ) and granzyme B ( Fig.11D ), comparison between the inhibitor pretreatment group and the control group. Cell samples were collected on the last day (day 15). The cells were then co-cultured with the indicated concentrations of the target peptide (WT1) for 24 h and then subjected to flow cytometry analysis. ( Fig.11E ) Comparison of proliferation analysis between the inhibitor pretreatment group and the control group. Cell samples were collected on the last day (day 15). The cells were then labeled with CFSE dye and co-cultured with the indicated concentrations of the target peptide (WT1) for 72 h, followed by flow cytometry analysis. ( Fig.11F ) Cell killing analysis to compare T cell function between inhibitor pretreatment group and control group. Cell samples were collected on the last day (day 15) and then co-cultured with target peptide pre-labeled T2 target cells at a specified ratio for 20 hours. Flow cytometry was used to measure the target cell apoptosis level by counting annexin V and 7-aminoactinomycin D (7-AAD) double positive populations, corrected by the target cell group only.

[0027] Figures 12A to 12BThe inhibitor pretreatment group and the control group had comparable T cell exhaustion profiles. Tim3 ( Fig. 12A ) and PD-1( Fig. 12B ) median fluorescence intensity (MFI). Cell samples were collected on the last day of the process (day 15). The cells were then co-cultured with the target peptide (WT1) at the indicated concentration for 24 hours and then subjected to flow cytometry analysis.

[0028] Figures 13A to 13C In three different donors, T cell cultures pretreated with the BX795 inhibitor prior to transfection displayed functional potential similar to that of untreated controls. Additional donors (Donors 1 to 3) were analyzed separately for T cell function, similar to Figures 11A to 11F As shown in. The cytokine expression of activation markers CD137, IFNγ, TNF-α, granzyme B and perforin expression in the inhibitor pretreatment group and the control group was measured. Cell samples were collected on the last day (day 15) of the process. The cells were then co-cultured with the target peptide (WT1) at a specified concentration for 24 hours, and then flow cytometry analysis was performed. In order to compare the proliferation analysis between the inhibitor pretreatment group and the control group, samples were collected on the 15th day, and then the cells were labeled with CFSE dye and co-cultured with the target peptide (WT1) at a specified concentration for 72 hours, and then flow cytometry analysis was performed. For cell killing analysis, T cell function between the inhibitor pretreatment group and the control group was compared. Cell samples were collected on the 15th day and co-cultured with T2 target cells pre-labeled with the target peptide at a specified ratio for 20 hours. Flow cytometry was used to measure the level of target cell apoptosis by calculating the double positive population of annexin V and 7-aminoactinomycin D (7-AAD), and correction was performed by the target cell group only.

[0029] Figures 14A to 14C . Plasmid DNA transfection efficiency depends on the electroporation (pulse coding) program used. The inhibitor BX795 enhances cell editing efficiency (knock-in) throughout the 15-day culture period. ( Fig.14A )Comparison of the percentage of GFP-positive CD8+ T cells between the different electroporation pulse codes used. EH115 (strong), EW113 (medium), EW100 (weak). DNA uptake was analyzed using 1ug / ml GFP plasmid according to the supplier's instructions. The percentage of GFP-positive CD8+ T cells was measured by flow cytometry 24 hours after electroporation. EH115 provides the harshest conditions of the three pulse codes, while EW100 is the basic pulse code recommended by the supplier and has the mildest transfection conditions. ( Figures 14B to 14C ) from the untreated control group ( Fig. 14B ) and BX795 (2.5uM) pretreatment groups ( Fig. 14C) according to the date of T cell knock-in and knock-out data. Transfection was completed by the 2nd day, and measurement started from the 4th day (48 hours after transfection). The knock-in ratio was identified by MHC-peptide dextramer staining and TCR staining double positive by flow cytometry. Knockout represents MHC-peptide dextramer and TCR double negative population. DETAILED DESCRIPTION

[0030] Although various embodiments and aspects of the present invention have been shown and described, it is obvious to those skilled in the art that such embodiments and aspects are provided only as examples. Without departing from the present invention, those skilled in the art will now expect many changes, variations and substitutions. It should be understood that various alternatives of the embodiments of the present invention described herein may be adopted in practicing the present invention.

[0031] The section headings used herein are for organizational purposes only and are not to 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, articles, books, manuals, and treatises, are expressly incorporated by reference in their entirety for any purpose.

[0032] Abbreviations used herein have their conventional meanings in the chemical and biological arts.The chemical structures and formulae described herein are constructed according to standard rules of chemical valency known in the chemical arts.

[0033] Unless otherwise defined, the scientific and technical terms used herein have the same meaning as those generally understood by those of ordinary skill in the art. Methods, devices and materials similar or equivalent to any method, device and material described herein can be used in the practice of the present invention. 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.

[0034] "Nucleic acid" refers to nucleotides (e.g., deoxyribonucleotides or ribonucleotides) and polymers thereof, or their complements, in single-stranded, double-stranded, or multi-stranded form; or nucleosides (e.g., deoxyribonucleotides or ribonucleotides). Examples of polynucleotides contemplated herein include single-stranded and double-stranded DNA, single-stranded and double-stranded RNA, and hybrid molecules having a mixture of single-stranded and double-stranded DNA and RNA. Examples of nucleic acids, such as 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 fragments thereof.

[0035] As used herein, the term "gene editing reagent" refers to components required 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 required for clustered regularly interspaced short palindromic repeats system (CRISPR / Cas) gene editing.

[0036] As used herein, "zinc finger protein" (ZFP) refers to a chimeric protein comprising a nuclease domain and a nucleic acid (e.g., DNA) binding domain that is stabilized by zinc. Each DNA binding domain is often referred to as a "finger," so that a zinc finger protein or polypeptide has at least one finger, more typically two fingers, or three fingers, or even four or five fingers, to at least six or more fingers. Each finger typically binds two to four base pairs of DNA. Each finger can include a zinc-chelating DNA binding region of about 30 amino acids (see, e.g., U.S. Patent Publication No. 2012 / 0329067A1, the disclosure of which is incorporated herein by reference).

[0037] As used herein, "transcription activator-like effector" (TALE) refers to a protein composed of more than one TAL repeat sequence and capable of binding to nucleic acids in a sequence-specific manner. TALE represents a class of DNA-binding proteins secreted by plant pathogenic bacteria of species such as Xanthomonas and Ralstonia via their type III secretion system after infecting plant cells. Natural TALEs have been shown to bind specifically to plant promoter sequences, thereby regulating gene expression and activating effector-specific host genes to promote bacterial reproduction ( 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 TAL allows the DNA binding domain to be combined with effector molecules (such as nucleases). In particular, TALE nucleases allow the development of new genome engineering tools.

[0038] Natural TALEs are typically characterized by a central repeat domain and a carboxyl-terminal nuclear localization signal sequence (NLS) and a transcriptional activation domain (AD). The central repeat domain is typically composed of a variable amount of between 1.5 and 33.5 amino acid repeats, typically 33 to 35 residues in length, with the exception of the typically shorter carboxyl-terminal repeats (called half-repeats). The repeats are mostly identical, but some hypervariable residues differ. The DNA recognition specificity of TALE is mediated by hypervariable residues, typically at positions 12 and 13 of each repeat, the so-called repeat variable diresidues (RVDs), where each RVD targets a specific nucleotide in a given DNA sequence. Therefore, the sequential order of repeats in TAL proteins is often related to the defined linear order of nucleotides in a given DNA sequence. Potential RVD codes for some naturally occurring TALEs have been identified, allowing prediction of the order of consecutive repeats required to bind to a given DNA sequence (Boch, J. et al., Science 326: 1509-1512 (2009); Moscou, MJ et al., Science 326: 1501 (2009)). In addition, TAL effectors generated with new repeat combinations have been shown to bind to target sequences predicted by the code. It has been shown that target DNA sequences typically begin with a 5' thymine base to be recognized by TAL proteins.

[0039] The terms "RNA-guided DNA nuclease" or "RNA-guided DNA endonuclease" and the like refer in their usual and customary sense to an enzyme that cleaves phosphodiester bonds within a DNA polynucleotide chain, wherein recognition of the phosphodiester bonds is facilitated by a single RNA sequence (e.g., a single guide RNA).

[0040] The term "class II CRISPR endonuclease" refers to an endonuclease having endonuclease activity similar to Cas9 and participating in a class II CRISPR system. An exemplary class II CRISPR system is a class II CRISPR locus from Streptococcus pyogenes SF370, which contains a cluster of four genes Cas9, Cas1, Cas2, and Csn1, and two non-coding RNA elements: tracrRNA and a characteristic array of repeat sequences (forward repeat sequences) spaced apart by short segments of non-repetitive sequences (spacers, each about 30 bp). The Cpf1 enzyme belongs to the assumed V-type CRISPR-Cas system. Both type II and type V systems are included in the class II of the CRISPR-Cas system. The C2c1 ("class 2 candidate 1") enzyme is a class II VB type enzyme. The C2c2 ("class 2 candidate 2") enzyme is a class II VI-A type enzyme. The C2c3 ("class 2 candidate 3") enzyme is a class II VC type enzyme. 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, 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.

[0041] As referred to herein, "CRISPR-associated protein 9", "Cas9", "Csn1" or "Cas9 protein" includes any of the recombinant or naturally occurring forms of a Cas9 endonuclease or a variant or homolog thereof that maintains Cas9 endonuclease activity (e.g., within at least 50%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% activity compared to Cas9). In some aspects, 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 50, 100, 150 or 200 contiguous amino acid portion) compared to the naturally occurring Cas9 protein. In some aspects, the Cas9 protein is substantially identical to the protein identified by UniProt reference number Q99ZW2 or a variant or homolog thereof having substantial identity. In some aspects, the Cas9 protein has at least 75% sequence identity with the amino acid sequence of the protein identified by UniProt reference number Q99ZW2. In some aspects, the Cas9 protein has at least 80% sequence identity with the amino acid sequence of the protein identified by UniProt reference number Q99ZW2. In some aspects, the Cas9 protein has at least 85% sequence identity with the amino acid sequence of the protein identified by UniProt reference number Q99ZW2. In some aspects, the Cas9 protein has at least 90% sequence identity with the amino acid sequence of the protein identified by UniProt reference number Q99ZW2. In some aspects, the Cas9 protein has at least 95% sequence identity with the amino acid sequence of the protein identified by UniProt reference number Q99ZW2.

[0042] As referred to herein, "CRISPR-associated endonuclease Cas12a", "Cas12a", "Cas12" or "Cas12 protein" includes any of the recombinant or naturally occurring forms of a Cas12 endonuclease or a variant or homolog thereof that maintains Cas12 endonuclease activity (e.g., within at least 50%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% activity compared to Cas12). In some aspects, 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., 50, 100, 150 or 200 consecutive amino acid portions) compared to the naturally occurring Cas12 protein. In some aspects, the Cas12 protein is substantially identical to a protein identified by UniProt reference number A0Q7Q2 or a variant or homolog thereof having substantial identity.

[0043] As referred to herein, "Cfp1" or "Cfp1 protein" includes any of a recombinant or naturally occurring form of a Cfp1 (CxxC finger protein 1) endonuclease or a variant or homolog thereof that maintains Cfp1 endonuclease enzymatic activity (e.g., within at least 50%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% activity compared to Cfp1). In some aspects, 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 50, 100, 150 or 200 contiguous amino acid portion) compared to the naturally occurring Cfp1 protein. In an embodiment, the Cfp1 protein is substantially identical to the protein identified by UniProt reference number Q9P0U4, or a variant or homolog thereof having substantial identity.

[0044] The terms "RNA-guided RNA nuclease" or "RNA-guided RNase" and the like refer in the usual and customary sense to an RNA-guided nuclease that targets a specific phosphodiester bond within an RNA polynucleotide, wherein recognition of the phosphodiester bond is facilitated by a separate polynucleotide sequence (e.g., an RNA sequence (e.g., a single guide RNA (sgRNA), a guide RNA (gRNA)). Typically, the RNA-guided RNase targets single-stranded RNA. In aspects, the RNA-guided RNase is Cas13 (e.g., Cas13a, Cas13b).

[0045] As referred to herein, "Cas13a" or "Cas13a protein" includes any of a recombinant or naturally occurring form of a variant or homolog of Cas13a (CRISPR-associated endoribonuclease Cas13a) endoribonuclease (also referred to as CRISPR-associated endoribonuclease C2c2, C2c2) or a variant or homolog thereof that maintains Cas13a endoribonuclease activity (e.g., at least 50%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% activity compared to Cas13a). In some aspects, 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., 50, 100, 150 or 200 consecutive amino acid portions) compared to a naturally occurring Cas13a protein. In an embodiment, the Cas13a protein is substantially identical to the protein identified by UniProt reference number C7NBY4, or a variant or homolog thereof having substantial identity.

[0046] As referred to herein, "Cas13b" or "Cas13b protein" includes any of the recombinant or naturally occurring forms of Cas13b (CRISPR-associated RNA-guided ribonuclease Cas13b) endonucleases or variants or homologs thereof that maintain Cas13b nuclease enzymatic activity (e.g., at least 50%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% activity compared to Cas13b. In some aspects, 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., 50, 100, 150 or 200 consecutive amino acid portions) compared to naturally occurring Cas13b proteins. In an embodiment, the Cas13b protein is substantially identical to a protein identified by UniProt reference number A0A8G0P913 or a variant or homolog having substantial identity thereto.

[0047] In an embodiment, the gene editing reagent comprises Cas-CLOVER. In an embodiment, Cas-CLOVER comprises a Clo051 nuclease domain fused to a catalytically inactivated Cas9. See, for example, U.S. Patent Publication No. US2021 / 0107993 and Madison et al., Molecular Therapy Nucleic Acids, Vol. 29, pp. 979-995, September 13, 2022, the full text of each of which is incorporated herein by reference. In an embodiment, the gene editing reagent comprises a nickase, such as nCas9 (nickase Cas9). Nickase is an engineered Cas protein that can introduce single-stranded cleavage with the same specificity as conventional CRISPR / Cas nucleases. See, for example, PCT Publication No. WO2014093694, which is incorporated herein by reference in its entirety.

[0048] The terms "guide RNA" and "gRNA" are used interchangeably and refer to a polynucleotide sequence including a crRNA sequence and an optional tracrRNA sequence. In an embodiment, the gRNA includes a crRNA sequence and a tracrRNA sequence (e.g., "single guide RNA" or "sgRNA"). In an embodiment, the gRNA does not include a tracrRNA sequence. The crRNA sequence includes a guide sequence (i.e., "guide" or "spacer") and a tracr matching sequence (i.e., a forward repeat sequence). The term "guide sequence" refers to a sequence that specifies a target site. In general, a tracr matching sequence includes any sequence that has sufficient complementarity with a tracrRNA sequence to promote one or more of the following: (1) excision of a guide sequence flanked by a tracr matching sequence in a cell containing a corresponding tracr sequence; and (2) forming a complex (e.g., a CRISPR complex) at the target sequence, wherein the complex (e.g., a CRISPR complex) includes a tracr matching sequence hybridized to a tracr sequence.

[0049] In an embodiment, the gRNA is a single-stranded ribonucleic acid. In one aspect, the length of the gRNA is about 10 to about 200 nucleic acid residues. In one aspect, the length of the gRNA is about 50 to about 150 nucleic acid residues. In one aspect, the length of the gRNA is about 80 to about 140 nucleic acid residues. In one aspect, the length of the gRNA is about 90 to about 130 nucleic acid residues. In one aspect, the length of the gRNA is about 100 to about 120 nucleic acid residues.

[0050] Typically, a guide sequence is any polynucleotide sequence that has sufficient complementarity to a target polynucleotide sequence to hybridize with the target sequence and guide the CRISPR complex to specifically bind to the target sequence sequence. In some embodiments, the degree of complementarity between a guide sequence and its corresponding target sequence is about or greater than about 50%, 60%, 75%, 80%, 85%, 90%, 95%, 97.5%, 99% or more when optimally aligned using a suitable alignment algorithm. Any suitable algorithm for aligning sequences can be used to determine optimal alignment, non-limiting examples of which include the Smith-Waterman algorithm, the Needleman-Wunsch algorithm, an algorithm based on the Burrows-Wheeler transform (e.g., Burrows WheelerAligner), ClustalW, Clustal X, BLAT, Novoalign (Novocraft Technologies, ELAND (Illumina, San Diego, CA), SOAP (available at soap.genomics.org.cn), and Maq (available at maq.sourceforge.net). In embodiments, the guide sequence is about or greater than 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 embodiments, the guide sequence is less than about 75, 50, 45, 40, 35, 30, 25, 20, 15, 12 or fewer nucleotides in length. The guide sequence directs the CRISPR complex to The ability of sequence-specific binding of a target sequence can be assessed by any suitable assay. For example, components of a CRISPR system sufficient to form a CRISPR complex, including a guide sequence to be tested, can be provided to a host cell having a corresponding target sequence, such as by transfection with a vector encoding the CRISPR sequence components, followed by assessment of preferential cleavage within the target sequence, such as by the Surveyor assay described herein. Similarly, the cutting of a target polynucleotide sequence can be assessed in a test tube by providing a target sequence, components of a CRISPR complex (including a guide sequence to be tested and a control guide sequence 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 are conceivable to those skilled in the art.

[0051] 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 a genetic modification method (e.g., CRISPR). For example, the donor DNA may have homology arms homologous to the gene region into which the donor DNA is to be inserted. For example, the donor DNA may form a complex with the Cas protein. In some cases, cells may be transfected with gene editing reagents and donor DNA. In an embodiment, the donor DNA is a part of a plasmid, a vector, or an expression vector that facilitates the delivery of the donor DNA to the cell. In an embodiment, the donor DNA is a part of a circular DNA. In an embodiment, the donor DNA is a part of a linear DNA. In an embodiment, the donor DNA may include one or more modifications.

[0052] The nucleic acid (such as donor DNA) used in the present method can be modified.For example, nucleic acid can include synthetic, naturally occurring and non-naturally occurring known nucleotide analogs or modified backbone residues or bonds, which have binding properties similar to reference nucleic acids, and are metabolized in a manner similar to reference nucleotides.The example of such analogs includes but is not limited to phosphodiester derivatives, including for example phosphoramidate, phosphorodiamidate, phosphorothioate (also referred to as the thio-oligodeoxynucleotide with double bond sulfur substituted phosphate oxygen), phosphorodithioate, phosphonocarboxylic acid, phosphonocarboxylate, phosphonoacetic acid, phosphonoformic acid, methylphosphonate, phosphonic acid boron or O-methylphosphoramidite bond (see Eckstein, OLIGONUCLEOTIDES AND ANALOGUES:APRACTICAL APPROACH, Oxford University Press) and modification of nucleotide bases (such as 5-methylcytidine or pseudouridine); and peptide nucleic acid backbone and bond. Other analog nucleic acids include those with positive backbones; non-ionic backbones, modified sugars, and non-ribose backbones (e.g., phosphorodiamidate morpholino oligonucleotides or locked nucleic acids (LNAs) known in the art), including those described in U.S. Pat. Nos. 5,235,033 and 5,034,506 and Chapters 6 and 7 of ASC Symposium Series 580, CARBOHYDRATE MODIFICATIONS INANTISENSE RESEARCH, Sanghui & Cook, eds. Nucleic acids containing one or more carbocyclic sugars are also included in a definition of nucleic acids. Mixtures of naturally occurring nucleic acids and analogs can be prepared; alternatively, mixtures of different nucleic acid analogs can be prepared, as well as mixtures of naturally occurring nucleic acids and analogs. In an embodiment, the internucleotide bond in the DNA is a phosphodiester, a phosphodiester derivative, or a combination of the two.

[0053] As used herein, "TANK binding kinase 1 protein" or "TBK1" includes any of a recombinant or naturally occurring form of a TANK binding kinase 1 (TBK1) (also known as serine / threonine protein kinase TBK1, NF-κ-B activating kinase, T2K) or a variant or homolog thereof that maintains TANK binding kinase 1 (TBK1) activity (e.g., within at least 50%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% activity compared to TBK1). In aspects, 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 50, 100, 150 or 200 contiguous amino acid portion) compared to a naturally occurring TBK1 protein. In some aspects, the TBK1 protein is substantially identical to the protein identified by UniProt reference number Q9UHD2, or a variant or homolog thereof having substantial identity.

[0054] As used herein, "cyclic GMP-AMP synthase protein" or "cGAS" includes any of the recombinant or naturally occurring forms of cyclic GMP-AMP synthase protein (cGAS) or a variant or homolog thereof that maintains cGAS activity (e.g., within at least 50%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% activity compared to cGAS). In aspects, 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 50, 100, 150 or 200 contiguous amino acid portion) compared to the naturally occurring cGAS protein. In aspects, the cGAS protein is substantially identical to the protein identified by UniProt reference number Q8N884 or a variant or homolog thereof having substantial identity.

[0055] As used herein, "stimulator of interferon genes protein" or "STING" includes any of the recombinant or naturally occurring forms of stimulator of interferon genes protein (STING) (also known as endoplasmic reticulum interferon stimulator, IRF3 activation mediator, transmembrane protein 173) or its variants or homologs that maintain STING activity (e.g., within at least 50%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% activity compared to STING). In some aspects, 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., 50, 100, 150 or 200 consecutive amino acid portions) compared to the naturally occurring STING protein. In some aspects, the STING protein is substantially identical to the protein identified by UniProt reference number Q86WV6 or a variant or homolog having substantial identity thereto.

[0056] As used herein, "interferon regulatory factor 3 protein" or "IRF3" includes any of a recombinant or naturally occurring form of an interferon regulatory factor 3 (IRF3) or a variant or homolog thereof that maintains IRF3 activity (e.g., within at least 50%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% activity compared to IRF3). In some aspects, 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., 50, 100, 150 or 200 consecutive amino acid portions) compared to the naturally occurring IRF3 protein. In some aspects, the IRF3 protein is substantially identical to the protein identified by UniProt reference number Q14653 or a variant or homolog thereof having substantial identity.

[0057] The term "gene" means a DNA segment involved in the production of a protein; it includes the regions before and after the coding region (leader and trailer regions) and the intervening sequences (introns) between the individual coding segments (exons). The leader, trailer, and introns include regulatory elements necessary for gene transcription and translation. Further, a "protein gene product" is a protein expressed by a specific gene.

[0058] The term "plasmid", "vector" or "expression vector" refers to a nucleic acid molecule encoding a gene and / or regulatory elements necessary for gene expression. In an embodiment, the plasmid, vector or expression vector is a circular nucleic acid. In an embodiment, the plasmid, vector or expression vector is not a linear nucleic acid. In an embodiment, the plasmid, vector or expression vector is a linear nucleic acid.

[0059] As used herein, the term "nanoplasmid" is used to refer to a circular nucleic acid containing at least a target nucleic acid sequence, a mini-replication origin (e.g., R6K) and a selectable marker (e.g., a small RNA selectable marker, RNA-OUT). In an embodiment, the nanoplasmid contains a prokaryotic DNA less than 500bp.

[0060] As used herein, the term "minicircle" refers to a circular nucleic acid, typically about 200 bases to about 5 kilobases in length. In embodiments, the length of the minicircle is about 2 kilobases to about 5 kilobases. In embodiments, the minicircle does not include prokaryotic DNA. Therefore, in embodiments, the minicircle includes at least the target nucleic acid sequence and the elements necessary for the expression of the nucleic acid sequence.

[0061] As used herein, the term "T cell engineering" or "T cell genetic engineering" refers to a type of genetic modification in which DNA is inserted, deleted, modified or replaced at one or more specific locations in the T cell genome. Unlike early 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 for T cell engineering, such as generating double-strand breaks at specific points in genes or genomes where DNA is inserted. Gene editing reagents may include, for example, a regularly clustered, interspaced short palindromic repeat system (CRISPR / Cas), ZFN or TALEN. Therefore, "engineered T cells" are T cells in which DNA is inserted, deleted, modified or replaced at one or more specific locations in the T cell genome.

[0062] The term "recombinant" when used in 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 by the alteration of a native nucleic acid or protein, or that the cell is derived from a cell so modified. In certain cases, a recombinant cell expresses genes that are not present in the native (non-recombinant) form of the cell or expresses native genes that are otherwise abnormally expressed, underexpressed or not expressed at all. Transgenic cells are those cells and plants that express heterologous genes or coding sequences, usually as a result of recombinant methods.

[0063] The term "heterologous" when used in 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, having two or more sequences from unrelated genes that are arranged to produce a new functional nucleic acid, such as 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).

[0064] 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 in which it is expressed. In contrast, the term "endogenous" or "endogenous promoter" refers to a molecule or substance that is produced or derived from a given cell or organism.

[0065] The term "isolated" when applied to a nucleic acid or protein means that the nucleic acid or protein is substantially free of other cellular components with which it is associated in the native state. For example, it can be in a homogeneous state and can be a dry solution or 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.

[0066] As used herein, the terms "electroporation," "electroperization," and "electrotransfer" are used in their ordinary sense and refer to a technique in which an electric field is applied to cells in order to increase the permeability of the cell membrane, allowing for the introduction of chemicals, drugs, proteins or nucleic acids, or a combination thereof, into the cells.

[0067] 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 gene sequence encoding a complete protein or a functional portion thereof. Non-viral transfection methods 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, nuclear transfection, sonoporation, transfection by heat shock, magnetofection and electroporation. In some embodiments, electroporation is used to introduce nucleic acid molecules into cells 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, adenoviral, lentiviral and adeno-associated viral vectors. In some embodiments, adenoviral vectors are used to introduce nucleic acid molecules into cells according to standard procedures well known in the art. The term "transfection" or "transduction" also refers to the introduction of proteins from the external environment into cells. In embodiments, transduction or transfection of proteins relies on the ability of a peptide or protein to cross the cell membrane and attach to the protein of interest. See, e.g., Ford et al. (2001) Gene Therapy 8:1-4 and Prochiantz (2007) Nat. Methods 4:119-20.

[0068] "Transduction" or "transduction" is used according to its ordinary meaning and refers to the process of introducing one or more exogenous nucleic acids (i.e., DNA not naturally present in the cell) into a cell. Transduction can be performed by introducing a virus or viral vector (e.g., an adenoviral vector) into the cell.

[0069] As used herein, the words "expression" or "expressed" referring to a gene means the transcription and / or translation product of the gene (e.g., TCR-α, TCR-β, etc.). The expression level of a DNA molecule in a cell can be determined based on the amount of corresponding mRNA in the cell or the amount of protein encoded by the DNA produced by the cell. The expression level of a nucleic acid molecule can be detected by standard methods, including PCR or Northern blotting methods well known in the art. See, Sambrook et al., 1989 Molecular Cloning: A Laboratory Manual, 18.1-18.88.

[0070] "Contacting" is used according to its ordinary meaning and refers to a process of allowing at least two different species (e.g., chemical compounds including biomolecules or cells) to become close enough to react, interact, or physically contact. The two species can be, for example, a cGAS-STING pathway inhibitor and a T cell as provided herein. In an embodiment, contacting includes, for example, allowing a cGAS-STING pathway inhibitor described herein to physically contact a T cell. In an embodiment, contacting can result in the delivery of a compound to a cell. For example, contacting may result in the delivery of a cGAS-STING inhibitor to a cell. In an embodiment, contacting can result in the delivery of a nucleic acid to a cell. In an embodiment, "contacting" or "contacted" includes culturing T cells in the presence of a species, such as a cGAS-STING pathway inhibitor.

[0071] "Control" or "standard control" refers to a sample, measurement result or value used as a reference (usually a known reference) for comparison with a test sample, measurement result or value. For example, a standard control can be an engineered T cell prepared without contacting the T cell with one or more cGAS-STING inhibitors provided herein (including its embodiments). In an embodiment, a standard control can be an engineered T cell population prepared without contacting a T cell population with one or more cGAS-STING inhibitors provided herein (including its embodiments). Therefore, a standard control can be an engineered T cell prepared by contacting a T cell with a nucleic acid in the absence of one or more cGAS-STING inhibitors. A standard control can be an engineered T cell population prepared by contacting a T cell population with a nucleic acid in the absence of one or more cGAS-STING inhibitors. Controls are also valuable for determining the significance of data. For example, if the value of a given parameter varies greatly in the control, the change in the test sample will not be considered significant. The technician will recognize that standard controls can be designed to assess any number of parameters (e.g., cell viability, cell expansion, total edited cell number, gene editing efficiency, etc.).

[0072] Those skilled in the art will understand which standard controls are most appropriate in a given situation and can analyze data based on comparisons with standard control values. Standard controls are also valuable for determining the significance (e.g., statistical significance) of data. For example, if the value of a given parameter varies greatly in the standard control, the change in the test sample will not be considered significant.

[0073] As used herein, "T cells" or "T lymphocytes" are lymphocyte types (subtypes of white blood cells) that play 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 T helper cells. Different types of T cells can be distinguished by using T cell detection agents.

[0074] The terms "inhibition", "inhibit", "inhibiting", etc., as defined herein with respect to cell proliferation (e.g., cancer cell proliferation), mean to negatively affect cells (e.g., reduce cell proliferation) or kill cells. In some embodiments, inhibition refers to a reduction in a disease or disease symptom (e.g., cancer, cancer cell proliferation). In embodiments, an "inhibitor" is a compound or protein that inhibits a receptor or another protein, such as by binding, partially or completely blocking, reducing, preventing, delaying, inactivating, desensitizing, or downregulating an activity (e.g., receptor activity or protein activity).

[0075] The term "disease" or "condition" refers to a physical state or health condition of a patient or subject that can be treated with the compounds or methods provided herein. The disease can be cancer. In some other cases, "cancer" refers to human cancer. In embodiments, the cancer is lymphoma, melanoma, or leukemia.

[0076] In the context of a substance or substance activity or function associated with a disease (e.g., cancer), the term "associated with" or "associated with..." means that it is caused (in whole or in part) by the substance or substance activity or function, or the symptoms of the disease are caused (in whole or in part) by the substance or substance activity or function.

[0077] "Patient" or "subject in need thereof" refers to a living organism suffering from or susceptible to a disease (e.g., cancer, etc.) or condition that can be treated by administering a composition or pharmaceutical composition as provided herein. Non-limiting examples include humans, other mammals, cattle, rats, mice, dogs, monkeys, goats, sheep, cows, deer, and other non-mammalian animals. In some embodiments, the patient is a human.

[0078] As used herein, the term "cancer" refers to all types of cancers, tumors or malignancies found in mammals (e.g., humans), including leukemias, lymphomas, carcinomas and sarcomas. Exemplary cancers that can be treated with compounds or methods provided herein include brain cancer, gliomas, glioblastomas, neuroblastomas, prostate cancer, colorectal cancer, pancreatic cancer, medulloblastomas, melanomas, cervical cancer, gastric cancer, ovarian cancer, lung cancer, head cancer, Hodgkin's disease and non-Hodgkin's lymphomas. Exemplary cancers that can be treated with compounds or methods provided herein include thyroid cancer, endocrine system cancer, brain cancer, breast cancer, cervical cancer, colon cancer, head and neck cancer, liver cancer, kidney cancer, lung cancer, ovarian cancer, pancreatic cancer, rectal cancer, gastric cancer and uterine cancer. Additional examples include thyroid cancer, bile duct cancer, pancreatic cancer, skin melanoma, colon adenocarcinoma, rectal adenocarcinoma, stomach adenocarcinoma, esophageal cancer, head and neck squamous cell carcinoma, breast invasive cancer, lung adenocarcinoma, lung squamous cell carcinoma, non-small cell lung cancer, mesothelioma, multiple myeloma, neuroblastoma, glioma, glioblastoma multiforme, ovarian cancer, rhabdomyosarcoma, essential thrombocythemia, essential macroglobulinemia, primary brain tumor, malignant pancreatic insulinoma, malignant carcinoid, bladder cancer, precancerous skin lesions, testicular cancer, thyroid cancer, neuroblastoma, esophageal cancer, genitourinary tract cancer, malignant hypercalcemia, endometrial cancer, adrenocortical carcinoma, endocrine or exocrine pancreatic tumors, medullary thyroid cancer, medullary thyroid cancer, melanoma, colorectal cancer, papillary thyroid cancer, hepatocellular carcinoma, or prostate cancer.

[0079] As used herein, "treating" or "treatment of" a condition, disease or illness, or symptoms associated with a condition, disease or illness, refers to a method for obtaining a beneficial or desired result, including a clinical result. Beneficial or desired clinical results may include, but are not limited to, alleviation or improvement of one or more symptoms or conditions, reduction in the extent of a condition, disease or disease, stabilization of the state of a condition, disease or disease, prevention of the development of a condition, disease or disease, prevention of the spread of a condition, disease or disease, delay or slowing of the progression of a condition, disease or disease, delay or slowing of the onset of a condition, disease or disease, improvement or alleviation of the state of a condition, disease or disease, and relief, whether partial or complete. "Treatment" may also mean causing the survival of a subject to exceed the survival expected in the absence of treatment. "Treatment" may also mean inhibiting the progression of a condition, disease or disease, temporarily slowing the progression of a condition, disease or disease, although in some cases, it involves permanently stopping the progression of a condition, disease or disease.

[0080] The terms "dose" and "dosage" are used interchangeably herein. Dose refers to the amount of active ingredient given to an individual per administration. The dose will vary according to a variety of factors, including the normal dose range for a given therapy, the frequency of administration; the size and tolerance of the individual; the severity of the condition; the risk of side effects; and the route of administration. Those skilled in the art will recognize that the dose may be modified based on the above factors or based on the progress of the treatment. The term "dosage form" refers to a specific form of a drug or pharmaceutical composition and depends on the route of administration. For example, a dosage form may be in liquid form, such as for injection.

[0081] As used herein, "therapeutically effective dose or amount" means a dose that produces the effect of its administration (e.g., treatment of a disease). The exact dosage and formulation will depend on the purpose of the treatment and can be determined by those skilled in the art using known techniques. For example, for a given parameter, a therapeutically effective dose will show an increase or decrease of at least 5%, 10%, 15%, 20%, 25%, 40%, 50%, 60%, 75%, 80%, 90% or at least 100%. The therapeutic effect can also be expressed as a "fold" of increase or decrease. For example, a therapeutically effective dose can have an effect of at least 1.2 times, 1.5 times, 2 times, 5 times or more compared to a standard control. A therapeutically effective dose or amount can improve one or more symptoms of a disease.

[0082] The term "administering" as used herein is used in its simple and ordinary sense and includes any administration suitable for cell therapy. Parenteral administration includes, for example, intravenous, intramuscular, intraarteriolar, intradermal, subcutaneous, intraperitoneal, intraventricular, and intracranial. In an embodiment, administration is intravenous.

[0083] As used herein, the term "signaling pathway" refers to a series of interactions between cellular components and optional extracellular components (e.g., proteins, nucleic acids, small molecules, ions, lipids) that transmit changes in one component to one or more other components, which in turn can transmit changes to additional components, thereby optionally propagating to other signaling pathway components. In an embodiment, the signaling pathway is a cyclic GMP-AMP synthase (cGAS)-stimulator of interferon genes (STING) pathway. The signaling pathway can be activated by, for example, the presence of intracellular compounds (e.g., circular nucleic acids). For example, the signaling pathway can be activated by the presence of circular nucleic acids (e.g., plasmids) in the cytoplasm of a cell. In certain cases, the signaling pathway can be activated by one or more conditions experienced by the cell (e.g., transduction, transfection, or electroporation of nucleic acids into the cell).

[0084] method

[0085] In particular, methods for engineering T cells are provided herein, the method comprising contacting T cells with one or more cyclic GMP-AMP synthase (cGAS)-interferon gene stimulator (STING) pathway inhibitors. The methods provided herein allow the formation of engineered T cells while inhibiting or reducing the cytotoxicity associated with previously known engineering T cell methods. For example, the methods provided herein are considered to effectively reduce or inhibit the toxicity associated with the presence of DNA (e.g., plasmid DNA) in the cytoplasm. For example, the methods provided herein effectively reduce or inhibit the cGAS-STING pathway, thereby reducing or inhibiting cell death. As used herein, "cyclic GMP-AMP synthase-interferon gene stimulator pathway inhibitors" or "cGAS-STING pathway inhibitors" refer to compounds that inhibit or downregulate the activity or production of any of the components in the cGAS-STING axis. For example, a cGAS-STING pathway inhibitor can inhibit or downregulate the activity or production of any of the components of the cGAS pathway. In an embodiment, a cGAS-STING pathway inhibitor inhibits the binding of cGAS to double-stranded DNA (e.g., plasmid DNA). In an embodiment, a cGAS-STING pathway inhibitor inhibits the activity of cGAMP. In another example, a cGAS-STING pathway inhibitor may inhibit or downregulate the activity or production of any one of the components of the STING pathway. In an embodiment, a cGAS-STING pathway inhibitor may inhibit STING oligomerization. In another example, a cGAS-STING pathway inhibitor may inhibit or downregulate the activity or production of TANK binding kinase 1 (TBK1). In an embodiment, a cGAS-STING pathway inhibitor inhibits TBK1 phosphorylation or STING phosphorylation. In an embodiment, a cGAS-STING pathway inhibitor inhibits phosphorylation of interferon regulatory factor 3 (IRF-3). Therefore, in an embodiment, a cGAS-STING pathway inhibitor includes a kinase inhibitor (e.g., BX795, Aml, MRT, etc.). In an embodiment, a cGAS-STING pathway inhibitor is a kinase inhibitor. In one aspect, a method of engineering a T cell is provided, the method 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.

[0086] In an embodiment, the length of nucleic acid is about 20 bases to about 30,000 bases. In an embodiment, the length of nucleic acid is about 1000 bases to about 30,000 bases. In an embodiment, the length of nucleic acid is about 2000 bases to about 30,000 bases. In an embodiment, the length of nucleic acid is about 3000 bases to about 30,000 bases. In an embodiment, the length of nucleic acid is about 4000 bases to about 30,000 bases. In an embodiment, the length of nucleic acid is about 5000 bases to about 30,000 bases. In an embodiment, the length of nucleic acid is about 6000 bases to about 30,000 bases. In an embodiment, the length of nucleic acid is about 7000 bases to about 30,000 bases. In an embodiment, the length of nucleic acid is about 8000 bases to about 30,000 bases. In an embodiment, the length of nucleic acid is about 9000 bases to about 30,000 bases. In an embodiment, the length of nucleic acid is about 10000 bases to about 30000 bases. In an embodiment, the length of nucleic acid is about 11000 bases to about 30000 bases. In an embodiment, the length of nucleic acid is about 12000 bases to about 30000 bases. In an embodiment, the length of nucleic acid is about 13000 bases to about 30000 bases. In an embodiment, the length of nucleic acid is about 14000 bases to about 30000 bases. In an embodiment, the length of nucleic acid is about 15000 bases to about 30000 bases. In an embodiment, the length of nucleic acid is about 16000 bases to about 30000 bases. In an embodiment, the length of nucleic acid is about 17000 bases to about 30000 bases. In an embodiment, the length of nucleic acid is about 18000 bases to about 30000 bases. In an embodiment, the length of the nucleic acid is about 19000 bases to about 30000 bases. In an embodiment, the length of the nucleic acid is about 20000 bases to about 30000 bases. In an embodiment, the length of the nucleic acid is about 21000 bases to about 30000 bases. In an embodiment, the length of the nucleic acid is about 22000 bases to about 30000 bases. In an embodiment, the length of the nucleic acid is about 23000 bases to about 30000 bases. In an embodiment, the length of the nucleic acid is about 24000 bases to about 30000 bases. In an embodiment, the length of the nucleic acid is about 25000 bases to about 30000 bases. In an embodiment, the length of the nucleic acid is about 26000 bases to about 30000 bases. In an embodiment, the length of the nucleic acid is about 27000 bases to about 30000 bases. In embodiments, the nucleic acid is about 28,000 bases to about 30,000 bases in length. In embodiments, the nucleic acid is about 29,000 bases to about 30,000 bases in length.

[0087] In an embodiment, the length of the nucleic acid is about 20 bases to about 29000 bases. In an embodiment, the length of the nucleic acid is about 20 bases to about 28000 bases. In an embodiment, the length of the nucleic acid is about 20 bases to about 27000 bases. In an embodiment, the length of the nucleic acid is about 20 bases to about 26000 bases. In an embodiment, the length of the nucleic acid is about 20 bases to about 25000 bases. In an embodiment, the length of the nucleic acid is about 20 bases to about 24000 bases. In an embodiment, the length of the nucleic acid is about 20 bases to about 23000 bases. In an embodiment, the length of the nucleic acid is about 20 bases to about 22000 bases. In an embodiment, the length of the nucleic acid is about 20 bases to about 21000 bases. In an embodiment, the length of the nucleic acid is about 20 bases to about 20000 bases. In an embodiment, the length of the nucleic acid is about 20 bases to about 19000 bases. In an embodiment, the length of the nucleic acid is about 20 bases to about 18000 bases. In an embodiment, the length of the nucleic acid is about 20 bases to about 17000 bases. In an embodiment, the length of the nucleic acid is about 20 bases to about 16000 bases. In an embodiment, the length of the nucleic acid is about 20 bases to about 15000 bases. In an embodiment, the length of the nucleic acid is about 20 bases to about 14000 bases. In an embodiment, the length of the nucleic acid is about 20 bases to about 13000 bases. In an embodiment, the length of the nucleic acid is about 20 bases to about 12000 bases. In an embodiment, the length of the nucleic acid is about 20 bases to about 11000 bases. In an embodiment, the length of the nucleic acid is about 20 bases to about 10000 bases. In an embodiment, the length of the nucleic acid is about 20 bases to about 9000 bases. In an embodiment, the length of the nucleic acid is about 20 bases to about 8000 bases. In an embodiment, the length of the nucleic acid is about 20 bases to about 7000 bases. In an embodiment, the length of the nucleic acid is about 20 bases to about 6000 bases. In an embodiment, the length of the nucleic acid is about 20 bases to about 5000 bases. In an embodiment, the length of the nucleic acid is about 20 bases to about 4000 bases. In an embodiment, the length of the nucleic acid is about 20 bases to about 3000 bases. In an embodiment, the length of the nucleic acid is about 20 bases to about 2000 bases. In an embodiment, the length of the nucleic acid is about 20 bases to about 1000 bases.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.

[0088] In an embodiment, the length of nucleic acid is about 20 bases to about 5000 bases. In an embodiment, the length of nucleic acid is about 50 bases to about 5000 bases. In an embodiment, the length of nucleic acid is about 100 bases to about 5000 bases. In an embodiment, the length of nucleic acid is about 150 bases to about 5000 bases. In an embodiment, the length of nucleic acid is about 200 bases to about 5000 bases. In an embodiment, the length of nucleic acid is about 250 bases to about 5000 bases. In an embodiment, the length of nucleic acid is about 300 bases to about 5000 bases. In an embodiment, the length of nucleic acid is about 350 bases to about 5000 bases. In an embodiment, the length of nucleic acid is about 400 bases to about 5000 bases. In an embodiment, the length of nucleic acid is about 450 bases to about 5000 bases. In an embodiment, the length of nucleic acid is about 500 bases to about 5000 bases. In an embodiment, the length of the nucleic acid is about 550 bases to about 5000 bases. In an embodiment, the length of the nucleic acid is about 600 bases to about 5000 bases. In an embodiment, the length of the nucleic acid is about 650 bases to about 5000 bases. In an embodiment, the length of the nucleic acid is about 700 bases to about 5000 bases. In an embodiment, the length of the nucleic acid is about 750 bases to about 5000 bases. In an embodiment, the length of the nucleic acid is about 800 bases to about 5000 bases. In an embodiment, the length of the nucleic acid is about 850 bases to about 5000 bases. In an embodiment, the length of the nucleic acid is about 900 bases to about 5000 bases. In an embodiment, the length of the nucleic acid is about 950 bases to about 5000 bases. In an embodiment, the length of the nucleic acid is about 1000 bases to about 5000 bases. In an embodiment, the length of the nucleic acid is about 1050 bases to about 5000 bases. In an embodiment, the length of nucleic acid is about 1100 bases to about 5000 bases. In an embodiment, the length of nucleic acid is about 1150 bases to about 5000 bases. In an embodiment, the length of nucleic acid is about 1200 bases to about 5000 bases. In an embodiment, the length of nucleic acid is about 1250 bases to about 5000 bases. In an embodiment, the length of nucleic acid is about 1300 bases to about 5000 bases. In an embodiment, the length of nucleic acid is about 1350 bases to about 5000 bases. In an embodiment, the length of nucleic acid is about 1400 bases to about 5000 bases. In an embodiment, the length of nucleic acid is about 1450 bases to about 5000 bases. In an embodiment, the length of nucleic acid is about 1500 bases to about 5000 bases. In an embodiment, the length of nucleic acid is about 1550 bases to about 5000 bases. In embodiments, the nucleic acid is about 1600 bases to about 5000 bases in length. In embodiments, the nucleic acid is about 1650 bases to about 5000 bases in length.In an embodiment, the length of nucleic acid is about 1700 bases to about 5000 bases. In an embodiment, the length of nucleic acid is about 1750 bases to about 5000 bases. In an embodiment, the length of nucleic acid is about 1800 bases to about 5000 bases. In an embodiment, the length of nucleic acid is about 1850 bases to about 5000 bases. In an embodiment, the length of nucleic acid is about 1900 bases to about 5000 bases. In an embodiment, the length of nucleic acid is about 1950 bases to about 5000 bases. In an embodiment, the length of nucleic acid is about 2000 bases to about 5000 bases. In an embodiment, the length of nucleic acid is about 2050 bases to about 5000 bases. In an embodiment, the length of nucleic acid is about 2100 bases to about 5000 bases. In an embodiment, the length of nucleic acid is about 2150 bases to about 5000 bases. In an embodiment, the length of nucleic acid is about 2200 bases to about 5000 bases. In an embodiment, the length of nucleic acid is about 2250 bases to about 5000 bases. In an embodiment, the length of nucleic acid is about 2300 bases to about 5000 bases. In an embodiment, the length of nucleic acid is about 2350 bases to about 5000 bases. In an embodiment, the length of nucleic acid is about 2400 bases to about 5000 bases. In an embodiment, the length of nucleic acid is about 2450 bases to about 5000 bases. In an embodiment, the length of nucleic acid is about 2500 bases to about 5000 bases. In an embodiment, the length of nucleic acid is about 2550 bases to about 5000 bases. In an embodiment, the length of nucleic acid is about 2600 bases to about 5000 bases. In an embodiment, the length of nucleic acid is about 2650 bases to about 5000 bases. In an embodiment, the length of nucleic acid is about 2700 bases to about 5000 bases. In an embodiment, the length of nucleic acid is about 2750 bases to about 5000 bases. In an embodiment, the length of nucleic acid is about 2800 bases to about 5000 bases. In an embodiment, the length of nucleic acid is about 2850 bases to about 5000 bases. In an embodiment, the length of nucleic acid is about 2900 bases to about 5000 bases. In an embodiment, the length of nucleic acid is about 2950 bases to about 5000 bases. In an embodiment, the length of nucleic acid is about 3000 bases to about 5000 bases. In an embodiment, the length of nucleic acid is about 3050 bases to about 5000 bases. In an embodiment, the length of nucleic acid is about 3100 bases to about 5000 bases. In an embodiment, the length of nucleic acid is about 3150 bases to about 5000 bases. In embodiments, the nucleic acid has a length of about 3200 bases to about 5000 bases. In embodiments, the nucleic acid has a length of about 3250 bases to about 5000 bases. In embodiments, the nucleic acid has a length of about 3300 bases to about 5000 bases.In an embodiment, the length of nucleic acid is about 3350 bases to about 5000 bases. In an embodiment, the length of nucleic acid is about 3400 bases to about 5000 bases. In an embodiment, the length of nucleic acid is about 3450 bases to about 5000 bases. In an embodiment, the length of nucleic acid is about 3500 bases to about 5000 bases. In an embodiment, the length of nucleic acid is about 3550 bases to about 5000 bases. In an embodiment, the length of nucleic acid is about 3600 bases to about 5000 bases. In an embodiment, the length of nucleic acid is about 3650 bases to about 5000 bases. In an embodiment, the length of nucleic acid is about 3700 bases to about 5000 bases. In an embodiment, the length of nucleic acid is about 3750 bases to about 5000 bases. In an embodiment, the length of nucleic acid is about 3800 bases to about 5000 bases. In an embodiment, the length of nucleic acid is about 3850 bases to about 5000 bases. In an embodiment, the length of nucleic acid is about 3900 bases to about 5000 bases. In an embodiment, the length of nucleic acid is about 3950 bases to about 5000 bases. In an embodiment, the length of nucleic acid is about 4000 bases to about 5000 bases. In an embodiment, the length of nucleic acid is about 4050 bases to about 5000 bases. In an embodiment, the length of nucleic acid is about 4100 bases to about 5000 bases. In an embodiment, the length of nucleic acid is about 4150 bases to about 5000 bases. In an embodiment, the length of nucleic acid is about 4200 bases to about 5000 bases. In an embodiment, the length of nucleic acid is about 4250 bases to about 5000 bases. In an embodiment, the length of nucleic acid is about 4300 bases to about 5000 bases. In an embodiment, the length of nucleic acid is about 4350 bases to about 5000 bases. In an embodiment, the length of nucleic acid is about 4400 bases to about 5000 bases. In an embodiment, the length of nucleic acid is about 4450 bases to about 5000 bases. In an embodiment, the length of nucleic acid is about 4500 bases to about 5000 bases. In an embodiment, the length of nucleic acid is about 4550 bases to about 5000 bases. In an embodiment, the length of nucleic acid is about 4600 bases to about 5000 bases. In an embodiment, the length of nucleic acid is about 4650 bases to about 5000 bases. In an embodiment, the length of nucleic acid is about 4700 bases to about 5000 bases. In an embodiment, the length of nucleic acid is about 4750 bases to about 5000 bases. In an embodiment, the length of nucleic acid is about 4800 bases to about 5000 bases. In embodiments, the nucleic acid is about 4850 bases to about 5000 bases in length. In embodiments, the nucleic acid is about 4900 bases to about 5000 bases in length. In embodiments, the nucleic acid is about 4950 bases to about 5000 bases in length.

[0089] In an embodiment, the length of the nucleic acid is about 20 bases to about 4950 bases. In an embodiment, the length of the nucleic acid is about 20 bases to about 4900 bases. In an embodiment, the length of the nucleic acid is about 20 bases to about 4850 bases. In an embodiment, the length of the nucleic acid is about 20 bases to about 4800 bases. In an embodiment, the length of the nucleic acid is about 20 bases to about 4750 bases. In an embodiment, the length of the nucleic acid is about 20 bases to about 4700 bases. In an embodiment, the length of the nucleic acid is about 20 bases to about 4650 bases. In an embodiment, the length of the nucleic acid is about 20 bases to about 4600 bases. In an embodiment, the length of the nucleic acid is about 20 bases to about 4550 bases. In an embodiment, the length of the nucleic acid is about 20 bases to about 4500 bases. In an embodiment, the length of the nucleic acid is about 20 bases to about 4450 bases. In an embodiment, the length of the nucleic acid is about 20 bases to about 4400 bases. In an embodiment, the length of the nucleic acid is about 20 bases to about 4350 bases. In an embodiment, the length of the nucleic acid is about 20 bases to about 4300 bases. In an embodiment, the length of the nucleic acid is about 20 bases to about 4250 bases. In an embodiment, the length of the nucleic acid is about 20 bases to about 4200 bases. In an embodiment, the length of the nucleic acid is about 20 bases to about 4150 bases. In an embodiment, the length of the nucleic acid is about 20 bases to about 4100 bases. In an embodiment, the length of the nucleic acid is about 20 bases to about 4050 bases. In an embodiment, the length of the nucleic acid is about 20 bases to about 4000 bases. In an embodiment, the length of the nucleic acid is about 20 bases to about 3950 bases. In an embodiment, the length of the nucleic acid is about 20 bases to about 3900 bases. In an embodiment, the length of the nucleic acid is about 20 bases to about 3850 bases. In an embodiment, the length of the nucleic acid is about 20 bases to about 3800 bases. In an embodiment, the length of the nucleic acid is about 20 bases to about 3750 bases. In an embodiment, the length of the nucleic acid is about 20 bases to about 3700 bases. In an embodiment, the length of the nucleic acid is about 20 bases to about 3650 bases. In an embodiment, the length of the nucleic acid is about 20 bases to about 3600 bases. In an embodiment, the length of the nucleic acid is about 20 bases to about 3550 bases. In an embodiment, the length of the nucleic acid is about 20 bases to about 3500 bases. In an embodiment, the length of the nucleic acid is about 20 bases to about 3450 bases. In an embodiment, the length of the nucleic acid is about 20 bases to about 3400 bases. In an embodiment, the length of the nucleic acid is about 20 bases to about 3350 bases. In embodiments, the nucleic acid is about 20 bases to about 3300 bases in length. In embodiments, the nucleic acid is about 20 bases to about 3250 bases in length.In an embodiment, the length of the nucleic acid is about 20 bases to about 3200 bases. In an embodiment, the length of the nucleic acid is about 20 bases to about 3150 bases. In an embodiment, the length of the nucleic acid is about 20 bases to about 3100 bases. In an embodiment, the length of the nucleic acid is about 20 bases to about 3050 bases. In an embodiment, the length of the nucleic acid is about 20 bases to about 3000 bases. In an embodiment, the length of the nucleic acid is about 20 bases to about 2950 bases. In an embodiment, the length of the nucleic acid is about 20 bases to about 2900 bases. In an embodiment, the length of the nucleic acid is about 20 bases to about 2850 bases. In an embodiment, the length of the nucleic acid is about 20 bases to about 2800 bases. In an embodiment, the length of the nucleic acid is about 20 bases to about 2750 bases. In an embodiment, the length of the nucleic acid is about 20 bases to about 2700 bases. In an embodiment, the length of the nucleic acid is about 20 bases to about 2650 bases. In an embodiment, the length of the nucleic acid is about 20 bases to about 2600 bases. In an embodiment, the length of the nucleic acid is about 20 bases to about 2550 bases. In an embodiment, the length of the nucleic acid is about 20 bases to about 2500 bases. In an embodiment, the length of the nucleic acid is about 20 bases to about 2450 bases. In an embodiment, the length of the nucleic acid is about 20 bases to about 2400 bases. In an embodiment, the length of the nucleic acid is about 20 bases to about 2350 bases. In an embodiment, the length of the nucleic acid is about 20 bases to about 2300 bases. In an embodiment, the length of the nucleic acid is about 20 bases to about 2250 bases. In an embodiment, the length of the nucleic acid is about 20 bases to about 2200 bases. In an embodiment, the length of the nucleic acid is about 20 bases to about 2150 bases. In an embodiment, the length of the nucleic acid is about 20 bases to about 2100 bases. In an embodiment, the length of the nucleic acid is about 20 bases to about 2050 bases. In an embodiment, the length of the nucleic acid is about 20 bases to about 2000 bases. In an embodiment, the length of the nucleic acid is about 20 bases to about 1950 bases. In an embodiment, the length of the nucleic acid is about 20 bases to about 1900 bases. In an embodiment, the length of the nucleic acid is about 20 bases to about 1850 bases. In an embodiment, the length of the nucleic acid is about 20 bases to about 1800 bases. In an embodiment, the length of the nucleic acid is about 20 bases to about 1750 bases. In an embodiment, the length of the nucleic acid is about 20 bases to about 1700 bases. In an embodiment, the length of the nucleic acid is about 20 bases to about 1650 bases. In an embodiment, the length of the nucleic acid is about 20 bases to about 1600 bases. In embodiments, the nucleic acid is about 20 bases to about 1550 bases in length. In embodiments, the nucleic acid is about 20 bases to about 1500 bases in length.In an embodiment, the length of the nucleic acid is about 20 bases to about 1450 bases. In an embodiment, the length of the nucleic acid is about 20 bases to about 1400 bases. In an embodiment, the length of the nucleic acid is about 20 bases to about 1350 bases. In an embodiment, the length of the nucleic acid is about 20 bases to about 1300 bases. In an embodiment, the length of the nucleic acid is about 20 bases to about 1250 bases. In an embodiment, the length of the nucleic acid is about 20 bases to about 1200 bases. In an embodiment, the length of the nucleic acid is about 20 bases to about 1150 bases. In an embodiment, the length of the nucleic acid is about 20 bases to about 1100 bases. In an embodiment, the length of the nucleic acid is about 20 bases to about 1050 bases. In an embodiment, the length of the nucleic acid is about 20 bases to about 1000 bases. In an embodiment, the length of the nucleic acid is about 20 bases to about 950 bases. In an embodiment, the length of the nucleic acid is about 20 bases to about 900 bases. In an embodiment, the length of the nucleic acid is about 20 bases to about 850 bases. In an embodiment, the length of the nucleic acid is about 20 bases to about 800 bases. In an embodiment, the length of the nucleic acid is about 20 bases to about 750 bases. In an embodiment, the length of the nucleic acid is about 20 bases to about 700 bases. In an embodiment, the length of the nucleic acid is about 20 bases to about 650 bases. In an embodiment, the length of the nucleic acid is about 20 bases to about 600 bases. In an embodiment, the length of the nucleic acid is about 20 bases to about 550 bases. In an embodiment, the length of the nucleic acid is about 20 bases to about 500 bases. In an embodiment, the length of the nucleic acid is about 20 bases to about 450 bases. In an embodiment, the length of the nucleic acid is about 20 bases to about 400 bases. In an embodiment, the length of the nucleic acid is about 20 bases to about 350 bases. In an embodiment, the length of the nucleic acid is about 20 bases to about 300 bases. In an embodiment, the length of the nucleic acid is about 20 bases to about 250 bases. In an embodiment, the length of the nucleic acid is about 20 bases to about 200 bases. In an embodiment, the length of the nucleic acid is about 20 bases to about 150 bases. In an embodiment, the length of the nucleic acid is about 20 bases to about 100 bases. In an embodiment, the length of the nucleic acid is about 20 bases to about 50 bases.In embodiments, the length of the nucleic acid is about 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, 400, 4200, 4250, 4300, 4350, 4400, 4450, 4500, 4550, 4600, 4650, 4700, 4750, 4800, 4850, 4900, 4950, 5000, 5050, 5100, 5200, 5300, 5400, 5500, 5600, 5700, 5750, 5800, 5850, 5900, 5950, 6000, 6500, 6600, 6700, 6800, 6900, 7000, 7100, 7150, 7100

[0090] In an embodiment, the length of nucleic acid is about 20 bases to about 1000 bases. In an embodiment, the length of nucleic acid is about 100 bases to about 1000 bases. In an embodiment, the length of nucleic acid is about 200 bases to about 1000 bases. In an embodiment, the length of nucleic acid is about 300 bases to about 1000 bases. In an embodiment, the length of nucleic acid is about 400 bases to about 1000 bases. In an embodiment, the length of nucleic acid is about 500 bases to about 1000 bases. In an embodiment, the length of nucleic acid is about 600 bases to about 1000 bases. In an embodiment, the length of nucleic acid is about 700 bases to about 1000 bases. In an embodiment, the length of nucleic acid is about 800 bases to about 1000 bases. In an embodiment, the length of nucleic acid is about 900 bases to about 1000 bases.

[0091] In an embodiment, the length of the nucleic acid is about 20 bases to about 900 bases. In an embodiment, the length of the nucleic acid is about 20 bases to about 800 bases. In an embodiment, the length of the nucleic acid is about 20 bases to about 700 bases. In an embodiment, the length of the nucleic acid is about 20 bases to about 600 bases. In an embodiment, the length of the nucleic acid is about 20 bases to about 500 bases. In an embodiment, the length of the nucleic acid is about 20 bases to about 400 bases. In an embodiment, the length of the nucleic acid is about 20 bases to about 300 bases. In an embodiment, the length of the nucleic acid is about 20 bases to about 200 bases. In an embodiment, the length of the nucleic acid is about 20 bases to about 100 bases. In an embodiment, the length of the nucleic acid is about 20, 100, 200, 300, 400, 500, 600, 700, 800, 900 or 1000 bases.

[0092] For methods provided herein, in an embodiment, nucleic acid includes donor DNA, which is inserted into the genome of T cells under certain circumstances, thereby producing engineered T cells. Therefore, in an embodiment, nucleic acid includes donor DNA. In an embodiment, nucleic acid is double-stranded circular DNA (e.g., plasmid). Various methods can be used to deliver donor DNA to cells, including transfection, electroporation, and viral transduction methods. Therefore, in an embodiment, nucleic acid is a plasmid, nanoplasmid, microcircle, linear plasmid or viral vector including donor DNA. In an embodiment, nucleic acid is a plasmid including donor DNA. In an embodiment, nucleic acid is a nanoplasmid including donor DNA. In an embodiment, nucleic acid is a microcircle including donor DNA. In an embodiment, nucleic acid is a viral vector including donor DNA. In an embodiment, DNA is not linear DNA (e.g., double-stranded linear DNA). In an embodiment, DNA is circular DNA (e.g., double-stranded circular DNA). In an embodiment, DNA is linear DNA. In an embodiment, DNA is single-stranded DNA.

[0093] The method provided herein can be used to prepare T cell receptor (TCR) engineered T cells. For example, the method can be used to replace (e.g., knock out) endogenous TCR with a TCR (e.g., knock-in) associated with a tumor-specific antigen in a T cell. Therefore, in an embodiment, donor DNA encodes exogenous T cell receptor (TCR) -α or its fragment, exogenous TCR-β or its fragment, or a combination thereof. In an embodiment, donor DNA encodes exogenous T cell receptor (TCR) -α or its fragment. In an embodiment, donor DNA encodes exogenous TCR-β or its fragment.

[0094] In an embodiment, the length of the donor DNA is about 5 to about 1000 bases. In an embodiment, the length of the donor DNA is about 100 to about 1000 bases. In an embodiment, the length of the donor DNA is about 200 to about 1000 bases. In an embodiment, the length of the donor DNA is about 300 to about 1000 bases. In an embodiment, the length of the donor DNA is about 400 to about 1000 bases. In an embodiment, the length of the donor DNA is about 500 to about 1000 bases. In an embodiment, the length of the donor DNA is about 600 to about 1000 bases. In an embodiment, the length of the donor DNA is about 700 to about 1000 bases. In an embodiment, the length of the donor DNA is about 800 to about 1000 bases. In an embodiment, the length of the donor DNA is about 900 to about 1000 bases.

[0095] In an embodiment, the length of the donor DNA is about 5 to about 900 bases. In an embodiment, the length of the donor DNA is about 5 to about 800 bases. In an embodiment, the length of the donor DNA is about 5 to about 700 bases. In an embodiment, the length of the donor DNA is about 5 to about 600 bases. In an embodiment, the length of the donor DNA is about 5 to about 500 bases. In an embodiment, the length of the donor DNA is about 5 to about 400 bases. In an embodiment, the length of the donor DNA is about 5 to about 300 bases. In an embodiment, the length of the donor DNA is about 5 to about 200 bases. In an embodiment, the length of the donor DNA is about 5 to about 100 bases. In an embodiment, the length of the donor DNA is about 5, 100, 200, 300, 400, 500, 600, 700, 800, 900 or 1000 bases.

[0096] In an embodiment, the length of the donor DNA is about 50 to about 600 bases. In an embodiment, the length of the donor DNA is about 100 to about 600 bases. In an embodiment, the length of the donor DNA is about 150 to about 600 bases. In an embodiment, the length of the donor DNA is about 200 to about 600 bases. In an embodiment, the length of the donor DNA is about 250 to about 600 bases. In an embodiment, the length of the donor DNA is about 300 to about 600 bases. In an embodiment, the length of the donor DNA is about 350 to about 600 bases. In an embodiment, the length of the donor DNA is about 400 to about 600 bases. In an embodiment, the length of the donor DNA is about 450 to about 600 bases. In an embodiment, the length of the donor DNA is about 500 to about 600 bases. In an embodiment, the length of the donor DNA is about 550 to about 600 bases.

[0097] In an embodiment, the length of the donor DNA is about 50 to about 550 bases. In an embodiment, the length of the donor DNA is about 50 to about 500 bases. In an embodiment, the length of the donor DNA is about 50 to about 450 bases. In an embodiment, the length of the donor DNA is about 50 to about 400 bases. In an embodiment, the length of the donor DNA is about 50 to about 350 bases. In an embodiment, the length of the donor DNA is about 50 to about 300 bases. In an embodiment, the length of the donor DNA is about 50 to about 250 bases. In an embodiment, the length of the donor DNA is about 50 to about 200 bases. In an embodiment, the length of the donor DNA is about 50 to about 150 bases. In an embodiment, the length of the donor DNA is about 150 to about 100 bases. In embodiments, the donor DNA is about 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, or 600 bases in length.

[0098] In an embodiment, the length of the donor DNA is about 20 bases to about 5000 bases. In an embodiment, the length of the donor DNA is about 250 bases to about 5000 bases. In an embodiment, the length of the donor DNA is about 500 bases to about 5000 bases. In an embodiment, the length of the donor DNA is about 750 bases to about 5000 bases. In an embodiment, the length of the donor DNA is about 1000 bases to about 5000 bases. In an embodiment, the length of the donor DNA is about 1250 bases to about 5000 bases. In an embodiment, the length of the donor DNA is about 1500 bases to about 5000 bases. In an embodiment, the length of the donor DNA is about 1750 bases to about 5000 bases. In an embodiment, the length of the donor DNA is about 2000 bases to about 5000 bases. In an embodiment, the length of the donor DNA is about 2250 bases to about 5000 bases. In an embodiment, the length of the donor DNA is about 2500 bases to about 5000 bases. In an embodiment, the length of the donor DNA is about 2750 bases to about 5000 bases. In an embodiment, the length of the donor DNA is about 3000 bases to about 5000 bases. In an embodiment, the length of the donor DNA is about 3250 bases to about 5000 bases. In an embodiment, the length of the donor DNA is about 3500 bases to about 5000 bases. In an embodiment, the length of the donor DNA is about 3750 bases to about 5000 bases. In an embodiment, the length of the donor DNA is about 4000 bases to about 5000 bases. In an embodiment, the length of the donor DNA is about 4250 bases to about 5000 bases. In an embodiment, the length of the donor DNA is about 4500 bases to about 5000 bases. In embodiments, the donor DNA is about 4750 bases to about 5000 bases in length.

[0099] In an embodiment, the length of the donor DNA is about 20 bases to about 4750 bases. In an embodiment, the length of the donor DNA is about 20 bases to about 4500 bases. In an embodiment, the length of the donor DNA is about 20 bases to about 4250 bases. In an embodiment, the length of the donor DNA is about 20 bases to about 4000 bases. In an embodiment, the length of the donor DNA is about 20 bases to about 3750 bases. In an embodiment, the length of the donor DNA is about 20 bases to about 3500 bases. In an embodiment, the length of the donor DNA is about 20 bases to about 3250 bases. In an embodiment, the length of the donor DNA is about 20 bases to about 3000 bases. In an embodiment, the length of the donor DNA is about 20 bases to about 2750 bases. In an embodiment, the length of the donor DNA is about 20 bases to about 2500 bases. In an embodiment, the length of the donor DNA is about 20 bases to about 2250 bases. In an embodiment, the length of the donor DNA is about 20 bases to about 2000 bases. In an embodiment, the length of the donor DNA is about 20 bases to about 1750 bases. In an embodiment, the length of the donor DNA is about 20 bases to about 1500 bases. In an embodiment, the length of the donor DNA is about 20 bases to about 1250 bases. In an embodiment, the length of the donor DNA is about 20 bases to about 1000 bases. In an embodiment, the length of the donor DNA is about 20 bases to about 750 bases. In an embodiment, the length of the donor DNA is about 20 bases to about 500 bases. In an embodiment, the length of the donor DNA is about 20 bases to about 250 bases. 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.

[0100] In certain cases, delivery of nucleic acid into T cells can be promoted by delivery vehicles. Delivery vehicles can promote the interaction of nucleic acid and T cell membrane, thereby allowing nucleic acid to enter T cells. In one example, nucleic acid can be encapsulated in a delivery vehicle. In another example, nucleic acid can be non-covalently associated with a delivery vehicle. Therefore, in an embodiment, nucleic acid is associated with a delivery vehicle. In an embodiment, the delivery vehicle is a lipid particle or a nanoparticle. In an embodiment, the delivery vehicle is a lipid particle. In an embodiment, the delivery vehicle is a nanoparticle. In an embodiment, the delivery vehicle is a liposome or a lipid nanoparticle.

[0101] For the methods provided herein, in embodiments, the T cells are primary T cells. "Primary T cells" are used according to their ordinary meaning in the biological field and refer to T cells that are directly expanded from T cells extracted from a subject.

[0102] The method provided herein (including its embodiments) may include contacting T cells with gene editing reagents, thereby allowing editing of target genes in T cells. For example, gene editing reagents can promote the knockout of endogenous genes (such as endogenous TCRs) and the knock-in of tumor antigen-specific TCRs. Therefore, in an embodiment, the method further includes contacting T cells with gene editing reagents. In an embodiment, contacting T cells with gene editing reagents includes contacting T cells with nucleic acid sequences encoding gene editing reagents. In an embodiment, T cells are contacted with nucleic acids in the presence of gene editing agents or nucleic acid sequences encoding gene editing reagents. In an embodiment, T cells are contacted with nucleic acids in the presence of gene editing reagents. In an embodiment, T cells are contacted with nucleic acids in the presence of nucleic acid sequences encoding gene editing reagents. In an embodiment, T cells are contacted with nucleic acids in the presence of nucleic acid sequences encoding gene editing reagents.

[0103] In an embodiment, the gene editing reagent comprises an RNA-guided nuclease. In an embodiment, the RNA-guided nuclease is a CRISPR-Cas system. In an embodiment, the CRISPR-Cas system comprises 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, 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 an embodiment, the CRISPR-Cas system comprises Cas1. In embodiments, the CRISPR-Cas system comprises Cas1B. In embodiments, the CRISPR-Cas system comprises Cas2. In embodiments, the CRISPR-Cas system comprises Cas3. In embodiments, the CRISPR-Cas system comprises Cas4. In 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 an embodiment, the CRISPR-Cas system comprises Cse2. In an embodiment, the CRISPR-Cas system comprises Csc1. In an embodiment, the CRISPR-Cas system comprises Csc2. In an embodiment, the CRISPR-Cas system comprises Csm2. In an embodiment, the CRISPR-Cas system comprises Csm3. In an embodiment, the CRISPR-Cas system comprises Csm4. In an embodiment, 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 an embodiment, the CRISPR-Cas system comprises Csf1. In an embodiment, the CRISPR-Cas system comprises Csf2. In an embodiment, the CRISPR-Cas system comprises Csf3. In an embodiment, the CRISPR-Cas system comprises Csf4. In an embodiment, the gene editing reagent further comprises a guide RNA (gRNA).

[0104] In an embodiment, the gene editing agent is MAD7, TALEN or ZFN. In an embodiment, the gene editing agent is MAD7. In an embodiment, the gene editing agent is TALEN. In an embodiment, the gene editing agent is ZFN. MAD7 is an engineered nuclease of the class 2 VA type CRISPR-Cas (Cas12a / Cpf1) family (refseq WP_055225123.1). See, e.g., CRISPR J. April 2020; 3(2):97–108, which is incorporated herein by reference in its entirety.

[0105] As described in the entire specification, including the drawings and examples, donor DNA can be inserted into the TCR locus of T cells, thereby forming engineered T cells. Therefore, for the methods provided herein, in an embodiment, donor DNA is inserted into an endogenous TCR locus. In an embodiment, the endogenous TCR locus is an endogenous TCR-α locus, an endogenous TCR-β locus, or a combination thereof. In an embodiment, the endogenous TCR locus is an endogenous TCR-α locus. In an embodiment, the endogenous TCR locus is an endogenous TCR-β locus.

[0106] For the methods provided herein, various methods known in the art can be used, including but not limited to electroporation and transfection methods, to deliver gene editing reagents and nucleic acids into T cells. In an embodiment, contacting T cells with gene editing reagents includes transfecting T cells with gene editing reagents. In an embodiment, contacting T cells with nucleic acids includes transfecting T cells with nucleic acids.

[0107] As described above, one or more cGAS-STING pathway inhibitors may be any compound that inhibits or downregulates the activity or production of any component of the cGAS-STING axis (including components of the cGAS pathway, the STING pathway, or TBK1). In an embodiment, one or more cGAS-STING pathway inhibitors include cGAS inhibitors. In an embodiment, one or more cGAS-STING pathway inhibitors include STING inhibitors. In an embodiment, one or more cGAS-STING pathway inhibitors include TANK binding kinase 1 (TBK1) inhibitors. cGAS, STING or TBK1 inhibitors may include kinase inhibitors. 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 an embodiment, a TBK1 inhibitor is Amlexanox, BX795, or MRT67307.

[0108] In an embodiment, one or more cGAS-STING pathway inhibitors are aml, MRT67307 (MRT), BX795, H151, ODN-A151 (ODN151), Ru.521, G140 or a combination thereof. In an embodiment, one or more cGAS-STING pathway inhibitors include Aml. In an embodiment, one or more cGAS-STING pathway inhibitors include MRT. In an embodiment, one or more cGAS-STING pathway inhibitors include BX795. In an embodiment, one or more cGAS-STING pathway inhibitors include H151. In an embodiment, one or more cGAS-STING pathway inhibitors include ODN151. In an embodiment, one or more cGAS-STING pathway inhibitors include Ru.521. In an embodiment, one or more cGAS-STING pathway inhibitors include G140. In an embodiment, one or more cGAS-STING pathway inhibitors include compounds listed in Table 5.

[0109] As used herein, ODN-A151 (ODN151) refers to a nucleic acid sequence comprising 5'-TTAGGGTTAGGGTTAGGGTTAGGG-3 (SEQ ID NO: 1). In an embodiment, ODN-A151 is a nucleic acid sequence of SEQ ID NO: 1. In an embodiment, one or more internucleotide bonds of SEQ ID NO: 1 are generated by a phosphorothioate moiety (phosphorothioate). The phosphorothioate moiety can be a monothiophosphate (-P(O)3(S) 3- -) or phosphorodithioate (-P(O)2(S)2 3- -). In an embodiment, the phosphorothioate moiety is a monothioate phosphorothioate (-P(O)3(S) 3- -).

[0110] In embodiments, one or more cGAS-STING pathway inhibitors are Aml. In embodiments, one or more cGAS-STING pathway inhibitors are MRT. In embodiments, one or more cGAS-STING pathway inhibitors are BX795. In embodiments, one or more cGAS-STING pathway inhibitors are H151. In embodiments, one or more cGAS-STING pathway inhibitors are ODN151. In embodiments, one or more cGAS-STING pathway inhibitors are Ru.521. In embodiments, one or more cGAS-STING pathway inhibitors are G140.

[0111] In embodiments, the cGAS-STING pathway inhibitor is Aml. 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 Aml and there are no other cGAS-STING inhibitors. In embodiments, the cGAS-STING pathway inhibitor is MRT and there are no other cGAS-STING inhibitors. In embodiments, the cGAS-STING pathway inhibitor is BX795 and there are no other cGAS-STING inhibitors. In embodiments, the cGAS-STING pathway inhibitor is H151 and there are no other cGAS-STING inhibitors. In embodiments, the cGAS-STING pathway inhibitor is ODN151 and no other cGAS-STING inhibitors. In embodiments, the cGAS-STING pathway inhibitor is Ru.521 and no other cGAS-STING inhibitors. In embodiments, the cGAS-STING pathway inhibitor is G140 and no other cGAS-STING inhibitors.

[0112] In an embodiment, one or more cGAS-STING pathway inhibitors are selected from: Aml, BX795, ODN151 and MRT. In an embodiment, one or more cGAS-STING pathway inhibitors are ODN151. In an embodiment, one or more cGAS-STING pathway inhibitors are BX795. In an embodiment, one or more cGAS-STING pathway inhibitors include at least one cGAS-STING pathway inhibitor. In an embodiment, one or more cGAS-STING pathway inhibitors include a combination of cGAS-STING pathway inhibitors. In an embodiment, the cGAS-STING pathway inhibitor does not include more than one cGAS-STING pathway inhibitor.

[0113] Applicants have discovered that treatment with one or more cGAS-STING pathway inhibitors prior to contacting T cells with nucleic acids or in the presence of nucleic acids is effective for generating engineered T cells. In an embodiment, T cells are contacted with nucleic acids in the presence of one or more cGAS-STING pathway inhibitors. For example, T cells can be transfected with nucleic acids in the presence of one or more cGAS-STING pathway inhibitors. In another example, T cells can be electroporated with nucleic acids in the presence of one or more cGAS-STING pathway inhibitors. In an embodiment, T cells are contacted with nucleic acids and one or more cGAS-STING pathway inhibitors in sequence. In an embodiment, T cells are contacted with one or more cGAS-STING pathway inhibitors prior to contacting with nucleic acids. For example, one or more cGAS-STING pathway inhibitors can be added to a T cell culture before transfecting T cells with nucleic acids.

[0114] In embodiments, T cells are contacted with one or more cGAS-STING pathway inhibitors for up to about 10 hours. In embodiments, T cells are contacted with one or more cGAS-STING pathway inhibitors for about 1 hour to about 10 hours. In embodiments, T cells are contacted with one or more cGAS-STING pathway inhibitors for about 2 hours to about 10 hours. In embodiments, T cells are contacted with one or more cGAS-STING pathway inhibitors for about 3 hours to about 10 hours. In embodiments, T cells are contacted with one or more cGAS-STING pathway inhibitors for about 4 hours to about 10 hours. In embodiments, T cells are contacted with one or more cGAS-STING pathway inhibitors for about 5 hours to about 10 hours. In embodiments, T cells are contacted with one or more cGAS-STING pathway inhibitors for about 6 hours to about 10 hours. In embodiments, T cells are contacted with one or more cGAS-STING pathway inhibitors for about 7 hours to about 10 hours. In embodiments, T cells are contacted with one or more cGAS-STING pathway inhibitors for about 8 hours to about 10 hours. In embodiments, T cells are contacted with one or more cGAS-STING pathway inhibitors for about 9 hours to about 10 hours.

[0115] In embodiments, T cells are contacted with one or more cGAS-STING pathway inhibitors for up to about 9 hours. In embodiments, T cells are contacted with one or more cGAS-STING pathway inhibitors for about 1 hour to about 9 hours. In embodiments, T cells are contacted with one or more cGAS-STING pathway inhibitors for about 2 hours to about 9 hours. In embodiments, T cells are contacted with one or more cGAS-STING pathway inhibitors for about 2 hours to about 8 hours. In embodiments, T cells are contacted with one or more cGAS-STING pathway inhibitors for about 2 hours to about 7 hours. In embodiments, T cells are contacted with one or more cGAS-STING pathway inhibitors for about 2 hours to about 6 hours. In embodiments, T cells are contacted with one or more cGAS-STING pathway inhibitors for about 2 hours to about 5 hours. In embodiments, T cells are contacted with one or more cGAS-STING pathway inhibitors for about 2 hours to about 4 hours. In embodiments, T cells are contacted with one or more cGAS-STING pathway inhibitors for about 2 hours to about 3 hours. In embodiments, T cells are contacted with 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, T cells are contacted with one or more cGAS-STING pathway inhibitors for about 6 hours. In embodiments, T cells are contacted with one or more cGAS-STING pathway inhibitors for 6 hours.

[0116] For the methods provided herein, in embodiments, T cells are contacted with one cGAS-STING pathway inhibitor. In embodiments, T cells are contacted with one cGAS-STING pathway inhibitor and are not contacted with other cGAS-STING pathway inhibitors. 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.

[0117] In particular, provided herein is a method for increasing the viability of engineered T cells, the method comprising contacting T cells with nucleic acids and one or more cGAS-STING pathway inhibitors, thereby forming engineered T cells, wherein the engineered T cells have increased cell viability relative to engineered T cells formed without contacting T cells with one or more cGAS-STING pathway inhibitors. The methods provided herein can be used to overcome toxicity associated with engineered T cells, including toxicity associated with delivering nucleic acids to T cells or with the presence of nucleic acids in the cytoplasm of T cells. Therefore, it is expected that the method improves the viability of engineered T cells. "Cell viability" is used in accordance with its ordinary meaning in the art and refers to the number or proportion of living 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 nucleic acid-binding dyes that only enter cells with damaged or damaged cell membranes. Cell viability can be measured by contacting cells with reagents that react with enzymes in living cells or reagents that detect the redox potential of cells. For example, cell viability can be measured using fluorescence detection assays, including assays using one or more of acridine orange, 4',6-diamidino-2-phenylindole (DAPI), propidium iodide (PI), or SYTOX Blue nucleic acid stains, etc. In an embodiment, cell viability can be measured by fluorescence microscopy or flow cytometry. Therefore, in one aspect, a method for increasing the cell viability of an engineered T cell population is provided, the method comprising contacting a T cell population with one or more cyclic GMP-AMP synthase (cGAS)-interferon gene stimulator (STING) pathway inhibitors and nucleic acids, thereby forming the population of engineered T cells, wherein the engineered T cell population has increased cell viability relative to an engineered T cell population that is not contacted with one or more cGAS-STING pathway inhibitors.

[0118] In an embodiment, the nucleic acid comprises a donor DNA. In an embodiment, the nucleic acid is a double-stranded circular DNA (e.g., a plasmid). In an embodiment, the nucleic acid is a plasmid, a nanoplasmid, a minicircle, or a viral vector comprising a donor DNA. In an embodiment, the donor DNA encodes an exogenous T cell receptor (TCR)-β or a fragment thereof, an exogenous TCR-α or a fragment thereof, or a combination thereof. In an embodiment, the nucleic acid is associated with a delivery vehicle. In an embodiment, the delivery vehicle is a lipid particle or a nanoparticle.

[0119] For the methods provided herein, in embodiments, the T cells are primary T cells.

[0120] For the methods provided herein, in embodiments, the method further comprises contacting a T cell population with a gene editing agent. In embodiments, contacting a T cell population with a gene editing agent comprises contacting a T cell population with a nucleic acid sequence encoding a gene editing agent. In embodiments, T cells are contacted with nucleic acids in the presence of a gene editing agent or a nucleic acid sequence encoding a gene editing agent.

[0121] 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.

[0122] In embodiments, contacting the T cells with the gene editing agent comprises transfecting the T cells with the gene editing agent.

[0123] In an embodiment, one or more cGAS-STING pathway inhibitors include cGAS inhibitors. In an embodiment, one or more cGAS-STING pathway inhibitors include STING inhibitors. In an embodiment, one or more cGAS-STING pathway inhibitors include TANK binding kinase 1 (TBK1) inhibitors. In an embodiment, one or more cGAS-STING pathway inhibitors are aml, MRT67307 (MRT), BX795, H151, ODN-A151 (ODN151), Ru.521, G140 or a combination thereof. In an embodiment, one or more cGAS-STING pathway inhibitors include Aml. In an embodiment, one or more cGAS-STING pathway inhibitors include MRT. In an embodiment, one or more cGAS-STING pathway inhibitors include BX795. In an embodiment, one or more cGAS-STING pathway inhibitors include H151. In an embodiment, one or more cGAS-STING pathway inhibitors include ODN-A151. In an embodiment, one or more cGAS-STING pathway inhibitors include Ru.521. In an embodiment, one or more cGAS-STING pathway inhibitors include G140. In an embodiment, one or more cGAS-STING pathway inhibitors are Aml. In an embodiment, one or more cGAS-STING pathway inhibitors are MRT. In an embodiment, one or more cGAS-STING pathway inhibitors are BX795. In an embodiment, one or more cGAS-STING pathway inhibitors are H151. In an embodiment, one or more cGAS-STING pathway inhibitors are ODN-A151 (ODN151). In an embodiment, one or more cGAS-STING pathway inhibitors are Ru.521. In an embodiment, one or more cGAS-STING pathway inhibitors are G140.

[0124] In embodiments, a population of T cells is independently contacted with one or more cGAS-STING pathway inhibitors of about 0.1uM to about 50uM. In embodiments, a population of T cells is independently contacted with one or more cGAS-STING pathway inhibitors of about 1uM to about 50uM. In embodiments, a population of T cells is independently contacted with one or more cGAS-STING pathway inhibitors of about 2uM to about 50uM. In embodiments, a population of T cells is independently contacted with one or more cGAS-STING pathway inhibitors of about 3uM to about 50uM. In embodiments, a population of T cells is independently contacted with one or more cGAS-STING pathway inhibitors of about 4uM to about 50uM. In embodiments, a population of T cells is independently contacted with one or more cGAS-STING pathway inhibitors of about 5uM to about 50uM. In embodiments, a population of T cells is independently contacted with one or more cGAS-STING pathway inhibitors of about 6uM to about 50uM. In embodiments, a population of T cells is independently contacted with one or more cGAS-STING pathway inhibitors of about 7uM to about 50uM. In embodiments, a population of T cells is independently contacted with one or more cGAS-STING pathway inhibitors of about 8uM to about 50uM. In embodiments, a population of T cells is independently contacted with one or more cGAS-STING pathway inhibitors of about 9uM to about 50uM. In embodiments, a population of T cells is independently contacted with one or more cGAS-STING pathway inhibitors of about 10uM to about 50uM. In embodiments, a population of T cells is independently contacted with one or more cGAS-STING pathway inhibitors of about 11uM to about 50uM. In embodiments, a population of T cells is independently contacted with one or more cGAS-STING pathway inhibitors of about 12uM to about 50uM. In embodiments, a population of T cells is independently contacted with one or more cGAS-STING pathway inhibitors of about 13uM to about 50uM. In embodiments, a population of T cells is independently contacted with one or more cGAS-STING pathway inhibitors of about 14uM to about 50uM. In embodiments, a population of T cells is independently contacted with one or more cGAS-STING pathway inhibitors of about 15uM to about 50uM. In embodiments, a population of T cells is independently contacted with about 16uM to about 50uM of one or more cGAS-STING pathway inhibitors. In embodiments, a population of T cells is independently contacted with about 17uM to about 50uM of one or more cGAS-STING pathway inhibitors. In embodiments, a population of T cells is independently contacted with about 18uM to about 50uM of one or more cGAS-STING pathway inhibitors. In embodiments, a population of T cells is independently contacted with about 19uM to about 50uM of one or more cGAS-STING pathway inhibitors.In embodiments, a population of T cells is independently contacted with about 20uM to about 50uM of one or more cGAS-STING pathway inhibitors. In embodiments, a population of T cells is independently contacted with about 21uM to about 50uM of one or more cGAS-STING pathway inhibitors. In embodiments, a population of T cells is independently contacted with about 22uM to about 50uM of one or more cGAS-STING pathway inhibitors. In embodiments, a population of T cells is independently contacted with about 23uM to about 50uM of one or more cGAS-STING pathway inhibitors. In embodiments, a population of T cells is independently contacted with about 24uM to about 50uM of one or more cGAS-STING pathway inhibitors. In embodiments, a population of T cells is independently contacted with about 25uM to about 50uM of one or more cGAS-STING pathway inhibitors. In embodiments, a population of T cells is independently contacted with about 26uM to about 50uM of one or more cGAS-STING pathway inhibitors. In embodiments, a population of T cells is independently contacted with one or more cGAS-STING pathway inhibitors at about 27uM to about 50uM. In embodiments, a population of T cells is independently contacted with one or more cGAS-STING pathway inhibitors at about 28uM to about 50uM. In embodiments, a population of T cells is independently contacted with one or more cGAS-STING pathway inhibitors at about 29uM to about 50uM. In embodiments, a population of T cells is independently contacted with one or more cGAS-STING pathway inhibitors at about 30uM to about 50uM. In embodiments, a population of T cells is independently contacted with one or more cGAS-STING pathway inhibitors at about 31uM to about 50uM. In embodiments, a population of T cells is independently contacted with one or more cGAS-STING pathway inhibitors at about 32uM to about 50uM. In embodiments, a population of T cells is independently contacted with one or more cGAS-STING pathway inhibitors at about 33uM to about 50uM. In embodiments, a population of T cells is independently contacted with about 34uM to about 50uM of one or more cGAS-STING pathway inhibitors. In embodiments, a population of T cells is independently contacted with about 35uM to about 50uM of one or more cGAS-STING pathway inhibitors. In embodiments, a population of T cells is independently contacted with about 36uM to about 50uM of one or more cGAS-STING pathway inhibitors. In embodiments, a population of T cells is independently contacted with about 37uM to about 50uM of one or more cGAS-STING pathway inhibitors. In embodiments, a population of T cells is independently contacted with about 38uM to about 50uM of one or more cGAS-STING pathway inhibitors. In embodiments, a population of T cells is independently contacted with about 39uM to about 50uM of one or more cGAS-STING pathway inhibitors.In embodiments, a population of T cells is independently contacted with about 40uM to about 50uM of one or more cGAS-STING pathway inhibitors. In embodiments, a population of T cells is independently contacted with about 41uM to about 50uM of one or more cGAS-STING pathway inhibitors. In embodiments, a population of T cells is independently contacted with about 42uM to about 50uM of one or more cGAS-STING pathway inhibitors. In embodiments, a population of T cells is independently contacted with about 43uM to about 50uM of one or more cGAS-STING pathway inhibitors. In embodiments, a population of T cells is independently contacted with about 44uM to about 50uM of one or more cGAS-STING pathway inhibitors. In embodiments, a population of T cells is independently contacted with about 45uM to about 50uM of one or more cGAS-STING pathway inhibitors. In embodiments, a population of T cells is independently contacted with about 46uM to about 50uM of one or more cGAS-STING pathway inhibitors. In embodiments, populations of T cells are independently contacted with about 47uM to about 50uM of one or more cGAS-STING pathway inhibitors. In embodiments, populations of T cells are independently contacted with about 48uM to about 50uM of one or more cGAS-STING pathway inhibitors. In embodiments, populations of T cells are independently contacted with about 49uM to about 50uM of one or more cGAS-STING pathway inhibitors.

[0125] In embodiments, a population of T cells is independently contacted with one or more cGAS-STING pathway inhibitors from about 0.1uM to about 49uM. In embodiments, a population of T cells is independently contacted with one or more cGAS-STING pathway inhibitors from about 0.1uM to about 48uM. In embodiments, a population of T cells is independently contacted with one or more cGAS-STING pathway inhibitors from about 0.1uM to about 47uM. In embodiments, a population of T cells is independently contacted with one or more cGAS-STING pathway inhibitors from about 0.1uM to about 46uM. In embodiments, a population of T cells is independently contacted with one or more cGAS-STING pathway inhibitors from about 0.1uM to about 45uM. In embodiments, a population of T cells is independently contacted with one or more cGAS-STING pathway inhibitors from about 0.1uM to about 44uM. In embodiments, a population of T cells is independently contacted with one or more cGAS-STING pathway inhibitors from about 0.1uM to about 43uM. In embodiments, a population of T cells is independently contacted with one or more cGAS-STING pathway inhibitors from about 0.1uM to about 42uM. In embodiments, a population of T cells is independently contacted with one or more cGAS-STING pathway inhibitors from about 0.1uM to about 41uM. In embodiments, a population of T cells is independently contacted with one or more cGAS-STING pathway inhibitors from about 0.1uM to about 40uM. In embodiments, a population of T cells is independently contacted with one or more cGAS-STING pathway inhibitors from about 0.1uM to about 39uM. In embodiments, a population of T cells is independently contacted with one or more cGAS-STING pathway inhibitors from about 0.1uM to about 38uM. In embodiments, a population of T cells is independently contacted with one or more cGAS-STING pathway inhibitors from about 0.1uM to about 37uM. In embodiments, a population of T cells is independently contacted with one or more cGAS-STING pathway inhibitors from about 0.1uM to about 36uM. In embodiments, a population of T cells is independently contacted with about 0.1uM to about 35uM of one or more cGAS-STING pathway inhibitors. In embodiments, a population of T cells is independently contacted with about 0.1uM to about 34uM of one or more cGAS-STING pathway inhibitors. In embodiments, a population of T cells is independently contacted with about 0.1uM to about 33uM of one or more cGAS-STING pathway inhibitors. In embodiments, a population of T cells is independently contacted with about 0.1uM to about 32uM of one or more cGAS-STING pathway inhibitors. In embodiments, a population of T cells is independently contacted with about 0.1uM to about 31uM of one or more cGAS-STING pathway inhibitors.In embodiments, a population of T cells is independently contacted with one or more cGAS-STING pathway inhibitors of about 0.1uM to about 30uM. In embodiments, a population of T cells is independently contacted with one or more cGAS-STING pathway inhibitors of about 0.1uM to about 29uM. In embodiments, a population of T cells is independently contacted with one or more cGAS-STING pathway inhibitors of about 0.1uM to about 28uM. In embodiments, a population of T cells is independently contacted with one or more cGAS-STING pathway inhibitors of about 0.1uM to about 27uM. In embodiments, a population of T cells is independently contacted with one or more cGAS-STING pathway inhibitors of about 0.1uM to about 26uM. In embodiments, a population of T cells is independently contacted with one or more cGAS-STING pathway inhibitors of about 0.1uM to about 25uM. In embodiments, a population of T cells is independently contacted with one or more cGAS-STING pathway inhibitors of about 0.1uM to about 24uM. In embodiments, a population of T cells is independently contacted with one or more cGAS-STING pathway inhibitors from about 0.1uM to about 23uM. In embodiments, a population of T cells is independently contacted with one or more cGAS-STING pathway inhibitors from about 0.1uM to about 22uM. In embodiments, a population of T cells is independently contacted with one or more cGAS-STING pathway inhibitors from about 0.1uM to about 21uM. In embodiments, a population of T cells is independently contacted with one or more cGAS-STING pathway inhibitors from about 0.1uM to about 20uM. In embodiments, a population of T cells is independently contacted with one or more cGAS-STING pathway inhibitors from about 0.1uM to about 19uM. In embodiments, a population of T cells is independently contacted with one or more cGAS-STING pathway inhibitors from about 0.1uM to about 18uM. In embodiments, a population of T cells is independently contacted with one or more cGAS-STING pathway inhibitors from about 0.1uM to about 17uM. In embodiments, a population of T cells is independently contacted with about 0.1 uM to about 16 uM of one or more cGAS-STING pathway inhibitors. In embodiments, a population of T cells is independently contacted with about 0.1 uM to about 15 uM of one or more cGAS-STING pathway inhibitors. In embodiments, a population of T cells is independently contacted with about 0.1 uM to about 14 uM of one or more cGAS-STING pathway inhibitors. In embodiments, a population of T cells is independently contacted with about 0.1 uM to about 13 uM of one or more cGAS-STING pathway inhibitors. In embodiments, a population of T cells is independently contacted with about 0.1 uM to about 12 uM of one or more cGAS-STING pathway inhibitors.In embodiments, a population of T cells is independently contacted with one or more cGAS-STING pathway inhibitors of about 0.1uM to about 11uM. In embodiments, a population of T cells is independently contacted with one or more cGAS-STING pathway inhibitors of about 0.1uM to about 10uM. In embodiments, a population of T cells is independently contacted with one or more cGAS-STING pathway inhibitors of about 0.1uM to about 9uM. In embodiments, a population of T cells is independently contacted with one or more cGAS-STING pathway inhibitors of about 0.1uM to about 8uM. In embodiments, a population of T cells is independently contacted with one or more cGAS-STING pathway inhibitors of about 0.1uM to about 7uM. In embodiments, a population of T cells is independently contacted with one or more cGAS-STING pathway inhibitors of about 0.1uM to about 6uM. In embodiments, a population of T cells is independently contacted with one or more cGAS-STING pathway inhibitors of about 0.1uM to about 5uM. In embodiments, populations of T cells are independently contacted with about 0.1 uM to about 4 uM of one or more cGAS-STING pathway inhibitors. In embodiments, populations of T cells are independently contacted with about 0.1 uM to about 3 uM of one or more cGAS-STING pathway inhibitors. In embodiments, populations of T cells are independently contacted with about 0.1 uM to about 2 uM of one or more cGAS-STING pathway inhibitors. In embodiments, populations of T cells are independently contacted with about 0.1 uM to about 1 uM of one or more cGAS-STING pathway inhibitors. In embodiments, a population of T cells is independently modified with about 0.1uM, 1uM, 2uM, 3uM, 4uM, 5uM, 6uM, 7uM, 8uM, 9uM, 10uM, 11uM, 12uM, 13uM, 14uM, 15uM, 16uM, 17uM, 18uM, 19uM, 20uM, 21uM, 22uM, 23uM, 24uM, 25uM, 26uM, 47uM, 48uM, 49uM or 50uM of one or more cGAS-STING pathway inhibitors.

[0126] In embodiments, a population of T cells is contacted with a nucleic acid in the presence of one or more cGAS-STING pathway inhibitors. In embodiments, a population of T cells is contacted with a nucleic acid and one or more cGAS-STING pathway inhibitors sequentially. In embodiments, a population of T cells is contacted with one or more cGAS-STING pathway inhibitors prior to contacting with the nucleic acid. In embodiments, a population of T cells is contacted with one cGAS-STING pathway inhibitor.

[0127] In an embodiment, a T cell population is contacted with about 1uM to about 10uM BX795. In an embodiment, a T cell population is contacted with about 1.5uM to about 10uM BX795. In an embodiment, a T cell population is contacted with about 2uM to about 10uM BX795. In an embodiment, a T cell population is contacted with about 2.5uM to about 10uM BX795. In an embodiment, a T cell population is contacted with about 3uM to about 10uM BX795. In an embodiment, a T cell population is contacted with about 3.5uM to about 10uM BX795. In an embodiment, a T cell population is contacted with about 4uM to about 10uM BX795. In an embodiment, a T cell population is contacted with about 4.5uM to about 10uM BX795. In an embodiment, a T cell population is contacted with about 5uM to about 10uM BX795. In an embodiment, a T cell population is contacted with about 5.5uM to about 10uM BX795. In an embodiment, a T cell population is contacted with about 6uM to about 10uM BX795. In an embodiment, a T cell population is contacted with about 6.5uM to about 10uM BX795. In an embodiment, a T cell population is contacted with about 7uM to about 10uM BX795. In an embodiment, a T cell population is contacted with about 7.5uM to about 10uM BX795. In an embodiment, a T cell population is contacted with about 8uM to about 10uM BX795. In an embodiment, a T cell population is contacted with about 8.5uM to about 10uM BX795. In an embodiment, a T cell population is contacted with about 9uM to about 10uM BX795. In an embodiment, a T cell population is contacted with about 9.5uM to about 10uM BX795.

[0128] In an embodiment, a T cell population is contacted with about 1uM to about 9.5uM BX795. In an embodiment, a T cell population is contacted with about 1uM to about 9uM BX795. In an embodiment, a T cell population is contacted with about 1uM to about 8.5uM BX795. In an embodiment, a T cell population is contacted with about 1uM to about 8uM BX795. In an embodiment, a T cell population is contacted with about 1uM to about 7.5uM BX795. In an embodiment, a T cell population is contacted with about 1uM to about 7uM BX795. In an embodiment, a T cell population is contacted with about 1uM to about 6.5uM BX795. In an embodiment, a T cell population is contacted with about 1uM to about 6uMBX795. In an embodiment, a T cell population is contacted with about 1uM to about 5.5uM BX795. In an embodiment, a T cell population is contacted with about 1uM to about 5uM BX795. In an embodiment, a T cell population is contacted with about 1uM to about 4.5uM BX795. In an embodiment, a T cell population is contacted with about 1uM to about 4uM BX795. In an embodiment, a T cell population is contacted with about 1uM to about 3.5uM BX795. In an embodiment, a T cell population is contacted with about 1uM to about 3uM BX795. In an embodiment, a T cell population is contacted with about 1uM to about 2.5uM BX795. In an embodiment, a T cell population is contacted with about 1uM to about 2uM BX795. In an embodiment, a T cell population is contacted with about 1uM to about 1.5uM BX795. In embodiments, a T cell population 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 embodiments, a T cell population is contacted with about 2.5 uM BX795. In embodiments, a T cell population is contacted with about 5 uM BX795.

[0129] In an embodiment, a T cell population is contacted with about 0.1uM to about 8uM ODN151. In an embodiment, a T cell population is contacted with about 0.5uM to about 8uM ODN151. In an embodiment, a T cell population is contacted with about 1uM to about 8uM ODN151. In an embodiment, a T cell population is contacted with about 1.5uM to about 8uM ODN151. In an embodiment, a T cell population is contacted with about 2uM to about 8uM ODN151. In an embodiment, a T cell population is contacted with about 2.5uM to about 8uM ODN151. In an embodiment, a T cell population is contacted with about 3uM to about 8uM ODN151. In an embodiment, a T cell population is contacted with about 3.5uM to about 8uM ODN151. In an embodiment, a T cell population is contacted with about 4uM to about 8uM ODN151. In an embodiment, a T cell population is contacted with about 4.5uM to about 8uM ODN151. In an embodiment, a T cell colony is contacted with about 5uM to about 8uM ODN151. In an embodiment, a T cell colony is contacted with about 5.5uM to about 8uM ODN151. In an embodiment, a T cell colony is contacted with about 6uM to about 8uM ODN151. In an embodiment, a T cell colony is contacted with about 6.5uM to about 8uM ODN151. In an embodiment, a T cell colony is contacted with about 7uM to about 8uM ODN151. In an embodiment, a T cell colony is contacted with about 7.5uM to about 8uM ODN151.

[0130] In an embodiment, a T cell population is contacted with about 0.1uM to about 7.5uM ODN151. In an embodiment, a T cell population is contacted with about 0.1uM to about 7uM ODN151. In an embodiment, a T cell population is contacted with about 0.1uM to about 6.5uM ODN151. In an embodiment, a T cell population is contacted with about 0.1uM to about 6uM ODN151. In an embodiment, a T cell population is contacted with about 0.1uM to about 5.5uM ODN151. In an embodiment, a T cell population is contacted with about 0.1uM to about 5uM ODN151. In an embodiment, a T cell population is contacted with about 0.1uM to about 4.5uM ODN151. In an embodiment, a T cell population is contacted with about 0.1uM to about 4uM ODN151. In an embodiment, a T cell population is contacted with about 0.1uM to about 3.5uM ODN151. In an embodiment, a T cell population is contacted with about 0.1uM to about 3uM ODN151. In an embodiment, a T cell population is contacted with about 0.1uM to about 2.5uM ODN151. In an embodiment, a T cell population is contacted with about 0.1uM to about 2uM ODN151. In an embodiment, a T cell population is contacted with about 0.1uM to about 1.5uM ODN151. In an embodiment, a T cell population is contacted with about 0.1uM to about 1uM ODN151. In an embodiment, a T cell population is contacted with about 0.1uM to about 0.5uM ODN151. In embodiments, a population of T cells is contacted with about 0.1 uM, 0.5 uM, 1 uM, 1.5 uM, 2uM, 2.5 uM, 3uM, 3.5 uM, 4uM, 4.5 uM, 5uM, 5.5uM, 6uM, 6.5uM, 7uM, 7.5uM, 8uM, 8.5uM, 9uM, 9.5uM or 10uM of D151.

[0131] As described above, compared to engineered T cells prepared without treatment with one or more cGAS-STING pathway inhibitors, the methods provided herein (including embodiments thereof) effectively increase the cell viability of engineered T cells. For example, compared to engineered T cell populations prepared without contacting T cells with one or more cGAS-STING pathway inhibitors, the engineered T cell populations provided herein have increased cell viability. In an embodiment, the cell viability of an engineered T cell population is increased by at least about 0.5 times to at least about 5 times relative to an engineered T cell population not contacted with one or more cGAS-STING pathway inhibitors. In an embodiment, the cell viability of an engineered T cell population is increased by at least about 1 times to at least about 5 times relative to an engineered T cell population not contacted with one or more cGAS-STING pathway inhibitors. In an embodiment, the cell viability of an engineered T cell population is increased by at least about 1.5 times to at least about 5 times relative to an engineered T cell population not contacted with one or more cGAS-STING pathway inhibitors. In an embodiment, the cell viability of the engineered T cell population is increased by at least about 2-fold to at least about 5-fold relative to an engineered T cell population that is not contacted with one or more cGAS-STING pathway inhibitors. In an embodiment, the cell viability of the engineered T cell population is increased by at least about 2.5-fold to at least about 5-fold relative to an engineered T cell population that is not contacted with one or more cGAS-STING pathway inhibitors. In an embodiment, the cell viability of the engineered T cell population is increased by at least about 3-fold to at least about 5-fold relative to an engineered T cell population that is not contacted with one or more cGAS-STING pathway inhibitors. In an embodiment, the cell viability of the engineered T cell population is increased by at least about 3.5-fold to at least about 5-fold relative to an engineered T cell population that is not contacted with one or more cGAS-STING pathway inhibitors. In an embodiment, the cell viability of the engineered T cell population is increased by at least about 4-fold to at least about 5-fold relative to an engineered T cell population that is not contacted with one or more cGAS-STING pathway inhibitors.

[0132] In an embodiment, the cell viability of the engineered T cell population is increased by at least about 0.5 times to at least about 4.5 times relative to an engineered T cell population that is not contacted with one or more cGAS-STING pathway inhibitors. In an embodiment, the cell viability of the engineered T cell population is increased by at least about 0.5 times to at least about 4 times relative to an engineered T cell population that is not contacted with one or more cGAS-STING pathway inhibitors. In an embodiment, the cell viability of the engineered T cell population is increased by at least about 0.5 times to at least about 3.5 times relative to an engineered T cell population that is not contacted with one or more cGAS-STING pathway inhibitors. In an embodiment, the cell viability of the engineered T cell population is increased by at least about 0.5 times to at least about 3 times relative to an engineered T cell population that is not contacted with one or more cGAS-STING pathway inhibitors. In an embodiment, the cell viability of the engineered T cell population is increased by at least about 0.5 times to at least about 2.5 times relative to an engineered T cell population that is not contacted with one or more cGAS-STING pathway inhibitors. In an embodiment, the cell viability of the engineered T cell population is increased by at least about 0.5 times to at least about 2 times relative to an engineered T cell population that is not contacted with one or more cGAS-STING pathway inhibitors. In an embodiment, the cell viability of the engineered T cell population is increased by at least about 0.5 times to at least about 1.5 times relative to an engineered T cell population that is not contacted with one or more cGAS-STING pathway inhibitors. In an embodiment, the cell viability of the engineered T cell population is increased by at least about 0.5 times to at least about 1 times relative to an engineered T cell population that is not contacted with one or more cGAS-STING pathway inhibitors. In an embodiment, the cell viability of the engineered T cell population is increased by about 0.5 times, 1 times, 1.5 times, 2 times, 2.5 times, 3 times, 3.5 times, 4 times, 4.5 times or 5 times relative to an engineered T cell population that is not contacted with one or more cGAS-STING pathway inhibitors. In an embodiment, the cell viability is increased by about 2 times relative to an engineered T cell population that is not contacted with one or more cGAS-STING pathway inhibitors. In embodiments, cell viability is increased 2-fold relative to a population of engineered T cells not contacted with one or more cGAS-STING pathway inhibitors.

[0133] In embodiments, the cell viability of the engineered T cell population is increased by about 30% to about 95% relative to an engineered T cell population that has not been contacted with one or more cGAS-STING pathway inhibitors. In embodiments, the cell viability of the engineered T cell population is increased by about 35% to about 95% relative to an engineered T cell population that has not been contacted with one or more cGAS-STING pathway inhibitors. In embodiments, the cell viability of the engineered T cell population is increased by about 40% to about 95% relative to an engineered T cell population that has not been contacted with one or more cGAS-STING pathway inhibitors. In embodiments, the cell viability of the engineered T cell population is increased by about 45% to about 95% relative to an engineered T cell population that has not been contacted with one or more cGAS-STING pathway inhibitors. In embodiments, the cell viability of the engineered T cell population is increased by about 50% to about 95% relative to an engineered T cell population that has not been contacted with one or more cGAS-STING pathway inhibitors. In embodiments, the cell viability of the engineered T cell population is increased by about 55% to about 95% relative to an engineered T cell population that has not been contacted with one or more cGAS-STING pathway inhibitors. In embodiments, the cell viability of the engineered T cell population is increased by about 60% to about 95% relative to an engineered T cell population that is not contacted with one or more cGAS-STING pathway inhibitors. In embodiments, the cell viability of the engineered T cell population is increased by about 65% to about 95% relative to an engineered T cell population that is not contacted with one or more cGAS-STING pathway inhibitors. In embodiments, the cell viability of the engineered T cell population is increased by about 70% to about 95% relative to an engineered T cell population that is not contacted with one or more cGAS-STING pathway inhibitors. In embodiments, the cell viability of the engineered T cell population is increased by about 75% to about 95% relative to an engineered T cell population that is not contacted with one or more cGAS-STING pathway inhibitors. In embodiments, the cell viability of the engineered T cell population is increased by about 80% to about 95% relative to an engineered T cell population that is not contacted with one or more cGAS-STING pathway inhibitors. In embodiments, the cell viability of the engineered T cell population is increased by about 85% to about 95% relative to an engineered T cell population that is not contacted with one or more cGAS-STING pathway inhibitors. In embodiments, the cell viability of the engineered T cell population is increased by about 90% to about 95% relative to an engineered T cell population not contacted with one or more cGAS-STING pathway inhibitors.

[0134] In embodiments, the cell viability of the engineered T cell population is increased by about 35% to about 90% relative to an engineered T cell population that is not contacted with one or more cGAS-STING pathway inhibitors. In embodiments, the cell viability of the engineered T cell population is increased by about 35% to about 85% relative to an engineered T cell population that is not contacted with one or more cGAS-STING pathway inhibitors. In embodiments, the cell viability of the engineered T cell population is increased by about 35% to about 80% relative to an engineered T cell population that is not contacted with one or more cGAS-STING pathway inhibitors. In embodiments, the cell viability of the engineered T cell population is increased by about 35% to about 75% relative to an engineered T cell population that is not contacted with one or more cGAS-STING pathway inhibitors. In embodiments, the cell viability of the engineered T cell population is increased by about 35% to about 70% relative to an engineered T cell population that is not contacted with one or more cGAS-STING pathway inhibitors. In embodiments, the cell viability of the engineered T cell population is increased by about 35% to about 65% relative to an engineered T cell population that is not contacted with one or more cGAS-STING pathway inhibitors. In embodiments, the cell viability of the engineered T cell population is increased by about 35% to about 60% relative to an engineered T cell population that has not been contacted with one or more cGAS-STING pathway inhibitors. In embodiments, the cell viability of the engineered T cell population is increased by about 35% to about 55% relative to an engineered T cell population that has not been contacted with one or more cGAS-STING pathway inhibitors. In embodiments, the cell viability of the engineered T cell population is increased by about 35% to about 50% relative to an engineered T cell population that has not been contacted with one or more cGAS-STING pathway inhibitors. In embodiments, the cell viability of the engineered T cell population is increased by about 35% to about 45% relative to an engineered T cell population that has not been contacted with one or more cGAS-STING pathway inhibitors. In embodiments, the cell viability of the engineered T cell population is increased by about 35% to about 40% relative to an engineered T cell population that has not been contacted with one or more cGAS-STING pathway inhibitors. In embodiments, the cell viability of the engineered T cell population is increased by about 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95% relative to an engineered T cell population not contacted with one or more cGAS-STING pathway inhibitors.

[0135] The method provided herein effectively improves the gene editing efficiency in T cells, thereby increasing the total number of engineered T cells. For example, the method can increase the efficiency of gene knockout and / or gene knock-in, thereby improving the gene editing efficiency of T cells. The method can thus improve the yield of engineered T cells generated by T cell colonies. For example, relative to the engineered T cell colonies in which T cells are not contacted with one or more cGAS-STING pathway inhibitors, the method provided herein can generate an increased engineered T cell colony from a T cell colony. Therefore, in one aspect, a method for increasing the gene editing efficiency in a T cell colony is provided, the method including contacting a T cell colony with one or more cyclic GMP-AMP synthase (cGAS)-interferon gene stimulator (STING) pathway inhibitors and nucleic acids, thereby forming an engineered T cell colony, wherein the T cell colony has an increased gene editing efficiency relative to a T cell colony not contacted with one or more cGAS-STING pathway inhibitors.

[0136] In an embodiment, the nucleic acid comprises a donor DNA. In an embodiment, the nucleic acid is a plasmid, nanoplasmid, minicircle or viral vector comprising a donor DNA. In an embodiment, the donor DNA encodes an exogenous T cell receptor (TCR)-β or a fragment thereof, an exogenous TCR-α or a fragment thereof, or a combination thereof. In an embodiment, the nucleic acid is associated with a delivery vehicle. In an embodiment, the delivery vehicle is a lipid particle or a nanoparticle.

[0137] In embodiments, the T cell population comprises primary T cells.

[0138] In an embodiment, the method further comprises contacting the T cell population with a gene editing reagent. In an embodiment, contacting the T cell population with a gene editing reagent comprises contacting the T cell population with a nucleic acid sequence encoding a gene editing reagent. In an embodiment, the T cell population is contacted with a nucleic acid in the presence of a gene editing agent or a nucleic acid sequence encoding a gene editing agent. In an embodiment, the gene editing reagent comprises an RNA-guided nuclease. In an embodiment, the RNA-guided nuclease is a CRISPR-Cas system.

[0139] 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.

[0140] In embodiments, contacting a population of T cells with a gene editing agent comprises transfecting a population of T cells with a gene editing agent. In embodiments, contacting a population of T cells with a nucleic acid comprises transfecting a population of T cells with a nucleic acid.

[0141] In an embodiment, one or more cGAS-STING pathway inhibitors include cGAS inhibitors. In an embodiment, one or more cGAS-STING pathway inhibitors include STING inhibitors. In an embodiment, one or more cGAS-STING pathway inhibitors include TANK binding kinase 1 (TBK1) inhibitors. In an embodiment, one or more cGAS-STING pathway inhibitors are aml, MRT67307 (MRT), BX795, H151, ODN-A151 (ODN151), Ru.521, G140 or a combination thereof. In an embodiment, one or more cGAS-STING pathway inhibitors include Aml. In an embodiment, one or more cGAS-STING pathway inhibitors include MRT. In an embodiment, one or more cGAS-STING pathway inhibitors include BX795. In an embodiment, one or more cGAS-STING pathway inhibitors include H151. In an embodiment, one or more cGAS-STING pathway inhibitors include ODN151. In an embodiment, one or more cGAS-STING pathway inhibitors include Ru.521. In embodiments, the one or more cGAS-STING pathway inhibitors include G140.

[0142] In embodiments, one or more cGAS-STING pathway inhibitors are Aml. In embodiments, one or more cGAS-STING pathway inhibitors are MRT. In embodiments, one or more cGAS-STING pathway inhibitors are BX795. In embodiments, one or more cGAS-STING pathway inhibitors are H151. In embodiments, one or more cGAS-STING pathway inhibitors are ODN151. In embodiments, one or more cGAS-STING pathway inhibitors are Ru.521. In embodiments, one or more cGAS-STING pathway inhibitors are G140. In embodiments, the cGAS-STING pathway inhibitor is Aml and does not include any other cGAS-STING pathway inhibitor. In embodiments, the cGAS-STING pathway inhibitor is MRT and does not include any other cGAS-STING pathway inhibitor. In embodiments, the cGAS-STING pathway inhibitor is BX795 and does not include any other cGAS-STING pathway inhibitor. In embodiments, the cGAS-STING pathway inhibitor is H151 and does not include any other cGAS-STING pathway inhibitor. In embodiments, the cGAS-STING pathway inhibitor is ODN151 and does not include any other cGAS-STING pathway inhibitor. In embodiments, the cGAS-STING pathway inhibitor is Ru.521 and does not include any other cGAS-STING pathway inhibitor. In embodiments, the cGAS-STING pathway inhibitor is G140 and does not include any other cGAS-STING pathway inhibitor.

[0143] In embodiments, a population of T cells is independently contacted with one or more cGAS-STING pathway inhibitors of about 0.1uM to about 50uM. In embodiments, a population of T cells is independently contacted with one or more cGAS-STING pathway inhibitors of about 2uM to about 50uM. In embodiments, a population of T cells is independently contacted with one or more cGAS-STING pathway inhibitors of about 4uM to about 50uM. In embodiments, a population of T cells is independently contacted with one or more cGAS-STING pathway inhibitors of about 6uM to about 50uM. In embodiments, a population of T cells is independently contacted with one or more cGAS-STING pathway inhibitors of about 8uM to about 50uM. In embodiments, a population of T cells is independently contacted with one or more cGAS-STING pathway inhibitors of about 10uM to about 50uM. In embodiments, a population of T cells is independently contacted with one or more cGAS-STING pathway inhibitors of about 12uM to about 50uM. In embodiments, a population of T cells is independently contacted with one or more cGAS-STING pathway inhibitors of about 14uM to about 50uM. In embodiments, a population of T cells is independently contacted with one or more cGAS-STING pathway inhibitors at about 16uM to about 50uM. In embodiments, a population of T cells is independently contacted with one or more cGAS-STING pathway inhibitors at about 18uM to about 50uM. In embodiments, a population of T cells is independently contacted with one or more cGAS-STING pathway inhibitors at about 20uM to about 50uM. In embodiments, a population of T cells is independently contacted with one or more cGAS-STING pathway inhibitors at about 22uM to about 50uM. In embodiments, a population of T cells is independently contacted with one or more cGAS-STING pathway inhibitors at about 24uM to about 50uM. In embodiments, a population of T cells is independently contacted with one or more cGAS-STING pathway inhibitors at about 26uM to about 50uM. In embodiments, a population of T cells is independently contacted with one or more cGAS-STING pathway inhibitors at about 28uM to about 50uM. In embodiments, a population of T cells is independently contacted with about 30uM to about 50uM of one or more cGAS-STING pathway inhibitors. In embodiments, a population of T cells is independently contacted with about 32uM to about 50uM of one or more cGAS-STING pathway inhibitors. In embodiments, a population of T cells is independently contacted with about 34uM to about 50uM of one or more cGAS-STING pathway inhibitors. In embodiments, a population of T cells is independently contacted with about 36uM to about 50uM of one or more cGAS-STING pathway inhibitors. In embodiments, a population of T cells is independently contacted with about 38uM to about 50uM of one or more cGAS-STING pathway inhibitors.In embodiments, a population of T cells is independently contacted with about 40uM to about 50uM of one or more cGAS-STING pathway inhibitors. In embodiments, a population of T cells is independently contacted with about 42uM to about 50uM of one or more cGAS-STING pathway inhibitors. In embodiments, a population of T cells is independently contacted with about 44uM to about 50uM of one or more cGAS-STING pathway inhibitors. In embodiments, a population of T cells is independently contacted with about 46uM to about 50uM of one or more cGAS-STING pathway inhibitors. In embodiments, a population of T cells is independently contacted with about 48uM to about 50uM of one or more cGAS-STING pathway inhibitors.

[0144] In embodiments, a population of T cells is independently contacted with one or more cGAS-STING pathway inhibitors from about 0.1uM to about 48uM. In embodiments, a population of T cells is independently contacted with one or more cGAS-STING pathway inhibitors from about 0.1uM to about 46uM. In embodiments, a population of T cells is independently contacted with one or more cGAS-STING pathway inhibitors from about 0.1uM to about 44uM. In embodiments, a population of T cells is independently contacted with one or more cGAS-STING pathway inhibitors from about 0.1uM to about 42uM. In embodiments, a population of T cells is independently contacted with one or more cGAS-STING pathway inhibitors from about 0.1uM to about 40uM. In embodiments, a population of T cells is independently contacted with one or more cGAS-STING pathway inhibitors from about 0.1uM to about 38uM. In embodiments, a population of T cells is independently contacted with one or more cGAS-STING pathway inhibitors from about 0.1uM to about 36uM. In embodiments, a population of T cells is independently contacted with one or more cGAS-STING pathway inhibitors from about 0.1uM to about 34uM. In embodiments, a population of T cells is independently contacted with one or more cGAS-STING pathway inhibitors from about 0.1uM to about 32uM. In embodiments, a population of T cells is independently contacted with one or more cGAS-STING pathway inhibitors from about 0.1uM to about 30uM. In embodiments, a population of T cells is independently contacted with one or more cGAS-STING pathway inhibitors from about 0.1uM to about 28uM. In embodiments, a population of T cells is independently contacted with one or more cGAS-STING pathway inhibitors from about 0.1uM to about 26uM. In embodiments, a population of T cells is independently contacted with one or more cGAS-STING pathway inhibitors from about 0.1uM to about 24uM. In embodiments, a population of T cells is independently contacted with one or more cGAS-STING pathway inhibitors from about 0.1uM to about 22uM. In embodiments, a population of T cells is independently contacted with about 0.1uM to about 20uM of one or more cGAS-STING pathway inhibitors. In embodiments, a population of T cells is independently contacted with about 0.1uM to about 18uM of one or more cGAS-STING pathway inhibitors. In embodiments, a population of T cells is independently contacted with about 0.1uM to about 16uM of one or more cGAS-STING pathway inhibitors. In embodiments, a population of T cells is independently contacted with about 0.1uM to about 14uM of one or more cGAS-STING pathway inhibitors. In embodiments, a population of T cells is independently contacted with about 0.1uM to about 12uM of one or more cGAS-STING pathway inhibitors.In embodiments, a population of T cells is independently contacted with about 0.1 uM to about 10 uM of one or more cGAS-STING pathway inhibitors. In embodiments, a population of T cells is independently contacted with about 0.1 uM to about 8 uM of one or more cGAS-STING pathway inhibitors. In embodiments, a population of T cells is independently contacted with about 0.1 uM to about 6 uM of one or more cGAS-STING pathway inhibitors. In embodiments, a population of T cells is independently contacted with about 0.1 uM to about 4 uM of one or more cGAS-STING pathway inhibitors. In embodiments, a population of T cells is independently contacted with about 0.1 uM to about 2 uM of one or more cGAS-STING pathway inhibitors. In embodiments, populations of T cells are independently contacted with about 0.1 uM, 2uM, 4uM, 6uM, 8uM, 10uM, 12uM, 14uM, 16uM, 18uM, 20uM, 22uM, 24uM, 26uM, 28uM, 30uM, 32uM, 34uM, 36uM, 38uM, 40uM, 42uM, 44uM, 46uM, 48uM or 50uM of one or more cGAS-STING pathway inhibitors.

[0145] In embodiments, a population of T cells is contacted with a nucleic acid in the presence of one or more cGAS-STING pathway inhibitors. In embodiments, a population of T cells is contacted with a nucleic acid and one or more cGAS-STING pathway inhibitors sequentially. In embodiments, a population of T cells is contacted with one or more cGAS-STING pathway inhibitors prior to contact with a nucleic acid. In embodiments, a population of T cells is contacted with a cGAS-STING pathway inhibitor provided herein and is not contacted with other cGAS-STING pathway inhibitors.

[0146] In an embodiment, a T cell population is contacted with about 2uM to about 8uM BX795. In an embodiment, a T cell population is contacted with about 2.5uM to about 8uM BX795. In an embodiment, a T cell population is contacted with about 3uM to about 8uM BX795. In an embodiment, a T cell population is contacted with about 3.5uM to about 8uM BX795. In an embodiment, a T cell population is contacted with about 4uM to about 8uM BX795. In an embodiment, a T cell population is contacted with about 4.5uM to about 8uM BX795. In an embodiment, a T cell population is contacted with about 5uM to about 8uM BX795. In an embodiment, a T cell population is contacted with about 5.5uM to about 8uM BX795. In an embodiment, a T cell population is contacted with about 6uM to about 8uM BX795. In an embodiment, a T cell population is contacted with about 6.5uM to about 8uM BX795. In embodiments, a population of T cells is contacted with about 7 uM to about 8 uM BX795. In embodiments, a population of T cells is contacted with about 7.5 uM to about 8 uM BX795.

[0147] In an embodiment, a T cell population is contacted with about 2uM to about 7.5uM BX795. In an embodiment, a T cell population is contacted with about 2uM to about 7uM BX795. In an embodiment, a T cell population is contacted with about 2uM to about 6.5uM BX795. In an embodiment, a T cell population is contacted with about 2uM to about 6uM BX795. In an embodiment, a T cell population is contacted with about 2uM to about 5.5uM BX795. In an embodiment, a T cell population is contacted with about 2uM to about 5uM BX795. In an embodiment, a T cell population is contacted with about 2uM to about 4.5uM BX795. In an embodiment, a T cell population is contacted with about 2uM to about 4uM BX795. In an embodiment, a T cell population is contacted with about 2uM to about 3.5uM BX795. In an embodiment, a T cell population is contacted with about 2uM to about 3uM BX795. In embodiments, a T cell population is contacted with about 2uM to about 2.5uM BX795. In embodiments, a T cell population is contacted with about 2uM, 2.5uM, 3uM, 3.5uM, 4uM, 4.5uM, 5uM, 5.5uM, 6uM, 6.5uM, 7uM, 7.5uM or 8uM BX795.

[0148] In embodiments, a T cell population is contacted with about 0.1 uM to about 5 uM ODNA151. In embodiments, a T cell population is contacted with about 0.25 uM to about 5 uM ODNA151. In embodiments, a T cell population is contacted with about 0.5 uM to about 5 uM ODNA151. In embodiments, a T cell population is contacted with about 0.75 uM to about 5 uM ODNA151. In embodiments, a T cell population is contacted with about 1 uM to about 5 uM ODNA151. In embodiments, a T cell population is contacted with about 1.25 uM to about 5 uM ODNA151. In embodiments, a T cell population is contacted with about 1.5 uM to about 5 uM ODNA151. In embodiments, a T cell population is contacted with about 1.75 uM to about 5 uM ODNA151. In embodiments, a T cell population is contacted with about 2 uM to about 5 uM ODNA151. In embodiments, a T cell population is contacted with about 2.25uM to about 5uM ODNA151. In embodiments, a T cell population is contacted with about 2.5uM to about 5uM ODNA151. In embodiments, a T cell population is contacted with about 2.75uM to about 5uM ODNA151. In embodiments, a T cell population is contacted with about 3uM to about 5uM ODNA151. In embodiments, a T cell population is contacted with about 3.25uM to about 5uM ODNA151. In embodiments, a T cell population is contacted with about 3.5uM to about 5uM ODNA151. In embodiments, a T cell population is contacted with about 3.75uM to about 5uM ODNA151. In embodiments, a T cell population is contacted with about 4uM to about 5uM ODNA151. In embodiments, a T cell population is contacted with about 4.25uM to about 5uM ODNA151. In embodiments, a population of T cells is contacted with about 4.5 uM to about 5 uM ODNA151. In embodiments, a population of T cells is contacted with about 4.75 uM to about 5 uM ODNA151.

[0149] In embodiments, a T cell population is contacted with about 0.1 uM to about 4.75 uM ODNA151. In embodiments, a T cell population is contacted with about 0.1 uM to about 4.5 uM ODNA151. In embodiments, a T cell population is contacted with about 0.1 uM to about 4.25 uM ODNA151. In embodiments, a T cell population is contacted with about 0.1 uM to about 4 uM ODNA151. In embodiments, a T cell population is contacted with about 0.1 uM to about 3.75 uM ODNA151. In embodiments, a T cell population is contacted with about 0.1 uM to about 3.5 uM ODNA151. In embodiments, a T cell population is contacted with about 0.1 uM to about 3.25 uM ODNA151. In embodiments, a T cell population is contacted with about 0.1 uM to about 3 uM ODNA151. In embodiments, a T cell population is contacted with about 0.1 uM to about 2.75 uM ODNA151. In embodiments, a T cell population is contacted with about 0.1 uM to about 2.5 uM ODNA151. In embodiments, a T cell population is contacted with about 0.1 uM to about 2.25 uM ODNA151. In embodiments, a T cell population is contacted with about 0.1 uM to about 2 uM ODNA151. In embodiments, a T cell population is contacted with about 0.1 uM to about 1.75 uM ODNA151. In embodiments, a T cell population is contacted with about 0.1 uM to about 1.5 uM ODNA151. In embodiments, a T cell population is contacted with about 0.1 uM to about 1.25 uM ODNA151. In embodiments, a T cell population is contacted with about 0.1 uM to about 1 uM ODNA151. In embodiments, a T cell population is contacted with about 0.1 uM to about 0.75 uM ODNA151. In embodiments, a T cell population is contacted with about 0.1 uM to about 0.5 uM ODNA151. In embodiments, a population of T cells is contacted with about 0.1 uM to about 0.25 uM ODNA151. In embodiments, a 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 ODNA151.

[0150] In an embodiment, a T cell population is contacted with about 10uM to about 50uM Aml. In an embodiment, a T cell population is contacted with about 12uM to about 50uM Aml. In an embodiment, a T cell population is contacted with about 14uM to about 50uM Aml. In an embodiment, a T cell population is contacted with about 16uM to about 50uM Aml. In an embodiment, a T cell population is contacted with about 18uM to about 50uM Aml. In an embodiment, a T cell population is contacted with about 20uM to about 50uM Aml. In an embodiment, a T cell population is contacted with about 22uM to about 50uM Aml. In an embodiment, a T cell population is contacted with about 24uM to about 50uM Aml. In an embodiment, a T cell population is contacted with about 26uM to about 50uM Aml. In an embodiment, a T cell population is contacted with about 28uM to about 50uM Aml. In an embodiment, a T cell population is contacted with about 30uM to about 50uM Aml. In an embodiment, a T cell population is contacted with about 32uM to about 50uM Aml. In an embodiment, a T cell population is contacted with about 34uM to about 50uM Aml. In an embodiment, a T cell population is contacted with about 36uM to about 50uM Aml. In an embodiment, a T cell population is contacted with about 38uM to about 50uM Aml. In an embodiment, a T cell population is contacted with about 40uM to about 50uM Aml. In an embodiment, a T cell population is contacted with about 42uM to about 50uM Aml. In an embodiment, a T cell population is contacted with about 44uM to about 50uM Aml. In an embodiment, a T cell population is contacted with about 46uM to about 50uM Aml. In an embodiment, a T cell population is contacted with about 48uM to about 50uM Aml.

[0151] In an embodiment, a T cell population is contacted with about 10uM to about 48uM Aml. In an embodiment, a T cell population is contacted with about 10uM to about 46uM Aml. In an embodiment, a T cell population is contacted with about 10uM to about 44uM Aml. In an embodiment, a T cell population is contacted with about 10uM to about 42uM Aml. In an embodiment, a T cell population is contacted with about 10uM to about 40uM Aml. In an embodiment, a T cell population is contacted with about 10uM to about 38uM Aml. In an embodiment, a T cell population is contacted with about 10uM to about 36uM Aml. In an embodiment, a T cell population is contacted with about 10uM to about 34uM Aml. In an embodiment, a T cell population is contacted with about 10uM to about 32uM Aml. In an embodiment, a T cell population is contacted with about 10uM to about 30uM Aml. In an embodiment, a T cell population is contacted with about 10uM to about 28uM Aml. In an embodiment, a T cell population is contacted with about 10uM to about 26uM Aml. In an embodiment, a T cell population is contacted with about 10uM to about 24uM Aml. In an embodiment, a T cell population is contacted with about 10uM to about 22uM Aml. In an embodiment, a T cell population is contacted with about 10uM to about 20uM Aml. In an embodiment, a T cell population is contacted with about 10uM to about 18uM Aml. In an embodiment, a T cell population is contacted with about 10uM to about 16uM Aml. In an embodiment, a T cell population is contacted with about 10uM to about 14uM Aml. In an embodiment, a T cell population is contacted with about 10uM to about 12uM Aml. In embodiments, a population of T cells is contacted with about 10uM, 12uM, 14uM, 16uM, 18uM, 20uM, 22uM, 24uM, 26uM, 28uM, 30uM, 32uM, 34uM, 36uM, 38uM, 40uM, 42uM, 44uM, 46uM, 48uM, or 50uM Aml.

[0152] In an embodiment, a T cell population is contacted with about 1uM to about 10uM MRT. In an embodiment, a T cell population is contacted with about 1.5uM to about 10uM MRT. In an embodiment, a T cell population is contacted with about 2uM to about 10uM MRT. In an embodiment, a T cell population is contacted with about 2.5uM to about 10uM MRT. In an embodiment, a T cell population is contacted with about 3uM to about 10uM MRT. In an embodiment, a T cell population is contacted with about 3.5uM to about 10uM MRT. In an embodiment, a T cell population is contacted with about 4uM to about 10uM MRT. In an embodiment, a T cell population is contacted with about 4.5uM to about 10uM MRT. In an embodiment, a T cell population is contacted with about 5uM to about 10uM MRT. In an embodiment, a T cell population is contacted with about 5.5uM to about 10uM MRT. In an embodiment, a T cell population is contacted with about 6uM to about 10uM MRT. In an embodiment, a T cell population is contacted with about 6.5uM to about 10uM MRT. In an embodiment, a T cell population is contacted with about 7uM to about 10uM MRT. In an embodiment, a T cell population is contacted with about 7.5uM to about 10uM MRT. In an embodiment, a T cell population is contacted with about 8uM to about 10uM MRT. In an embodiment, a T cell population is contacted with about 8.5uM to about 10uM MRT. In an embodiment, a T cell population is contacted with about 9uM to about 10uM MRT. In an embodiment, a T cell population is contacted with about 9.5uM to about 10uM MRT.

[0153] In an embodiment, a T cell population is contacted with about 1uM to about 9.5uM MRT. In an embodiment, a T cell population is contacted with about 1uM to about 9uM MRT. In an embodiment, a T cell population is contacted with about 1uM to about 8.5uM MRT. In an embodiment, a T cell population is contacted with about 1uM to about 8uM MRT. In an embodiment, a T cell population is contacted with about 1uM to about 7.5uM MRT. In an embodiment, a T cell population is contacted with about 1uM to about 7uM MRT. In an embodiment, a T cell population is contacted with about 1uM to about 6.5uM MRT. In an embodiment, a T cell population is contacted with about 1uM to about 6uM MRT. In an embodiment, a T cell population is contacted with about 1uM to about 5.5uM MRT. In an embodiment, a T cell population is contacted with about 1uM to about 5uM MRT. In an embodiment, a T cell population is contacted with about 1uM to about 4.5uM MRT. In an embodiment, a T cell population is contacted with about 1uM to about 4uM MRT. In an embodiment, a T cell population is contacted with about 1uM to about 3.5uM MRT. In an embodiment, a T cell population is contacted with about 1uM to about 3uM MRT. In an embodiment, a T cell population is contacted with about 1uM to about 2.5uM MRT. In an embodiment, a T cell population is contacted with about 1uM to about 2uM MRT. In an embodiment, a T cell population is contacted with about 1uM to about 1.5uM MRT. In an embodiment, a T cell population is contacted with about 1uM, 1.5uM, 2uM, 2.5uM, 3uM, 3.5uM, 4uM, 4.5uM, 5uM, 5.5uM, 6uM, 6.5uM, 7uM, 7.5uM, 8uM, 8.5uM, 9uM, 9.5uM or 10uM MRT.

[0154] In an embodiment, the gene editing efficiency of the T cell population is increased by at least about 0.5 times to at least about 5 times compared to a T cell population that is not contacted with one or more cGAS-STING pathway inhibitors. In an embodiment, the gene editing efficiency of the T cell population is increased by at least about 1 times to at least about 5 times relative to a T cell population that is not contacted with one or more cGAS-STING pathway inhibitors. In an embodiment, the gene editing efficiency of the T cell population is increased by at least about 1.5 times to at least about 5 times compared to a T cell population that is not contacted with one or more cGAS-STING pathway inhibitors. In an embodiment, the gene editing efficiency of the T cell population is increased by at least about 2 times to at least about 5 times compared to a T cell population that is not contacted with one or more cGAS-STING pathway inhibitors. In an embodiment, the gene editing efficiency of the T cell population is increased by at least about 2.5 times to at least about 5 times compared to a T cell population that is not contacted with one or more cGAS-STING pathway inhibitors. In an embodiment, the gene editing efficiency of the T cell population is increased by at least about 3 times to at least about 5 times compared to a T cell population that is not contacted with one or more cGAS-STING pathway inhibitors. In an embodiment, the gene editing efficiency of the T cell population is increased by at least about 3.5 times to at least about 4 times compared to a T cell population not contacted with one or more cGAS-STING pathway inhibitors. In an embodiment, the gene editing efficiency of the T cell population is increased by at least about 4 times to at least about 5 times compared to a T cell population not contacted with one or more cGAS-STING pathway inhibitors. In an embodiment, the gene editing efficiency of the T cell population is increased by at least about 4.5 times to at least about 5 times compared to a T cell population not contacted with one or more cGAS-STING pathway inhibitors.

[0155] In an embodiment, the gene editing efficiency of the T cell population is increased by at least about 0.5 times to at least about 4.5 times compared to a T cell population that is not contacted with one or more cGAS-STING pathway inhibitors. In an embodiment, the gene editing efficiency of the T cell population is increased by at least about 0.5 times to at least about 4 times compared to a T cell population that is not contacted with one or more cGAS-STING pathway inhibitors. In an embodiment, the gene editing efficiency of the T cell population is increased by at least about 0.5 times to at least about 3.5 times compared to a T cell population that is not contacted with one or more cGAS-STING pathway inhibitors. In an embodiment, the gene editing efficiency of the T cell population is increased by at least about 0.5 times to at least about 3 times compared to a T cell population that is not contacted with one or more cGAS-STING pathway inhibitors. In an embodiment, the gene editing efficiency of the T cell population is increased by at least about 0.5 times to at least about 2.5 times compared to a T cell population that is not contacted with one or more cGAS-STING pathway inhibitors. In an embodiment, the gene editing efficiency of the T cell population is increased by at least about 0.5 times to at least about 2 times compared to a T cell population not contacted with one or more cGAS-STING pathway inhibitors. In an embodiment, the gene editing efficiency of the T cell population is increased by at least about 0.5 times to at least about 1.5 times compared to a T cell population not contacted with one or more cGAS-STING pathway inhibitors. In an embodiment, the gene editing efficiency of the T cell population is increased by at least about 0.5 times to at least about 1 times compared to a T cell population not contacted with one or more cGAS-STING pathway inhibitors. In an embodiment, the gene editing efficiency of the T cell population is increased by at least about 0.5 times, 1 times, 1.5 times, 2 times, 2.5 times, 3 times, 3.5 times, 4 times, 4.5 times or 5 times relative to a T cell population not contacted with one or more cGAS-STING pathway inhibitors.

[0156] In an embodiment, the gene editing efficiency of the T cell population is increased by about 2 times to about 3 times relative to a T cell population that is not contacted with one or more cGAS-STING pathway inhibitors. In an embodiment, the gene editing efficiency of the T cell population is increased by about 2.2 times to about 3 times relative to a T cell population that is not contacted with one or more cGAS-STING pathway inhibitors. In an embodiment, the gene editing efficiency of the T cell population is increased by about 2.4 times to about 3 times relative to a T cell population that is not contacted with one or more cGAS-STING pathway inhibitors. In an embodiment, the gene editing efficiency of the T cell population is increased by about 2.6 times to about 3 times relative to a T cell population that is not contacted with one or more cGAS-STING pathway inhibitors. In an embodiment, the gene editing efficiency of the T cell population is increased by about 2.8 times to about 3 times relative to a T cell population that is not contacted with one or more cGAS-STING pathway inhibitors.

[0157] In an embodiment, the gene editing efficiency of the T cell population is increased by about 2 times to about 2.8 times relative to a T cell population that is not contacted with one or more cGAS-STING pathway inhibitors. In an embodiment, the gene editing efficiency of the T cell population is increased by about 2 times to about 2.6 times relative to a T cell population that is not contacted with one or more cGAS-STING pathway inhibitors. In an embodiment, the gene editing efficiency of the T cell population is increased by about 2 times to about 2.4 times relative to a T cell population that is not contacted with one or more cGAS-STING pathway inhibitors. In an embodiment, the gene editing efficiency of the T cell population is increased by about 2 times to about 2.2 times relative to a T cell population that is not contacted with one or more cGAS-STING pathway inhibitors. In an embodiment, the gene editing efficiency of the T cell population is increased by about 2 times, 2.2 times, 2.4 times, 2.6 times, 2.8 times or 3 times relative to a T cell population that is not contacted with one or more cGAS-STING pathway inhibitors.

[0158] In embodiments, the gene editing efficiency of the T cell population is increased by about 60% to about 99% relative to a T cell population that has not been contacted with one or more cGAS-STING pathway inhibitors. In embodiments, the gene editing efficiency of the T cell population is increased by about 63% to about 99% relative to a T cell population that has not been contacted with one or more cGAS-STING pathway inhibitors. In embodiments, the gene editing efficiency of the T cell population is increased by about 66% to about 99% relative to a T cell population that has not been contacted with one or more cGAS-STING pathway inhibitors. In embodiments, the gene editing efficiency of the T cell population is increased by about 69% to about 99% relative to a T cell population that has not been contacted with one or more cGAS-STING pathway inhibitors. In embodiments, the gene editing efficiency of the T cell population is increased by about 72% to about 99% relative to a T cell population that has not been contacted with one or more cGAS-STING pathway inhibitors. In embodiments, the gene editing efficiency of the T cell population is increased by about 75% to about 99% relative to a T cell population that has not been contacted with one or more cGAS-STING pathway inhibitors. In embodiments, the gene editing efficiency of the T cell population is increased by about 78% to about 99% relative to a T cell population that is not contacted with one or more cGAS-STING pathway inhibitors. In embodiments, the gene editing efficiency of the T cell population is increased by about 81% to about 99% relative to a T cell population that is not contacted with one or more cGAS-STING pathway inhibitors. In embodiments, the gene editing efficiency of the T cell population is increased by about 84% to about 99% relative to a T cell population that is not contacted with one or more cGAS-STING pathway inhibitors. In embodiments, the knockout efficiency of the T cell population is increased by about 87% to about 99% relative to a T cell population that is not contacted with one or more cGAS-STING pathway inhibitors. In embodiments, the gene editing efficiency of the T cell population is increased by about 90% to about 99% relative to a T cell population that is not contacted with one or more cGAS-STING pathway inhibitors. In embodiments, the gene editing efficiency of the T cell population is increased by about 93% to about 99% relative to a T cell population that is not contacted with one or more cGAS-STING pathway inhibitors. In embodiments, the gene editing efficiency of the T cell population is increased by about 96% to about 99% relative to a T cell population not contacted with one or more cGAS-STING pathway inhibitors.

[0159] In embodiments, the gene editing efficiency of the T cell population is increased by about 60% to about 96% relative to a T cell population that is not contacted with one or more cGAS-STING pathway inhibitors. In embodiments, the gene editing efficiency of the T cell population is increased by about 60% to about 93% relative to a T cell population that is not contacted with one or more cGAS-STING pathway inhibitors. In embodiments, the gene editing efficiency of the T cell population is increased by about 60% to about 90% relative to a T cell population that is not contacted with one or more cGAS-STING pathway inhibitors. In embodiments, the gene editing efficiency of the T cell population is increased by about 60% to about 87% relative to a T cell population that is not contacted with one or more cGAS-STING pathway inhibitors. In embodiments, the gene editing efficiency of the T cell population is increased by about 60% to about 84% relative to a T cell population that is not contacted with one or more cGAS-STING pathway inhibitors. In embodiments, the gene editing efficiency of the T cell population is increased by about 60% to about 81% relative to a T cell population that is not contacted with one or more cGAS-STING pathway inhibitors. In embodiments, the gene editing efficiency of the T cell population is increased by about 60% to about 78% relative to a T cell population that has not been contacted with one or more cGAS-STING pathway inhibitors. In embodiments, the gene editing efficiency of the T cell population is increased by about 60% to about 75% relative to a T cell population that has not been contacted with one or more cGAS-STING pathway inhibitors. In embodiments, the gene editing efficiency of the T cell population is increased by about 60% to about 72% relative to a T cell population that has not been contacted with one or more cGAS-STING pathway inhibitors. In embodiments, the gene editing efficiency of the T cell population is increased by about 60% to about 69% relative to a T cell population that has not been contacted with one or more cGAS-STING pathway inhibitors. In embodiments, the gene editing efficiency of the T cell population is increased by about 60% to about 66% relative to a T cell population that has not been contacted with one or more cGAS-STING pathway inhibitors. In embodiments, the gene editing efficiency of the T cell population is increased by about 60% to about 63% relative to a T cell population that has not been contacted with one or more cGAS-STING pathway inhibitors. In embodiments, the gene editing efficiency of the T cell population is increased by about 60%, 63%, 66%, 69%, 72%, 75%, 78%, 81%, 84%, 87%, 90%, 93%, 96% or 99% relative to a T cell population not contacted with one or more cGAS-STING pathway inhibitors.

[0160] In an embodiment, the knockout efficiency of the engineered T cell population is about 20% to about 100%. In an embodiment, the knockout efficiency of the engineered T cell population is about 30% to about 100%. In an embodiment, the knockout efficiency of the engineered T cell population is about 40% to about 100%. In an embodiment, the knockout efficiency of the engineered T cell population is about 50% to about 100%. In an embodiment, the knockout efficiency of the engineered T cell population is about 60% to about 100%. In an embodiment, the knockout efficiency of the engineered T cell population is about 70% to about 100%. In an embodiment, the knockout efficiency of the engineered T cell population is about 70% to about 99%. In an embodiment, the knockout efficiency of the engineered T cell population is about 72% to about 99%. In an embodiment, the knockout efficiency of the engineered T cell population is about 74% to about 99%. In an embodiment, the knockout efficiency of the engineered T cell population is about 76% to about 99%. In an embodiment, the knockout efficiency of the engineered T cell population is about 78% to about 99%. In an embodiment, the knockout efficiency of the engineered T cell population is about 80% to about 99%. In an embodiment, the knockout efficiency of the engineered T cell population is about 82% to about 99%. In an embodiment, the knockout efficiency of the engineered T cell population is about 84% to about 99%. In an embodiment, the knockout efficiency of the engineered T cell population is about 86% to about 99%. In an embodiment, the knockout efficiency of the engineered T cell population is about 88% to about 99%. In an embodiment, the knockout efficiency of the engineered T cell population is about 90% to about 99%. In an embodiment, the knockout efficiency of the engineered T cell population is about 92% to about 99%. In an embodiment, the knockout efficiency of the engineered T cell population is about 94% to about 99%. In an embodiment, the knockout efficiency of the engineered T cell population is about 96% to about 99%. In an embodiment, the knockout efficiency of the engineered T cell population is about 98% to about 99%.

[0161] In an embodiment, the knockout efficiency of the engineered T cell population is about 50% to about 98%. In an embodiment, the knockout efficiency of the engineered T cell population is about 60% to about 98%. In an embodiment, the knockout efficiency of the engineered T cell population is about 70% to about 98%. In an embodiment, the knockout efficiency of the engineered T cell population is about 70% to about 96%. In an embodiment, the knockout efficiency of the engineered T cell population is about 70% to about 94%. In an embodiment, the knockout efficiency of the engineered T cell population is about 70% to about 92%. In an embodiment, the knockout efficiency of the engineered T cell population is about 70% to about 90%. In an embodiment, the knockout efficiency of the engineered T cell population is about 70% to about 88%. In an embodiment, the knockout efficiency of the engineered T cell population is about 70% to about 86%. In an embodiment, the knockout efficiency of the engineered T cell population is about 70% to about 84%. In an embodiment, the knockout efficiency of the engineered T cell population is about 70% to about 82%. In an embodiment, the knockout efficiency of the engineered T cell population is about 70% to about 80%. In an embodiment, the knockout efficiency of the engineered T cell population is about 70% to about 78%. In an embodiment, the knockout efficiency of the engineered T cell population is about 70% to about 76%. In an embodiment, the knockout efficiency of the engineered T cell population is about 70% to about 74%. In an embodiment, the knockout efficiency of the engineered T cell population is about 70% to about 72%. In an embodiment, the knockout efficiency of the engineered T cell population 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 an embodiment, the knockout efficiency of the T cell population is about 90%. In embodiments, the knockout efficiency of the T cell population is 90%.

[0162] In an embodiment, the knock-in efficiency of a T cell population is about 20% to about 100%. In an embodiment, the knock-in efficiency of a T cell population is about 20% to about 99%. In an embodiment, the knock-in efficiency of a T cell population is about 25% to about 99%. In an embodiment, the knock-in efficiency of a T cell population is about 30% to about 99%. In an embodiment, the knock-in efficiency of a T cell population is about 35% to about 99%. In an embodiment, the knock-in efficiency of a T cell population is about 40% to about 99%. In an embodiment, the knock-in efficiency of a T cell population is about 45% to about 99%. In an embodiment, the knock-in efficiency of a T cell population is about 50% to about 99%. In an embodiment, the knock-in efficiency of a T cell population is about 55% to about 99%. In an embodiment, the knock-in efficiency of a T cell population is about 60% to about 99%. In an embodiment, the knock-in efficiency of a T cell population is about 65% to about 99%. In an embodiment, the knock-in efficiency of a T cell population is about 70% to about 99%. In embodiments, the knock-in efficiency of a T cell population is about 75% to about 99%. In embodiments, the knock-in efficiency of a T cell population is about 80% to about 99%. In embodiments, the knock-in efficiency of a T cell population is about 85% to about 99%. In embodiments, the knock-in efficiency of a T cell population is about 90% to about 99%. In embodiments, the knock-in efficiency of a T cell population is about 95% to about 99%.

[0163] In an embodiment, the knock-in efficiency of a T cell population is about 20% to about 95%. In an embodiment, the knock-in efficiency of a T cell population is about 20% to about 90%. In an embodiment, the knock-in efficiency of a T cell population is about 20% to about 85%. In an embodiment, the knock-in efficiency of a T cell population is about 20% to about 80%. In an embodiment, the knock-in efficiency of a T cell population is about 20% to about 75%. In an embodiment, the knock-in efficiency of a T cell population is about 20% to about 70%. In an embodiment, the knock-in efficiency of a T cell population is about 20% to about 65%. In an embodiment, the knock-in efficiency of a T cell population is about 20% to about 60%. In an embodiment, the knock-in efficiency of a T cell population is about 20% to about 55%. In an embodiment, the knock-in efficiency of a T cell population is about 20% to about 50%. In an embodiment, the knock-in efficiency of a T cell population is about 20% to about 45%. In an embodiment, the knock-in efficiency of a T cell population is about 20% to about 40%. In an embodiment, the knock-in efficiency of a T cell population is about 20% to about 35%. In an embodiment, the knock-in efficiency of a T cell population is about 20% to about 30%. In an embodiment, the knock-in efficiency of a T cell population is about 20% to about 25%. In an embodiment, the knock-in efficiency of a T cell population is about 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or 100%. In an embodiment, the knock-in efficiency is about 60%. In an embodiment, the knock-in efficiency is 60%.

[0164] In particular, a method for increasing the expansion of engineered T cells is provided herein. It is expected that the method provided herein effectively improves the expansion of engineered T cells, thereby increasing the total number of engineered T cells in culture. The expansion of the engineered T cells is improved compared to the engineered T cells generated without contact with one or more cGAS-STING pathway inhibitors. Therefore, in one aspect, a method for increasing the expansion of an engineered T cell population is provided, including i) contacting a T cell population with one or more cyclic GMP-AMP synthase (cGAS)-interferon gene stimulator (STING) pathway inhibitors and nucleic acids to form an engineered T cell population, and ii) amplifying the engineered T cell population to form an amplified engineered T cell population, wherein relative to the engineered T cell population in which the T cell population in step i) is not contacted with one or more cGAS-STING pathway inhibitors, one or more cGAS-STING pathway inhibitors increase the amplified engineered T cell population.

[0165] In an embodiment, the nucleic acid comprises a donor DNA. In an embodiment, the nucleic acid is a plasmid, nanoplasmid, minicircle or viral vector comprising the donor DNA. In an embodiment, the donor DNA encodes an exogenous T cell receptor (TCR)-β or a fragment thereof, an exogenous TCR-α or a fragment thereof, or a combination thereof. In an embodiment, the nucleic acid is associated with a delivery vehicle. In an embodiment, the delivery vehicle is a lipid particle or a nanoparticle.

[0166] In embodiments, the T cell population comprises primary T cells.

[0167] For the methods provided herein, in embodiments, step i) further comprises contacting the T cells with a gene editing agent. In embodiments, contacting the T cells with a gene editing agent comprises contacting the T cells with a nucleic acid sequence encoding a gene editing agent. In embodiments, the T cells are contacted with nucleic acid in the presence of a gene editing agent or a nucleic acid sequence encoding a gene editing agent.

[0168] 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.

[0169] In embodiments, contacting the T cell with a gene editing agent comprises transfecting the T cell with the gene editing agent. In embodiments, contacting the T cell with a nucleic acid comprises transfecting the T cell with a nucleic acid.

[0170] In an embodiment, one or more cGAS-STING pathway inhibitors include a cGAS inhibitor. In an embodiment, one or more cGAS-STING pathway inhibitors include a TANK binding kinase 1 (TBK1) inhibitor. In an embodiment, one or more cGAS-STING pathway inhibitors are aml, MRT67307 (MRT), BX795, H151, ODN-A151 (ODN151), Ru.521, G140 or a combination thereof. In an embodiment, one or more cGAS-STING pathway inhibitors include Aml. In an embodiment, one or more cGAS-STING pathway inhibitors include MRT. In an embodiment, one or more cGAS-STING pathway inhibitors include BX795. In an embodiment, one or more cGAS-STING pathway inhibitors include H151. In an embodiment, one or more cGAS-STING pathway inhibitors include ODN-A151. In an embodiment, one or more cGAS-STING pathway inhibitors include ODN151. In an embodiment, one or more cGAS-STING pathway inhibitors include Ru.521. In embodiments, the one or more cGAS-STING pathway inhibitors include G140.

[0171] In embodiments, the cGAS-STING pathway inhibitor is Aml. 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 Aml, and there are no other cGAS-STING pathway inhibitors. In embodiments, the cGAS-STING pathway inhibitor is MRT, and there are no other cGAS-STING pathway inhibitors. In embodiments, the cGAS-STING pathway inhibitor is BX795, and there are no other cGAS-STING pathway inhibitors. In embodiments, the cGAS-STING pathway inhibitor is H151, and there are no other cGAS-STING pathway inhibitors. In embodiments, the cGAS-STING pathway inhibitor is ODN-A151, and there are no other cGAS-STING pathway inhibitors. In embodiments, the cGAS-STING pathway inhibitor is ODN151, and there are no other cGAS-STING pathway inhibitors. In embodiments, the cGAS-STING pathway inhibitor is Ru.521, and there are no other cGAS-STING pathway inhibitors. In embodiments, the cGAS-STING pathway inhibitor is G140, and there are no other cGAS-STING pathway inhibitors.

[0172] In embodiments, a population of T cells is independently contacted with one or more cGAS-STING pathway inhibitors at about 1uM to about 50uM. In embodiments, a population of T cells is independently contacted with one or more cGAS-STING pathway inhibitors at about 2uM to about 50uM. In embodiments, a population of T cells is independently contacted with one or more cGAS-STING pathway inhibitors at about 4uM to about 50uM. In embodiments, a population of T cells is independently contacted with one or more cGAS-STING pathway inhibitors at about 6uM to about 50uM. In embodiments, a population of T cells is independently contacted with one or more cGAS-STING pathway inhibitors at about 8uM to about 50uM. In embodiments, a population of T cells is independently contacted with one or more cGAS-STING pathway inhibitors at about 10uM to about 50uM. In embodiments, a population of T cells is independently contacted with one or more cGAS-STING pathway inhibitors at about 12uM to about 50uM. In embodiments, a population of T cells is independently contacted with one or more cGAS-STING pathway inhibitors at about 14uM to about 50uM. In embodiments, a population of T cells is independently contacted with one or more cGAS-STING pathway inhibitors at about 16uM to about 50uM. In embodiments, a population of T cells is independently contacted with one or more cGAS-STING pathway inhibitors at about 18uM to about 50uM. In embodiments, a population of T cells is independently contacted with one or more cGAS-STING pathway inhibitors at about 20uM to about 50uM. In embodiments, a population of T cells is independently contacted with one or more cGAS-STING pathway inhibitors at about 22uM to about 50uM. In embodiments, a population of T cells is independently contacted with one or more cGAS-STING pathway inhibitors at about 24uM to about 50uM. In embodiments, a population of T cells is independently contacted with one or more cGAS-STING pathway inhibitors at about 26uM to about 50uM. In embodiments, a population of T cells is independently contacted with one or more cGAS-STING pathway inhibitors at about 28uM to about 50uM. In embodiments, a population of T cells is independently contacted with about 30uM to about 50uM of one or more cGAS-STING pathway inhibitors. In embodiments, a population of T cells is independently contacted with about 32uM to about 50uM of one or more cGAS-STING pathway inhibitors. In embodiments, a population of T cells is independently contacted with about 34uM to about 50uM of one or more cGAS-STING pathway inhibitors. In embodiments, a population of T cells is independently contacted with about 36uM to about 50uM of one or more cGAS-STING pathway inhibitors. In embodiments, a population of T cells is independently contacted with about 38uM to about 50uM of one or more cGAS-STING pathway inhibitors.In embodiments, a population of T cells is independently contacted with about 40uM to about 50uM of one or more cGAS-STING pathway inhibitors. In embodiments, a population of T cells is independently contacted with about 42uM to about 50uM of one or more cGAS-STING pathway inhibitors. In embodiments, a population of T cells is independently contacted with about 44uM to about 50uM of one or more cGAS-STING pathway inhibitors. In embodiments, a population of T cells is independently contacted with about 46uM to about 50uM of one or more cGAS-STING pathway inhibitors. In embodiments, a population of T cells is independently contacted with about 48uM to about 50uM of one or more cGAS-STING pathway inhibitors.

[0173] In embodiments, a population of T cells is independently contacted with one or more cGAS-STING pathway inhibitors from about 1uM to about 48uM. In embodiments, a population of T cells is independently contacted with one or more cGAS-STING pathway inhibitors from about 1uM to about 46uM. In embodiments, a population of T cells is independently contacted with one or more cGAS-STING pathway inhibitors from about 1uM to about 44uM. In embodiments, a population of T cells is independently contacted with one or more cGAS-STING pathway inhibitors from about 1uM to about 42uM. In embodiments, a population of T cells is independently contacted with one or more cGAS-STING pathway inhibitors from about 1uM to about 40uM. In embodiments, a population of T cells is independently contacted with one or more cGAS-STING pathway inhibitors from about 1uM to about 38uM. In embodiments, a population of T cells is independently contacted with one or more cGAS-STING pathway inhibitors from about 1uM to about 36uM. In embodiments, a population of T cells is independently contacted with one or more cGAS-STING pathway inhibitors from about 1uM to about 32uM. In embodiments, a population of T cells is independently contacted with one or more cGAS-STING pathway inhibitors from about 1uM to about 30uM. In embodiments, a population of T cells is independently contacted with one or more cGAS-STING pathway inhibitors from about 1uM to about 28uM. In embodiments, a population of T cells is independently contacted with one or more cGAS-STING pathway inhibitors from about 1uM to about 26uM. In embodiments, a population of T cells is independently contacted with one or more cGAS-STING pathway inhibitors from about 1uM to about 24uM. In embodiments, a population of T cells is independently contacted with one or more cGAS-STING pathway inhibitors from about 1uM to about 22uM. In embodiments, a population of T cells is independently contacted with one or more cGAS-STING pathway inhibitors from about 1uM to about 20uM. In embodiments, a population of T cells is independently contacted with one or more cGAS-STING pathway inhibitors from about 1uM to about 18uM. In embodiments, a population of T cells is independently contacted with one or more cGAS-STING pathway inhibitors from about 1uM to about 16uM. In embodiments, populations of T cells are independently contacted with about 1 uM to about 14 uM of one or more cGAS-STING pathway inhibitors. In embodiments, populations of T cells are independently contacted with about 1 uM to about 12 uM of one or more cGAS-STING pathway inhibitors. In embodiments, populations of T cells are independently contacted with about 1 uM to about 10 uM of one or more cGAS-STING pathway inhibitors. In embodiments, populations of T cells are independently contacted with about 1 uM to about 8 uM of one or more cGAS-STING pathway inhibitors.In embodiments, a population of T cells is independently contacted with about 1uM to about 6uM of one or more cGAS-STING pathway inhibitors. In embodiments, a population of T cells is independently contacted with about 1uM to about 4uM of one or more cGAS-STING pathway inhibitors. In embodiments, a population of T cells is independently contacted with about 1uM to about 2uM of one or more cGAS-STING pathway inhibitors. In embodiments, a population of T cells is independently contacted with about 1uM, 2uM, 4uM, 6uM, 8uM, 10uM, 12uM, 14uM, 16uM, 18uM, 20uM, 22uM, 24uM, 26uM, 28uM, 30uM, 32uM, 34uM, 36uM, 38uM, 40uM, 42uM, 44uM, 46uM, 48uM or 50uM of one or more cGAS-STING pathway inhibitors.

[0174] In embodiments, a population of T cells is contacted with a nucleic acid in the presence of one or more cGAS-STING pathway inhibitors. In embodiments, a population of T cells is contacted with a nucleic acid and one or more cGAS-STING pathway inhibitors sequentially. In embodiments, a population of T cells is contacted with one or more cGAS-STING pathway inhibitors prior to contacting with the nucleic acid.

[0175] In embodiments, T cells are contacted with one cGAS-STING pathway inhibitor. In embodiments, T cells are contacted with one cGAS-STING pathway inhibitor provided herein and no other cGAS-STING pathway inhibitor.

[0176] In an embodiment, a T cell population is contacted with about 1uM to about 10uM BX795. In an embodiment, a T cell population is contacted with about 0.5uM to about 10uM BX795. In an embodiment, a T cell population is contacted with about 2uM to about 10uM BX795. In an embodiment, a T cell population is contacted with about 2.5uM to about 10uM BX795. In an embodiment, a T cell population is contacted with about 3uM to about 10uM BX795. In an embodiment, a T cell population is contacted with about 3.5uM to about 10uM BX795. In an embodiment, a T cell population is contacted with about 4uM to about 10uM BX795. In an embodiment, a T cell population is contacted with about 4.5uM to about 10uM BX795. In an embodiment, a T cell population is contacted with about 5uM to about 10uM BX795. In an embodiment, a T cell population is contacted with about 5.5uM to about 10uM BX795. In an embodiment, a T cell population is contacted with about 6uM to about 10uM BX795. In an embodiment, a T cell population is contacted with about 6.5uM to about 10uM BX795. In an embodiment, a T cell population is contacted with about 7uM to about 10uM BX795. In an embodiment, a T cell population is contacted with about 7.5uM to about 10uM BX795. In an embodiment, a T cell population is contacted with about 8uM to about 10uM BX795. In an embodiment, a T cell population is contacted with about 8.5uM to about 10uM BX795. In an embodiment, a T cell population is contacted with about 8uM to about 10uM BX795. In an embodiment, a T cell population is contacted with about 8.5uM to about 10uM BX795. In an embodiment, a T cell population is contacted with about 8.5uM to about 10uM BX795. In embodiments, a population of T cells is contacted with about 9 uM to about 10 uM BX795. In embodiments, a population of T cells is contacted with about 9.5 uM to about 10 uM BX795.

[0177] In an embodiment, a T cell population is contacted with about 1uM to about 9.5uM BX795. In an embodiment, a T cell population is contacted with about 0.5uM to about 9uM BX795. In an embodiment, a T cell population is contacted with about 1uM to about 8.5uM BX795. In an embodiment, a T cell population is contacted with about 0.5uM to about 8uM BX795. In an embodiment, a T cell population is contacted with about 1uM to about 7.5uM BX795. In an embodiment, a T cell population is contacted with about 0.5uM to about 7uM BX795. In an embodiment, a T cell population is contacted with about 1uM to about 6.5uM BX795. In an embodiment, a T cell population is contacted with about 0.5uM to about 6uM BX795. In an embodiment, a T cell population is contacted with about 1uM to about 5.5uM BX795. In an embodiment, a T cell population is contacted with about 0.5uM to about 5uM BX795. In an embodiment, a T cell population is contacted with about 1uM to about 4.5uM BX795. In an embodiment, a T cell population is contacted with about 0.5uM to about 4uM BX795. In an embodiment, a T cell population is contacted with about 1uM to about 3.5uM BX795. In an embodiment, a T cell population is contacted with about 0.5uM to about 3uM BX795. In an embodiment, a T cell population is contacted with about 1uM to about 2.5uM BX795. In an embodiment, a T cell population is contacted with about 0.5uM to about 2uM BX795. In an embodiment, a T cell population is contacted with about 1uM to about 1.5uM BX795. In an embodiment, a T cell population is contacted with about 0.5uM to about 1uM BX795. In embodiments, a 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.

[0178] In embodiments, a T cell population is contacted with about 0.1 uM to about 8 uM ODNA151. In embodiments, a T cell population is contacted with about 0.5 uM to about 8 uM ODNA151. In embodiments, a T cell population is contacted with about 1 uM to about 8 uM ODNA151. In embodiments, a T cell population is contacted with about 1.5 uM to about 8 uM ODNA151. In embodiments, a T cell population is contacted with about 2 uM to about 8 uM ODNA151. In embodiments, a T cell population is contacted with about 2.5 uM to about 8 uM ODNA151. In embodiments, a T cell population is contacted with about 3 uM to about 8 uM ODNA151. In embodiments, a T cell population is contacted with about 3.5 uM to about 8 uM ODNA151. In embodiments, a T cell population is contacted with about 4 uM to about 8 uM ODNA151. In embodiments, a T cell population is contacted with about 4.5 uM to about 8 uM ODNA151. In embodiments, a T cell population is contacted with about 5uM to about 8uM ODNA151. In embodiments, a T cell population is contacted with about 5.5uM to about 8uM ODNA151. In embodiments, a T cell population is contacted with about 6uM to about 8uM ODNA151. In embodiments, a T cell population is contacted with about 6.5uM to about 8uM ODNA151. In embodiments, a T cell population is contacted with about 7uM to about 8uM ODNA151. In embodiments, a T cell population is contacted with about 7.5uM to about 8uM ODNA151.

[0179] In embodiments, a T cell population is contacted with about 0.1 uM to about 7.5 uM ODNA151. In embodiments, a T cell population is contacted with about 0.1 uM to about 7 uM ODNA151. In embodiments, a T cell population is contacted with about 0.1 uM to about 7.5 uM ODNA151. In embodiments, a T cell population is contacted with about 0.1 uM to about 7 uM ODNA151. In embodiments, a T cell population is contacted with about 0.1 uM to about 6.5 uM ODNA151. In embodiments, a T cell population is contacted with about 0.1 uM to about 6 uM ODNA151. In embodiments, a T cell population is contacted with about 0.1 uM to about 5.5 uM ODNA151. In embodiments, a T cell population is contacted with about 0.1 uM to about 5 uM ODNA151. In embodiments, a T cell population is contacted with about 0.1 uM to about 4.5 uM ODNA151. In embodiments, a T cell population is contacted with about 0.1 uM to about 4 uM ODNA151. In embodiments, a T cell population is contacted with about 0.1 uM to about 3.5 uM ODNA151. In embodiments, a T cell population is contacted with about 0.1 uM to about 3 uM ODNA151. In embodiments, a T cell population is contacted with about 0.1 uM to about 2.5 uM ODNA151. In embodiments, a T cell population is contacted with about 0.1 uM to about 2 uM ODNA151. In embodiments, a T cell population is contacted with about 0.1 uM to about 1.5 uM ODNA151. In embodiments, a T cell population is contacted with about 0.1 uM to about 1 uM ODNA151. In embodiments, a T cell population is contacted with about 0.1 uM to about 0.5 uM ODNA151. In embodiments, a 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 ODNA151.

[0180] In an embodiment, a T cell population is contacted with about 5uM to about 50uM AML. In an embodiment, a T cell population is contacted with about 15uM to about 50uM AML. In an embodiment, a T cell population is contacted with about 20uM to about 50uM AML. In an embodiment, a T cell population is contacted with about 25uM to about 50uM AML. In an embodiment, a T cell population is contacted with about 30uM to about 50uM AML. In an embodiment, a T cell population is contacted with about 35uM to about 50uM AML. In an embodiment, a T cell population is contacted with about 40uM to about 50uM AML. In an embodiment, a T cell population is contacted with about 45uM to about 50uM AML.

[0181] In an embodiment, a T cell population is contacted with about 5uM to about 45uM AML. In an embodiment, a T cell population is contacted with about 5uM to about 40uM AML. In an embodiment, a T cell population is contacted with about 5uM to about 35uM AML. In an embodiment, a T cell population is contacted with about 5uM to about 30uM AML. In an embodiment, a T cell population is contacted with about 5uM to about 25uM AML. In an embodiment, a T cell population is contacted with about 5uM to about 20uM AML. In an embodiment, a T cell population is contacted with about 5uM to about 15uM AML. In an embodiment, a T cell population is contacted with about 5uM to about 10uM AML. In an embodiment, a T cell population is contacted with about 5uM, 10uM, 15uM, 20uM, 25uM, 30uM, 35uM, 40uM, 45uM or 50uM AML.

[0182] In an embodiment, a T cell population is contacted with about 1uM to about 25uM MRT. In an embodiment, a T cell population is contacted with about 2uM to about 25uM MRT. In an embodiment, a T cell population is contacted with about 3uM to about 25uM MRT. In an embodiment, a T cell population is contacted with about 4uM to about 25uM MRT. In an embodiment, a T cell population is contacted with about 5uM to about 25uM MRT. In an embodiment, a T cell population is contacted with about 6uM to about 25uM MRT. In an embodiment, a T cell population is contacted with about 7uM to about 25uM MRT. In an embodiment, a T cell population is contacted with about 8uM to about 25uM MRT. In an embodiment, a T cell population is contacted with about 9uM to about 25uM MRT. In an embodiment, a T cell population is contacted with about 10uM to about 25uM MRT. In an embodiment, a T cell population is contacted with about 11uM to about 25uM MRT. In an embodiment, a T cell population is contacted with about 12uM to about 25uM MRT. In an embodiment, a T cell population is contacted with about 13uM to about 25uM MRT. In an embodiment, a T cell population is contacted with about 14uM to about 25uM MRT. In an embodiment, a T cell population is contacted with about 15uM to about 25uM MRT. In an embodiment, a T cell population is contacted with about 16uM to about 25uM MRT. In an embodiment, a T cell population is contacted with about 17uM to about 25uM MRT. In an embodiment, a T cell population is contacted with about 18uM to about 25uM MRT. In an embodiment, a T cell population is contacted with about 19uM to about 25uM MRT. In an embodiment, a T cell population is contacted with about 20uM to about 25uM MRT. In an embodiment, a T cell population is contacted with about 21uM to about 25uM MRT. In an embodiment, a T cell population is contacted with about 22uM to about 25uM MRT. In embodiments, a population of T cells is contacted with about 23uM to about 25uM MRT. In embodiments, a population of T cells is contacted with about 24uM to about 25uM MRT.

[0183] In an embodiment, a T cell population is contacted with about 1uM to about 24uM MRT. In an embodiment, a T cell population is contacted with about 1uM to about 23uM MRT. In an embodiment, a T cell population is contacted with about 1uM to about 22uM MRT. In an embodiment, a T cell population is contacted with about 1uM to about 21uM MRT. In an embodiment, a T cell population is contacted with about 1uM to about 20uM MRT. In an embodiment, a T cell population is contacted with about 1uM to about 19uM MRT. In an embodiment, a T cell population is contacted with about 1uM to about 18uM MRT. In an embodiment, a T cell population is contacted with about 1uM to about 17uM MRT. In an embodiment, a T cell population is contacted with about 1uM to about 16uM MRT. In an embodiment, a T cell population is contacted with about 1uM to about 15uM MRT. In an embodiment, a T cell population is contacted with about 1uM to about 14uM MRT. In an embodiment, a T cell population is contacted with about 1uM to about 13uM MRT. In an embodiment, a T cell population is contacted with about 1uM to about 12uM MRT. In an embodiment, a T cell population is contacted with about 1uM to about 11uM MRT. In an embodiment, a T cell population is contacted with about 1uM to about 10uM MRT. In an embodiment, a T cell population is contacted with about 1uM to about 9uM MRT. In an embodiment, a T cell population is contacted with about 1uM to about 8uM MRT. In an embodiment, a T cell population is contacted with about 1uM to about 7uM MRT. In an embodiment, a T cell population is contacted with about 1uM to about 6uM MRT. In an embodiment, a T cell population is contacted with about 1uM to about 5uM MRT. In an embodiment, a T cell population is contacted with about 1uM to about 4uM MRT. In an embodiment, a T cell population is contacted with about 1uM to about 3uM MRT. In an embodiment, a T cell population is contacted with about 1uM to about 2uM MRT. In embodiments, a population of T cells is contacted with about 1 uM, 2uM, 3uM, 4uM, 5uM, 6uM, 7uM, 8uM, 9uM, 10uM, 11uM, 12uM, 13uM, 14uM, 15uM, 16uM, 17uM, 18uM, 19uM, 20uM, 21uM, 22uM, 23uM, 24uM or 25uM MRT.

[0184] In embodiments, the expanded engineered T cell population increases by at least about 0.5 times to at least about 5 times relative to the engineered T cell population in which the T cell population of step i) is not contacted with one or more cGAS-STING pathway inhibitors. In embodiments, the expanded engineered T cell population increases by at least about 1 times to at least about 5 times relative to the engineered T cell population in which the T cell population of step i) is not contacted with one or more cGAS-STING pathway inhibitors. In embodiments, the expanded engineered T cell population increases by at least about 1.5 times to at least about 5 times relative to the engineered T cell population in which the T cell population of step i) is not contacted with one or more cGAS-STING pathway inhibitors. In embodiments, the expanded engineered T cell population increases by at least about 2 times to at least about 5 times relative to the engineered T cell population in which the T cell population of step i) is not contacted with one or more cGAS-STING pathway inhibitors. In an embodiment, the expanded engineered T cell population increases by at least about 2.5 times to at least about 5 times relative to the engineered T cell population in which the T cell population of step i) is not contacted with one or more cGAS-STING pathway inhibitors. In an embodiment, the expanded engineered T cell population increases by at least about 3 times to at least about 5 times relative to the engineered T cell population in which the T cell population of step i) is not contacted with one or more cGAS-STING pathway inhibitors. In an embodiment, the expanded engineered T cell population increases by at least about 3.5 times to at least about 5 times relative to the engineered T cell population in which the T cell population of step i) is not contacted with one or more cGAS-STING pathway inhibitors. In an embodiment, the expanded engineered T cell population increases by at least about 4 times to at least about 5 times relative to the engineered T cell population in which the T cell population of step i) is not contacted with one or more cGAS-STING pathway inhibitors. In embodiments, the expanded engineered T cell population is increased by at least about 4.5-fold to at least about 5-fold relative to an engineered T cell population in which the T cell population of step i) is not contacted with one or more cGAS-STING pathway inhibitors.

[0185] In an embodiment, the expanded engineered T cell population increases by at least about 0.5 times to at least about 4.5 times relative to the engineered T cell population in which the T cell population of step i) is not contacted with one or more cGAS-STING pathway inhibitors. In an embodiment, the expanded engineered T cell population increases by at least about 0.5 times to at least about 4 times relative to the engineered T cell population in which the T cell population of step i) is not contacted with one or more cGAS-STING pathway inhibitors. In an embodiment, the expanded engineered T cell population increases by at least about 0.5 times to at least about 3.5 times relative to the engineered T cell population in which the T cell population of step i) is not contacted with one or more cGAS-STING pathway inhibitors. In an embodiment, the expanded engineered T cell population increases by at least about 0.5 times to at least about 3 times relative to the engineered T cell population in which the T cell population of step i) is not contacted with one or more cGAS-STING pathway inhibitors. In an embodiment, the expanded engineered T cell population increases by at least about 0.5 times to at least about 2.5 times relative to the engineered T cell population in which the T cell population of step i) is not contacted with one or more cGAS-STING pathway inhibitors. In an embodiment, the expanded engineered T cell population increases by at least about 0.5 times to at least about 2 times relative to the engineered T cell population in which the T cell population of step i) is not contacted with one or more cGAS-STING pathway inhibitors. In an embodiment, the expanded engineered T cell population increases by at least about 0.5 times to at least about 1.5 times relative to the engineered T cell population in which the T cell population of step i) is not contacted with one or more cGAS-STING pathway inhibitors. In an embodiment, the expanded engineered T cell population increases by at least about 0.5 times to at least about 1 times relative to the engineered T cell population in which the T cell population of step i) is not contacted with one or more cGAS-STING pathway inhibitors. In embodiments, the expanded engineered T cell population 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 relative to an engineered T cell population in which the T cell population of step i) is not contacted with one or more cGAS-STING pathway inhibitors.

[0186] For the methods provided herein, in embodiments, the expanded engineered T cell population is increased by about 2-fold to about 3-fold relative to an engineered T cell population in which the T cell population of step i) is not contacted with one or more cGAS-STING pathway inhibitors.

[0187] In embodiments, the engineered T cell population is expanded at least about 0.5-fold to at least about 100-fold relative to an engineered T cell population in which the T cell population of step i) is not contacted with one or more cGAS-STING pathway inhibitors. In embodiments, the engineered T cell population is expanded at least about 5-fold to at least about 100-fold relative to an engineered T cell population in which the T cell population of step i) is not contacted with one or more cGAS-STING pathway inhibitors. In embodiments, the engineered T cell population is expanded at least about 10-fold to at least about 100-fold relative to an engineered T cell population in which the T cell population of step i) is not contacted with one or more cGAS-STING pathway inhibitors. In embodiments, the engineered T cell population is expanded at least about 15-fold to at least about 100-fold relative to an engineered T cell population in which the T cell population of step i) is not contacted with one or more cGAS-STING pathway inhibitors. In embodiments, the engineered T cell population is expanded at least about 20-fold to at least about 100-fold relative to an engineered T cell population in which the T cell population of step i) is not contacted with one or more cGAS-STING pathway inhibitors. In embodiments, the engineered T cell population is expanded at least about 25-fold to at least about 100-fold relative to an engineered T cell population in which the T cell population of step i) is not contacted with one or more cGAS-STING pathway inhibitors. In embodiments, the engineered T cell population is expanded at least about 30-fold to at least about 100-fold relative to an engineered T cell population in which the T cell population of step i) is not contacted with one or more cGAS-STING pathway inhibitors. In embodiments, the engineered T cell population is expanded at least about 35-fold to at least about 100-fold relative to an engineered T cell population in which the T cell population of step i) is not contacted with one or more cGAS-STING pathway inhibitors. In embodiments, the engineered T cell population is expanded at least about 40-fold to at least about 100-fold relative to an engineered T cell population in which the T cell population of step i) is not contacted with one or more cGAS-STING pathway inhibitors. In embodiments, the engineered T cell population is expanded at least about 45-fold to at least about 100-fold relative to an engineered T cell population in which the T cell population of step i) is not contacted with one or more cGAS-STING pathway inhibitors. In embodiments, the engineered T cell population is expanded at least about 50-fold to at least about 100-fold relative to an engineered T cell population in which the T cell population of step i) is not contacted with one or more cGAS-STING pathway inhibitors. In embodiments, the engineered T cell population is expanded at least about 55-fold to at least about 100-fold relative to an engineered T cell population in which the T cell population of step i) is not contacted with one or more cGAS-STING pathway inhibitors.In embodiments, the engineered T cell population is expanded at least about 60-fold to at least about 100-fold relative to an engineered T cell population in which the T cell population of step i) is not contacted with one or more cGAS-STING pathway inhibitors. In embodiments, the engineered T cell population is expanded at least about 65-fold to at least about 100-fold relative to an engineered T cell population in which the T cell population of step i) is not contacted with one or more cGAS-STING pathway inhibitors. In embodiments, the engineered T cell population is expanded at least about 70-fold to at least about 100-fold relative to an engineered T cell population in which the T cell population of step i) is not contacted with one or more cGAS-STING pathway inhibitors. In embodiments, the engineered T cell population is expanded at least about 75-fold to at least about 100-fold relative to an engineered T cell population in which the T cell population of step i) is not contacted with one or more cGAS-STING pathway inhibitors. In embodiments, the engineered T cell population is expanded at least about 80-fold to at least about 100-fold relative to an engineered T cell population in which the T cell population of step i) is not contacted with one or more cGAS-STING pathway inhibitors. In embodiments, the engineered T cell population is expanded at least about 85-fold to at least about 100-fold relative to an engineered T cell population in which the T cell population of step i) is not contacted with one or more cGAS-STING pathway inhibitors. In embodiments, the engineered T cell population is expanded at least about 90-fold to at least about 100-fold relative to an engineered T cell population in which the T cell population of step i) is not contacted with one or more cGAS-STING pathway inhibitors. In embodiments, the engineered T cell population is expanded at least about 95-fold to at least about 100-fold relative to an engineered T cell population in which the T cell population of step i) is not contacted with one or more cGAS-STING pathway inhibitors.

[0188] In embodiments, the engineered T cell population is expanded at least about 0.5-fold to at least about 95-fold relative to an engineered T cell population in which the T cell population of step i) is not contacted with one or more cGAS-STING pathway inhibitors. In embodiments, the engineered T cell population is expanded at least about 0.5-fold to at least about 90-fold relative to an engineered T cell population in which the T cell population of step i) is not contacted with one or more cGAS-STING pathway inhibitors. In embodiments, the engineered T cell population is expanded at least about 0.5-fold to at least about 85-fold relative to an engineered T cell population in which the T cell population of step i) is not contacted with one or more cGAS-STING pathway inhibitors. In embodiments, the engineered T cell population is expanded at least about 0.5-fold to at least about 80-fold relative to an engineered T cell population in which the T cell population of step i) is not contacted with one or more cGAS-STING pathway inhibitors. In embodiments, the engineered T cell population is expanded at least about 0.5-fold to at least about 75-fold relative to an engineered T cell population in which the T cell population of step i) is not contacted with one or more cGAS-STING pathway inhibitors. In embodiments, the engineered T cell population is expanded at least about 0.5-fold to at least about 70-fold relative to an engineered T cell population in which the T cell population of step i) is not contacted with one or more cGAS-STING pathway inhibitors. In embodiments, the engineered T cell population is expanded at least about 0.5-fold to at least about 65-fold relative to an engineered T cell population in which the T cell population of step i) is not contacted with one or more cGAS-STING pathway inhibitors. In embodiments, the engineered T cell population is expanded at least about 0.5-fold to at least about 60-fold relative to an engineered T cell population in which the T cell population of step i) is not contacted with one or more cGAS-STING pathway inhibitors. In embodiments, the engineered T cell population is expanded at least about 0.5-fold to at least about 55-fold relative to an engineered T cell population in which the T cell population of step i) is not contacted with one or more cGAS-STING pathway inhibitors. In embodiments, the engineered T cell population is expanded at least about 0.5-fold to at least about 50-fold relative to an engineered T cell population in which the T cell population of step i) is not contacted with one or more cGAS-STING pathway inhibitors. In embodiments, the engineered T cell population is expanded at least about 0.5-fold to at least about 45-fold relative to an engineered T cell population in which the T cell population of step i) is not contacted with one or more cGAS-STING pathway inhibitors. In embodiments, the engineered T cell population is expanded at least about 0.5-fold to at least about 40-fold relative to an engineered T cell population in which the T cell population of step i) is not contacted with one or more cGAS-STING pathway inhibitors.In embodiments, the engineered T cell population is expanded at least about 0.5-fold to at least about 35-fold relative to an engineered T cell population in which the T cell population of step i) is not contacted with one or more cGAS-STING pathway inhibitors. In embodiments, the engineered T cell population is expanded at least about 0.5-fold to at least about 30-fold relative to an engineered T cell population in which the T cell population of step i) is not contacted with one or more cGAS-STING pathway inhibitors. In embodiments, the engineered T cell population is expanded at least about 0.5-fold to at least about 25-fold relative to an engineered T cell population in which the T cell population of step i) is not contacted with one or more cGAS-STING pathway inhibitors. In embodiments, the engineered T cell population is expanded at least about 0.5-fold to at least about 20-fold relative to an engineered T cell population in which the T cell population of step i) is not contacted with one or more cGAS-STING pathway inhibitors. In embodiments, the engineered T cell population is expanded at least about 0.5-fold to at least about 15-fold relative to an engineered T cell population in which the T cell population of step i) is not contacted with one or more cGAS-STING pathway inhibitors. In embodiments, the engineered T cell population is expanded at least about 0.5-fold to at least about 10-fold relative to an engineered T cell population in which the T cell population of step i) is not contacted with one or more cGAS-STING pathway inhibitors. In embodiments, the engineered T cell population is expanded at least about 0.5-fold to at least about 5-fold relative to an engineered T cell population in which the T cell population of step i) is not contacted with one or more cGAS-STING pathway inhibitors. In embodiments, the engineered T cell population is expanded 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 relative to an engineered T cell population in which the T cell population of step i) is not contacted with one or more cGAS-STING pathway inhibitors.

[0189] Engineered T cell compositions

[0190] In particular, compositions comprising engineered T cells prepared by the methods provided herein (including the examples thereof) are provided herein. In one aspect, an engineered T cell prepared by the methods provided herein (including the examples thereof) is provided.

[0191] In particular, compositions comprising engineered T cell populations prepared by the methods provided herein (including embodiments thereof) are provided herein. The engineered T cell population may have increased viability and / or amplification compared to an engineered T cell population prepared by a method in which the T cell population is not contacted with a cGAS-STING pathway inhibitor before generating the engineered T cell population. Therefore, in one aspect, there is provided an engineered T cell population prepared by contacting a T cell population with a nucleic acid and one or more cyclic GMP-AMP synthase (cGAS)-interferon gene stimulator (STING) pathway inhibitors.

[0192] In an embodiment, the nucleic acid comprises a donor DNA. In an embodiment, the nucleic acid is a plasmid, nanoplasmid, minicircle or viral vector comprising a donor DNA. In an embodiment, the donor DNA encodes an exogenous T cell receptor (TCR)-α or a fragment thereof, an exogenous TCR-β or a fragment thereof, or a combination thereof. In an embodiment, the nucleic acid is associated with a delivery vehicle. In an embodiment, the delivery vehicle is a lipid particle or a nanoparticle.

[0193] In embodiments, the population of T cells comprises primary T cells.

[0194] For the engineered T cell populations provided herein, in embodiments, the T cell population is further contacted with a gene editing reagent. In embodiments, the donor DNA is inserted into an endogenous TCR locus. In embodiments, the endogenous TCR locus is an endogenous TCR-α locus, an endogenous TCR-β locus, or a combination thereof.

[0195] In embodiments, contacting the T cell with a gene editing agent comprises transfecting the T cell with the gene editing agent. In embodiments, contacting the T cell with a nucleic acid comprises transfecting the T cell with a nucleic acid.

[0196] In embodiments, one or more cGAS-STING pathway inhibitors include cGAS inhibitors. In embodiments, one or more cGAS-STING pathway inhibitors include STING inhibitors. In embodiments, one or more cGAS-STING pathway inhibitors include TANK binding kinase 1 (TBK1) inhibitors.

[0197] In an embodiment, one or more cGAS-STING pathway inhibitors are Aml, MRT67307 (MRT), BX795, H151, ODN-A151 (ODN151), Ru.521, G140 or a combination thereof. In an embodiment, one or more cGAS-STING pathway inhibitors are selected from: Aml, BX795, ODN151 and MRT. In an embodiment, one or more cGAS-STING pathway inhibitors are ODN151. In an embodiment, one or more cGAS-STING pathway inhibitors are BX795.

[0198] In embodiments, a population of T cells is contacted with a nucleic acid in the presence of one or more cGAS-STING pathway inhibitors. In embodiments, a population of T cells is contacted with a nucleic acid and one or more cGAS-STING pathway inhibitors sequentially. In embodiments, a population of T cells is contacted with one or more cGAS-STING pathway inhibitors prior to contacting with the nucleic acid.

[0199] In embodiments, a T cell population is contacted with one or more cGAS-STING pathway inhibitors for up to about 10 hours, such as about 2 hours to about 10 hours. In embodiments, a T cell population is contacted with one or more cGAS-STING pathway inhibitors for about 3 hours to about 10 hours. In embodiments, a T cell population is contacted with one or more cGAS-STING pathway inhibitors for about 4 hours to about 10 hours. In embodiments, a T cell population is contacted with one or more cGAS-STING pathway inhibitors for about 5 hours to about 10 hours. In embodiments, a T cell population is contacted with one or more cGAS-STING pathway inhibitors for about 6 hours to about 10 hours. In embodiments, a T cell population is contacted with one or more cGAS-STING pathway inhibitors for about 7 hours to about 10 hours. In embodiments, a T cell population is contacted with one or more cGAS-STING pathway inhibitors for about 8 hours to about 10 hours. In embodiments, a T cell population is contacted with one or more cGAS-STING pathway inhibitors for about 9 hours to about 10 hours.

[0200] In embodiments, a T cell population is contacted with one or more cGAS-STING pathway inhibitors for up to about 9 hours, such as about 2 hours to about 9 hours. In embodiments, a T cell population is contacted with one or more cGAS-STING pathway inhibitors for about 2 hours to about 8 hours. In embodiments, a T cell population is contacted with one or more cGAS-STING pathway inhibitors for about 2 hours to about 7 hours. In embodiments, a T cell population is contacted with one or more cGAS-STING pathway inhibitors for about 2 hours to about 6 hours. In embodiments, a T cell population is contacted with one or more cGAS-STING pathway inhibitors for about 2 hours to about 5 hours. In embodiments, a T cell population is contacted with one or more cGAS-STING pathway inhibitors for about 2 hours to about 4 hours. In embodiments, a T cell population is contacted with one or more cGAS-STING pathway inhibitors for about 2 hours to about 3 hours. In embodiments, a T cell population is contacted with 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, T cells are contacted with one or more cGAS-STING pathway inhibitors for about 6 hours. In embodiments, T cells are contacted with one or more cGAS-STING pathway inhibitors for 6 hours.

[0201] In embodiments, a population of T cells is contacted with one cGAS-STING pathway inhibitor. In embodiments, a population of T cells is contacted with one cGAS-STING pathway inhibitor and no other cGAS-STING pathway inhibitor.

[0202] T cell composition

[0203] Provided herein are compositions comprising a T cell population and one or more cGAS-STING inhibitors, wherein the composition can be used to generate an engineered T cell population. Applicants have demonstrated that one or more cGAS-STING inhibitors increase gene editing efficiency in T cells. The present application has further demonstrated that, compared with compositions not including one or more cGAS-STING inhibitors, compositions provided herein (including embodiments thereof) produce engineered T cell populations with increased cell viability and amplification. Therefore, in one aspect, a composition is provided comprising a T cell population, a nucleic acid, and one or more cyclic GMP-AMP synthase (cGAS)-interferon gene stimulator (STING) pathway inhibitors.

[0204] In an embodiment, the nucleic acid comprises a donor DNA. In an embodiment, the nucleic acid is a plasmid, nanoplasmid, minicircle or viral vector comprising a donor DNA. In an embodiment, the donor DNA encodes an exogenous T cell receptor (TCR)-α or a fragment thereof, an exogenous TCR-β or a fragment thereof, or a combination thereof. In an embodiment, the nucleic acid is associated with a delivery vehicle. In an embodiment, the delivery vehicle is a lipid particle or a nanoparticle.

[0205] In embodiments, the T cell population comprises primary T cells.

[0206] In an embodiment, the composition further comprises a gene editing agent.

[0207] In embodiments, one or more cGAS-STING pathway inhibitors include cGAS inhibitors. In embodiments, one or more cGAS-STING pathway inhibitors include STING inhibitors. In embodiments, one or more cGAS-STING pathway inhibitors include TANK binding kinase 1 (TBK1) inhibitors.

[0208] In an embodiment, one or more cGAS-STING pathway inhibitors are aml, MRT67307 (MRT), BX795, H151, ODN-A151 (ODN151), Ru.521, G140 or a combination thereof. In an embodiment, one or more cGAS-STING pathway inhibitors include Aml. In an embodiment, one or more cGAS-STING pathway inhibitors include MRT. In an embodiment, one or more cGAS-STING pathway inhibitors include BX795. In an embodiment, one or more cGAS-STING pathway inhibitors include H151. In an embodiment, one or more cGAS-STING pathway inhibitors include ODN151. In an embodiment, one or more cGAS-STING pathway inhibitors include Ru.521. In an embodiment, one or more cGAS-STING pathway inhibitors include G140.

[0209] In embodiments, the cGAS-STING pathway inhibitor is Aml. 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 Aml, and there are no other cGAS-STING pathway inhibitors. In embodiments, the cGAS-STING pathway inhibitor is MRT, and there are no other cGAS-STING pathway inhibitors. In embodiments, the cGAS-STING pathway inhibitor is BX795, and there are no other cGAS-STING pathway inhibitors. In embodiments, the cGAS-STING pathway inhibitor is H151, and there are no other cGAS-STING pathway inhibitors. In embodiments, the cGAS-STING pathway inhibitor is ODN151, and there are no other cGAS-STING pathway inhibitors. In embodiments, the cGAS-STING pathway inhibitor is Ru.521, and there are no other cGAS-STING pathway inhibitors. In embodiments, the cGAS-STING pathway inhibitor is G140, and there are no other cGAS-STING pathway inhibitors.

[0210] In an embodiment, the one or more cGAS-STING pathway inhibitors are selected from: Aml, BX795, ODN151 and MRT. In an embodiment, the one or more cGAS-STING pathway inhibitors are ODN151. In an embodiment, the one or more cGAS-STING pathway inhibitors are BX795.

[0211] Pharmaceutical composition

[0212] It is expected that the compositions provided herein, including T cell compositions and engineered T cell compositions, are effective in treating diseases (e.g., cancer). For example, the engineered T cells provided herein may include exogenous T cell receptors specific for cancer cell antigens. Therefore, in one aspect, a pharmaceutical composition is provided, which includes the engineered T cells provided herein (including embodiments thereof). In an embodiment, the pharmaceutical composition further includes a pharmaceutical excipient (e.g., saline).

[0213] Treatment

[0214] It is expected that the engineered T cells provided herein (including its embodiments) have specificity to disease-specific antigens (such as cancer cell antigens), thereby allowing effective targeting of cancer cells. Engineered T cells may include, for example, one or more exogenous T cell receptors, which are engineered to have specificity to individual cancer cells, thereby allowing personalized and specific targeting of cancer cells. Therefore, in one aspect, a method for treating a disease of a subject in need thereof is provided, the method including the use of an engineered T cell provided herein (including its embodiments) or a pharmaceutical composition provided herein (including its embodiments) of a therapeutically effective amount. In an embodiment, the method includes the use of an engineered T cell provided herein (including its embodiments) of a therapeutically effective amount. In an embodiment, the method includes the use of a pharmaceutical composition provided herein (including its embodiments) of a therapeutically effective amount.

[0215] For the methods provided herein, in an embodiment, engineered T cells can be generated by a subject. For example, T cells can be extracted from a subject, contacted in vitro with nucleic acids (e.g., donor nucleic acids) and one or more cGAS-STING inhibitors, thereby generating engineered T cells, and administering them back to the subject. Therefore, in an embodiment, engineered T cells are autologous T cells. In an embodiment, engineered T cells can be generated by T cells that are not taken from a subject. For example, engineered T cells can be generated by healthy subjects (e.g., subjects without cancer). Therefore, in an embodiment, engineered T cells are allogeneic T cells.

[0216] 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.

[0217] It should be understood that the examples and embodiments described herein are for illustrative purposes only, and that various modifications or variations thereof will be suggested to those skilled in the art and will be included within the spirit and limits of the present application and within the scope of the appended claims. All publications, patents, and patent applications cited herein are incorporated herein by reference in their entirety for all purposes.

[0218] Examples

[0219] Example 1: Case Study Introduction

[0220] Over the past decade, a better understanding of tumor immunotherapy combined with the development of human cell engineering technology has made adoptive cell therapy (ACT) a viable treatment option. ACT is a highly personalized cancer treatment method that relies on ex vivo engineering of cancer-targeting lymphocytes and then administering these cancer cell-killing T cells back to the patient in vivo. Unlike other forms of cancer immunotherapy that rely on the development of anti-tumor lymphocytes in the patient's body, which lack specificity and efficiency in most cases, ACT has multiple advantages such as high efficiency, specificity, high number of lymphocytes, and long-lasting circulation time. Currently, three forms of ACT are being developed and used for clinical applications: tumor infiltrating lymphocytes (TIL), chimeric antigen receptor T cells (CAR-T), and T cell receptor engineered T cells (TCR-T). TIL and CAR-T have shown potential and achieved encouraging clinical results in patients with melanoma, lymphoma, and leukemia, thereby improving the quality of life and life expectancy of patients. However, serious side effects, such as severe cytokine release syndrome (CRS), cardiovascular toxicity, and unexpected neurological complications remain to be resolved. In addition, the success of TIL and CAR-T approaches in the treatment of solid tumors has been limited. To overcome these challenges, TCR-engineered T cells have been used over the past two decades, and a large number of clinical and preclinical studies have demonstrated their impact on mediating tumor lysis and eradication in multiple tumor types. For example, in early 2022, Immunocore Inc.'s Kimtrak, the first TCR-engineered T cell therapy for the treatment of melanoma, was approved by the FDA as a treatment option.

[0221] The T cell receptor (TCR) consists of alpha (α) and beta (β) chains that associate with the CD3 complex on the surface of T cells. TCRs non-covalently bind to peptide histocompatibility complex class I (MHC-I) molecules on the surface of tumor cells in a highly specific manner because they are able to distinguish MHC-I molecules loaded with non-self peptides or self-peptides with mutations from MHC-I molecules loaded with self-peptides. However, in some cases, immune surveillance evasion by tumor cells, reduced surface expression of MHC-I molecules, or low-level expression of mutant self-peptides on the cell surface can inhibit T cell-mediated tumor cell killing. TCR engineering approaches aim to address this issue by knocking in tumor-specific antigen-associated TCRs while knocking out endogenous TCRs. Today, somatic point mutations in tumor tissues are screened by utilizing whole genome or exome sequencing technologies, and unique peptide antigens with MHC-I presentation potential (called neoantigens) are identified. Proprietary algorithms are then used to engineer TCRs that can recognize MHC-I / neoantigen complexes, with the goal of improving the specificity of T cell-mediated tumor cell killing. By further optimizing TCR subtypes and intrinsic characteristics, TCR-engineered T cells may offer unique therapeutic opportunities that could benefit many patients with diseases such as synovial cell sarcoma, melanoma, and myeloma.

[0222] Many methods have been developed to improve T cell engineering efficiency. 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 may result from such methods cannot be ignored. The use of adeno-associated virus as an advanced version of viral editing technology seems to improve safety issues, however, it has limitations in gene size and site-specific delivery of target transgenes. Since CAR-T engineering has no strict requirements for genomic insertion sites, viral-based gene editing methods have been widely used in CAR-T methods. However, more accurate genome editing technologies that allow site-specific insertion of transgenes are highly needed but have not yet been fully discovered, and the technical needs for site-specific insertion of transgenes have not yet been achieved.

[0223] On the other hand, non-viral gene editing can allow convenient, safe and effective reprogramming of cultured cells and primary cells. Compared with the above-mentioned traditional recombinant viral vectors, 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, reduce safety risks, and enable highly specific genome editing / insertion, and can integrate larger DNA sequences. Through the homology-directed repair (HDR) mechanism, the DNA template can be integrated into the precise site of the CRISPR-Cas ribonucleoprotein (RNP, composed of cas9 protein and gRNA) incision in the T cell genome. Although Roth et al. and others have adopted this method to target human T cells, NK cells and induced pluripotent stem cells (iPSC), the use of this method in clinical settings has not yet been widely used because editing efficiency and off-target activity levels remain to be determined. In addition, the functional effects of the electroporation process on cultured T cells have not been fully tested. Previous studies have shown that up to 80% of electroporated cells are negatively affected after electroporation, showing loss of viability, reduced growth, and changes in gene expression. Although there is some correlation between cytotoxicity and DNA format (ssDNA, dsDNA, cDNA), length, and delivery method, the root cause of post-electroporation cytotoxicity observed in T cell cultures has not been fully characterized. Therefore, there is an urgent need to mitigate the cytotoxicity of electroporation during the TCR engineering process to improve gene editing efficiency and cell growth conditions.

[0224] In this study, we describe an efficient, safe, and convenient method that significantly improves electroporation-mediated T cell engineering efficiency, expansion, and thus total edited cell (TEC) number levels during TCR engineering. Using WT1 peptide-associated TCR as DNA template, we generated a clinically relevant model system in which the effects of various components of the electroporation-based DNA delivery method were tested. We investigated the potential molecular mechanisms of cytotoxicity during the T cell engineering process and identified double-stranded DNA as the most potent inducer of cellular DNA genotoxicity in cultured T cells. Subsequently, we screened several small molecule inhibitors of DNA genotoxicity pathways and analyzed their effects on T cell growth, viability, and TEC number to improve TCR engineering efficiency. In addition, the identified top DNA genotoxicity inhibitors had no negative impact on the phenotype and function of the final drug product (FDP), such as tumor cell killing ability. The above DNA genotoxicity mitigation method has been shown to be effective on lymphocyte samples from different healthy donors, implying the potential for broad application in clinical research.

[0225] Example 2: Inhibition of genomic stress significantly improves T cell engineering efficiency

[0226] result

[0227] DNA plasmids activate the cGAS-STING pathway and negatively affect T cell viability and expansion after electroporation

[0228] To achieve high gene editing efficiency and low toxicity during TCR engineering, we co-electroporated human primary CD8+ T cells with CRISPR-Cas9 ribonucleoprotein (RNP) and nanoplasmid DNA templates, as circular DNA has been reported to be less toxic than linear DNA [2,3]. By homology-directed repair, a 1572 bp WT1 peptide-specific TCR template was targeted to TRAC exon 1 ( Figure 1A ). Figure 1B An overview of the optimized 15-day process that includes T cell activation, electroporation, expansion, and harvesting is shown. To address the root cause of the decreased viability and low growth rate after electroporation, we electroporated T cells with different components of the CRISPR / Cas9 gene targeting reagent (RNP, DNA template, etc.). EH115 pulse encoding was used with a 100ul cuvette in a Lonza Nucleofector instrument according to the manufacturer's protocol. Viability, T cell growth rate, and knock-in (KI) / knock-out (KO) efficiency were measured during this process. Our data show that the presence of plasmid DNA during electroporation (DNA only and DNA+RNP) is associated with a substantial decrease in viability and reduced growth of T cells ( Figure 1C , 1D , Figures 2A to 2C ). No severe 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 (non-TFX) group. The RNP+DNA group exhibited a more delayed recovery and lower growth / expansion rate compared to the DNA-only group. These findings suggest that neither the electroporation process nor RNPs are the primary cause of reduced viability and growth in T cell cultures, whereas DNA was identified as the primary cause of reduced T cell growth and viability during the TCR engineering process. Notably, the knock-in / knock-out (KI / KO) data suggest that the most optimized electroporation procedure can achieve at least 85% to 90% KO and 50% KI / KO efficiencies ( Figure 1E ), and there were no non-specific KI / KO populations from RNP-only, no RNP, or no DNA arms.

[0229] Based on reports on monocytes [4,5], we hypothesized that the genotoxic stress after transfection due to the presence of plasmid DNA in T cells would trigger a decrease in viability and reduce culture expansion. To confirm this, Western blot analysis was performed on cell samples from day 2 to day 15, and indeed both the DNA-only group and the DNA+RNP group activated the cGAS-STING pathway, as evidenced by the corresponding phosphorylation of STING, TBK1, and IRF3 ( Figure 1F ). Although the RNP group alone achieved a high knockout percentage ( Figure 1E ), but did not activate the cGAS-STING pathway ( Figure 1F To exclude the possibility that other potential pathways could affect culture growth or expansion in the presence of single-stranded DNA (ssDNA) or double-stranded DNA (dsDNA), we investigated the activation of inflammasome-related pathways (Aim2 / IL-1B) and DNA endosomal activation pathways (TLR9 / MyD88) in transfected T cells. Our results showed that none of these pathways were activated ( Figure 3A , 3B ), who outlined the differences between viral-based and nanoparticle-based DNA delivery methods [6,7,8].

[0230] Pretreatment with BX795, AML, and MRT during the TCR engineering process increased T cell viability, expansion, and and total edited cell numbers

[0231] Since plasmid DNA appears to activate the cGAS-STING pathway, leading to decreased viability and growth inhibition of 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 circumvent potential contamination and nonspecific immune responses caused by exposure to human serum, chemically defined medium without serum was used in this study. T cells were treated with the specified concentrations of these inhibitors (Table 1) only before transfection and for specific durations, as adding inhibitors after transfection had a negative impact on culture growth. Pretreatment of T cells with some of these inhibitors at optimized concentrations significantly improved culture viability 48 hours after transfection ( Figure 4A , 4D , 4G, Figure 5A , 5D On average, when BX795 (5uM), Aml (25uM), and MRT (2.5uM) inhibitors were used, the culture viability was approximately 2-fold that of the untreated control group (CTR) ( Fig. 6AIn addition, the final T cell product collected on day 15 showed higher cell expansion compared to the control group, which had an average expansion of only 6.6-fold ( Figure 4B , 4E , 4H, Figure 5B , 5E ). Cells pretreated with BX795 (5uM), Aml (25uM), and MRT (5uM) achieved 20.3, 14.5, and 17.2-fold expansion, respectively. On average, treatment of T cells with the aforementioned inhibitors (at a given concentration) resulted in an approximately 2- to 3-fold improvement in culture expansion, which was consistent for all donors ( Figure 6B ).

[0232] Since inhibition of the cGAS-STING pathway should only mitigate plasmid DNA-mediated genotoxicity, no effect on gene editing efficiency is expected, and thus TCR-targeted knock-in / knock-out (KO+KI) rates are roughly equivalent in all donors (Figure 7). The only exception is donors 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, better expansion of cells with a KO+KI phenotype (Figure 7). Figure 7C ). This will be discussed further in the following section.

[0233] The total number of edited cells (TEC) in the final drug product is arguably the most important metric for adoptive cell transfer (ACT), and among other conditions, it is considered one of the most important criteria for cell therapy purposes because it is expected to translate into clinical therapeutic effects. Using the total number of cells in the final cell product on day 15 multiplied by the knock-in percentage, we obtain the TEC that represents the output of our T cell engineering and culture. As shown in the control condition (CTR), at a starting seeding density of 2 million cells and without inhibitor treatment, the number of TECs averaged about 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 , 4F 、4I, Figure 5C , 5F ), which is consistent with the cell proliferation data. Pretreatment with BX795 (2.5uM), Aml (25uM) and MRT (5uM) increased the number of TEC from 5.24 million to 21.1 million, 14.7 million and 17.4 million, respectively, resulting in an average increase of TEC by 303.4%, 267.0% and 180% ( Figure 6C ).

[0234] 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 expansion rates. Interestingly, however, for T cells pretreated with medium or high concentrations (2.5uM and 5um) of the inhibitor BX795, TEC analysis revealed a statistically significant improvement compared to the control group ( Figure 6C ), resulting in a more than 3-fold improvement in TEC.

[0235] BX795 pretreatment improved cell viability and proliferation rate by inhibiting IRF3 phosphorylation.

[0236] According to the data, BX795 (2.5uM and 5uM) was most effective in improving culture viability and growth and was therefore used for further studies. 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 at both medium and high concentrations successfully attenuated the phosphorylation of IRF3, a downstream target of TBK1, after transfection ( Fig. 8A ). Thus, downstream cytokine expression, such as type 1 interferons (IFNa, IFNB) and proinflammatory cytokines (IL-6), was reduced after pretreatment with BX795 ( Fig.8F ), indicating that the cGAS-STING pathway was successfully inhibited during the TCR engineering process. However, TBK1 phosphorylation itself was not attenuated and was actually enhanced after BX795 pretreatment, which is consistent with a previous report that TBK1 phosphorylation was enhanced 2-fold after BX795 treatment [9]. As previously shown ( Figures 4A to 4I and Figures 5A to 5F ), BX795 treatment resulted in a higher percentage of viability ( Figure 8B ), increased T cell proliferation rate ( Figure 8C ), equal or better KI / KO ratio ( Fig. 8E ) and improved TEC quantity.

[0237] BX795 inhibitor treatment improved the knockout and knockin ratios of the final T cell products.

[0238] Pretreatment of cultured T cells with the BX795 inhibitor is not expected to negatively affect the targeted cleavage of the target gene mediated by the Cas9 protein, nor should it affect the homology-directed repair process. However, data from the final cell product showed that T cells pretreated with BX795 had a significantly higher overall knockout percentage than the control group ( Fig.9A Compared with the control group with approximately 70% KO efficiency, BX795-pretreated T cells achieved knockout efficiencies of 86.7% (BX795-M) and 83.5% (BX795-H), with statistically significantly higher editing values ​​( Fig. 9B ). Notably, BX795 pretreatment of T cells also showed a higher knock-in percentage in the final cell product, increasing the knock-in ratio from 49.5% (CTR) to 61% (BX795-M) ( Fig. 9C ), which was statistically significant compared with the control group without inhibitor treatment ( Fig.9D Pretreatment of T cells with the BX795 inhibitor is unlikely to enhance Cas9 protein-mediated target gene cleavage or improve plasmid DNA-based homology-directed repair processes. The improvement in knock-in and knock-out ratios observed in the final T cell product ( Figure 4C , 4F , 4I, 5C, 5F, 6C) may be due to the survival and growth of edited T cells, as the improved trends observed in TECs in cultures pretreated with BX795 were expansion fold and higher viability ( Figure 6C ). T cells with a successful KO+KI ratio may have lower survival and expansion potential due to changes in metabolism and higher cellular stress, and pre-treatment of T cells with BX795 may partially alleviate some of these cellular stresses.

[0239] BX795 inhibitor treatment did not affect memory T cell phenotype.

[0240] It is expected that the number of memory T cells in the final T cell product is closely related to the clinical efficacy during T cell therapy, because memory T cells have the potential to proliferate and survive for a long time once they are reintroduced into the patient [14,15]. The balance of glycolysis to fatty acid oxidation metabolism and growth-related signaling pathways (Jak-STAT pathway, etc.) in T cells can affect T cell phenotype

[16] . In our optimized T cell activation and culture method, at least 95% of the cells in the final cell product have a central memory T cell (TCM) or stem cell memory T cell (TSCM) phenotype ( Figures 10A to 10F ). A high percentage of memory T cell phenotype may promote higher proliferation and enhance the tumor killing ability of T cells. Pretreatment of T cell cultures with the BX795 inhibitor did not adversely affect the T cell phenotype compared with untreated controls from different donors ( Figures 10A to 10F ). This is a very important criterion for inhibitor selection / application, as the higher growth rates observed in inhibitor-treated groups may alter cell metabolism, cytokine concentrations, and levels of metabolites in the culture medium, leading to changes in T cell phenotype. Fortunately, BX795 treatment had no effect on T cell phenotype.

[0241] CD8+ T cells from cultures treated with the BX795 inhibitor showed similar activity to untreated controls. Proper activation, proliferation and target cell killing.

[0242] The cGAS-STING pathway is essential for detecting and neutralizing genotoxic stress. It is a major component of the innate immune system that can trigger inflammation in response to cytoplasmic DNA detection [17,18]. By blocking the cGAS-STING pathway, we observed significantly improved culture viability, cell expansion rate, and thus the total number of edited cells. To ensure that treatment of cells with BX795 did not have any adverse effects on the functionality of T cell products, we used cells from four independent donors and followed the activation, transfection, and culture protocols (as described). T cells harvested from inhibitor-treated and control groups were pulsed with the specified concentrations of a specific peptide (WT1) that binds to the engineered TCR for 24 hours. Flow cytometry data showed that with respect to CD137 ( Fig.11A )、IFNg( Fig. 11B )、TNFa( Fig. 11C ) and granzyme B expression ( Fig.11D ), T cells pretreated with BX795 had an activation profile comparable to untreated control cells. The data showed that pretreatment of T cells with the specified concentration of BX795 inhibitor did not affect T cell activation and cytokine expression. Another major issue is the exhaustion of engineered T cell cultures. To measure the level of T cell exhaustion, the expression of exhaustion markers such as Tim3 and PD-1 was assessed. No significant differences in the levels of T cell exhaustion markers were observed in the inhibitor-treated or untreated groups at medium or low peptide concentrations compared to the negative control ( Fig. 12A , 12B As expected, T cells pretreated with the inhibitor had comparable Tim3 and PD-1 expression levels to untreated controls ( Fig. 12A , 12B ).

[0243] We next investigated the proliferation of T cells after treatment with BX795. The final T cell products obtained from the BX795 pre-treated or untreated groups were labeled with CFSE Far Red and co-cultured with the indicated concentrations of WT1 peptide for 72 h. As analyzed by flow cytometry, T cell proliferation in response to antigen-specific stimulation was comparable between BX795-treated and untreated samples ( Fig.11E). T cells treated with higher concentrations of WT1 peptide achieved a high (about 60%) proliferation rate, implying a robust and antigen-specific proliferation of the final T cell product. Finally, we compared T cells isolated from BX795 pre-treated and untreated control cultures in a target cell killing assay. Target cells T2 (T) were labeled with CFSE Far Red and then pulsed with 20uM WT1 peptide and then co-incubated with our final T cell product (E) in a ratio-dependent manner. Our data confirm that at all E:T ratios, T cells pre-treated with inhibitors have target cell killing capabilities comparable to those of untreated controls ( Fig.11F ). Notably, our T cells achieved high killing efficiency at a much lower E:T ratio compared to CTLs generated by traditional vaccine strategies [19,20], which outlines the potential advantages of our TCR-engineered T cells in antigen-specific tumor cell killing. In summary, our data have demonstrated the safety and feasibility of using the BX795 cGAS-STING pathway inhibitor for the T cell engineering process, outlining the potential broad impact of such inhibitors in the field of adoptive T cell therapy. The use of cGAS-STING inhibitors significantly improved T cell engineering efficiency, cell expansion rate, and therefore the total number of edited cells. By improving T cell viability and growth after electroporation, TECs that were two to three times that of the control group could be obtained, with activation, proliferation, and target cell killing capabilities comparable to those of the control group. Notably, we showed that this phenomenon was not donor-specific or limited to certain antigens, as similar favorable results were observed for T cells obtained from all donors in this study ( Figures 4A to 4I , Figures 5A to 5F , Figures 13A to 13C ).

[0244] discuss

[0245] In this study, we have introduced a simple, efficient and factory-friendly solution to improve the viability and growth of T cells during the TCR engineering process. Pretreatment of T cells with a TBK1 inhibitor resulted in higher T cell expansion and improved TCR editing efficiency without affecting the expected phenotype of the T cell final drug product (FDP). We successfully performed TCR engineering by electroporating RNPs and long DNA sequences into T cells and simultaneously improved editing efficiency, electroporation scale and cell expansion rate. Our data revealed a direct correlation between the electroporation procedure used (pulse coding) and T cell viability and expansion rate. We hypothesized that this phenomenon is related to the amount of DNA introduced into T cells during the electroporation process, where stronger pulse coding (e.g., EH115) results in more plasmid DNA constructs being delivered to T cells compared to milder pulse coding (e.g., EW100). We validated this by using a GFP plasmid construct during T cell transfection ( Fig.14A ). This also explains why using stronger pulse codes resulted in reduced viability, as our data indicate that plasmid DNA is the main cause of cell death and reduced growth after electroporation. When using the TBK1 inhibitor, we observed better T cell expansion that could be detected in multiple pulse codes (EW113, EH115, etc.). In addition, the BX795 inhibitor performed better in the EH115 group, which may be due to the introduction of more plasmid DNA, thus triggering higher levels of genotoxicity.

[0246] To our knowledge, this is the first report showing that the TBK1 inhibitor BX795 enhances TCR editing efficacy in a non-viral cell editing approach. Since BX795 has not been reported to directly affect homology-directed DNA repair (HDR) or DNA internalization processes, the improvement in 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 proposed mechanism has not been described. We hypothesize that BX795 enhances the survival of edited T cells during electroporation and subsequent post-transfection culture by reducing genotoxicity. This hypothesis is supported by time-graded TCR-dextramer flow cytometric analysis ( Fig. 14B , 14C ), which showed that BX795 treatment increased exogenous TCR expression both early (days 4 and 5) and throughout the culture process (days 6 to 15) compared with the control group, with a higher increase curve.

[0247] The mechanisms of genotoxic stress are complex. Although our western blot and qPCR data showed that cGAS / STING pathway activation was significantly reduced after BX795 treatment ( Fig. 8A), but we suspect that BX795 treatment does not completely resolve plasmid DNA-mediated genotoxic stress. Levels of type 1 interferons (such as TNFα and TNFβ) did not show a significant decrease after BX795 treatment, which may be due to the removal of the BX795 inhibitor before transfection. Exposing T cells to TBK1 inhibitors before and after electroporation or extending the treatment time may resolve this issue.

[0248] CD3 / CD28 stimulation or exposure to TCR-specific peptide antigens displayed on MHC-I molecules can significantly enhance the proliferation of transfected T cell cultures. However, this inadvertently causes T cell exhaustion and a high percentage of effector T cells in FDP. Previous CAR-T exploration studies have shown that memory phenotype CD8+T cells (TCM, TSCM, etc.) maintain better circulation time, proliferation potential, and target tumor cell killing ability when re-infused back into patients. In this study, we used IL-7 / IL-15 cytokines to enhance the generation of memory CD8+T cells, resulting in more than 95% memory TCM or TSCM in FDP ( Figures 10A to 10F However, this had a negative impact on T cell growth rate, where only an average of 5.24-fold T cell expansion was achieved. Pre-treatment of these T cell cultures with a TBK1 inhibitor prior to electroporation significantly improved the expansion rate of T cell cultures, up to 20.3-fold ( Figures 4A to 4I , Figures 5A to 5F , Figures 6A to 6C ), with no detectable adverse effects on T cell phenotype.

[0249] There is no doubt that non-viral 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 have enabled the synthesis of gRNA and DNA plasmid templates within a few days. However, the use of electroporation as the most efficient non-viral gene delivery / editing method requires overcoming various burdens, such as severe effects on cultured cells and genotoxic stress mediated by plasmid DNA. In this regard, many parameters, such as electroporation instruments, buffers, and electroporation procedures, need to be tested and optimized to find a balance between knock-in efficiency, cell expansion, and overall T cell phenotype. Our work introduces a new method to effectively address genotoxic stress responses in T cells from different donors in a simple and cost-effective manner. Treatment of T cell cultures with cGAS inhibitors before transfection can improve cell viability, promote cell growth, and enhance editing efficiency. Our process is scalable, cost-effective, and applicable not only to T cell engineering but also to any non-viral cell engineering process.

[0250] method

[0251] RNA ribonucleoprotein and DNA plasmid

[0252] The single guide RNA sequences of both TRAC and TRBC were derived according to the description of Oh, Senger et al. and ordered from Synthego (Menlo Park, CA, USA). SpyFi Cas9 protein was purchased from Aldevron (Fargo, ND, USA) and used for electroporation at a final concentration of 0.05 mg / ml. For the 100ul electroporation procedure, 2.5ug Cas9 protein was pre-compounded with 3 molar excess sgRNA for each knockout site and then mixed before delivery. Nanoplasmids with WT1 TCR sequences were purchased from Nature Technologies (Lincoln, NE, USA) and used for electroporation at a working concentration of 150ug / ml.

[0253] Cell isolation and activation

[0254] Peripheral blood mononuclear cells (PBMC) were separated from cryopreserved leukopak using Ficoll gradient centrifugation (400rmp, 25 minutes). All samples were obtained from healthy donors using HLA A*02:01MHC class I complexes for genotyping. The CD8+T cells were then separated using Miltenyi AutoMACS cell separator according to the manufacturer's protocol. The CD8+T cells separated were then mixed with Miltenyi T cell TransACT reagent (1:100) (Cat. No. 130-111-160), 25ng / mL IL-7 (Cat. No. 130-095-367) and 50ng / mL IL-15 (Cat. No. 130-095-760) in Fuji Prime-XV culture medium (IrvineScientific, Cat. No. 91154) for 48 hours to activate.

[0255] Electroporation

[0256] Cells were electroporated using the 4D-Nucleofector system (Lonza) according to the manufacturer's protocol. After activation and inhibitor treatment, cells were washed and resuspended in 10 x 10 6 (or 10 million) CD8+ T cells were then mixed with premixed RNPs and DNA plasmids and transferred to a Lonza 100-μL cuvette. After electroporation, 400ul FUJI Prime-XV medium was added to the cuvette and incubated for 15 minutes before cells were seeded into culture flasks. In all cases, the pulse code used in this manuscript was EH115.

[0257] Cell culture

[0258] 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 carefully transferred to 24-well gREX (Wilsonwolf, catalog: 80192M) at a seeding density of 1 to 2 million cells / cm 2 8 ml of complete medium was added to each well, and 50% of the medium was replaced after 6 days. The cells were incubated at 37°C and 5% CO2. NucleoCounter NC-200 automated cell counter with Via2-Casette was used for cell counting and viability analysis in this study.

[0259] Flow cytometry

[0260] All surface and intracellular staining antibodies were purchased from Biolegend and BD Biosciences as listed in Table 2. Dextramer antibodies used for knock-in analysis were purchased from Immudex (Catalog: WB3469-PE, WT1) and used according to the manufacturer's protocol. Surface staining, CFSE staining, 7-AAD / Annexin V staining, and intracellular staining were performed as described previously

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

[0261] Western blotting

[0262] Antibodies used 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 blotting experiments, and data analysis were performed as previously described (Tang, JBC, 2020).

[0263] qPCR

[0264] RNeasy mini kit (catalog number 74106) was purchased from Qiagen. TaqMan RNA-to-CT 1-step kit (catalog number 4392938) and Taqman primer-probe assay using reporter dye FAM and MGB quencher were purchased from Thermo Fisher (assay information is listed in Table 4). After RNA isolation, 2ng RNA was mixed with Taqman primer-probe mixture, TaqMan RNA-to-CT 1-step master mix and RT enzyme mixture in a 10μL qPCR system according to the manufacturer's protocol. The reaction readings were measured using a QuantStudio 6Flex machine, and the data were analyzed using QuantStudio real-time PCR software v1.2. PCR cycle conditions were 50°C for 30 minutes, 95°C for 10 minutes, 95°C for 40 cycles of 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).

[0265] Data analysis and statistics

[0266] Western blot data were analyzed using 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 created and presented using Graphpad Prism 10. For all statistical analyses, data are presented as mean ± SEM.

[0267] Table 1. Inhibitor treatment concentrations used

[0268] 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 with FUJI Prime-XV complete medium without inhibitors.

[0269] Inhibitors Target Low dose Moderate dose High dose G140 cGAS 1uM 2.5uM 5uM Ru.521 cGAS 1uM 2.5uM 5uM H151 STING 1uM 2.5uM 5uM BX795 TBK-1 1uM 2.5uM 5uM Amlexanox TBK-1 25uM 50uM 100uM MRT67307 TBK-1 1uM 2.5uM 5uM

[0270] Table 2. List of flow cytometry antibodies

[0271]

[0272] Antibody Targets Fluorescence Supplier Name Catalog Number Tim3 BV605 BioLegend 345018 TNF-α BV605 BioLegend 502936

[0273] Table 3. Western blot antibody list

[0274] Antibody Targets Supplier Name Catalog Number cGAS Cell Signaling 15102s STING Cell Signaling 13647s P-STING Cell Signaling 50907s TBK1 Cell Signaling 3504s P-TBK1 Cell Signaling 5483s IRF3 Cell Signaling 4302s P-IRF3 Cell Signaling 29047s Aim2 Cell Signaling 12948s Caspase 1 Cell Signaling 3866s Cleaved caspase 1 Cell Signaling 4199s IL-1β Cell Signaling 12703s Cleaved IL-1β Cell Signaling 83186s MyD88 Cell Signaling 4283s TRAF6 Cell Signaling 8028s IRAK4 Cell Signaling 4363s P-IRAK4 Cell Signaling 11927s GAPDH Cell Signaling 2118s GAPDH-HRP Cell Signaling 3683s

[0275] Table 4. qPCR primer list

[0276] qPCR Targets Assay ID Catalog Number IFN-α Hs00265051_s1 4331182 IFN-β Hs01077958_s1 4331182 IFN-γ Hs00989291_m1 4331182 IL-6 Hs00985639_m1 4331182 RNA18S Hs03928990_g1 4331182

[0277] Table 5. Exemplary cGAS-STING pathway inhibitors

[0278]

[0279]

[0280] References

[0281] [1]Roth T. et al., Reprogramming human T cell function and specificity with non-viral genome targeting. Nature.559,405–409(2018)

[0282] [2] Cornu, TI, Mussolino, C. & Cathomen, T. Refining strategies to translate genome editing to the clinic. Nat. Med. 23, 415–423 (2017).

[0283] [3] Hornung, V. & Latz, E. Intracellular DNA recognition. Nat. Rev. Immunol. 10, 123–130 (2010).

[0284] [4] Luecke, S. et al., cGAS is activated by DNA in a length-dependentmanner. EMBO Rep. 18, 1707-1715 (2017)

[0285] [5]Hansen, K. et al., Listeria monocytogenes induces IFNβexpression through an IFI16-, cGAS-and STING-dependent pathway. EMBO J. 33, 1654-1666 (2014)

[0286] [6] Rathiam V. et al., The AIM2 inflammasome is essential for host defense against cytosolic bacteria and DNA viruses. Nature Immunology. 11, pp. 395–402 (2010)

[0287] [7] Ferrer J. et al., Dual Toll-Like Receptor Targeting Liposomal Spherical Nucleic Acids. Bioconjugate Chem. 30, 944–951 (2019)

[0288] [8] Radovic-Moreno A. et al., Immunomodulatory spherical nucleic acids. Proc. Natl. Acad. Sci. U.S.A., 112, 3892-3897 (2015)

[0289] [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)

[0290]

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

[0291]

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

[0292]

[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)

[0293]

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

[0294]

[14] Lugli E. et al., Superior T memory stem cell persistence supports long-lived T cell memory. J Clin Invest. 123, 594 - 599 (2013)

[0295]

[15] Toews K. et al., Central memory phenotype drives success of checkpoint inhibition in combination with CAR T cells. Molecular Carcinogenesis. 59, 724–735 (2020)

[0296]

[16] Kaech S. et al., Transcriptional control of effector and memory CD8+ T cell differentiation. Nature Reviews Immunology. 12, 749–761 (2012)

[0297]

[17] Zhang X. et al., Structures and Mechanisms in the cGAS-STING Innate Immunity Pathway. Immunity. 53, 43 - 53 (2020)

[0298]

[18] Cheng W. et al., The cGas–Sting Signaling Pathway Is Required for the Innate Immune Response Against Ectromelia Virus. Front. Immunol., 14, doi:10.3389 / fimmu.2018.01297 (2018)

[0299]

[19] Wang S. et al., Rational vaccinology with spherical nucleic acids. Proc. Natl. Acad. Sci. U.S.A., 116, 10473-10481 (2019)

Claims

1. A method for engineering T cells, the method comprising contacting the T cells 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. The method according to claim 1 or 2, wherein the nucleic acid is a double-stranded circular nucleic acid.

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

5. The method according to any one of claims 2 to 4, wherein the donor DNA encodes an exogenous T cell receptor (TCR)-α or a fragment thereof, an exogenous TCR-β 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. The method of claim 6, wherein the delivery vehicle is a lipid particle or a nanoparticle.

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

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

10. The method of claim 9, wherein contacting the T cells with the gene editing agent comprises contacting the T cells with a nucleic acid sequence encoding the gene editing agent.

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

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 method of claim 9, wherein the CRISPR-Cas system comprises 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, 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. The method of claim 15, wherein the CRISPR-Cas system comprises Cas9.

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

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

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

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

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

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

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

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

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

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

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 comprise 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. 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. 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. The method of claim 31, wherein the one or more cGAS-STING pathway inhibitors are selected from the group consisting of: Aml, BX795, ODN151, and MRT.

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

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

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

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

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

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

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

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

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

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

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

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

49. The method of any one of claims 41 to 48, further comprising contacting the T cell population with a gene editing agent.

50. The method of claim 49, wherein contacting the T cell population with the gene editing agent comprises contacting the T cell population with a nucleic acid sequence encoding the gene editing agent.

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

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

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

54. The method of claim 53, wherein the CRISPR-Cas system comprises 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, 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. The method of claim 54, wherein the CRISPR-Cas system comprises Cas9, nCas9, or Cas-CLOVER.

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

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

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

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

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

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

62. The method of claim 60, wherein the gene editing reagent comprises TALEN.

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

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

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

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

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

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

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 contacting with the nucleic acid.

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

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

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

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

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

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

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

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

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

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

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

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

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

90. The method of any one of claims 82 to 89, further comprising contacting the T cell population with a gene editing agent.

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

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

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

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

95. The method of claim 94, wherein the CRISPR-Cas system comprises 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, 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. The method of claim 95, wherein the CRISPR-Cas system comprises Cas9, nCas9, or Cas-CLOVER.

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

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

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

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

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

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

103. The method of claim 101, wherein the gene editing reagent comprises TALEN.

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

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

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

107. The method of any one of claims 90 to 106, wherein contacting the T cell population with the gene editing agent comprises transfecting the T cell population with the gene editing agent.

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

109. The method of any one of claims 82 to 92, wherein the one or more cGAS-STING pathway inhibitors comprise 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. The method of any one of claims 82 to 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 to 112, wherein the T cell populations 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 to 113, wherein the T cell population 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 to 113, wherein the population of T cells is contacted sequentially with the nucleic acid and the one or more cGAS-STING pathway inhibitors.

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

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

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

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

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

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

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

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

124. The method of any one of claims 82 to 121, wherein the gene editing efficiency of the engineered T cell population is about 60% to about 99%.

125. The method of any one of claims 82 to 121, wherein the knockout efficiency of the engineered T cell population is about 10% to about 100%.

126. The method of claim 109, wherein the knockout efficiency of the engineered T cell population is about 30% to about 100%.

127. The method of claim 109, wherein the knockout efficiency of the engineered T cell population is about 50% to about 100%.

128. The method of claim 109, wherein the knockout efficiency of the engineered T cell population is about 60% to about 100%.

129. The method of claim 109, wherein the knockout efficiency of the engineered T cell population is about 60% to about 99%.

130. The method of claim 109, wherein the knockout efficiency of the engineered T cell population is about 60% to about 95%.

131. 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. The method of any one of claims 82 to 112, wherein the knock-in efficiency of the engineered T cell population is about 20% to about 99%.

133. The method of claim 113, wherein the knock-in efficiency of the engineered T cell population is about 30% to about 99%.

134. The method of claim 113, wherein the knock-in efficiency of the engineered T cell population is about 40% to about 99%.

135. The method of claim 113, wherein the knock-in efficiency of the engineered T cell population is about 50% to about 99%.

136. 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. A method for increasing the expansion of an engineered T cell population, the method comprising: i) contacting the T cell population with one or more cyclic GMP-AMP synthase (cGAS)-stimulator of interferon genes (STING) pathway inhibitors and a nucleic acid, thereby forming the engineered T cell population, and ii) expanding the engineered T cell population, thereby forming an expanded engineered T cell population, wherein the one or more cGAS-STING pathway inhibitors increase the expanded engineered T cell population relative to an engineered T cell population in which the T cell population of step i) is not contacted with the one or more cGAS-STING pathway inhibitors.

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

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

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

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

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

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

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

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

146. The method of claim 145, wherein contacting the T cells with the gene editing agent comprises contacting the T cells with a nucleic acid sequence encoding the gene editing agent.

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

148. The method of any one of claims 145 to 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 method of claim 149, wherein the CRISPR-Cas system comprises 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, 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. The method of claim 150, wherein the CRISPR-Cas system comprises Cas9, nCas9, or Cas-CLOVER.

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

179. The method of any one of claims 137 to 176, wherein the engineered T cell population is expanded at least about 0.5-fold to at least about 100-fold.

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

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

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

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

184. An engineered T cell population according to claim 182 or 183, wherein the nucleic acid is a double-stranded circular nucleic acid.

185. An engineered T cell population according to claim 183 or 184, wherein the nucleic acid is a plasmid, nanoplasmid, minicircle or viral vector comprising donor DNA.

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

187. An engineered T cell population according to any one of claims 182 to 186, wherein the nucleic acid is associated with a delivery vehicle.

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

189. An engineered T cell population according to any one of claims 182 to 188, wherein the T cells are primary T cells.

190. An engineered T cell population according to any one of claims 182 to 189, wherein the T cell population is further contacted with a gene editing agent.

191. An engineered T cell population according to any one of claims 183 to 190, wherein the donor DNA is inserted into an endogenous TCR locus.

192. The engineered T cell population of claim 191, wherein the endogenous TCR locus is an endogenous TCR-α locus, an endogenous TCR-β locus, or a combination thereof.

193. The engineered T cell population of any one of claims 190 to 192, wherein contacting the T cells with the gene editing agent comprises transfecting the T cells with the gene editing agent.

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

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

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

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

198. An engineered T cell population according to any one of claims 182 to 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. The engineered T cell population of claim 198, wherein the one or more cGAS-STING pathway inhibitors are selected from: Aml, BX795, ODN151 and MRT.

200. The engineered T cell population of claim 198, wherein the one or more cGAS-STING pathway inhibitors are ODN151.

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

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

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

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

205. The engineered T cell population of any one of claims 182 to 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. The engineered T cell population of claim 205, wherein the T cells are contacted with the one or more cGAS-STING pathway inhibitors for about 6 hours.

207. The engineered T cell population of any one of claims 182 to 206, wherein the T cells are contacted with a 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 comprising donor DNA.

211. The composition of claim 209 or 210, wherein the donor DNA encodes an exogenous T cell receptor (TCR)-α or a fragment thereof, an exogenous TCR-β 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. A composition according to any one of claims 208 to 213, wherein the T cell population comprises primary T cells.

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

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

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

218. The composition of any one of claims 208 to 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 to 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 the group consisting of: Aml, 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. A pharmaceutical composition comprising the engineered T cells according to claim 181.

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

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

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

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