Insulin treatment to improve T cell engineering

Abstract

Provided herein, inter alia, are methods and compositions for engineering T cells. The methods include culturing T cells with insulin during engineering of the T cells. The methods provided herein contemplate to increase cell viability, expansion and gene editing efficiency, allowing for an increase in the total number of engineered T cells.

Description

[0001] Cross - Reference to Related Applications

[0002] This application is an international application that claims the priority and benefit of U.S. Provisional Application No. 63 / 387,068, filed on December 12, 2022, and U.S. Provisional Application No. 63 / 608,697, filed on December 11, 2023. The content of this application is incorporated herein by reference in its entirety for all purposes. Background Art

[0003] Genetic engineering has almost unlimited potential as a tool for improving human health. In fact, genetic engineering has introduced and transformed therapies in all aspects of current medical practice. Particular attention has been paid to the genetic engineering of T cells. T - cell - based therapies have become powerful new drugs, especially in the fields of cancer and immune system regulation. However, significant obstacles still remain regarding improving the viability and expansion capacity of genetically engineered T cells.

[0004] Specifically disclosed herein are solutions to these and other problems in the art. Summary of the Invention

[0005] In one aspect, provided herein is a method of editing an endogenous gene in a population of T cells, the method comprising: contacting the population of T cells with a gene - editing reagent or the polynucleotide encoding the gene - editing reagent under conditions that permit entry of the polynucleotide or gene - editing reagent into the cells; and culturing the population of T cells in the presence of one or more of the following before and / or during and / or after the contacting step to obtain an engineered population of T cells: insulin, insulin analogs, insulin agonists, and / or insulin partial agonists.

[0006] In another aspect, provided herein is a method of monitoring the cell viability of an engineered population of T cells, the method comprising measuring mitochondrial function and cell metabolism over time.

[0007] In another aspect, provided herein is a method of monitoring the cell viability of an engineered population of T cells, the method comprising measuring cell - metabolism markers over time.

[0008] In another aspect, provided herein is a method of increasing the cell viability of an engineered population of T cells, the method comprising contacting a population of T cells in the presence of insulin, insulin analogs, insulin agonists, insulin partial agonists, a gene - editing reagent, or a polynucleotide encoding a gene - editing reagent to form the engineered population of T cells, wherein the engineered population of T cells has increased cell viability, growth, and / or gene - editing efficiency relative to an engineered population of T cells not contacted with insulin, insulin analogs, insulin agonists, or insulin partial agonists, and wherein the engineered population of T cells is administered to a subject in need thereof.

[0009] In another aspect, the present disclosure provides a method for increasing the gene editing efficiency of an engineered T cell population, the method comprising contacting a T cell population with insulin, an insulin analog, an insulin agonist, an insulin partial agonist, a gene editing reagent, and a polynucleotide to form the engineered T cell population, wherein the engineered T cell population has an increased gene editing efficiency relative to an engineered T cell population not contacted with insulin, an insulin analog, an insulin agonist, or an insulin partial agonist.

[0010] A method for increasing the expansion of an engineered T cell population, the method comprising: (i) contacting a T cell population with insulin, an insulin analog, an insulin agonist, and / or an insulin partial agonist and a polynucleotide to form the engineered T cell population, and (ii) expanding the engineered T cell population to form an expanded engineered T cell population, wherein the insulin, insulin analog, insulin agonist, and / or insulin partial agonist increases the expanded engineered T cell population relative to an engineered T cell population not contacted with insulin, an insulin analog, an insulin agonist, and / or an insulin partial agonist.

[0011] In another aspect, the present disclosure provides an engineered T cell population prepared by the methods provided herein (including embodiments thereof).

[0012] In another aspect, the present disclosure provides a pharmaceutical composition comprising the engineered T cells provided herein (including embodiments thereof).

[0013] In another aspect, the present disclosure provides a method for treating a disease in a subject in need thereof, the method comprising administering a therapeutically effective amount of the engineered T cells provided herein (including embodiments thereof) or the pharmaceutical composition provided herein (including embodiments thereof). BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 Insulin treatment stimulates Akt phosphorylation in T cells. Protein immunoblot analysis of Akt, Erk, and STAT5 and their corresponding phosphorylated forms in activated human CD8+ T cells pretreated with (+ insulin) or without (control) insulin. Samples were obtained at 15 minutes, 30 minutes, and 180 minutes (min) after insulin treatment in glucose-free medium. All measurements were performed using Image Lab software. C-Cas3: cleaved caspase 3.

[0015] Figures 2A to 2F Insulin pretreatment (24 hours) of cultured T cells prior to electroporation improves culture viability, expansion, and the level of all edited cells (TEC).Figures 2A to 2C Shows the T cell final drug product (FDP) amplification fold calculated based on the seeding density on day 2 after 15 days of engineering T cells using different pre - transfection (TFX) insulin treatment concentrations. Data were obtained from 3 individual donors pre - treated with different concentrations of insulin. Figures 2D to 2F Shows Figures 2A to 2C The number of all edited cells (TEC) in the FDP calculated for engineered T cells as shown. Numbers in each row are rounded up. TEC is calculated by multiplying the amplification fold by the knock - in percentage and is based on a seeding density of 1 million cells per group after transfection. Insulin concentrations used: low: 1 μg / ml, medium: 5 μg / ml, high: 25 μg / ml. CTR: control.

[0016] Figures 3A to 3E Insulin pretreatment enhances the expression / activation of Akt and Erk signaling pathways in T cells. Results of Western blotting of Akt and Erk expression and phosphorylation with (insulin 24 - hour pretreatment) or without (CTR) insulin pretreatment ( Figure 3A ). Figures 3B to 3E Shows the quantification of Western blotting data of Akt and Erk expression and phosphorylation. Total Akt ( Figure 3B ) and Erk ( Figure 3D ) expression is calculated based on the ratio of the protein band intensity to the internal control (actin) band intensity. Protein phosphorylation levels are calculated based on the ratio of the phosphorylated Akt ( Figure 3C ) or Erk ( Figure 3E ) band intensity to the total Akt or Erk protein band intensity. 0N, 2N represent day 0 and day 2 (before transfection) respectively; 2, 3, 4, 6, 8, 11, 15 (or 2T, 3T, 4T, 6T, 8T, 11T, 15T) represent the number of days after 0N (TFX on day 2 of culture). Western blotting results were imaged using Image Lab software and band intensities were analyzed by ImageJ.

[0017] Figures 4A to 4E Insulin treatment enhances the survival of edited T cells and thus increases the level of T cell editing in FDP before and after TFX. Figure 4A And 4B Show the knock - in percentages when cultures from 2 independent donors are treated with the indicated insulin concentrations before TFX compared to untreated controls (CTR). Figure 4C And 4DShows the percentage of knock-in when cultures from 2 independent donors are treated with the specified insulin concentration after TFX, compared to untreated control (CTR). The specified insulin dose before TFX was used 24 hours before transfection (pretreatment) or the specified insulin dose after TFX was used 6 days after transfection (post-treatment). Samples were collected on day 15 and analyzed by flow cytometry. The knock-in ratio was measured by using MHC-peptide dextramer staining (Dex) during flow cytometry. Figure 4E Shows the flow cytometry dot plot data gating strategy for analyzing the knock-in levels at three tested insulin concentrations (as Figure 4A ). Insulin concentrations used: low: 1 μg / ml, medium: 5 μg / ml, high: 25 μg / ml. CTR: control.

[0018] Figures 5A to 5G . Insulin pretreatment enhances T cell metabolism and mitochondrial function. Figure 5A and 5B Show that insulin treatment significantly enhances T cell metabolism and glucose uptake before transfection ( Figure 5A ) and 48 hours after transfection ( Figure 5B ). Figure 5C and 5D Show the mitochondrial membrane potential (MMP) in T cells treated with insulin (insulin-2N) or untreated (CTR) before transfection ( Figure 5C ) and 48 hours after transfection ( Figure 5D ). Figure 5E and 5F Show the mitochondrial mass in insulin-treated T cells or T cells in the CTR group. In insulin-treated T cells, the mitochondrial mass was significantly higher 48 hours after transfection ( Figure 5F ), while insulin-treated and untreated cultures had comparable mitochondrial mass before transfection ( Figure 5E ). Figure 5G Shows the qPCR analysis of the relative expression of Bcl-2L1 transcript levels in CTR and insulin-treated groups before transfection. Insulin treatment: treated with 25 μg / mL insulin 24 hours before transfection. ***P<0.001; **P<0.01; *P<0.05 (compared to CTR).

[0019] Figures 6A to 6D . Insulin pretreatment does not affect FDP characteristics and T cell function. Pretreating T cell cultures with insulin before transfection has no effect on the phenotype and function of the final T cell product. Figure 6A and 6BT cell phenotype data collected from two independent donors are shown. Insulin doses were used as indicated and insulin was added 24 hours before TFX in all cases. Cell samples were collected on day 15 and phenotype ratios were measured by flow cytometry. T stem cell memory (TSCM: CD45RA + CD45RO - CD95 + CD27 + ), T central memory (TCM: CD45RA + CD45RO + CD95 + CD27 + ), T cell effector memory (TEM: CD45RA + CD45RO + CD95 + CD27 - ), and effector T cell (TE: CD45RA + CD45RO - CD95 + CD27 - ) phenotypes are shown. All donors were subjected to similar treatments with respect to insulin pretreatment, electroporation procedures, and culture conditions and methods. Figure 6C Comparison of CD137 expression, an activation marker, between the insulin pretreatment (Insulin-TFX) group and the control (CTR-TFX) group is shown. Cell samples were collected on the last day (day 15). The cells were then co-cultured with the indicated concentration of the target peptide (WT1) for 24 hours, followed by flow cytometry analysis. Figure 6D Cell killing assays are shown to compare the functions of T cells obtained from the insulin pretreatment group and the control group. T cell samples were collected on the last day (day 15) and co-cultured with T2 target cells pre-labeled with the target peptide at the indicated T cell:target cell ratio for 20 hours. The level of target cell apoptosis was measured by calculating the annexin V and 7-aminoactinomycin D (7-AAD) double-positive population in samples of T cell / target cell co-cultures and in cultures of target cells alone.

[0020] Figure 7A and 7B . Schematic diagrams of TCR editing and T cell culture procedures. Figure 7A Exemplary schematic diagrams of exogenous T cell receptor DNA templates for CRISPR / Cas9-mediated homology-directed repair at the TCR-α locus are shown. DNA templates containing the TCR-α variant chain and the TCR-β chain were designed to be inserted into the TCR-α constant (TRAC) locus while the endogenous TCR-α (VJ) and TCR-β loci had been disrupted. Figure 7BExemplary schematic showing a 15-day workflow of T cell activation, engineering, and cell culture processes as described herein. The entire process is carried out in chemically defined medium.

[0021] Figure 8A and 8B . The Akt and Erk signaling pathways are activated during the engineering of T cells and may be associated with the amplification rate and loss of viability. Figure 8A Protein immunoblot analysis showing Akt and Erk phosphorylation of T cell samples obtained during the engineering of T cells in the presence of RNP and DNA for two independent donors. Figure 8B Showing T cell viability and amplification profiles for two independent donors during 15 days of T cell engineering and culture; T cell viability data and cell amplification were measured by NucleoCounter NC-200.

[0022] Figures 9A to 9F . Pretreatment durations of insulin for all tested cases before transfection enhanced T cell amplification and TEC levels (three donors each treated with 3 different insulin treatment time points). Figures 9A to 9C Showing the T cell final drug product (FDP) fold amplification for three different donors after 15 days of T cell engineering. Different TFX pre-insulin treatment time points (6 hours, 24 hours, and 48 hours) were tested and calculated based on the number of seeded cells on day 2 compared to untreated control (CTR). Figures 9D to 9F Showing for Figures 9A to 9C the number of total edited cells (TEC) in the FDP of engineered T cells as shown in. TEC was calculated based on a seeding density of 1 million cells per group after transfection, rounded up. The number of total edited cells was calculated by multiplying the fold amplification by the knock-in percentage. In all cases, insulin was used at 25 μg / mL.

[0023] Figure 10A and 10B . Insulin treatment before transfection had the most significant effect on T cell amplification and TEC levels. Insulin was added to activated CD8+ T cells 24 hours before TFX (pre-TFX), or 6 days after TFX (post-TFX), or both 24 hours before TFX and 6 days after TFX (pre + post). Figure 10A Showing the T cell final drug product (FDP) fold amplification calculated based on the number of seeded cells on day 2 after 15 days of T cell engineering using different insulin treatment regimens and concentrations. Figure 10BShow the total number of edited cells (TEC) in FDP. TEC is calculated based on a seeding density of 1 million cells per group after transfection, rounded up. TEC is calculated by multiplying the amplification factor by the knock-in percentage. Insulin doses used: NA: 0 μM, medium: 5 μM, high: 10 μM.

[0024] Figures 11A - 11F . Test the effect of insulin pretreatment on T cell metabolism and mitochondrial function in the second donor. Figure 11A and 11B Show T cell metabolism data, showing that insulin treatment significantly enhances T cell glucose uptake before transfection ( Figure 11A ) and 48 hours after transfection ( Figure 11B ), as measured by 2-NBDG glucose fluorescence. Figure 11C and 11D Show that when measured 96 hours after transfection, the mitochondrial membrane potential (MMP) is higher in insulin-treated T cells (Insulin-2N) compared to the control group (CTR) ( Figure 11D ), while before transfection, the two groups have comparable MMP ( Figure 11C ). Figure 11E and 11F Show the mitochondrial mass in insulin-treated T cells, confirming that the mitochondrial mass is higher in insulin-treated samples measured 48 hours after transfection ( Figure 11F ), while before transfection, the two groups have comparable mitochondrial mass. ***P < 0.001; **P < 0.01; *P < 0.05 (compared to the control group).

[0025] Figures 12A to 12F . Insulin treatment before TFX does not affect FDP characteristics and T cell function under pre-TFX or post-TFX treatment conditions (3 independent donors). T cells treated with insulin before and after TFX have comparable cell phenotypes to the control group. Figure 12A and 12B Show T cell phenotype data collected from donor 1 for insulin treatment before TFX ( Figure 12A ) and insulin treatment after TFX ( Figure 12B ). Figure 12C and 12D Show T cell phenotype data collected from donor 2 for insulin treatment before TFX ( Figure 12C ) and insulin treatment after TFX ( Figure 12D ). Figure 12E and 12F Show T cell phenotype data collected from donor 3 for insulin treatment before TFX ( Figure 12E ) and insulin treatment after TFX ( Figure 12FT cell phenotypic data collected from donor 3. Insulin doses were used as indicated; pre-TFX indicates insulin treatment 24 hours before TFX, and post-TFX indicates insulin treatment 6 days after TFX. Cell samples were collected on day 15, and different T cell phenotypes were measured by flow cytometry. T stem cell memory (TSCM: CD45RA+CD45RO-CD95+CD27+), T central memory (TCM: CD45RA+CD45RO+CD95+CD27+), T cell effector memory (TEM: CD45RA+CD45RO+CD95+CD27-), and effector T cell (TE: CD45RA+CD45RO-CD95+CD27-) phenotypes were measured as shown. All donors were subjected to similar treatments in terms of insulin pretreatment, electroporation procedure, and culture conditions and methods.

[0026] Figures 13A to 13F . Insulin pretreatment of shorter duration enhances TCR-engineered T cell expansion and total edited cell count (TEC). Figures 13A to 13C Shows the fold expansion of T cell final drug product (FDP) for three different donors after 15-day T cell engineering. Different pre-TFX insulin treatment time points (24 hours, 6 hours, 4 hours, 2 hours, and 30 minutes) were tested and calculated based on the number of seeded cells on day 2 compared to untreated control (CTR). Figures 13D to 13F Shows for Figures 13A to 13C the number of total edited cells (TEC) in the FDP of the engineered T cells shown in. TEC was calculated based on a seeding density of 1 million cells per group after transfection, rounded up. The total edited cell count was calculated by multiplying the fold expansion by the knock-in percentage. In all cases, insulin was used at 25 μg / mL.

[0027] Figures 14A to 14C . Insulin treatment enhances the total edited cell count (TEC) of T cells. Figure 14A Shows the change in TEC when T cells are treated with insulin before transfection, after transfection (e.g., during expansion), after the midpoint (e.g., after expansion), or a combination thereof. Figure 14B Shows the change in TEC when T cells are treated with low-dose (1 μg / ml), medium-dose (mid; 5 μg / ml), or high-dose (25 μg / ml) insulin before transfection. Figure 14C Shows the change in TEC when T cells are treated with high-dose insulin for 30 minutes, 2 hours, 4 hours, 6 hours, or 24 hours before transfection. On average, by insulin treatment for 2 hours and 6 hours, the T cell TEC is enhanced by 123% and 77% respectively.

[0028] Figures 15A to 15HUsing xenon transfection, insulin enhances T cell growth and total edited cell count (TEC). Figures 15A to 15C Shows the effect of insulin treatment for 2 hours and 6 hours on the expansion of T cells from donor 1 ( Figure 15A ), donor 2 ( Figure 15B ), and donor 3 ( Figure 15C ). Figures 15D to 15F Shows the effect of insulin treatment for 2 hours and 6 hours on the TEC of T cells from donor 1 ( Figure 15D ), donor 2 ( Figure 15E ), and donor 3 ( Figure 15F ). On average, by insulin treatment for 2 hours and 6 hours, T cell expansion is enhanced by 30% and 50% respectively ( Figure 15G ). On average, with insulin treatment for 2 hours and 6 hours, T cell TEC is enhanced by 22% and 47% respectively ( Figure 15H ).

[0029] Figures 16A to 16B Using xenon transfection in a Good Manufacturing Practice (GMP) scale system, insulin enhances T cell growth and total edited cell count (TEC). Figure 16A Shows the effect of insulin treatment for 6 hours on T cell expansion. Figure 16B Shows the effect of insulin treatment for 6 hours on T cell TEC. On average, for 100 million transfected cells, 6-hour insulin treatment enhances T cell expansion and TEC by 27.7% and 23.1% respectively.

[0030] Figures 17A to 17B Insulin enhances T cell growth and cell count in samples from diseased patients. Figure 17A Shows the results of T cell viability after transfection. Figure 17B Shows the results of T cell count after transfection. Compared with the group without insulin treatment, insulin treatment (6 hours) partially restores the viability and cell count of diseased patients after transfection.

[0031] Figures 18A to 18D Insulin improves T cell metabolism, mitochondrial function and reduces ER stress. Figure 18A Shows the effect of insulin treatment on glucose uptake level. Figure 18B Shows the effect of insulin treatment on mitochondrial potential. Figures 18C to 18D Shows the effect of insulin treatment on ER stress, as measured by the expression of ATF4 ( Figure 18C ) and IRE1 ( Figure 18D ).

[0032] Figures 19A to 19E Insulin treatment stimulates the T cell growth pathway during the process of engineering the TCR. Figures 19A to 19EWestern blot analysis of Akt, Erk, and their phosphorylated forms in T cells treated with (insulin) or without (CTR) insulin before transfection. 25 μg / ml insulin was used for 24-hour treatment. Figure 19A Shows an image of the Western blot gel. Figure 19B Shows total Akt normalized to actin expressed in T cells treated with and without insulin. Figure 19C Shows the ratio of phosphorylated Akt (p-Akt) to total Akt in T cells treated with and without insulin. Figure 19D Shows total Erk normalized to actin expressed in T cells treated with and without insulin. Figure 19E Shows the ratio of phosphorylated Erk (p-Erk) to total Erk in T cells treated with and without insulin. After insulin treatment, total Akt, total ERK, phosphorylated Akt, and phosphorylated ERK in T cells all increased after transfection.

[0033] Figures 20A to 20D Insulin treatment had no effect on the gene editing efficiency of knock-in (KI) or knock-out (KO). Figures 20A to 20B Shows the quantification of gene editing efficiency for knock-in experiments. Figures 20C to 20D Shows the quantification of gene editing efficiency for knock-out experiments.

[0034] Figures 21A to 21C Insulin treatment had no effect on the final cell product (FCP) phenotype. All groups tested maintained approximately 97% total memory phenotype (TCM + TSCM%).

[0035] Figures 22A to 22D Insulin treatment had no effect on the final cell product (FCP) function. Results from CD137 activation assay ( Figure 22A ) and IFN-g ( Figure 22B ) and TNF-a ( Figure 22C ) cytokine expression showed that insulin treatment and control had similar responses to target peptide stimulation at all peptide concentration gradients. Results from T cell cytotoxicity assay against target cells showed comparable killing ability at each E:T ratio between the insulin treatment group and the control group ( Figure 22D ).

[0036] Figures 23A to 23B Negligible insulin residue in T cell FCP medium. Figures 23A to 23B Shows the results obtained from ELISA analysis of cell culture medium collected on day 12. On day 12, the amount of insulin measured in the medium was negligible. Detailed Description

[0037] Although various embodiments and aspects of the present invention are shown and described herein, it will be apparent to those skilled in the art that such embodiments and aspects are provided by way of example only. Many variations, changes, and substitutions will now occur to those skilled in the art without departing from the invention. It should be understood that various alternatives to the embodiments of the invention described herein may be employed in practicing the invention.

[0038] The section headings used herein are for organizational purposes only and should not be construed as limiting the subject matter described. All documents or portions of documents cited in this application, including but not limited to patents, patent applications, articles, books, manuals, and papers, are hereby incorporated by reference in their entirety for any purpose.

[0039] The abbreviations used herein have their conventional meanings in the fields of chemistry and biology. The chemical structures and formulas described herein are constructed according to the standard rules of chemical valence known in the art of chemistry.

[0040] Unless otherwise defined, scientific and technical terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. Methods, devices, and materials similar or equivalent to those described herein may 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 disclosure.

[0041] "Nucleic acid" refers to nucleotides (e.g., deoxyribonucleotides or ribonucleotides) and their polymers in single-stranded, double-stranded, or multi-stranded form or their complementary forms; or nucleosides (e.g., deoxyribonucleosides or ribonucleosides). Examples of polynucleotides contemplated herein include single-stranded and double-stranded DNA, single-stranded and double-stranded RNA, and hybrid molecules having mixtures 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.

[0042] As used herein, the term "gene editing reagent" refers to the components required for a gene editing tool and may include enzymes, nucleoproteins, 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 repeat systems (CRISPR / Cas) gene editing.

[0043] As used herein, a "zinc finger protein" (ZFP) refers to a chimeric protein stabilized by zinc that contains a nuclease domain and a nucleic acid (e.g., DNA) binding domain. The individual DNA binding domains are typically referred to as "fingers" such 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, up to at least six or more fingers. Each finger typically binds two to four base pairs of DNA. Each finger may comprise a DNA binding region of about 30 amino acids that chelates zinc (see, e.g., U.S. Patent Publication No. 2012 / 0329067A1, the disclosure of which is incorporated herein by reference in its entirety).

[0044] As used herein, a "transcription activator-like effector" (TALE) refers to a protein composed of more than one TAL repeat and capable of binding to nucleic acids in a sequence-specific manner. TALEs represent a class of DNA binding proteins secreted by plant pathogenic bacteria of the genera Xanthomonas and Ralstonia via their type III secretion systems after infecting plant cells. Native TALEs have specifically been shown to bind to plant promoter sequences, thereby regulating gene expression and activating effector-specific host genes to promote bacterial proliferation ( 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 TALEs allows for the combination of DNA binding domains with effectors such as nucleases. In particular, TALE nucleases allow for the development of new genome engineering tools.

[0045] The characteristics of natural TALEs typically lie in the central repeat domain and the carboxy-terminal nuclear localization signal sequence (NLS) and transcriptional activation domain (AD). The central repeat domain is generally composed of a variable number of amino acid repeats between 1.5 and 33.5, with a length usually of 33 to 35 residues, except for the generally shorter carboxy-terminal repeats (referred to as half-repeats). The repeats are mostly identical, but certain hypervariable residues differ. The DNA recognition specificity of TALEs is mediated by hypervariable residues usually 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. Thus, the consecutive order of repeats in a TAL protein often correlates with the defined linear order of nucleotides in a given DNA sequence. The potential RVD codes of some naturally occurring TALEs have been identified, allowing prediction of the consecutive repeat order required to bind to a given DNA sequence (Boch, J. et al., Science 326:1509-1512 (2009); Moscou, M.J. et al., Science 326:1501 (2009)). In addition, TAL effectors generated with new repeat combinations have been shown to bind to the target sequences predicted by this code. It has been shown that the target DNA sequence generally starts with a 5'-thymine base to be recognized by the TAL protein.

[0046] The term "RNA-guided DNA nuclease" or "RNA-guided DNA endonuclease", etc. refers, in the usual and customary sense, to an enzyme that cleaves a phosphodiester bond within a DNA polynucleotide chain, where the recognition of the phosphodiester bond is facilitated by a separate RNA sequence (for example, a single guide RNA).

[0047] The term "Class 2 CRISPR endonuclease" refers to an endonuclease that has endonuclease activity similar to Cas9 and is involved in the Class 2 CRISPR system. An exemplary Class 2 CRISPR system is the Class 2 CRISPR locus from Streptococcus pyogenes SF370, which contains a cluster of four genes, Cas9, Cas1, Cas2, and Csn1, as well as two non-coding RNA elements: tracrRNA and an array of characteristic repeat sequences (direct repeats) separated by short segments of non-repeating sequences (spacers, each approximately 30 bp). The Cpf1 enzyme belongs to the putative Type V CRISPR-Cas system. Both Type II and Type V systems are included in Class 2 of the CRISPR-Cas systems. The C2c1 ("Class 2 candidate 1") enzyme is a Class 2 Type V-B enzyme. The C2c2 ("Class 2 candidate 2") enzyme is a Class 2 Type VI-A enzyme. The C2c3 ("Class 2 candidate 3") enzyme is a Class 2 Type V-C 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, Cas13, nCas9, and Cas-CLOVER. Class 2 CRISPR endonucleases can be further modified to be expressed as fusion proteins (e.g., fused to cytidine or adenine base editors).

[0048] As used herein, "CRISPR-associated protein 9", "Cas9", "Csn1", or "Cas9 protein" includes any recombinant or naturally occurring form of Cas9 endonuclease or a variant or homolog thereof that retains Cas9 endonuclease activity (e.g., having an activity that is at least 50%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% within that of Cas9). In certain 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-consecutive amino acid portion) compared to the naturally occurring Cas9 protein. In certain aspects, the Cas9 protein is substantially identical to the protein identified by UniProt accession number Q99ZW2 or a variant or homolog having substantial identity thereto. In certain aspects, the Cas9 protein has at least 75% sequence identity with the amino acid sequence of the protein identified by UniProt accession number Q99ZW2. In certain aspects, the Cas9 protein has at least 80% sequence identity with the amino acid sequence of the protein identified by UniProt accession number Q99ZW2. In certain aspects, the Cas9 protein has at least 85% sequence identity with the amino acid sequence of the protein identified by UniProt accession number Q99ZW2. In certain aspects, the Cas9 protein has at least 90% sequence identity with the amino acid sequence of the protein identified by UniProt accession number Q99ZW2. In certain aspects, the Cas9 protein has at least 95% sequence identity with the amino acid sequence of the protein identified by UniProt accession number Q99ZW2.

[0049] As used herein, "CRISPR-associated endonuclease Cas12a", "Cas12a", "Cas12", or "Cas12 protein" includes any recombinant or naturally occurring form of Cas12 endonuclease or a variant or homolog thereof that retains Cas12 endonuclease activity (e.g., having an activity that is at least 50%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% within that of Cas12). In certain 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-consecutive amino acid portion) compared to the naturally occurring Cas12 protein. In certain aspects, the Cas12 protein is substantially identical to the protein identified by UniProt accession number A0Q7Q2 or a variant or homolog having substantial identity thereto.

[0050] As used herein, "Cfp1" or "Cfp1 protein" includes any recombinant or naturally-occurring form of Cfp1 (CXXC finger protein 1) endonuclease or a variant or homolog thereof that retains Cfp1 endonuclease activity (e.g., having an activity at least within 50%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% 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-consecutive 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 accession number Q9P0U4 or a variant or homolog thereof having substantial identity thereto.

[0051] The terms "RNA-guided RNA nuclease" or "RNA-guided RNA enzyme", etc., in their ordinary and customary sense, refer 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))). Generally, an RNA-guided RNA enzyme targets single-stranded RNA. In certain aspects, the RNA-guided RNA enzyme is Cas13 (e.g., Cas13a, Cas13b).

[0052] As used herein, "Cas13a" or "Cas13a protein" includes Cas13a (CRISPR-associated ribonuclease Cas13a, also known as CRISPR-associated ribonuclease C2c2, C2c2) endonuclease, or any recombinant or naturally-occurring form of a variant or homolog thereof that retains Cas13a endonuclease activity (e.g., having an activity at least within 50%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% compared to Cas13a). 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-consecutive amino acid portion) compared to the naturally-occurring Cas13a protein. In an embodiment, the Cas13a protein is substantially identical to the protein identified by UniProt accession number C7NBY4 or a variant or homolog thereof having substantial identity thereto.

[0053] As used herein, "Cas13b" or "Cas13b protein" includes any recombinant or naturally occurring form of the Cas13b (CRISPR-associated RNA-guided ribonuclease Cas13b) endonuclease, or a variant or homolog thereof that retains Cas13b endonuclease activity (e.g., having an activity that is at least 50%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% within that of Cas13b). 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-consecutive amino acid portion) compared to the naturally occurring Cas13b protein. In embodiments, the Cas13b protein is substantially identical to the protein identified by UniProt accession number A0A8G0P913 or a variant or homolog having substantial identity thereto.

[0054] In embodiments, the gene editing reagent includes Cas-CLOVER. In embodiments, Cas-CLOVER comprises the Clo051 nuclease domain fused to catalytically inactive Cas9 (dead Cas9). See, e.g., U.S. Patent Publication No. US2021 / 0107993; and Madison et al., Molecular Therapy Nucleic Acids, Vol. 29, pp. 979-995, September 13, 2022, each of which is incorporated herein by reference in its entirety. In embodiments, the gene editing reagent includes a nickase, such as nCas9 (nickase Cas9). A nickase is an engineered Cas protein that is capable of introducing a single-strand cut and has the same specificity as a conventional CRISPR / Cas nuclease. See, e.g., PCT Publication No. WO2014093694, which is incorporated herein by reference in its entirety.

[0055] The terms "guide RNA" and "gRNA", "single guide RNA" and "sgRNA" are used interchangeably and refer to a polynucleotide sequence comprising a crRNA sequence and optionally an optional tracrRNA sequence. In embodiments, the gRNA comprises a crRNA sequence and a tracrRNA sequence. (e.g., "single guide RNA" or "sgRNA"). In embodiments, the gRNA does not comprise a tracrRNA sequence. The crRNA sequence comprises a guide sequence (i.e., "guide" or "spacer") and a tracr matching sequence (i.e., direct repeat). The term "guide sequence" refers to a sequence that specifies a target site. Generally, the tracr matching sequence comprises any sequence having sufficient complementarity to the tracrRNA sequence to facilitate one or more of the following: (1) excision of the guide sequence flanked by the tracr pairing sequence in a cell containing the corresponding tracr sequence; and (2) formation of a complex (e.g., a CRISPR complex) at the target sequence, wherein the complex (e.g., a CRISPR complex) comprises a tracr pairing sequence hybridized to the tracr sequence.

[0056] In embodiments, the gRNA is a single-stranded ribonucleic acid. In some aspects, the gRNA has a length of from about 10 to about 200 nucleic acid residues. In some aspects, the gRNA has a length of from about 50 to about 150 nucleic acid residues. In some aspects, the gRNA has a length of from about 80 to about 140 nucleic acid residues. In some aspects, the gRNA has a length of from about 90 to about 130 nucleic acid residues. In some aspects, the gRNA has a length of from about 100 to about 120 nucleic acid residues.

[0057] Generally, a guide sequence is any polynucleotide sequence that has sufficient complementarity to a target polynucleotide sequence to hybridize with the target sequence and direct sequence-specific binding of a CRISPR complex sequence to the target sequence. In some embodiments, the degree of complementarity between the guide sequence and its corresponding target sequence is about or more 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 the optimal alignment, non-limiting examples of which include the Smith-Waterman algorithm, the Needleman-Wunsch algorithm, algorithms based on the Burrows-Wheeler transform (e.g., Burrows Wheeler Aligner), ClustalW, Clustal X, BLAST, Novoalign (Novocraft Technologies), ELAND (Illumina, San Diego, Calif.), SOAP (available at soap.genomics.org.cn), and Maq (available at maq.sourceforge.net). In embodiments, the guide sequence is about or more 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 ability of the guide sequence to direct sequence-specific binding of the CRISPR complex to the target sequence can be assayed by any suitable assay. For example, the components of a CRISPR system sufficient to form a CRISPR complex, including the guide sequence to be tested, can be provided to a host cell having the corresponding target sequence, such as by transfection with a vector encoding the CRISPR sequence components, and subsequent preferential cleavage within the target sequence can be assayed, such as by the Surveyor assay described herein. Similarly, cleavage of the target polynucleotide sequence can be evaluated in vitro by providing the target sequence, the components of the CRISPR complex (including the guide sequence to be tested and a control guide sequence different from the test guide sequence), and comparing the binding or cleavage rates at the target sequence between the test and control guide sequence reactions. Other assays are possible and are contemplated by those of skill in the art.

[0058] 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 gene modification method (e.g., CRISPR). For example, the donor DNA can have homology arms that are homologous to the gene region into which the donor DNA is to be inserted. For example, the donor DNA can form a complex with a Cas protein. In some cases, cells can be transfected with a gene editing reagent and donor DNA. In an embodiment, the donor DNA is part of a plasmid, vector, or expression vector that facilitates delivery of the donor DNA into the cell. In an embodiment, the donor DNA is part of circular DNA.

[0059] In an embodiment, the donor DNA is part of linear DNA. In an embodiment, the donor DNA can comprise one or more modifications. Nucleic acids (such as donor DNA) used in the methods herein can be modified. For example, the nucleic acid can include synthetic, naturally occurring, and non-naturally occurring known nucleotide analogs or modified backbone residues or linkages that have binding properties similar to a reference nucleic acid and are metabolized in a manner similar to the reference nucleotide. Examples of such analogs include, but are not limited to, phosphodiester derivatives, including, for example, phosphoramidates, phosphorodiamidates, phosphorothioates (also known as phosphorothioate oligodeoxynucleotides with double bond sulfur substitution for oxygen in the phosphate), dithiophosphates, phosphonylcarboxylic acids, phosphonylcarboxylates, phosphonoacetic acid, phosphonoformic acid, methylphosphonates, phosphoborates, or O-methyl phosphoramidite linkages (see Eckstein, OLIGONUCLEOTIDES AND ANALOGUES: A PRACTICAL APPROACH, Oxford University Press) and modifications to nucleobases (such as 5-methylcytidine or pseudouridine); and peptide nucleic acid backbones and linkages. Other analog nucleic acids include analog nucleic acids having a positive backbone, a non-ionic backbone, modified sugars, and non-ribose backbones (e.g., phosphorodiamidate morpholino oligomers or locked nucleic acid (LNA) known in the art), including those described in U.S. Patent Nos. 5,235,033 and 5,034,506 and in Chapters 6 and 7 of ASC Symposium Series 580, CARBOHYDRATE MODIFICATIONSIN ANTISENSE RESEARCH, edited by Sanghui & Cook. Nucleic acids containing one or more carbocyclic sugars are also included in one definition of nucleic acids. Mixtures of naturally occurring nucleic acids and analogs can be prepared; alternatively, mixtures of different nucleic acid analogs, as well as mixtures of naturally occurring nucleic acids and analogs, can be prepared. In an embodiment, the internucleotide linkage in the DNA is a phosphodiester, a phosphodiester derivative, or a combination of both.

[0060] As used herein, "insulin" refers to the polypeptide hormone that is naturally encoded by the INS gene and is naturally produced in the β-cells of the pancreas. Insulin is also referred to as "proinsulin" and also includes any recombinant or naturally-occurring form of insulin or a variant or homolog thereof that retains insulin activity (e.g., has an activity that is at least within 50%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% compared to insulin). In certain aspects, a variant, analog, pharmaceutical, drug 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 compared to a naturally-occurring insulin polypeptide. In certain aspects, the insulin polypeptide is substantially the same as the polypeptide identified by UniProt accession number P01308 or a variant or homolog thereof that has substantial identity thereto.

[0061] As used herein, the terms "insulin analog", "insulin agonist" or "insulin partial agonist" are used interchangeably and refer to any molecule that mimics the activity of the naturally-occurring insulin polypeptide hormone. Unless explicitly excluded, when "insulin" is used herein, it is intended to encompass "insulin agonist", "insulin partial agonist" or "insulin analog".

[0062] The term "gene" means a DNA fragment that is related to the production of a protein; it includes the regions before and after the coding region (leader region and tail region) and the intervening sequences (introns) between the individual coding segments (exons). The leader region, tail region and introns include regulatory elements that are essential for gene transcription and translation. Further, a "protein gene product" is the protein expressed by a specific gene.

[0063] The terms "plasmid", "vector" or "expression vector" refer to a nucleic acid molecule that encodes a gene and / or regulatory components 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.

[0064] As used herein, the term "nano-plasmid" is used to refer to a circular nucleic acid that contains at least the nucleic acid sequence of interest, a mini-replication origin (e.g., R6K) and an optional marker (e.g., a small RNA selectable marker, RNA-OUT). The nano-plasmid contains less than 500 bp of prokaryotic DNA.

[0065] As used herein, the term "minicircle" or "mcDNA" is used to refer to a supercoiled, circular plasmid DNA that carries the gene of interest, is less than 4 kb, and has had all prokaryotic vector parts removed.

[0066] As used herein, terms such as "engineering T cells" or "T cell genetic engineering" refer to a class of genetic modifications in which DNA is inserted, deleted, modified, or replaced at one or more specific locations in the T cell genome. Different from early genetic engineering techniques that randomly insert genetic material into the host genome, T cell engineering targets gene modifications at site-specific locations. Gene editing reagents can be used to engineer T cells, for example, to introduce double-strand breaks at specific points within a gene or the genome where DNA is to be inserted. Gene editing reagents can include, for example but not limited to, the clustered regularly interspaced short palindromic repeats (CRISPR / Cas) system, ZFNs, or TALENs. Thus, "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.

[0067] When used with respect to, for example, a virus, cell, nucleic acid, protein, or vector, the term "recombinant" indicates that the cell (e.g., a T cell), nucleic acid, protein, or vector has been modified by the introduction of a heterologous nucleic acid or protein, or an 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 a gene that is not present in the native (non-recombinant) form of the cell or expresses a native gene that is otherwise abnormally expressed, under-expressed, or not expressed at all. Transgenic cells and plants are those that express a heterologous gene or coding sequence, typically as a result of recombinant methods.

[0068] When the term "heterologous" is used with respect to a portion of a nucleic acid, it refers to a nucleic acid that contains two or more subsequences that do not have the same relationship to each other in nature. For example, a nucleic acid can be recombinantly produced and have 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 refers to a protein that contains two or more subsequences that do not have the same relationship to each other in nature (e.g., a fusion protein).

[0069] The term "exogenous" refers to a molecule or substance (e.g., a compound, nucleic acid, or protein) that is derived from outside a given cell or organism. For example, an "exogenous promoter" as referred to herein is a promoter that is not derived 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.

[0070] When applied to nucleic acids or proteins, the term "isolated" means that the nucleic acid or protein is substantially free of other cellular components with which it is associated in its natural state. For example, it can be in a homogeneous state and can be a dry solution or an aqueous solution. Purity and homogeneity are generally determined using analytical chemistry techniques such as polyacrylamide gel electrophoresis or high performance liquid chromatography. A nucleic acid that is the major species present in a preparation is substantially purified.

[0071] As used herein, the terms "electroporation", "electropermeabilization", and "electrotransfer" are used in their simple general sense and refer to a technique of applying an electric field to a cell to increase the permeability of the cell membrane and allow the introduction of a chemical, drug, protein, or nucleic acid, or a combination thereof, into the cell.

[0072] The terms "transfection / transfecting" or "transduction / transducing" are used interchangeably and are defined as the process of introducing a nucleic acid molecule or a protein into a cell. Nucleic acids are introduced into cells using non-viral or virus-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 the nucleic acid molecule into the cell. Exemplary non-viral transfection methods include calcium phosphate transfection, liposome transfection, nucleofection, sonoporation, transfection by heat shock, magnetofection, and electroporation. In some embodiments, nucleic acid molecules are introduced into cells using electroporation according to standard procedures well known in the art. For virus-based transfection methods, any useful viral vector (e.g., an adenoviral vector) can be used in the methods described herein. Examples of viral vectors include, but are not limited to, retroviruses, adenoviruses, lentiviruses, and adeno-associated viral vectors. In some embodiments, nucleic acid molecules are introduced into cells using an adenoviral vector according to standard procedures well known in the art. The terms "transfection" or "transduction" also refer to the introduction of a protein from the external environment into a cell. In embodiments, the transduction or transfection of a protein 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.

[0073] "Transduce or transduction" is used in its simple general sense and refers to the process of introducing one or more foreign nucleic acids (i.e., DNA that is not naturally present in the cell) into a cell. Transduction can be carried out by introducing a virus or viral vector (e.g., an adenoviral vector) into the cell.

[0074] As used herein, the term "expression" or "expressed" with respect to a gene means the transcriptional and / or translational product of that 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 the corresponding mRNA in the cell or the amount of the 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, e.g., Sambrook et al., 1989 Molecular Cloning: A Laboratory Manual, 18.1-18.88.

[0075] "Contact" is used in its plain and ordinary meaning and refers to the 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 touch. The two substances can be, for example, insulin or an insulin analogue and a T cell. In embodiments, contact includes, for example, allowing the insulin or insulin analogue described herein to physically contact a T cell. In embodiments, contact can result in delivery of a compound into a cell. For example, contact can result in delivery of insulin or an insulin analogue into a cell. In embodiments, contact can result in delivery of a nucleic acid into a cell. In embodiments, "contact" or "contacted" includes culturing T cells in the presence of a substance (e.g., insulin or an insulin analogue).

[0076] "Control" or "standard control" refers to a sample, measurement result, or value used as a reference, typically a known reference, for comparing a test sample, measurement result, or value. For example, a standard control can be engineered T cells prepared without contacting (e.g., culturing) T cells with one or more of the insulins or insulin analogs (including their embodiments) provided herein. In an embodiment, the standard control can be a population of engineered T cells prepared without contacting (e.g., culturing) a population of T cells with one or more of the insulins or insulin analogs (including their embodiments) provided herein. Thus, a standard control can be engineered T cells prepared by contacting T cells with a nucleic acid without using one or more insulins or insulin analogs. A standard control can be a population of engineered T cells prepared by contacting a population of T cells with a nucleic acid without using one or more insulins or insulin analogs. Controls are also valuable for determining the significance of data. For example, if the value of a given parameter varies widely in the control, a change in the test sample will not be considered significant. One of ordinary skill in the art will recognize that standard controls can be designed to assess any number of parameters (e.g., cell viability, cell expansion, total number of edited cells, gene editing efficiency, etc.). Those of ordinary skill in the art will understand which standard controls are most appropriate in a given situation and can analyze data based on a comparison to the standard control values.

[0077] As used herein, "T cell" or "T lymphocyte" is a class of lymphocytes (a subtype of white blood cells) that play a central role in cell-mediated immunity. It 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 (CTL), regulatory T (Treg) cells, and T helper cells. Different types of T cells can be distinguished by using T cell detection agents.

[0078] As defined herein, terms such as "inhibition / inhibit / inhibiting" with respect to cell proliferation mean negatively affecting (e.g., reducing proliferation) or killing cells. In some embodiments, inhibition refers to a reduction in a disease or a disease symptom (e.g., cancer, cancer cell proliferation). In an embodiment, an "inhibitor" is, for example, a compound or protein that inhibits a receptor or another protein by binding, partially or completely blocking, reducing, preventing, delaying, inactivating, desensitizing, or downregulating its activity (e.g., receptor activity or protein activity).

[0079] The term "disease" or "condition" refers to the 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 additional cases, "cancer" refers to human cancer. In the examples, the cancer is lymphoma, melanoma or leukemia.

[0080] "Patient", "subject" or "subject in need thereof" refers to an organism suffering from or predisposed to a disease (e.g., cancer, etc.) or condition that can be treated by administering the compositions or pharmaceutical compositions 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 examples, the subject is human.

[0081] As used herein, the term "cancer" refers to all types of cancer, neoplasm or malignancy found in mammals (e.g., humans), including leukemia, lymphoma, carcinoma and sarcoma. Exemplary cancers that can be treated with the compounds or methods provided herein include brain cancer, glioma, glioblastoma, neuroblastoma, prostate cancer, colorectal cancer, pancreatic cancer, medulloblastoma, melanoma, cervical cancer, gastric cancer, ovarian cancer, lung cancer, head cancer, Hodgkin's disease and non-Hodgkin's lymphoma. Exemplary cancers that can be treated with the compounds or methods provided herein include 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, cholangiocarcinoma, pancreatic cancer, cutaneous melanoma, colon adenocarcinoma, rectal adenocarcinoma, gastric adenocarcinoma, esophageal cancer, head and neck squamous cell carcinoma, breast invasive carcinoma, lung adenocarcinoma, lung squamous cell carcinoma, non-small cell lung cancer, mesothelioma, multiple myeloma, neuroblastoma, glioma, glioblastoma multiforme, ovarian cancer, rhabdomyosarcoma, essential thrombocythemia, Waldenström's macroglobulinemia, primary brain tumor, malignant pancreatic insulinoma, malignant carcinoid, bladder cancer, precancerous skin lesions, testicular cancer, thyroid cancer, neuroblastoma, esophageal cancer, urogenital cancer, malignant hypercalcemia, endometrial cancer, adrenocortical carcinoma, endocrine or exocrine pancreatic tumors, medullary thyroid cancer, medullary thyroid carcinoma, melanoma, colorectal cancer, papillary thyroid cancer, hepatocellular carcinoma or prostate cancer.

[0082] As used herein, "treatment" or "treating" a condition, disease or disorder, or a symptom associated with a condition, disease or disorder, refers to a method for obtaining a beneficial or desired result, including a clinical result. Beneficial or desired clinical results can include, but are not limited to: alleviation or improvement of one or more symptoms or conditions; reduction in the severity of a condition, disorder or disease; stabilization of a condition, disorder or disease state; prevention of the spread of a condition, disorder or disease; delay or slowing of the progression of a condition, disorder or disease; postponement or slowing of the onset of a condition, disorder or disease; improvement or alleviation of a condition, disorder or disease state; and remission (whether partial or complete). "Treatment" can also mean prolonging the survival of a subject beyond that expected in the absence of treatment. "Treatment" can also mean inhibiting the progression of a disorder, condition or disease, temporarily slowing the progression of a disorder, condition or disease, although in some instances, it involves permanently halting the progression of a disorder, condition or disease.

[0083] The terms "dose" and "dosage" are used interchangeably herein. A dose refers to the amount of active ingredient administered to an individual at each administration. The dose will vary depending on a variety of factors, including the normal dosage 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 can be modified based on the above factors or on the progress of the treatment.

[0084] As used herein, a "therapeutically effective dose or amount" means a dose that produces the effect for which it is administered (e.g., treats or prevents a disease). The exact dose or formulation will depend on the purpose of the treatment and will be determined by those skilled in the art using known techniques (see, e.g., Lieberman, Pharmaceutical Dosage Forms (vols. 1-3, 1992); Lloyd, The Art, Science and Technology of Pharmaceutical Compounding (1999); Remington: The Science and Practice of Pharmacy, 20th ed., Gennaro ed. (2003); and Pickar, Dosage Calculations (1999)). For example, for a given parameter, a therapeutically effective amount will exhibit an increase or decrease of at least 5%, 10%, 15%, 20%, 25%, 40%, 50%, 60%, 75%, 80%, 90% or at least 100%. Efficacy can also be expressed as a "multiple" of increase or decrease. For example, a therapeutically effective amount can have at least 1.2-fold, 1.5-fold, 2-fold, 5-fold or more effect compared to a standard control. A therapeutically effective dose or amount can improve one or more symptoms of a disease.

[0085] As used herein, the term "administering" is used in its plain and ordinary meaning and includes any administration suitable for cell therapy. For example, the administration can be parenteral administration. Parenteral administration includes, for example, intravenous, intramuscular, intra-arterial, intradermal, subcutaneous, intraperitoneal, intraventricular, and intracranial. In an embodiment, the administration is intravenous.

[0086] Method

[0087] Provided herein are, in particular, methods of engineering T cells, which include contacting the T cells with insulin, insulin analogs, insulin agonists, and / or insulin partial agonists. Compared with previously known methods of engineering T cells, the methods provided herein allow for the formation of engineered T cells while improving the viability and growth of the T cells. For example, the methods provided herein are expected to effectively improve growth and viability by activating parallel growth signaling pathways.

[0088] In one aspect, provided herein is a method of editing an endogenous gene in a population of T cells, the method comprising: contacting the population of T cells with the gene editing reagent or the polynucleotide encoding the gene editing reagent under conditions that permit entry of the polynucleotide or the gene editing reagent encoding the gene editing reagent into the cells; and culturing the population of T cells in the presence of one or more of the following before and / or during and / or after the contacting step to obtain a population of engineered T cells: insulin, insulin analogs, insulin agonists, and / or insulin partial agonists.

[0089] For the methods provided herein, in an embodiment, further comprises contacting the population of T cells with donor DNA. In an embodiment, the polynucleotide encoding the gene editing reagent comprises: single-stranded DNA, double-stranded DNA, linear DNA strand, plasmid, nanoplasmid, or minicircle. In an embodiment, the polynucleotide encoding the gene editing reagent comprises single-stranded DNA. In an embodiment, the polynucleotide encoding the gene editing reagent comprises double-stranded DNA. In an embodiment, the polynucleotide encoding the gene editing reagent comprises a linear DNA strand. In an embodiment, the polynucleotide encoding the gene editing reagent comprises a plasmid. In an embodiment, the polynucleotide encoding the gene editing reagent comprises a nanoplasmid. In an embodiment, the polynucleotide encoding the gene editing reagent comprises a minicircle.

[0090] In an embodiment, the polynucleotide encoding the gene editing reagent comprises a plasmid, the plasmid comprising a plasmid backbone and a polynucleotide sequence encoding the gene editing reagent. In an embodiment, the plasmid further comprises donor DNA. In an embodiment, the donor DNA sequence comprises a polynucleotide encoding a gene product. In an embodiment, the population of T cells is obtained from a subject.

[0091] In embodiments, the gene product sequence comprises a chimeric antigen receptor (CAR), a T cell receptor (TCR), a human leukocyte antigen (HLA), or an allogeneic immune defense receptor (ADR) or a subunit thereof. In embodiments, the gene product sequence comprises a chimeric antigen receptor (CAR). In embodiments, the gene product sequence comprises a T cell receptor (TCR). In embodiments, the gene product sequence comprises a human leukocyte antigen (HLA). In embodiments, the gene product sequence comprises an allogeneic immune defense receptor (ADR).

[0092] In embodiments, the TCR sequence comprises an exogenous TCR-β subunit or a fragment thereof, and / or an exogenous TCR-α subunit or a fragment thereof, or a chimeric antigen receptor and / or a subunit thereof. In embodiments, the TCR sequence comprises an exogenous TCR-β subunit or a fragment thereof and an exogenous TCR-α subunit or a fragment thereof. In embodiments, the TCR sequence comprises an exogenous TCR-β subunit or a fragment thereof, or an exogenous TCR-α subunit or a fragment thereof. In embodiments, the TCR sequence comprises an exogenous TCR-β subunit or a fragment thereof. In embodiments, the TCR sequence comprises an exogenous TCR-α subunit or a fragment thereof. In embodiments, the TCR sequence comprises a chimeric antigen receptor and a subunit thereof. In embodiments, the TCR sequence comprises a chimeric antigen receptor or a subunit thereof. In embodiments, the TCR sequence is inserted into the TRAC or TRBC locus. In embodiments, the TCR sequence is inserted into the TRAC locus. In embodiments, the TCR sequence is inserted into the TRBC locus.

[0093] In embodiments, contacting a T cell population with a gene editing reagent or a polynucleotide encoding the gene editing reagent comprises transfecting the T cell population with the gene editing reagent or the polynucleotide encoding the gene editing reagent. In embodiments, contacting a T cell population with a gene editing reagent comprises transfecting the T cell population with the gene editing reagent. In embodiments, contacting a T cell population with a polynucleotide encoding a gene reagent comprises transfecting the T cell population with the polynucleotide encoding the gene editing reagent. In embodiments, transfection comprises electroporation. In embodiments, transfection comprises nucleofection. In embodiments, transfection comprises liposome transfection.

[0094] In some cases, delivery of a polynucleotide to a T cell can be facilitated by a delivery vehicle. The delivery vehicle can facilitate the interaction of the polynucleotide with the T cell membrane, thereby allowing the polynucleotide to enter the T cell. In one example, the polynucleotide can be encapsulated in the delivery vehicle. In another example, the polynucleotide can be non-covalently associated with the delivery vehicle. Thus, in embodiments, the polynucleotide is associated with the delivery vehicle. In embodiments, the delivery vehicle is a lipid particle or a nanoparticle. In embodiments, the delivery vehicle is a lipid particle. In embodiments, the delivery vehicle is a nanoparticle. In embodiments, the delivery vehicle is a liposome or a lipid nanoparticle.

[0095] For the methods provided herein, in an embodiment, the T cells are primary T cells. "Primary T cells" are used in accordance with their ordinary meaning in the biological arts and refer to T cells directly expanded from T cells isolated from a subject. Thus, "secondary T cells" are T cells expanded from primary T cells. For example, secondary T cells are T cells expanded via a primary T cell culture.

[0096] For the methods provided herein, a variety of methods known in the art (including but not limited to electroporation and transfection methods) can be used to deliver gene editing reagents and polynucleotides into T cells. In an embodiment, contacting the T cells with a gene editing reagent comprises transfecting the T cells with the gene editing reagent. In an embodiment, contacting the T cells with a polynucleotide comprises transfecting the T cells with the polynucleotide.

[0097] As described above, insulin is a naturally occurring insulin polypeptide. In some embodiments, insulin has a propeptide configuration. In some embodiments, insulin is mature, naturally disulfide-linked A-chain and B-chain insulin. In some embodiments, insulin is a sequence variant of a naturally occurring or wild-type insulin sequence. In some embodiments, insulin is purified from an animal source. In some embodiments, insulin is from a shark source. In some embodiments, insulin is from a fish source. In some embodiments, insulin is from a mammalian source. In some embodiments, insulin is recombinantly produced. In some embodiments, recombinantly produced insulin is a mammalian polypeptide sequence. In some embodiments, recombinantly produced insulin is a porcine polypeptide sequence. In some embodiments, recombinantly produced insulin is a human polypeptide sequence. In some embodiments, the human sequence is identical to the full sequence of P01308 (UniProtKB). In some embodiments, the human sequence is identical to a portion of P01308 (UniProtKB). In some embodiments, the human sequence comprises the A-chain and B-chain of P01308 (UniProtKB). In some embodiments, insulin is a pharmaceutical. In some embodiments, insulin is a commodity not suitable for use as a drug.

[0098] In an embodiment, the insulin is an insulin analogue (agonist). In an embodiment, when applied to T cells, the insulin analogue produces the same effect as naturally occurring insulin. In an embodiment, when applied to T cells, the insulin analogue produces an effect that is substantially similar to that of naturally occurring insulin. In an embodiment, the insulin analogue is long-acting or short-acting. In an embodiment, long-acting insulin analogues include detemir, glargine, degludec, or a combination thereof. In an embodiment, short-acting insulin analogues include aspart, lispro, glulisine, or a combination thereof. In an embodiment, one or more insulin analogues include detemir. In an embodiment, one or more insulin analogues include glargine. In an embodiment, one or more insulin analogues include degludec. In an embodiment, one or more insulin analogues include aspart. In an embodiment, one or more insulin analogues include lispro. In an embodiment, one or more insulin analogues include glulisine.

[0099] In an embodiment, the insulin analogue is detemir. In an embodiment, the insulin analogue is glargine. In an embodiment, the insulin analogue is degludec. In an embodiment, the insulin analogue is aspart. In an embodiment, the insulin analogue is lispro. In an embodiment, the insulin analogue is glulisine.

[0100] The applicant has unexpectedly found that treating T cells with insulin before or in the presence of a polynucleotide improves the effectiveness of engineering T cells. In an embodiment, T cells and a polynucleotide are contacted in the presence of insulin. For example, T cells can be transfected with a polynucleotide in the presence of insulin. In another example, T cells can be electroporated with a polynucleotide in the presence of insulin. In an embodiment, T cells are contacted with a polynucleotide and insulin sequentially. In an embodiment, T cells are contacted with insulin before the polynucleotide. For example, insulin can be added to the T cell culture before transfecting the T cells with the polynucleotide. In an embodiment, T cells are contacted with insulin after the polynucleotide. For example, insulin can be added to the T cell culture after transfecting the T cells with the polynucleotide.

[0101] In an embodiment, prior to the contacting step, a T cell population is cultured in the presence of insulin, an insulin analogue, an insulin agonist, and / or an insulin partial agonist. In an embodiment, prior to the contacting step, a T cell population is cultured in the presence of insulin, an insulin analogue, an insulin agonist, and an insulin partial agonist. In an embodiment, prior to the contacting step, a T cell population is cultured in the presence of insulin, an insulin analogue, an insulin agonist, or an insulin partial agonist. In an embodiment, prior to the contacting step, a T cell population is cultured in the presence of insulin. In an embodiment, prior to the contacting step, a T cell population is cultured in the presence of an insulin analogue. In an embodiment, prior to the contacting step, a T cell population is cultured in the presence of an insulin agonist. In an embodiment, prior to the contacting step, a T cell population is cultured in the presence of an insulin partial agonist. In an embodiment, it includes culturing a T cell population in the presence of insulin, an insulin analogue, an insulin agonist, and / or an insulin partial agonist for 48 hours, 24 hours, 12 hours, 6 hours, 4 hours, 2 hours, 1 hour, or 30 minutes prior to the contacting step. In an embodiment, it includes culturing a T cell population in the presence of insulin, an insulin analogue, an insulin agonist, and / or an insulin partial agonist for 48 hours prior to the contacting step. In an embodiment, it includes culturing a T cell population in the presence of insulin, an insulin analogue, an insulin agonist, and / or an insulin partial agonist for 24 hours prior to the contacting step. In an embodiment, it includes culturing a T cell population in the presence of insulin, an insulin analogue, an insulin agonist, and / or an insulin partial agonist for 12 hours prior to the contacting step. In an embodiment, it includes culturing a T cell population in the presence of insulin, an insulin analogue, an insulin agonist, and / or an insulin partial agonist for 6 hours prior to the contacting step. In an embodiment, it includes culturing a T cell population in the presence of insulin, an insulin analogue, an insulin agonist, and / or an insulin partial agonist for 4 hours prior to the contacting step. In an embodiment, it includes culturing a T cell population in the presence of insulin, an insulin analogue, an insulin agonist, and / or an insulin partial agonist for 2 hours prior to the contacting step. In an embodiment, it includes culturing a T cell population in the presence of insulin, an insulin analogue, an insulin agonist, and / or an insulin partial agonist for 1 hour prior to the contacting step. In an embodiment, it includes culturing a T cell population in the presence of insulin, an insulin analogue, an insulin agonist, and / or an insulin partial agonist for 30 minutes prior to the contacting step.

[0102] In an embodiment, after the contacting step, the T cell population is cultured in the presence of insulin, an insulin analogue, an insulin agonist, and / or an insulin partial agonist. In an embodiment, after the contacting step, the T cell population is cultured in the presence of insulin, an insulin analogue, an insulin agonist, and an insulin partial agonist. In an embodiment, after the contacting step, the T cell population is cultured in the presence of insulin, an insulin analogue, an insulin agonist, or an insulin partial agonist. In an embodiment, after the contacting step, the T cell population is cultured in the presence of insulin. In an embodiment, after a step similar to the contacting step, the T cell population is cultured in the presence of an insulin analogue. In an embodiment, after a step similar to the contacting step, the T cell population is cultured in the presence of an insulin agonist. In an embodiment, after a step similar to the contacting step, the T cell population is cultured in the presence of an insulin partial agonist. In an embodiment, it includes culturing the T cell population in the presence of insulin, an insulin analogue, an insulin agonist, and / or an insulin partial agonist for 48 hours after the contacting step. In an embodiment, it includes culturing the T cell population in the presence of insulin, an insulin analogue, an insulin agonist, and / or an insulin partial agonist for 24 hours after the contacting step. In an embodiment, it includes culturing the T cell population in the presence of insulin, an insulin analogue, an insulin agonist, and / or an insulin partial agonist for 12 hours after the contacting step. In an embodiment, it includes culturing the T cell population in the presence of insulin, an insulin analogue, an insulin agonist, and / or an insulin partial agonist for 6 hours after the contacting step. In an embodiment, it includes culturing the T cell population in the presence of insulin, an insulin analogue, an insulin agonist, and / or an insulin partial agonist for 4 hours after the contacting step. In an embodiment, it includes culturing the T cell population in the presence of insulin, an insulin analogue, an insulin agonist, and / or an insulin partial agonist for 2 hours after the contacting step. In an embodiment, it includes culturing the T cell population in the presence of insulin, an insulin analogue, an insulin agonist, and / or an insulin partial agonist for 1 hour after the contacting step. In an embodiment, it includes culturing the T cell population in the presence of insulin, an insulin analogue, an insulin agonist, and / or an insulin partial agonist for 30 minutes after the contacting step.

[0103] In an embodiment, before and after the contacting step, a T cell population is cultured in the presence of insulin, an insulin analogue, an insulin agonist, and / or an insulin partial agonist. In an embodiment, before and after the contacting step, a T cell population is cultured in the presence of insulin, an insulin analogue, an insulin agonist, and an insulin partial agonist. In an embodiment, before and after the contacting step, a T cell population is cultured in the presence of insulin, an insulin analogue, an insulin agonist, or an insulin partial agonist. In an embodiment, before and after the contacting step, a T cell population is cultured in the presence of insulin. In an embodiment, before and after the contacting step, a T cell population is cultured in the presence of an insulin analogue. In an embodiment, before and after the contacting step, a T cell population is cultured in the presence of an insulin agonist. In an embodiment, before and after the contacting step, a T cell population is cultured in the presence of an insulin partial agonist.

[0104] In an embodiment, it includes culturing a T cell population in the presence of insulin, an insulin analogue, an insulin agonist, and / or an insulin partial agonist for 48 hours, 24 hours, 12 hours, 6 hours, 4 hours, 2 hours, 1 hour, or 30 minutes before the contacting step. In an embodiment, it includes culturing a T cell population in the presence of insulin, an insulin analogue, an insulin agonist, and / or an insulin partial agonist for 48 hours before the contacting step. In an embodiment, it includes culturing a T cell population in the presence of insulin, an insulin analogue, an insulin agonist, and / or an insulin partial agonist for 24 hours before the contacting step. In an embodiment, it includes culturing a T cell population in the presence of insulin, an insulin analogue, an insulin agonist, and / or an insulin partial agonist for 12 hours before the contacting step. In an embodiment, it includes culturing a T cell population in the presence of insulin, an insulin analogue, an insulin agonist, and / or an insulin partial agonist for 6 hours before the contacting step. In an embodiment, it includes culturing a T cell population in the presence of insulin, an insulin analogue, an insulin agonist, and / or an insulin partial agonist for 4 hours before the contacting step. In an embodiment, it includes culturing a T cell population in the presence of insulin, an insulin analogue, an insulin agonist, and / or an insulin partial agonist for 2 hours before the contacting step. In an embodiment, it includes culturing a T cell population in the presence of insulin, an insulin analogue, an insulin agonist, and / or an insulin partial agonist for 1 hour before the contacting step. In an embodiment, it includes culturing a T cell population in the presence of insulin, an insulin analogue, an insulin agonist, and / or an insulin partial agonist for 30 minutes before the contacting step.

[0105] In an embodiment, it includes culturing a T cell population for 48 hours, 24 hours, 12 hours, 6 hours, 4 hours, 2 hours, 1 hour, or 30 minutes in the presence of insulin, an insulin analogue, an insulin agonist, and / or an insulin partial agonist after the contacting step. In an embodiment, it includes culturing a T cell population for 48 hours in the presence of insulin, an insulin analogue, an insulin agonist, and / or an insulin partial agonist after the contacting step. In an embodiment, it includes culturing a T cell population for 24 hours in the presence of insulin, an insulin analogue, an insulin agonist, and / or an insulin partial agonist after the contacting step. In an embodiment, it includes culturing a T cell population for 12 hours in the presence of insulin, an insulin analogue, an insulin agonist, and / or an insulin partial agonist after the contacting step. In an embodiment, it includes culturing a T cell population for 6 hours in the presence of insulin, an insulin analogue, an insulin agonist, and / or an insulin partial agonist after the contacting step. In an embodiment, it includes culturing a T cell population for 4 hours in the presence of insulin, an insulin analogue, an insulin agonist, and / or an insulin partial agonist after the contacting step. In an embodiment, it includes culturing a T cell population for 2 hours in the presence of insulin, an insulin analogue, an insulin agonist, and / or an insulin partial agonist after the contacting step. In an embodiment, it includes culturing a T cell population for 1 hour in the presence of insulin, an insulin analogue, an insulin agonist, and / or an insulin partial agonist after the contacting step. In an embodiment, it includes culturing a T cell population for 30 minutes in the presence of insulin, an insulin analogue, an insulin agonist, and / or an insulin partial agonist after the contacting step.

[0106] In an embodiment, insulin, an insulin analogue, an insulin agonist, and / or an insulin partial agonist is administered at a concentration of from about 1 μg / ml to about 50 μg / ml. In an embodiment, insulin, an insulin analogue, an insulin agonist, and / or an insulin partial agonist is administered at a concentration of from about 2 μg / ml to about 50 μg / ml. In an embodiment, insulin, an insulin analogue, an insulin agonist, and / or an insulin partial agonist is administered at a concentration of from about 3 μg / ml to about 50 μg / ml. In an embodiment, insulin, an insulin analogue, an insulin agonist, and / or an insulin partial agonist is administered at a concentration of from about 4 μg / ml to about 50 μg / ml. In an embodiment, insulin, an insulin analogue, an insulin agonist, and / or an insulin partial agonist is administered at a concentration of from about 5 μg / ml to about 50 μg / ml. In an embodiment, insulin, an insulin analogue, an insulin agonist, and / or an insulin partial agonist is administered at a concentration of from about 6 μg / ml to about 50 μg / ml. In an embodiment, insulin, an insulin analogue, an insulin agonist, and / or an insulin partial agonist is administered at a concentration of from about 7 μg / ml to about 50 μg / ml. In an embodiment, insulin, an insulin analogue, an insulin agonist, and / or an insulin partial agonist is administered at a concentration of from about 8 μg / ml to about 50 μg / ml. In an embodiment, insulin, an insulin analogue, an insulin agonist, and / or an insulin partial agonist is administered at a concentration of from about 9 μg / ml to about 50 μg / ml. In an embodiment, insulin, an insulin analogue, an insulin agonist, and / or an insulin partial agonist is administered at a concentration of from about 10 μg / ml to about 50 μg / ml. In an embodiment, insulin, an insulin analogue, an insulin agonist, and / or an insulin partial agonist is administered at a concentration of from about 15 μg / ml to about 50 μg / ml. In an embodiment, insulin, an insulin analogue, an insulin agonist, and / or an insulin partial agonist is administered at a concentration of from about 20 μg / ml to about 50 μg / ml. In an embodiment, insulin, an insulin analogue, an insulin agonist, and / or an insulin partial agonist is administered at a concentration of from about 25 μg / ml to about 50 μg / ml. In an embodiment, insulin, an insulin analogue, an insulin agonist, and / or an insulin partial agonist is administered at a concentration of from about 30 μg / ml to about 50 μg / ml. In an embodiment, insulin, an insulin analogue, an insulin agonist, and / or an insulin partial agonist is administered at a concentration of from about 35 μg / ml to about 50 μg / ml. In an embodiment, insulin, an insulin analogue, an insulin agonist, and / or an insulin partial agonist is administered at a concentration of from about 40 μg / ml to about 50 μg / ml. In an embodiment, insulin, an insulin analogue, an insulin agonist, and / or an insulin partial agonist is administered at a concentration of from about 45 μg / ml to about 50 μg / ml.

[0107] In embodiments, insulin, an insulin analogue, an insulin agonist, and / or an insulin partial agonist is administered at a concentration of from about 1 μg / ml to about 45 μg / ml. In embodiments, insulin, an insulin analogue, an insulin agonist, and / or an insulin partial agonist is administered at a concentration of from about 1 μg / ml to about 40 μg / ml. In embodiments, insulin, an insulin analogue, an insulin agonist, and / or an insulin partial agonist is administered at a concentration of from about 1 μg / ml to about 35 μg / ml. In embodiments, insulin, an insulin analogue, an insulin agonist, and / or an insulin partial agonist is administered at a concentration of from about 1 μg / ml to about 30 μg / ml. In embodiments, insulin, an insulin analogue, an insulin agonist, and / or an insulin partial agonist is administered at a concentration of from about 1 μg / ml to about 25 μg / ml. In embodiments, insulin, an insulin analogue, an insulin agonist, and / or an insulin partial agonist is administered at a concentration of from about 1 μg / ml to about 20 μg / ml. In embodiments, insulin, an insulin analogue, an insulin agonist, and / or an insulin partial agonist is administered at a concentration of from about 1 μg / ml to about 15 μg / ml. In embodiments, insulin, an insulin analogue, an insulin agonist, and / or an insulin partial agonist is administered at a concentration of from about 1 μg / ml to about 10 μg / ml. In embodiments, insulin, an insulin analogue, an insulin agonist, and / or an insulin partial agonist is administered at a concentration of from about 1 μg / ml to about 9 μg / ml. In embodiments, insulin, an insulin analogue, an insulin agonist, and / or an insulin partial agonist is administered at a concentration of from about 1 μg / ml to about 8 μg / ml. In embodiments, insulin, an insulin analogue, an insulin agonist, and / or an insulin partial agonist is administered at a concentration of from about 1 μg / ml to about 7 μg / ml. In embodiments, insulin, an insulin analogue, an insulin agonist, and / or an insulin partial agonist is administered at a concentration of from about 1 μg / ml to about 6 μg / ml. In embodiments, insulin, an insulin analogue, an insulin agonist, and / or an insulin partial agonist is administered at a concentration of from about 1 μg / ml to about 5 μg / ml. In embodiments, insulin, an insulin analogue, an insulin agonist, and / or an insulin partial agonist is administered at a concentration of from about 1 μg / ml to about 4 μg / ml. In embodiments, insulin, an insulin analogue, an insulin agonist, and / or an insulin partial agonist is administered at a concentration of from about 1 μg / ml to about 3 μg / ml. In embodiments, insulin, an insulin analogue, an insulin agonist, and / or an insulin partial agonist is administered at a concentration of from about 1 μg / ml to about 2 μg / ml.

[0108] In an embodiment, insulin, an insulin analogue, an insulin agonist, and / or an insulin partial agonist are administered at a concentration of from 1 μg / ml to about 50 μg / ml. In an embodiment, insulin, an insulin analogue, an insulin agonist, and / or an insulin partial agonist are administered at a concentration of from 2 μg / ml to about 50 μg / ml. In an embodiment, insulin, an insulin analogue, an insulin agonist, and / or an insulin partial agonist are administered at a concentration of from 3 μg / ml to about 50 μg / ml. In an embodiment, insulin, an insulin analogue, an insulin agonist, and / or an insulin partial agonist are administered at a concentration of from 4 μg / ml to about 50 μg / ml. In an embodiment, insulin, an insulin analogue, an insulin agonist, and / or an insulin partial agonist are administered at a concentration of from 5 μg / ml to about 50 μg / ml. In an embodiment, insulin, an insulin analogue, an insulin agonist, and / or an insulin partial agonist are administered at a concentration of from 6 μg / ml to about 50 μg / ml. In an embodiment, insulin, an insulin analogue, an insulin agonist, and / or an insulin partial agonist are administered at a concentration of from 7 μg / ml to about 50 μg / ml. In an embodiment, insulin, an insulin analogue, an insulin agonist, and / or an insulin partial agonist are administered at a concentration of from 8 μg / ml to about 50 μg / ml. In an embodiment, insulin, an insulin analogue, an insulin agonist, and / or an insulin partial agonist are administered at a concentration of from 9 μg / ml to about 50 μg / ml. In an embodiment, insulin, an insulin analogue, an insulin agonist, and / or an insulin partial agonist are administered at a concentration of from 10 μg / ml to about 50 μg / ml. In an embodiment, insulin, an insulin analogue, an insulin agonist, and / or an insulin partial agonist are administered at a concentration of from 15 μg / ml to about 50 μg / ml. In an embodiment, insulin, an insulin analogue, an insulin agonist, and / or an insulin partial agonist are administered at a concentration of from 20 μg / ml to about 50 μg / ml. In an embodiment, insulin, an insulin analogue, an insulin agonist, and / or an insulin partial agonist are administered at a concentration of from 25 μg / ml to about 50 μg / ml. In an embodiment, insulin, an insulin analogue, an insulin agonist, and / or an insulin partial agonist are administered at a concentration of from 30 μg / ml to about 50 μg / ml. In an embodiment, insulin, an insulin analogue, an insulin agonist, and / or an insulin partial agonist are administered at a concentration of from 35 μg / ml to about 50 μg / ml. In an embodiment, insulin, an insulin analogue, an insulin agonist, and / or an insulin partial agonist are administered at a concentration of from 40 μg / ml to about 50 μg / ml. In an embodiment, insulin, an insulin analogue, an insulin agonist, and / or an insulin partial agonist are administered at a concentration of from 45 μg / ml to about 50 μg / ml.

[0109] In an embodiment, insulin, an insulin analogue, an insulin agonist, and / or an insulin partial agonist are administered at a concentration of 1 μg / ml to about 45 μg / ml. In an embodiment, insulin, an insulin analogue, an insulin agonist, and / or an insulin partial agonist are administered at a concentration of 1 μg / ml to about 40 μg / ml. In an embodiment, insulin, an insulin analogue, an insulin agonist, and / or an insulin partial agonist are administered at a concentration of 1 μg / ml to about 35 μg / ml. In an embodiment, insulin, an insulin analogue, an insulin agonist, and / or an insulin partial agonist are administered at a concentration of 1 μg / ml to about 30 μg / ml. In an embodiment, insulin, an insulin analogue, an insulin agonist, and / or an insulin partial agonist are administered at a concentration of 1 μg / ml to about 25 μg / ml. In an embodiment, insulin, an insulin analogue, an insulin agonist, and / or an insulin partial agonist are administered at a concentration of 1 μg / ml to about 20 μg / ml. In an embodiment, insulin, an insulin analogue, an insulin agonist, and / or an insulin partial agonist are administered at a concentration of 1 μg / ml to about 15 μg / ml. In an embodiment, insulin, an insulin analogue, an insulin agonist, and / or an insulin partial agonist are administered at a concentration of 1 μg / ml to about 10 μg / ml. In an embodiment, insulin, an insulin analogue, an insulin agonist, and / or an insulin partial agonist are administered at a concentration of 1 μg / ml to about 9 μg / ml. In an embodiment, insulin, an insulin analogue, an insulin agonist, and / or an insulin partial agonist are administered at a concentration of 1 μg / ml to about 8 μg / ml. In an embodiment, insulin, an insulin analogue, an insulin agonist, and / or an insulin partial agonist are administered at a concentration of 1 μg / ml to about 7 μg / ml. In an embodiment, insulin, an insulin analogue, an insulin agonist, and / or an insulin partial agonist are administered at a concentration of 1 μg / ml to about 6 μg / ml. In an embodiment, insulin, an insulin analogue, an insulin agonist, and / or an insulin partial agonist are administered at a concentration of 1 μg / ml to about 5 μg / ml. In an embodiment, insulin, an insulin analogue, an insulin agonist, and / or an insulin partial agonist are administered at a concentration of 1 μg / ml to about 4 μg / ml. In an embodiment, insulin, an insulin analogue, an insulin agonist, and / or an insulin partial agonist are administered at a concentration of 1 μg / ml to about 3 μg / ml. In an embodiment, insulin, an insulin analogue, an insulin agonist, and / or an insulin partial agonist are administered at a concentration of 1 μg / ml to about 2 μg / ml.

[0110] In embodiments, insulin, an insulin analogue, an insulin agonist, and / or an insulin partial agonist are administered at a concentration of about 1 μg / ml, about 5 μg / ml, or about 25 μg / ml. In embodiments, insulin, an insulin analogue, an insulin agonist, and / or an insulin partial agonist are administered at a concentration of about 1 μg / ml. In embodiments, insulin, an insulin analogue, an insulin agonist, and / or an insulin partial agonist are administered at a concentration of about 5 μg / ml. In embodiments, insulin, an insulin analogue, an insulin agonist, and / or an insulin partial agonist are administered at a concentration of about 25 μg / ml. In embodiments, insulin, an insulin analogue, an insulin agonist, and / or an insulin partial agonist are administered at a concentration of 1 μg / ml, 5 μg / ml, or 25 μg / ml. In embodiments, insulin, an insulin analogue, an insulin agonist, and / or an insulin partial agonist are administered at a concentration of 1 μg / ml. In embodiments, insulin, an insulin analogue, an insulin agonist, and / or an insulin partial agonist are administered at a concentration of 5 μg / ml. In embodiments, insulin, an insulin analogue, an insulin agonist, and / or an insulin partial agonist are administered at a concentration of 25 μg / ml.

[0111] The methods provided herein (including embodiments thereof) can include contacting a T cell with a gene editing reagent, thereby allowing editing of a target gene within the T cell. For example, the gene editing reagent can facilitate knockout of an endogenous gene (such as an endogenous TCR) and knock-in of a tumor antigen-specific TCR. Thus, in embodiments, the method further includes contacting a T cell with a gene editing reagent. In embodiments, contacting a T cell with a gene editing reagent includes contacting the T cell with a polynucleotide encoding the gene editing reagent. In embodiments, the T cell is contacted with a polynucleotide in the presence of the gene editing reagent or a polynucleotide encoding the gene editing reagent. In embodiments, the T cell is contacted with a polynucleotide in the presence of the gene editing reagent. In embodiments, the T cell is contacted with a polynucleotide in the presence of a polynucleotide encoding the gene editing reagent.

[0112] In an embodiment, the gene editing reagent includes an RNA-guided nuclease. In an embodiment, the RNA-guided nuclease is a CRISPR-Cas system. In an embodiment, the CRISPR-Cas system includes Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9, Cas10, Cas12, Cas13, nCas9, Cas-CLOVER, Csy1, Csy2, Csy3, Cse1, Cse2, Csc1, Csc2, Csa5, Csn2, 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 includes Cas1. In an embodiment, the CRISPR-Cas system includes Cas1B. In an embodiment, the CRISPR-Cas system includes Cas2. In an embodiment, the CRISPR-Cas system includes Cas3. In an embodiment, the CRISPR-Cas system includes Cas4. In an embodiment, the CRISPR-Cas system includes Cas5. In an embodiment, the CRISPR-Cas system includes Cas6. In an embodiment, the CRISPR-Cas system includes Cas7. In an embodiment, the CRISPR-Cas system includes Cas8. In an embodiment, the CRISPR-Cas system includes Cas9. In an embodiment, the CRISPR-Cas system includes Cas9 or a Cas9 variant. In an embodiment, the CRISPR-Cas system includes Cas9. In an embodiment, the CRISPR-Cas system includes a Cas9 variant. In an embodiment, the CRISPR-Cas system includes Cas10. In an embodiment, the CRISPR-Cas system includes Cas12. In an embodiment, the CRISPR-Cas system includes Cas13. In an embodiment, the CRISPR-Cas system includes Csy1. In an embodiment, the CRISPR-Cas system includes Csy2. In an embodiment, the CRISPR-Cas system includes Csy3. In an embodiment, the CRISPR-Cas system includes Cse1. In an embodiment, the CRISPR-Cas system includes Cse2. In an embodiment, the CRISPR-Cas system includes Csc1. In an embodiment, the CRISPR-Cas system includes Csc2. In an embodiment, the CRISPR-Cas system includes Csm2. In an embodiment, the CRISPR-Cas system includes Csm3. In an embodiment, the CRISPR-Cas system includes Csm4.In an embodiment, the CRISPR-Cas system includes Csm5. In an embodiment, the CRISPR-Cas system includes Csm6. In an embodiment, the CRISPR-Cas system includes Cmr1. In an embodiment, the CRISPR-Cas system includes Cmr3. In an embodiment, the CRISPR-Cas system includes Cmr4. In an embodiment, the CRISPR-Cas system includes Cmr5. In an embodiment, the CRISPR-Cas system includes Cmr6. In an embodiment, the CRISPR-Cas system includes Csb1. In an embodiment, the CRISPR-Cas system includes Csb3. In an embodiment, the CRISPR-Cas system includes Csx17. In an embodiment, the CRISPR-Cas system includes Csx14. In an embodiment, the CRISPR-Cas system includes Csx10. In an embodiment, the CRISPR-Cas system includes Csx16. In an embodiment, the CRISPR-Cas system includes CsaX. In an embodiment, the CRISPR-Cas system includes Csx3. In an embodiment, the CRISPR-Cas system includes Csx1. In an embodiment, the CRISPR-Cas system includes Csx15. In an embodiment, the CRISPR-Cas system includes Csf1. In an embodiment, the CRISPR-Cas system includes Csf2. In an embodiment, the CRISPR-Cas system includes Csf3. In an embodiment, the CRISPR-Cas system includes Csf4. In an embodiment, the CRISPR-Cas system includes Cas-CLOVER. In an embodiment, the CRISPR-Cas system includes nCas9. In an embodiment, the gene editing reagent includes a CRISPR-Cas system that includes a Cas protein and a guide RNA (gRNA).

[0113] In an embodiment, the gene editing reagent is MAD7, TALEN, or ZFN. In an embodiment, the gene editing reagent is MAD7. In an embodiment, the gene editing reagent is TALEN. In an embodiment, the gene editing reagent is ZFN. MAD7 is an engineered nuclease of the class 2 type V-A CRISPR-Cas (Cas12a / Cpf1) family (refseq WP_055225123.1). See, for example, CRISPR J. April 2020; 3(2):97–108, which is incorporated herein by reference in its entirety.

[0114] In a further embodiment, the CRISPR-Cas system comprises a Cas enzyme fusion protein. In some embodiments, the fusion protein comprises any of the above-described Cas enzymes. In some embodiments, the fusion protein comprises a Cas enzyme and comprises an exonuclease. In some embodiments, the fusion protein comprises a Cas enzyme and comprises a deaminase. In some embodiments, the fusion protein comprises a Cas enzyme and comprises a DNA repair protein. In some embodiments, the fusion protein comprises a Cas enzyme and a chromatin remodeling protein. In some embodiments, the fusion protein comprises a Cas enzyme and an NEHJ inhibitory protein. In some embodiments, more than one combination of Cas enzyme fusion proteins is included in the CRISPR-Cas system.

[0115] In an embodiment, at least 80% of the engineered T cells are TCM and / or TSCM. In an embodiment, at least 85% of the engineered T cells are TCM and / or TSCM. In an embodiment, at least 80% of the engineered T cells are TCM and / or TSCM. In an embodiment, at least 90% of the engineered T cells are TCM and / or TSCM. In an embodiment, at least 91% of the engineered T cells are TCM and / or TSCM. In an embodiment, at least 92% of the engineered T cells are TCM and / or TSCM. In an embodiment, at least 93% of the engineered T cells are TCM and / or TSCM. In an embodiment, at least 94% of the engineered T cells are TCM and / or TSCM. In an embodiment, at least 95% of the engineered T cells are TCM and / or TSCM. In an embodiment, at least 96% of the engineered T cells are TCM and / or TSCM. In an embodiment, at least 97% of the engineered T cells are TCM and / or TSCM. In an embodiment, at least 80% of the engineered T cells are TCM and / or TSCM. In an embodiment, at least 99% of the engineered T cells are TCM and / or TSCM.

[0116] In an embodiment, at least 80% of the engineered T cells are TCM and TSCM. In an embodiment, at least 85% of the engineered T cells are TCM and TSCM. In an embodiment, at least 80% of the engineered T cells are TCM and TSCM. In an embodiment, at least 90% of the engineered T cells are TCM and TSCM. In an embodiment, at least 91% of the engineered T cells are TCM and TSCM. In an embodiment, at least 92% of the engineered T cells are TCM and TSCM. In an embodiment, at least 93% of the engineered T cells are TCM and TSCM. In an embodiment, at least 94% of the engineered T cells are TCM and TSCM. In an embodiment, at least 95% of the engineered T cells are TCM and TSCM. In an embodiment, at least 96% of the engineered T cells are TCM and TSCM. In an embodiment, at least 97% of the engineered T cells are TCM and TSCM. In an embodiment, at least 80% of the engineered T cells are TCM and TSCM. In an embodiment, at least 99% of the engineered T cells are TCM and TSCM.

[0117] In an embodiment, at least 80% of the engineered T cells are TCM or TSCM. In an embodiment, at least 85% of the engineered T cells are TCM or TSCM. In an embodiment, at least 80% of the engineered T cells are TCM or TSCM. In an embodiment, at least 90% of the engineered T cells are TCM or TSCM. In an embodiment, at least 91% of the engineered T cells are TCM or TSCM. In an embodiment, at least 92% of the engineered T cells are TCM or TSCM. In an embodiment, at least 93% of the engineered T cells are TCM or TSCM. In an embodiment, at least 94% of the engineered T cells are TCM or TSCM. In an embodiment, at least 95% of the engineered T cells are TCM or TSCM. In an embodiment, at least 96% of the engineered T cells are TCM or TSCM. In an embodiment, at least 97% of the engineered T cells are TCM or TSCM. In an embodiment, at least 80% of the engineered T cells are TCM or TSCM. In an embodiment, at least 99% of the engineered T cells are TCM or TSCM.

[0118] In embodiments, at least 80% of the engineered T cells are TCM. In embodiments, at least 85% of the engineered T cells are TCM. In embodiments, at least 80% of the engineered T cells are TCM. In embodiments, at least 90% of the engineered T cells are TCM. In embodiments, at least 91% of the engineered T cells are TCM. In embodiments, at least 92% of the engineered T cells are TCM. In embodiments, at least 93% of the engineered T cells are TCM. In embodiments, at least 94% of the engineered T cells are TCM. In embodiments, at least 95% of the engineered T cells are TCM. In embodiments, at least 96% of the engineered T cells are TCM. In embodiments, at least 97% of the engineered T cells are TCM. In embodiments, at least 80% of the engineered T cells are TCM. In embodiments, at least 99% of the engineered T cells are TCM.

[0119] In embodiments, at least 80% of the engineered T cells are TSCM. In embodiments, at least 85% of the engineered T cells are TSCM. In embodiments, at least 80% of the engineered T cells are TSCM. In embodiments, at least 90% of the engineered T cells are TSCM. In embodiments, at least 91% of the engineered T cells are TSCM. In embodiments, at least 92% of the engineered T cells are TSCM. In embodiments, at least 93% of the engineered T cells are TSCM. In embodiments, at least 94% of the engineered T cells are TSCM. In embodiments, at least 95% of the engineered T cells are TSCM. In embodiments, at least 96% of the engineered T cells are TSCM. In embodiments, at least 97% of the engineered T cells are TSCM. In embodiments, at least 80% of the engineered T cells are TSCM. In embodiments, at least 99% of the engineered T cells are TSCM.

[0120] For the methods provided herein, in embodiments, T cells are cultured in the presence (contact) of insulin. In embodiments, T cells are cultured in the presence of an insulin analogue. In embodiments, the insulin analogue is an analogue as provided herein.

[0121] The present disclosure particularly provides methods for monitoring the cell viability of a population of engineered T cells, the method comprising measuring mitochondrial function and cell metabolism over time. In embodiments, the mitochondrial membrane potential is measured. In embodiments, a dye-based assay is used to measure the mitochondrial membrane potential. In some embodiments, the dye is JC-1 or JC-10. In embodiments, the dye is JC-1. In embodiments, the dye is JC-10.

[0122] The present disclosure particularly provides methods for increasing the cell viability of engineered T cell populations, which comprise contacting a T cell population in the presence of insulin, an insulin analog, an insulin agonist, an insulin partial agonist, a gene editing reagent, or a polynucleotide encoding a gene editing reagent, thereby forming the engineered T cell population, wherein the engineered T cell population has increased cell viability, growth, and / or gene editing efficiency relative to an engineered T cell population not contacted with insulin, an insulin analog, an insulin agonist, or an insulin partial agonist, and wherein the engineered T cell population is administered to a subject in need thereof. Accordingly, it is contemplated that the methods improve the viability of engineered T cells.

[0123] "Cell viability" is used according to its ordinary meaning in the art and refers to the number or proportion of live cells within 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 the cells with a nucleic acid-binding dye that only enters cells with a damaged or compromised cell membrane. Cell viability can be measured by contacting the cells with a reagent that reacts with an enzyme in live cells, or a reagent that measures cell glucose metabolism or mitochondrial viability. For example, mitochondrial viability can be determined by measuring mitochondrial membrane potential using fluorescence detection assays, which include assays using dye-based detection, wherein the dye includes JC-1 or JC-10. For example, glucose metabolism can be determined by measuring glucose consumption / uptake using glucose with a heavy isotope carbon (C13) or a glucose analog, wherein the glucose analog includes 2-NBDG. In the examples, cell viability can be measured by fluorescence microscopy or flow cytometry. Accordingly, in one aspect, provided is a method for increasing the cell viability of an engineered T cell population, which comprises contacting a T cell population with insulin and a polynucleotide, thereby forming the engineered T cell population, wherein the engineered T cell population has increased cell viability relative to an engineered T cell population not contacted with insulin.

[0124] For the methods provided herein, in the examples, further comprises contacting the T cell population with donor DNA. In the examples, the polynucleotide encoding a gene editing reagent comprises: single-stranded DNA, double-stranded DNA, linear DNA strand, plasmid, nanoplasmid, or minicircle. In the examples, the polynucleotide encoding a gene editing reagent comprises single-stranded DNA. In the examples, the polynucleotide encoding a gene editing reagent comprises double-stranded DNA. In the examples, the polynucleotide encoding a gene editing reagent comprises a linear DNA strand. In the examples, the polynucleotide encoding a gene editing reagent comprises a plasmid. In the examples, the polynucleotide encoding a gene editing reagent comprises a nanoplasmid. In the examples, the polynucleotide encoding a gene editing reagent comprises a minicircle.

[0125] In an embodiment, the polynucleotide encoding the gene editing reagent comprises a plasmid, which contains a plasmid backbone and a polynucleotide sequence encoding the gene editing reagent. In an embodiment, the plasmid further comprises donor DNA. In an embodiment, the donor DNA sequence comprises a polynucleotide encoding a gene product. In an embodiment, the gene product is autologous or allogeneic to the subject. In an embodiment, the gene product is autologous to the subject. In an embodiment, the gene product is allogeneic to the subject.

[0126] In an embodiment, the gene product sequence comprises a chimeric antigen receptor (CAR), a T cell receptor (TCR), a human leukocyte antigen (HLA), or an allogeneic immune defense receptor (ADR) or a subunit thereof. In an embodiment, the gene product sequence comprises a chimeric antigen receptor (CAR). In an embodiment, the gene product sequence comprises a T cell receptor (TCR). In an embodiment, the gene product sequence comprises a human leukocyte antigen (HLA). In an embodiment, the gene product sequence comprises an allogeneic immune defense receptor (ADR).

[0127] In an embodiment, the TCR sequence comprises an exogenous TCR-β subunit or a fragment thereof, and / or an exogenous TCR-α subunit or a fragment thereof, or a chimeric antigen receptor and / or a subunit thereof. In an embodiment, the TCR sequence comprises an exogenous TCR-β subunit or a fragment thereof and an exogenous TCR-α subunit or a fragment thereof. In an embodiment, the TCR sequence comprises an exogenous TCR-β subunit or a fragment thereof, or an exogenous TCR-α subunit or a fragment thereof. In an embodiment, the TCR sequence comprises an exogenous TCR-β subunit or a fragment thereof. In an embodiment, the TCR sequence comprises an exogenous TCR-α subunit or a fragment thereof. In an embodiment, the TCR sequence comprises a chimeric antigen receptor and a subunit thereof. In an embodiment, the TCR sequence comprises a chimeric antigen receptor or a subunit thereof. In an embodiment, the TCR sequence is inserted into the TRAC or TRBC locus. In an embodiment, the TCR sequence is inserted into the TRAC locus. In an embodiment, the TCR sequence is inserted into the TRBC locus.

[0128] In an embodiment, contacting a T cell population with the gene editing reagent or the polynucleotide encoding the gene editing reagent comprises transfecting the T cell population with the gene editing reagent or the polynucleotide encoding the gene editing reagent. In an embodiment, contacting a T cell population with the gene editing reagent comprises transfecting the T cell population with the gene editing reagent. In an embodiment, contacting a T cell population with the polynucleotide encoding the gene reagent comprises transfecting the T cell population with the polynucleotide encoding the gene editing reagent. In an embodiment, transfection comprises electroporation. In an embodiment, transfection comprises nucleofection. In an embodiment, transfection comprises liposomal transfection. In an embodiment, transfection comprises microfluidic transfection.

[0129] In an embodiment, prior to the contacting step, the T cell population is cultured in the presence of insulin, an insulin analogue, an insulin agonist, and / or an insulin partial agonist. In an embodiment, prior to the contacting step, the T cell population is cultured in the presence of insulin, an insulin analogue, an insulin agonist, and an insulin partial agonist. In an embodiment, prior to the contacting step, the T cell population is cultured in the presence of insulin, an insulin analogue, an insulin agonist, or an insulin partial agonist. In an embodiment, prior to the contacting step, the T cell population is cultured in the presence of insulin. In an embodiment, prior to the contacting step, the T cell population is cultured in the presence of an insulin analogue. In an embodiment, prior to the contacting step, the T cell population is cultured in the presence of an insulin agonist. In an embodiment, prior to the contacting step, the T cell population is cultured in the presence of an insulin partial agonist. In an embodiment, it includes culturing the T cell population in the presence of insulin, an insulin analogue, an insulin agonist, and / or an insulin partial agonist for 48 hours, 24 hours, 12 hours, 6 hours, 4 hours, 2 hours, 1 hour, or 30 minutes prior to the contacting step. In an embodiment, it includes culturing the T cell population in the presence of insulin, an insulin analogue, an insulin agonist, and / or an insulin partial agonist for 48 hours prior to the contacting step. In an embodiment, it includes culturing the T cell population in the presence of insulin, an insulin analogue, an insulin agonist, and / or an insulin partial agonist for 24 hours prior to the contacting step. In an embodiment, it includes culturing the T cell population in the presence of insulin, an insulin analogue, an insulin agonist, and / or an insulin partial agonist for 12 hours prior to the contacting step. In an embodiment, it includes culturing the T cell population in the presence of insulin, an insulin analogue, an insulin agonist, and / or an insulin partial agonist for 6 hours prior to the contacting step. In an embodiment, it includes culturing the T cell population in the presence of insulin, an insulin analogue, an insulin agonist, and / or an insulin partial agonist for 4 hours prior to the contacting step. In an embodiment, it includes culturing the T cell population in the presence of insulin, an insulin analogue, an insulin agonist, and / or an insulin partial agonist for 2 hours prior to the contacting step. In an embodiment, it includes culturing the T cell population in the presence of insulin, an insulin analogue, an insulin agonist, and / or an insulin partial agonist for 1 hour prior to the contacting step. In an embodiment, it includes culturing the T cell population in the presence of insulin, an insulin analogue, an insulin agonist, and / or an insulin partial agonist for 30 minutes prior to the contacting step.

[0130] In an embodiment, after the contacting step, the T cell population is cultured in the presence of insulin, an insulin analog, an insulin agonist, and / or an insulin partial agonist. In an embodiment, after the contacting step, the T cell population is cultured in the presence of insulin, an insulin analog, an insulin agonist, and an insulin partial agonist. In an embodiment, after the contacting step, the T cell population is cultured in the presence of insulin, an insulin analog, an insulin agonist, or an insulin partial agonist. In an embodiment, after the contacting step, the T cell population is cultured in the presence of insulin. In an embodiment, after a step similar to the contacting step, the T cell population is cultured in the presence of an insulin analog. In an embodiment, after a step similar to the contacting step, the T cell population is cultured in the presence of an insulin agonist. In an embodiment, after a step similar to the contacting step, the T cell population is cultured in the presence of an insulin partial agonist. In an embodiment, it includes culturing the T cell population in the presence of insulin, an insulin analog, an insulin agonist, and / or an insulin partial agonist for up to 48 hours after the contacting step. In an embodiment, it includes culturing the T cell population in the presence of insulin, an insulin analog, an insulin agonist, and / or an insulin partial agonist for up to 24 hours after the contacting step. In an embodiment, it includes culturing the T cell population in the presence of insulin, an insulin analog, an insulin agonist, and / or an insulin partial agonist for up to 12 hours after the contacting step. In an embodiment, it includes culturing the T cell population in the presence of insulin, an insulin analog, an insulin agonist, and / or an insulin partial agonist for up to 6 hours after the contacting step. In an embodiment, it includes culturing the T cell population in the presence of insulin, an insulin analog, an insulin agonist, and / or an insulin partial agonist for up to 4 hours after the contacting step. In an embodiment, it includes culturing the T cell population in the presence of insulin, an insulin analog, an insulin agonist, and / or an insulin partial agonist for up to 2 hours after the contacting step. In an embodiment, it includes culturing the T cell population in the presence of insulin, an insulin analog, an insulin agonist, and / or an insulin partial agonist for up to 1 hour after the contacting step. In an embodiment, it includes culturing the T cell population in the presence of insulin, an insulin analog, an insulin agonist, and / or an insulin partial agonist for up to 30 minutes after the contacting step.

[0131] In an embodiment, the T cell population is cultured in the presence of insulin, insulin analogs, insulin agonists, and / or insulin partial agonists before and after the contacting step. In an embodiment, the T cell population is cultured in the presence of insulin, insulin analogs, insulin agonists, and insulin partial agonists before and after the contacting step. In an embodiment, the T cell population is cultured in the presence of insulin, insulin analogs, insulin agonists, or insulin partial agonists before and after the contacting step. In an embodiment, the T cell population is cultured in the presence of insulin before and after the contacting step. In an embodiment, the T cell population is cultured in the presence of insulin analogs before and after the contacting step. In an embodiment, the T cell population is cultured in the presence of insulin agonists before and after the contacting step. In an embodiment, the T cell population is cultured in the presence of insulin partial agonists before and after the contacting step.

[0132] In an embodiment, it includes culturing the T cell population in the presence of insulin, insulin analogs, insulin agonists, and / or insulin partial agonists for 48 hours, 24 hours, 12 hours, 6 hours, 4 hours, 2 hours, 1 hour, or 30 minutes before the contacting step. In an embodiment, it includes culturing the T cell population in the presence of insulin, insulin analogs, insulin agonists, and / or insulin partial agonists for 48 hours before the contacting step. In an embodiment, it includes culturing the T cell population in the presence of insulin, insulin analogs, insulin agonists, and / or insulin partial agonists for 24 hours before the contacting step. In an embodiment, it includes culturing the T cell population in the presence of insulin, insulin analogs, insulin agonists, and / or insulin partial agonists for 12 hours before the contacting step. In an embodiment, it includes culturing the T cell population in the presence of insulin, insulin analogs, insulin agonists, and / or insulin partial agonists for 6 hours before the contacting step. In an embodiment, it includes culturing the T cell population in the presence of insulin, insulin analogs, insulin agonists, and / or insulin partial agonists for 4 hours before the contacting step. In an embodiment, it includes culturing the T cell population in the presence of insulin, insulin analogs, insulin agonists, and / or insulin partial agonists for 2 hours before the contacting step. In an embodiment, it includes culturing the T cell population in the presence of insulin, insulin analogs, insulin agonists, and / or insulin partial agonists for 1 hour before the contacting step. In an embodiment, it includes culturing the T cell population in the presence of insulin, insulin analogs, insulin agonists, and / or insulin partial agonists for 30 minutes before the contacting step.

[0133] In an embodiment, it includes culturing a T cell population in the presence of insulin, an insulin analog, an insulin agonist, and / or an insulin partial agonist for 48 hours, 24 hours, 12 hours, 6 hours, 4 hours, 2 hours, 1 hour, or 30 minutes after the contacting step. In an embodiment, it includes culturing a T cell population in the presence of insulin, an insulin analog, an insulin agonist, and / or an insulin partial agonist for 48 hours after the contacting step. In an embodiment, it includes culturing a T cell population in the presence of insulin, an insulin analog, an insulin agonist, and / or an insulin partial agonist for 24 hours after the contacting step. In an embodiment, it includes culturing a T cell population in the presence of insulin, an insulin analog, an insulin agonist, and / or an insulin partial agonist for 12 hours after the contacting step. In an embodiment, it includes culturing a T cell population in the presence of insulin, an insulin analog, an insulin agonist, and / or an insulin partial agonist for 6 hours after the contacting step. In an embodiment, it includes culturing a T cell population in the presence of insulin, an insulin analog, an insulin agonist, and / or an insulin partial agonist for 4 hours after the contacting step. In an embodiment, it includes culturing a T cell population in the presence of insulin, an insulin analog, an insulin agonist, and / or an insulin partial agonist for 2 hours after the contacting step. In an embodiment, it includes culturing a T cell population in the presence of insulin, an insulin analog, an insulin agonist, and / or an insulin partial agonist for 1 hour after the contacting step. In an embodiment, it includes culturing a T cell population in the presence of insulin, an insulin analog, an insulin agonist, and / or an insulin partial agonist for 30 minutes after the contacting step.

[0134] In embodiments, insulin, an insulin analog, an insulin agonist, and / or an insulin partial agonist are administered at a concentration of from about 1 μg / ml to about 50 μg / ml. In embodiments, insulin, an insulin analog, an insulin agonist, and / or an insulin partial agonist are administered at a concentration of from about 2 μg / ml to about 50 μg / ml. In embodiments, insulin, an insulin analog, an insulin agonist, and / or an insulin partial agonist are administered at a concentration of from about 3 μg / ml to about 50 μg / ml. In embodiments, insulin, an insulin analog, an insulin agonist, and / or an insulin partial agonist are administered at a concentration of from about 4 μg / ml to about 50 μg / ml. In embodiments, insulin, an insulin analog, an insulin agonist, and / or an insulin partial agonist are administered at a concentration of from about 5 μg / ml to about 50 μg / ml. In embodiments, insulin, an insulin analog, an insulin agonist, and / or an insulin partial agonist are administered at a concentration of from about 6 μg / ml to about 50 μg / ml. In embodiments, insulin, an insulin analog, an insulin agonist, and / or an insulin partial agonist are administered at a concentration of from about 7 μg / ml to about 50 μg / ml. In embodiments, insulin, an insulin analog, an insulin agonist, and / or an insulin partial agonist are administered at a concentration of from about 8 μg / ml to about 50 μg / ml. In embodiments, insulin, an insulin analog, an insulin agonist, and / or an insulin partial agonist are administered at a concentration of from about 9 μg / ml to about 50 μg / ml. In embodiments, insulin, an insulin analog, an insulin agonist, and / or an insulin partial agonist are administered at a concentration of from about 10 μg / ml to about 50 μg / ml. In embodiments, insulin, an insulin analog, an insulin agonist, and / or an insulin partial agonist are administered at a concentration of from about 15 μg / ml to about 50 μg / ml. In embodiments, insulin, an insulin analog, an insulin agonist, and / or an insulin partial agonist are administered at a concentration of from about 20 μg / ml to about 50 μg / ml. In embodiments, insulin, an insulin analog, an insulin agonist, and / or an insulin partial agonist are administered at a concentration of from about 25 μg / ml to about 50 μg / ml. In embodiments, insulin, an insulin analog, an insulin agonist, and / or an insulin partial agonist are administered at a concentration of from about 30 μg / ml to about 50 μg / ml. In embodiments, insulin, an insulin analog, an insulin agonist, and / or an insulin partial agonist are administered at a concentration of from about 35 μg / ml to about 50 μg / ml. In embodiments, insulin, an insulin analog, an insulin agonist, and / or an insulin partial agonist are administered at a concentration of from about 40 μg / ml to about 50 μg / ml. In embodiments, insulin, an insulin analog, an insulin agonist, and / or an insulin partial agonist are administered at a concentration of from about 45 μg / ml to about 50 μg / ml.

[0135] In an embodiment, insulin, an insulin analog, an insulin agonist, and / or an insulin partial agonist is administered at a concentration of about 1 μg / ml to about 45 μg / ml. In an embodiment, insulin, an insulin analog, an insulin agonist, and / or an insulin partial agonist is administered at a concentration of about 1 μg / ml to about 40 μg / ml. In an embodiment, insulin, an insulin analog, an insulin agonist, and / or an insulin partial agonist is administered at a concentration of about 1 μg / ml to about 35 μg / ml. In an embodiment, insulin, an insulin analog, an insulin agonist, and / or an insulin partial agonist is administered at a concentration of about 1 μg / ml to about 30 μg / ml. In an embodiment, insulin, an insulin analog, an insulin agonist, and / or an insulin partial agonist is administered at a concentration of about 1 μg / ml to about 25 μg / ml. In an embodiment, insulin, an insulin analog, an insulin agonist, and / or an insulin partial agonist is administered at a concentration of about 1 μg / ml to about 20 μg / ml. In an embodiment, insulin, an insulin analog, an insulin agonist, and / or an insulin partial agonist is administered at a concentration of about 1 μg / ml to about 15 μg / ml. In an embodiment, insulin, an insulin analog, an insulin agonist, and / or an insulin partial agonist is administered at a concentration of about 1 μg / ml to about 10 μg / ml. In an embodiment, insulin, an insulin analog, an insulin agonist, and / or an insulin partial agonist is administered at a concentration of about 1 μg / ml to about 9 μg / ml. In an embodiment, insulin, an insulin analog, an insulin agonist, and / or an insulin partial agonist is administered at a concentration of about 1 μg / ml to about 8 μg / ml. In an embodiment, insulin, an insulin analog, an insulin agonist, and / or an insulin partial agonist is administered at a concentration of about 1 μg / ml to about 7 μg / ml. In an embodiment, insulin, an insulin analog, an insulin agonist, and / or an insulin partial agonist is administered at a concentration of about 1 μg / ml to about 6 μg / ml. In an embodiment, insulin, an insulin analog, an insulin agonist, and / or an insulin partial agonist is administered at a concentration of about 1 μg / ml to about 5 μg / ml. In an embodiment, insulin, an insulin analog, an insulin agonist, and / or an insulin partial agonist is administered at a concentration of about 1 μg / ml to about 4 μg / ml. In an embodiment, insulin, an insulin analog, an insulin agonist, and / or an insulin partial agonist is administered at a concentration of about 1 μg / ml to about 3 μg / ml. In an embodiment, insulin, an insulin analog, an insulin agonist, and / or an insulin partial agonist is administered at a concentration of about 1 μg / ml to about 2 μg / ml.

[0136] In an embodiment, insulin, an insulin analogue, an insulin agonist, and / or an insulin partial agonist is administered at a concentration of from 1 μg / ml to about 50 μg / ml. In an embodiment, insulin, an insulin analogue, an insulin agonist, and / or an insulin partial agonist is administered at a concentration of from 2 μg / ml to about 50 μg / ml. In an embodiment, insulin, an insulin analogue, an insulin agonist, and / or an insulin partial agonist is administered at a concentration of from 3 μg / ml to about 50 μg / ml. In an embodiment, insulin, an insulin analogue, an insulin agonist, and / or an insulin partial agonist is administered at a concentration of from 4 μg / ml to about 50 μg / ml. In an embodiment, insulin, an insulin analogue, an insulin agonist, and / or an insulin partial agonist is administered at a concentration of from 5 μg / ml to about 50 μg / ml. In an embodiment, insulin, an insulin analogue, an insulin agonist, and / or an insulin partial agonist is administered at a concentration of from 6 μg / ml to about 50 μg / ml. In an embodiment, insulin, an insulin analogue, an insulin agonist, and / or an insulin partial agonist is administered at a concentration of from 7 μg / ml to about 50 μg / ml. In an embodiment, insulin, an insulin analogue, an insulin agonist, and / or an insulin partial agonist is administered at a concentration of from 8 μg / ml to about 50 μg / ml. In an embodiment, insulin, an insulin analogue, an insulin agonist, and / or an insulin partial agonist is administered at a concentration of from 9 μg / ml to about 50 μg / ml. In an embodiment, insulin, an insulin analogue, an insulin agonist, and / or an insulin partial agonist is administered at a concentration of from 10 μg / ml to about 50 μg / ml. In an embodiment, insulin, an insulin analogue, an insulin agonist, and / or an insulin partial agonist is administered at a concentration of from 15 μg / ml to about 50 μg / ml. In an embodiment, insulin, an insulin analogue, an insulin agonist, and / or an insulin partial agonist is administered at a concentration of from 20 μg / ml to about 50 μg / ml. In an embodiment, insulin, an insulin analogue, an insulin agonist, and / or an insulin partial agonist is administered at a concentration of from 25 μg / ml to about 50 μg / ml. In an embodiment, insulin, an insulin analogue, an insulin agonist, and / or an insulin partial agonist is administered at a concentration of from 30 μg / ml to about 50 μg / ml. In an embodiment, insulin, an insulin analogue, an insulin agonist, and / or an insulin partial agonist is administered at a concentration of from 35 μg / ml to about 50 μg / ml. In an embodiment, insulin, an insulin analogue, an insulin agonist, and / or an insulin partial agonist is administered at a concentration of from 40 μg / ml to about 50 μg / ml. In an embodiment, insulin, an insulin analogue, an insulin agonist, and / or an insulin partial agonist is administered at a concentration of from 45 μg / ml to about 50 μg / ml.

[0137] In an embodiment, insulin, an insulin analog, an insulin agonist, and / or an insulin partial agonist are administered at a concentration of from 1 μg / ml to about 45 μg / ml. In an embodiment, insulin, an insulin analog, an insulin agonist, and / or an insulin partial agonist are administered at a concentration of from 1 μg / ml to about 40 μg / ml. In an embodiment, insulin, an insulin analog, an insulin agonist, and / or an insulin partial agonist are administered at a concentration of from 1 μg / ml to about 35 μg / ml. In an embodiment, insulin, an insulin analog, an insulin agonist, and / or an insulin partial agonist are administered at a concentration of from 1 μg / ml to about 30 μg / ml. In an embodiment, insulin, an insulin analog, an insulin agonist, and / or an insulin partial agonist are administered at a concentration of from 1 μg / ml to about 25 μg / ml. In an embodiment, insulin, an insulin analog, an insulin agonist, and / or an insulin partial agonist are administered at a concentration of from 1 μg / ml to about 20 μg / ml. In an embodiment, insulin, an insulin analog, an insulin agonist, and / or an insulin partial agonist are administered at a concentration of from 1 μg / ml to about 15 μg / ml. In an embodiment, insulin, an insulin analog, an insulin agonist, and / or an insulin partial agonist are administered at a concentration of from 1 μg / ml to about 10 μg / ml. In an embodiment, insulin, an insulin analog, an insulin agonist, and / or an insulin partial agonist are administered at a concentration of from 1 μg / ml to about 9 μg / ml. In an embodiment, insulin, an insulin analog, an insulin agonist, and / or an insulin partial agonist are administered at a concentration of from 1 μg / ml to about 8 μg / ml. In an embodiment, insulin, an insulin analog, an insulin agonist, and / or an insulin partial agonist are administered at a concentration of from 1 μg / ml to about 7 μg / ml. In an embodiment, insulin, an insulin analog, an insulin agonist, and / or an insulin partial agonist are administered at a concentration of from 1 μg / ml to about 6 μg / ml. In an embodiment, insulin, an insulin analog, an insulin agonist, and / or an insulin partial agonist are administered at a concentration of from 1 μg / ml to about 5 μg / ml. In an embodiment, insulin, an insulin analog, an insulin agonist, and / or an insulin partial agonist are administered at a concentration of from 1 μg / ml to about 4 μg / ml. In an embodiment, insulin, an insulin analog, an insulin agonist, and / or an insulin partial agonist are administered at a concentration of from 1 μg / ml to about 3 μg / ml. In an embodiment, insulin, an insulin analog, an insulin agonist, and / or an insulin partial agonist are administered at a concentration of from 1 μg / ml to about 2 μg / ml. The concentration can be any value or sub-range within the recited range including the endpoints.

[0138] In embodiments, insulin, an insulin analog, an insulin agonist, and / or an insulin partial agonist are administered at a concentration of about 1 μg / ml, about 5 μg / ml, or about 25 μg / ml. In embodiments, insulin, an insulin analog, an insulin agonist, and / or an insulin partial agonist are administered at a concentration of about 1 μg / ml. In embodiments, insulin, an insulin analog, an insulin agonist, and / or an insulin partial agonist are administered at a concentration of about 5 μg / ml. In embodiments, insulin, an insulin analog, an insulin agonist, and / or an insulin partial agonist are administered at a concentration of about 25 μg / ml. In embodiments, insulin, an insulin analog, an insulin agonist, and / or an insulin partial agonist are administered at a concentration of 1 μg / ml, 5 μg / ml, or 25 μg / ml. In embodiments, insulin, an insulin analog, an insulin agonist, and / or an insulin partial agonist are administered at a concentration of 1 μg / ml. In embodiments, insulin, an insulin analog, an insulin agonist, and / or an insulin partial agonist are administered at a concentration of 5 μg / ml. In embodiments, insulin, an insulin analog, an insulin agonist, and / or an insulin partial agonist are administered at a concentration of 25 μg / ml.

[0139] In embodiments, a T cell population is transfected with donor DNA and a gene editing reagent or a polynucleotide encoding the gene editing reagent simultaneously. In embodiments, a T cell population is transfected with donor DNA and a gene editing reagent simultaneously. In embodiments, a T cell population is transfected with donor DNA and a polynucleotide encoding a gene editing reagent simultaneously. In embodiments, the gene editing reagent comprises an RNA-guided nuclease. In embodiments, the RNA-guided nuclease is a CRISPR-Cas system. In embodiments, the CRISPR-Cas system comprises Cas9 or a Cas9 variant. In embodiments, the CRISPR-Cas system comprises Cas9. In embodiments, the CRISPR-Cas system comprises a Cas9 variant. In embodiments, the gene editing reagent comprises a CRISPR-Cas system comprising a Cas protein and a guide RNA (gRNA).

[0140] In embodiments, at least 80% of the engineered T cells are TCM and / or TSCM. In embodiments, at least 85% of the engineered T cells are TCM and / or TSCM. In embodiments, at least 80% of the engineered T cells are TCM and / or TSCM. In embodiments, at least 90% of the engineered T cells are TCM and / or TSCM. In embodiments, at least 91% of the engineered T cells are TCM and / or TSCM. In embodiments, at least 92% of the engineered T cells are TCM and / or TSCM. In embodiments, at least 93% of the engineered T cells are TCM and / or TSCM. In embodiments, at least 94% of the engineered T cells are TCM and / or TSCM. In embodiments, at least 95% of the engineered T cells are TCM and / or TSCM. In embodiments, at least 96% of the engineered T cells are TCM and / or TSCM. In embodiments, at least 97% of the engineered T cells are TCM and / or TSCM. In embodiments, at least 80% of the engineered T cells are TCM and / or TSCM. In embodiments, at least 99% of the engineered T cells are TCM and / or TSCM.

[0141] In embodiments, at least 80% of the engineered T cells are TCM and TSCM. In embodiments, at least 85% of the engineered T cells are TCM and TSCM. In embodiments, at least 80% of the engineered T cells are TCM and TSCM. In embodiments, at least 90% of the engineered T cells are TCM and TSCM. In embodiments, at least 91% of the engineered T cells are TCM and TSCM. In embodiments, at least 92% of the engineered T cells are TCM and TSCM. In embodiments, at least 93% of the engineered T cells are TCM and TSCM. In embodiments, at least 94% of the engineered T cells are TCM and TSCM. In embodiments, at least 95% of the engineered T cells are TCM and TSCM. In embodiments, at least 96% of the engineered T cells are TCM and TSCM. In embodiments, at least 97% of the engineered T cells are TCM and TSCM. In embodiments, at least 80% of the engineered T cells are TCM and TSCM. In embodiments, at least 99% of the engineered T cells are TCM and TSCM.

[0142] In an embodiment, at least 80% of the engineered T cells are TCM or TSCM. In an embodiment, at least 85% of the engineered T cells are TCM or TSCM. In an embodiment, at least 80% of the engineered T cells are TCM or TSCM. In an embodiment, at least 90% of the engineered T cells are TCM or TSCM. In an embodiment, at least 91% of the engineered T cells are TCM or TSCM. In an embodiment, at least 92% of the engineered T cells are TCM or TSCM. In an embodiment, at least 93% of the engineered T cells are TCM or TSCM. In an embodiment, at least 94% of the engineered T cells are TCM or TSCM. In an embodiment, at least 95% of the engineered T cells are TCM or TSCM. In an embodiment, at least 96% of the engineered T cells are TCM or TSCM. In an embodiment, at least 97% of the engineered T cells are TCM or TSCM. In an embodiment, at least 80% of the engineered T cells are TCM or TSCM. In an embodiment, at least 99% of the engineered T cells are TCM or TSCM.

[0143] In an embodiment, at least 80% of the engineered T cells are TCM. In an embodiment, at least 85% of the engineered T cells are TCM. In an embodiment, at least 80% of the engineered T cells are TCM. In an embodiment, at least 90% of the engineered T cells are TCM. In an embodiment, at least 91% of the engineered T cells are TCM. In an embodiment, at least 92% of the engineered T cells are TCM. In an embodiment, at least 93% of the engineered T cells are TCM. In an embodiment, at least 94% of the engineered T cells are TCM. In an embodiment, at least 95% of the engineered T cells are TCM. In an embodiment, at least 96% of the engineered T cells are TCM. In an embodiment, at least 97% of the engineered T cells are TCM. In an embodiment, at least 80% of the engineered T cells are TCM. In an embodiment, at least 99% of the engineered T cells are TCM.

[0144] In an embodiment, at least 80% of the engineered T cells are TSCM. In an embodiment, at least 85% of the engineered T cells are TSCM. In an embodiment, at least 80% of the engineered T cells are TSCM. In an embodiment, at least 90% of the engineered T cells are TSCM. In an embodiment, at least 91% of the engineered T cells are TSCM. In an embodiment, at least 92% of the engineered T cells are TSCM. In an embodiment, at least 93% of the engineered T cells are TSCM. In an embodiment, at least 94% of the engineered T cells are TSCM. In an embodiment, at least 95% of the engineered T cells are TSCM. In an embodiment, at least 96% of the engineered T cells are TSCM. In an embodiment, at least 97% of the engineered T cells are TSCM. In an embodiment, at least 80% of the engineered T cells are TSCM. In an embodiment, at least 99% of the engineered T cells are TSCM.

[0145] In an embodiment, the cell viability and culture performance of an engineered T cell population are monitored, and the method includes measuring mitochondrial function and cell metabolism over time. In an embodiment, the mitochondrial membrane potential is measured. In an embodiment, a dye-based assay is used to measure the mitochondrial membrane potential. In some embodiments, the dye is JC-1 or JC-10. In an embodiment, the dye is JC-1. In an embodiment, the dye is JC-10. In an embodiment, the cell viability and culture performance of a T cell population are monitored, and the method includes measuring cell metabolism markers over time. In an embodiment, the method includes measuring the change in glucose metabolism, Bcl-2 expression, Bcl-XL expression, Bax expression, or Bad expression over time. In an embodiment, the method includes measuring the change in glucose metabolism over time. In an embodiment, the method includes measuring the change in Bcl-2 expression over time. In an embodiment, the method includes measuring the change in Bcl-XL expression over time. In an embodiment, the method includes measuring the change in Bax expression over time. In an embodiment, the method includes measuring the change in Bad expression over time. In an embodiment, a glucose analog is used to monitor the glucose metabolism of an engineered T cell population. In an embodiment, the analog is 2-NBDG.

[0146] In an embodiment, the cell viability of the engineered T cell population is increased by at least about 0.1-fold to at least about 5.0-fold relative to an engineered T cell population not cultured in the presence of insulin, an insulin analog, an insulin agonist, or an insulin partial agonist. In an embodiment, the cell viability of the engineered T cell population is increased by at least about 0.2-fold to at least about 5.0-fold relative to an engineered T cell population not cultured in the presence of insulin, an insulin analog, an insulin agonist, or an insulin partial agonist. In an embodiment, the cell viability of the engineered T cell population is increased by at least about 0.3-fold to at least about 5.0-fold relative to an engineered T cell population not cultured in the presence of insulin, an insulin analog, an insulin agonist, or an insulin partial agonist. In an embodiment, the cell viability of the engineered T cell population is increased by at least about 0.4-fold to at least about 5.0-fold relative to an engineered T cell population not cultured in the presence of insulin, an insulin analog, an insulin agonist, or an insulin partial agonist. In an embodiment, the cell viability of the engineered T cell population is increased by at least about 0.5-fold to at least about 5.0-fold relative to an engineered T cell population not cultured in the presence of insulin, an insulin analog, an insulin agonist, or an insulin partial agonist. In an embodiment, the cell viability of the engineered T cell population is increased by at least about 0.6-fold to at least about 5.0-fold relative to an engineered T cell population not cultured in the presence of insulin, an insulin analog, an insulin agonist, or an insulin partial agonist. In an embodiment, the cell viability of the engineered T cell population is increased by at least about 0.7-fold to at least about 5.0-fold relative to an engineered T cell population not cultured in the presence of insulin, an insulin analog, an insulin agonist, or an insulin partial agonist. In an embodiment, the cell viability of the engineered T cell population is increased by at least about 0.8-fold to at least about 5.0-fold relative to an engineered T cell population not cultured in the presence of insulin, an insulin analog, an insulin agonist, or an insulin partial agonist. In an embodiment, the cell viability of the engineered T cell population is increased by at least about 0.9-fold to at least about 5.0-fold relative to an engineered T cell population not cultured in the presence of insulin, an insulin analog, an insulin agonist, or an insulin partial agonist. In an embodiment, the cell viability of the engineered T cell population is increased by at least about 1.0-fold to at least about 5.0-fold relative to an engineered T cell population not cultured in the presence of insulin, an insulin analog, an insulin agonist, or an insulin partial agonist. In an embodiment, the cell viability of the engineered T cell population is increased by at least about 1.5-fold to at least about 5.0-fold relative to an engineered T cell population not cultured in the presence of insulin, an insulin analog, an insulin agonist, or an insulin partial agonist.In embodiments, the cell viability of the engineered T cell population is increased by at least about 2.0-fold to at least about 5.0-fold relative to an engineered T cell population not cultured in the presence of insulin, an insulin analog, an insulin agonist, or an insulin partial agonist. In embodiments, the cell viability of the engineered T cell population is increased by at least about 2.5-fold to at least about 5.0-fold relative to an engineered T cell population not cultured in the presence of insulin, an insulin analog, an insulin agonist, or an insulin partial agonist. In embodiments, the cell viability of the engineered T cell population is increased by at least about 3.0-fold to at least about 5.0-fold relative to an engineered T cell population not cultured in the presence of insulin, an insulin analog, an insulin agonist, or an insulin partial agonist. In embodiments, the cell viability of the engineered T cell population is increased by at least about 3.5-fold to at least about 5.0-fold relative to an engineered T cell population not cultured in the presence of insulin, an insulin analog, an insulin agonist, or an insulin partial agonist. In embodiments, the cell viability of the engineered T cell population is increased by at least about 4.0-fold to at least about 5.0-fold relative to an engineered T cell population not cultured in the presence of insulin, an insulin analog, an insulin agonist, or an insulin partial agonist. In embodiments, the cell viability of the engineered T cell population is increased by at least about 4.5-fold to at least about 5.0-fold relative to an engineered T cell population not cultured in the presence of insulin, an insulin analog, an insulin agonist, or an insulin partial agonist.

[0147] In an embodiment, the cell viability of the engineered T cell population is increased by at least about 0.1-fold to at least about 4.5-fold relative to an engineered T cell population not cultured in the presence of insulin, an insulin analog, an insulin agonist, or an insulin partial agonist. In an embodiment, the cell viability of the engineered T cell population is increased by at least about 0.1-fold to at least about 4.0-fold relative to an engineered T cell population not cultured in the presence of insulin, an insulin analog, an insulin agonist, or an insulin partial agonist. In an embodiment, the cell viability of the engineered T cell population is increased by at least about 0.1-fold to at least about 3.5-fold relative to an engineered T cell population not cultured in the presence of insulin, an insulin analog, an insulin agonist, or an insulin partial agonist. In an embodiment, the cell viability of the engineered T cell population is increased by at least about 0.1-fold to at least about 3.0-fold relative to an engineered T cell population not cultured in the presence of insulin, an insulin analog, an insulin agonist, or an insulin partial agonist. In an embodiment, the cell viability of the engineered T cell population is increased by at least about 0.1-fold to at least about 2.5-fold relative to an engineered T cell population not cultured in the presence of insulin, an insulin analog, an insulin agonist, or an insulin partial agonist. In an embodiment, the cell viability of the engineered T cell population is increased by at least about 0.1-fold to at least about 2.0-fold relative to an engineered T cell population not cultured in the presence of insulin, an insulin analog, an insulin agonist, or an insulin partial agonist. In an embodiment, the cell viability of the engineered T cell population is increased by at least about 0.1-fold to at least about 1.5-fold relative to an engineered T cell population not cultured in the presence of insulin, an insulin analog, an insulin agonist, or an insulin partial agonist. In an embodiment, the cell viability of the engineered T cell population is increased by at least about 0.1-fold to at least about 1.0-fold relative to an engineered T cell population not cultured in the presence of insulin, an insulin analog, an insulin agonist, or an insulin partial agonist. In an embodiment, the cell viability of the engineered T cell population is increased by at least about 0.1-fold to at least about 0.9-fold relative to an engineered T cell population not cultured in the presence of insulin, an insulin analog, an insulin agonist, or an insulin partial agonist. In an embodiment, the cell viability of the engineered T cell population is increased by at least about 0.1-fold to at least about 0.8-fold relative to an engineered T cell population not cultured in the presence of insulin, an insulin analog, an insulin agonist, or an insulin partial agonist. In an embodiment, the cell viability of the engineered T cell population is increased by at least about 0.1-fold to at least about 0.7-fold relative to an engineered T cell population not cultured in the presence of insulin, an insulin analog, an insulin agonist, or an insulin partial agonist.In an embodiment, the cell viability of the engineered T cell population is increased by at least about 0.1-fold to at least about 0.6-fold relative to an engineered T cell population not cultured in the presence of insulin, an insulin analogue, an insulin agonist, or an insulin partial agonist. In an embodiment, the cell viability of the engineered T cell population is increased by at least about 0.1-fold to at least about 0.5-fold relative to an engineered T cell population not cultured in the presence of insulin, an insulin analogue, an insulin agonist, or an insulin partial agonist. In an embodiment, the cell viability of the engineered T cell population is increased by at least about 0.1-fold to at least about 0.4-fold relative to an engineered T cell population not cultured in the presence of insulin, an insulin analogue, an insulin agonist, or an insulin partial agonist. In an embodiment, the cell viability of the engineered T cell population is increased by at least about 0.1-fold to at least about 0.3-fold relative to an engineered T cell population not cultured in the presence of insulin, an insulin analogue, an insulin agonist, or an insulin partial agonist. In an embodiment, the cell viability of the engineered T cell population is increased by at least about 0.1-fold to at least about 0.2-fold relative to an engineered T cell population not cultured in the presence of insulin, an insulin analogue, an insulin agonist, or an insulin partial agonist.

[0148] In an embodiment, the cell viability of the engineered T cell population is increased by at least 0.1-fold to at least 5.0-fold relative to an engineered T cell population not cultured in the presence of insulin, an insulin analog, an insulin agonist, or an insulin partial agonist. In an embodiment, the cell viability of the engineered T cell population is increased by at least 0.2-fold to at least 5.0-fold relative to an engineered T cell population not cultured in the presence of insulin, an insulin analog, an insulin agonist, or an insulin partial agonist. In an embodiment, the cell viability of the engineered T cell population is increased by at least 0.3-fold to at least 5.0-fold relative to an engineered T cell population not cultured in the presence of insulin, an insulin analog, an insulin agonist, or an insulin partial agonist. In an embodiment, the cell viability of the engineered T cell population is increased by at least 0.4-fold to at least 5.0-fold relative to an engineered T cell population not cultured in the presence of insulin, an insulin analog, an insulin agonist, or an insulin partial agonist. In an embodiment, the cell viability of the engineered T cell population is increased by at least 0.5-fold to at least 5.0-fold relative to an engineered T cell population not cultured in the presence of insulin, an insulin analog, an insulin agonist, or an insulin partial agonist. In an embodiment, the cell viability of the engineered T cell population is increased by at least 0.6-fold to at least 5.0-fold relative to an engineered T cell population not cultured in the presence of insulin, an insulin analog, an insulin agonist, or an insulin partial agonist. In an embodiment, the cell viability of the engineered T cell population is increased by at least 0.7-fold to at least 5.0-fold relative to an engineered T cell population not cultured in the presence of insulin, an insulin analog, an insulin agonist, or an insulin partial agonist. In an embodiment, the cell viability of the engineered T cell population is increased by at least 0.8-fold to at least 5.0-fold relative to an engineered T cell population not cultured in the presence of insulin, an insulin analog, an insulin agonist, or an insulin partial agonist. In an embodiment, the cell viability of the engineered T cell population is increased by at least 0.9-fold to at least 5.0-fold relative to an engineered T cell population not cultured in the presence of insulin, an insulin analog, an insulin agonist, or an insulin partial agonist. In an embodiment, the cell viability of the engineered T cell population is increased by at least 1.0-fold to at least 5.0-fold relative to an engineered T cell population not cultured in the presence of insulin, an insulin analog, an insulin agonist, or an insulin partial agonist. In an embodiment, the cell viability of the engineered T cell population is increased by at least 1.5-fold to at least 5.0-fold relative to an engineered T cell population not cultured in the presence of insulin, an insulin analog, an insulin agonist, or an insulin partial agonist. In an embodiment, the cell viability of the engineered T cell population is increased by at least 2.0-fold to at least 5.0-fold relative to an engineered T cell population not cultured in the presence of insulin, an insulin analog, an insulin agonist, or an insulin partial agonist.In an embodiment, the cell viability of the engineered T cell population is increased by at least 2.5-fold to at least 5.0-fold relative to an engineered T cell population not cultured in the presence of insulin, an insulin analogue, an insulin agonist, or an insulin partial agonist. In an embodiment, the cell viability of the engineered T cell population is increased by at least 3.0-fold to at least 5.0-fold relative to an engineered T cell population not cultured in the presence of insulin, an insulin analogue, an insulin agonist, or an insulin partial agonist. In an embodiment, the cell viability of the engineered T cell population is increased by at least 3.5-fold to at least 5.0-fold relative to an engineered T cell population not cultured in the presence of insulin, an insulin analogue, an insulin agonist, or an insulin partial agonist. In an embodiment, the cell viability of the engineered T cell population is increased by at least 4.0-fold to at least 5.0-fold relative to an engineered T cell population not cultured in the presence of insulin, an insulin analogue, an insulin agonist, or an insulin partial agonist. In an embodiment, the cell viability of the engineered T cell population is increased by at least 4.5-fold to at least 5.0-fold relative to an engineered T cell population not cultured in the presence of insulin, an insulin analogue, an insulin agonist, or an insulin partial agonist.

[0149] In an embodiment, the cell viability of the engineered T cell population is increased by at least 0.1-fold to at least 4.5-fold relative to an engineered T cell population not cultured in the presence of insulin, an insulin analog, an insulin agonist, or an insulin partial agonist. In an embodiment, the cell viability of the engineered T cell population is increased by at least 0.1-fold to at least 4.0-fold relative to an engineered T cell population not cultured in the presence of insulin, an insulin analog, an insulin agonist, or an insulin partial agonist. In an embodiment, the cell viability of the engineered T cell population is increased by at least 0.1-fold to at least 3.5-fold relative to an engineered T cell population not cultured in the presence of insulin, an insulin analog, an insulin agonist, or an insulin partial agonist. In an embodiment, the cell viability of the engineered T cell population is increased by at least 0.1-fold to at least 3.0-fold relative to an engineered T cell population not cultured in the presence of insulin, an insulin analog, an insulin agonist, or an insulin partial agonist. In an embodiment, the cell viability of the engineered T cell population is increased by at least 0.1-fold to at least 2.5-fold relative to an engineered T cell population not cultured in the presence of insulin, an insulin analog, an insulin agonist, or an insulin partial agonist. In an embodiment, the cell viability of the engineered T cell population is increased by at least 0.1-fold to at least 2.0-fold relative to an engineered T cell population not cultured in the presence of insulin, an insulin analog, an insulin agonist, or an insulin partial agonist. In an embodiment, the cell viability of the engineered T cell population is increased by at least 0.1-fold to at least 1.5-fold relative to an engineered T cell population not cultured in the presence of insulin, an insulin analog, an insulin agonist, or an insulin partial agonist. In an embodiment, the cell viability of the engineered T cell population is increased by at least 0.1-fold to at least 1.0-fold relative to an engineered T cell population not cultured in the presence of insulin, an insulin analog, an insulin agonist, or an insulin partial agonist. In an embodiment, the cell viability of the engineered T cell population is increased by at least 0.1-fold to at least 0.9-fold relative to an engineered T cell population not cultured in the presence of insulin, an insulin analog, an insulin agonist, or an insulin partial agonist. In an embodiment, the cell viability of the engineered T cell population is increased by at least 0.1-fold to at least 0.8-fold relative to an engineered T cell population not cultured in the presence of insulin, an insulin analog, an insulin agonist, or an insulin partial agonist. In an embodiment, the cell viability of the engineered T cell population is increased by at least 0.1-fold to at least 0.7-fold relative to an engineered T cell population not cultured in the presence of insulin, an insulin analog, an insulin agonist, or an insulin partial agonist. In an embodiment, the cell viability of the engineered T cell population is increased by at least 0.1-fold to at least 0.6-fold relative to an engineered T cell population not cultured in the presence of insulin, an insulin analog, an insulin agonist, or an insulin partial agonist.In an embodiment, the cell viability of the engineered T cell population is increased by at least 0.1-fold to at least 0.5-fold relative to an engineered T cell population not cultured in the presence of insulin, an insulin analog, an insulin agonist, or an insulin partial agonist. In an embodiment, the cell viability of the engineered T cell population is increased by at least 0.1-fold to at least 0.4-fold relative to an engineered T cell population not cultured in the presence of insulin, an insulin analog, an insulin agonist, or an insulin partial agonist. In an embodiment, the cell viability of the engineered T cell population is increased by at least 0.1-fold to at least 0.3-fold relative to an engineered T cell population not cultured in the presence of insulin, an insulin analog, an insulin agonist, or an insulin partial agonist. In an embodiment, the cell viability of the engineered T cell population is increased by at least 0.1-fold to at least 0.2-fold relative to an engineered T cell population not cultured in the presence of insulin, an insulin analog, an insulin agonist, or an insulin partial agonist. The fold increase can be any value or sub-range within the recited range including the endpoints.

[0150] In an embodiment, the cell viability is increased by about 2.0-fold. In an embodiment, the cell viability is increased 2.0-fold. The fold increase can be any value or sub-range within the recited range including the endpoints.

[0151] In an embodiment, the cell viability of the engineered T cell population is from about 30% to about 95%. In an embodiment, the cell viability of the engineered T cell population is from about 35% to about 95%. In an embodiment, the cell viability of the engineered T cell population is from about 40% to about 95%. In an embodiment, the cell viability of the engineered T cell population is from about 45% to about 95%. In an embodiment, the cell viability of the engineered T cell population is from about 50% to about 95%. In an embodiment, the cell viability of the engineered T cell population is from about 55% to about 95%. In an embodiment, the cell viability of the engineered T cell population is from about 60% to about 95%. In an embodiment, the cell viability of the engineered T cell population is from about 65% to about 95%. In an embodiment, the cell viability of the engineered T cell population is from about 70% to about 95%. In an embodiment, the cell viability of the engineered T cell population is from about 75% to about 95%. In an embodiment, the cell viability of the engineered T cell population is from about 80% to about 95%. In an embodiment, the cell viability of the engineered T cell population is from about 85% to about 95%. In an embodiment, the cell viability of the engineered T cell population is from about 90% to about 95%. In an embodiment, the cell viability of the engineered T cell population is from about 91% to about 95%. In an embodiment, the cell viability of the engineered T cell population is from about 92% to about 95%. In an embodiment, the cell viability of the engineered T cell population is from about 93% to about 95%. In an embodiment, the cell viability of the engineered T cell population is from about 94% to about 95%.

[0152] In an embodiment, the cell viability of the engineered T cell population is from about 30% to about 94%. In an embodiment, the cell viability of the engineered T cell population is from about 30% to about 93%. In an embodiment, the cell viability of the engineered T cell population is from about 30% to about 92%. In an embodiment, the cell viability of the engineered T cell population is from about 30% to about 91%. In an embodiment, the cell viability of the engineered T cell population is from about 30% to about 90%. In an embodiment, the cell viability of the engineered T cell population is from about 30% to about 85%. In an embodiment, the cell viability of the engineered T cell population is from about 30% to about 80%. In an embodiment, the cell viability of the engineered T cell population is from about 30% to about 75%. In an embodiment, the cell viability of the engineered T cell population is from about 30% to about 70%. In an embodiment, the cell viability of the engineered T cell population is from about 30% to about 65%. In an embodiment, the cell viability of the engineered T cell population is from about 30% to about 60%. In an embodiment, the cell viability of the engineered T cell population is from about 30% to about 55%. In an embodiment, the cell viability of the engineered T cell population is from about 30% to about 50%. In an embodiment, the cell viability of the engineered T cell population is from about 30% to about 45%. In an embodiment, the cell viability of the engineered T cell population is from about 30% to about 40%. In an embodiment, the cell viability of the engineered T cell population is from about 30% to about 35%.

[0153] In an embodiment, the cell viability of the engineered T cell population is from 30% to 95%. In an embodiment, the cell viability of the engineered T cell population is from 35% to 95%. In an embodiment, the cell viability of the engineered T cell population is from 40% to 95%. In an embodiment, the cell viability of the engineered T cell population is from 45% to 95%. In an embodiment, the cell viability of the engineered T cell population is from 50% to 95%. In an embodiment, the cell viability of the engineered T cell population is from 55% to 95%. In an embodiment, the cell viability of the engineered T cell population is from 60% to 95%. In an embodiment, the cell viability of the engineered T cell population is from 65% to 95%. In an embodiment, the cell viability of the engineered T cell population is from 70% to 95%. In an embodiment, the cell viability of the engineered T cell population is from 75% to 95%. In an embodiment, the cell viability of the engineered T cell population is from 80% to 95%. In an embodiment, the cell viability of the engineered T cell population is from 85% to 95%. In an embodiment, the cell viability of the engineered T cell population is from 90% to 95%. In an embodiment, the cell viability of the engineered T cell population is from 91% to 95%. In an embodiment, the cell viability of the engineered T cell population is from 92% to 95%. In an embodiment, the cell viability of the engineered T cell population is from 93% to 95%. In an embodiment, the cell viability of the engineered T cell population is from 94% to 95%.

[0154] In embodiments, the cell viability of the engineered T cell population is from 30% to 94%. In embodiments, the cell viability of the engineered T cell population is from 30% to 93%. In embodiments, the cell viability of the engineered T cell population is from 30% to 92%. In embodiments, the cell viability of the engineered T cell population is from 30% to 91%. In embodiments, the cell viability of the engineered T cell population is from 30% to 90%. In embodiments, the cell viability of the engineered T cell population is from 30% to 85%. In embodiments, the cell viability of the engineered T cell population is from 30% to 80%. In embodiments, the cell viability of the engineered T cell population is from 30% to 75%. In embodiments, the cell viability of the engineered T cell population is from 30% to 70%. In embodiments, the cell viability of the engineered T cell population is from 30% to 65%. In embodiments, the cell viability of the engineered T cell population is from 30% to 60%. In embodiments, the cell viability of the engineered T cell population is from 30% to 55%. In embodiments, the cell viability of the engineered T cell population is from 30% to 50%. In embodiments, the cell viability of the engineered T cell population is from 30% to 45%. In embodiments, the cell viability of the engineered T cell population is from 30% to 40%. In embodiments, the cell viability of the engineered T cell population is from 30% to 35%. The percent increase can be any value or sub-range within the recited range including the endpoints.

[0155] Provided herein are methods for increasing the gene editing efficiency of an engineered T cell population, which include contacting a T cell population with insulin, an insulin analog, an insulin agonist, an insulin partial agonist, a gene editing reagent, and a polynucleotide to form the engineered T cell population, wherein the engineered T cell population has an increased gene editing efficiency relative to an engineered T cell population that has not been contacted with insulin, an insulin analog, an insulin agonist, or an insulin partial agonist.

[0156] In embodiments, contacting the T cell population with the polynucleotide includes transfecting the T cell population with the polynucleotide. In embodiments, the polynucleotide is donor DNA. In embodiments, the polynucleotide includes: single-stranded DNA, double-stranded DNA, linear DNA strands, plasmids, nanoplasmids, or minicircles. In embodiments, the polynucleotide includes single-stranded DNA. In embodiments, the polynucleotide includes double-stranded DNA. In embodiments, the polynucleotide includes linear DNA strands. In embodiments, the polynucleotide includes plasmids. In embodiments, the polynucleotide includes nanoplasmids. In embodiments, the polynucleotide includes minicircles.

[0157] In an embodiment, it further includes contacting a T cell population with a gene editing reagent. In an embodiment, contacting the T cell population with the gene editing reagent includes transfecting the T cell population with the gene editing reagent or a polynucleotide encoding the gene editing reagent. In an embodiment, contacting the T cell population with the gene editing reagent includes transfecting the T cell population with the gene editing reagent. In an embodiment, contacting the T cell population with the gene editing reagent includes transfecting the T cell population with a polynucleotide encoding the gene editing reagent. In an embodiment, the T cell population is transfected simultaneously with a polynucleotide and the gene editing reagent or a polynucleotide encoding the gene editing reagent. In an embodiment, the T cell population is transfected simultaneously with a polynucleotide and the gene editing reagent. In an embodiment, the T cell population is transfected simultaneously with a polynucleotide and a polynucleotide encoding the gene editing reagent.

[0158] In an embodiment, insulin, an insulin analogue, an insulin agonist, and / or an insulin partial agonist are administered at a concentration of from about 1 μg / ml to about 50 μg / ml. In an embodiment, insulin, an insulin analogue, an insulin agonist, and / or an insulin partial agonist are administered at a concentration of from about 2 μg / ml to about 50 μg / ml. In an embodiment, insulin, an insulin analogue, an insulin agonist, and / or an insulin partial agonist are administered at a concentration of from about 3 μg / ml to about 50 μg / ml. In an embodiment, insulin, an insulin analogue, an insulin agonist, and / or an insulin partial agonist are administered at a concentration of from about 4 μg / ml to about 50 μg / ml. In an embodiment, insulin, an insulin analogue, an insulin agonist, and / or an insulin partial agonist are administered at a concentration of from about 5 μg / ml to about 50 μg / ml. In an embodiment, insulin, an insulin analogue, an insulin agonist, and / or an insulin partial agonist are administered at a concentration of from about 6 μg / ml to about 50 μg / ml. In an embodiment, insulin, an insulin analogue, an insulin agonist, and / or an insulin partial agonist are administered at a concentration of from about 7 μg / ml to about 50 μg / ml. In an embodiment, insulin, an insulin analogue, an insulin agonist, and / or an insulin partial agonist are administered at a concentration of from about 8 μg / ml to about 50 μg / ml. In an embodiment, insulin, an insulin analogue, an insulin agonist, and / or an insulin partial agonist are administered at a concentration of from about 9 μg / ml to about 50 μg / ml. In an embodiment, insulin, an insulin analogue, an insulin agonist, and / or an insulin partial agonist are administered at a concentration of from about 10 μg / ml to about 50 μg / ml. In an embodiment, insulin, an insulin analogue, an insulin agonist, and / or an insulin partial agonist are administered at a concentration of from about 15 μg / ml to about 50 μg / ml. In an embodiment, insulin, an insulin analogue, an insulin agonist, and / or an insulin partial agonist are administered at a concentration of from about 20 μg / ml to about 50 μg / ml. In an embodiment, insulin, an insulin analogue, an insulin agonist, and / or an insulin partial agonist are administered at a concentration of from about 25 μg / ml to about 50 μg / ml. In an embodiment, insulin, an insulin analogue, an insulin agonist, and / or an insulin partial agonist are administered at a concentration of from about 30 μg / ml to about 50 μg / ml. In an embodiment, insulin, an insulin analogue, an insulin agonist, and / or an insulin partial agonist are administered at a concentration of from about 35 μg / ml to about 50 μg / ml. In an embodiment, insulin, an insulin analogue, an insulin agonist, and / or an insulin partial agonist are administered at a concentration of from about 40 μg / ml to about 50 μg / ml. In an embodiment, insulin, an insulin analogue, an insulin agonist, and / or an insulin partial agonist are administered at a concentration of from about 45 μg / ml to about 50 μg / ml.

[0159] In embodiments, insulin, an insulin analogue, an insulin agonist, and / or an insulin partial agonist is administered at a concentration of from about 1 μg / ml to about 45 μg / ml. In embodiments, insulin, an insulin analogue, an insulin agonist, and / or an insulin partial agonist is administered at a concentration of from about 1 μg / ml to about 40 μg / ml. In embodiments, insulin, an insulin analogue, an insulin agonist, and / or an insulin partial agonist is administered at a concentration of from about 1 μg / ml to about 35 μg / ml. In embodiments, insulin, an insulin analogue, an insulin agonist, and / or an insulin partial agonist is administered at a concentration of from about 1 μg / ml to about 30 μg / ml. In embodiments, insulin, an insulin analogue, an insulin agonist, and / or an insulin partial agonist is administered at a concentration of from about 1 μg / ml to about 25 μg / ml. In embodiments, insulin, an insulin analogue, an insulin agonist, and / or an insulin partial agonist is administered at a concentration of from about 1 μg / ml to about 20 μg / ml. In embodiments, insulin, an insulin analogue, an insulin agonist, and / or an insulin partial agonist is administered at a concentration of from about 1 μg / ml to about 15 μg / ml. In embodiments, insulin, an insulin analogue, an insulin agonist, and / or an insulin partial agonist is administered at a concentration of from about 1 μg / ml to about 10 μg / ml. In embodiments, insulin, an insulin analogue, an insulin agonist, and / or an insulin partial agonist is administered at a concentration of from about 1 μg / ml to about 9 μg / ml. In embodiments, insulin, an insulin analogue, an insulin agonist, and / or an insulin partial agonist is administered at a concentration of from about 1 μg / ml to about 8 μg / ml. In embodiments, insulin, an insulin analogue, an insulin agonist, and / or an insulin partial agonist is administered at a concentration of from about 1 μg / ml to about 7 μg / ml. In embodiments, insulin, an insulin analogue, an insulin agonist, and / or an insulin partial agonist is administered at a concentration of from about 1 μg / ml to about 6 μg / ml. In embodiments, insulin, an insulin analogue, an insulin agonist, and / or an insulin partial agonist is administered at a concentration of from about 1 μg / ml to about 5 μg / ml. In embodiments, insulin, an insulin analogue, an insulin agonist, and / or an insulin partial agonist is administered at a concentration of from about 1 μg / ml to about 4 μg / ml. In embodiments, insulin, an insulin analogue, an insulin agonist, and / or an insulin partial agonist is administered at a concentration of from about 1 μg / ml to about 3 μg / ml. In embodiments, insulin, an insulin analogue, an insulin agonist, and / or an insulin partial agonist is administered at a concentration of from about 1 μg / ml to about 2 μg / ml.

[0160] In an embodiment, insulin, an insulin analogue, an insulin agonist, and / or an insulin partial agonist are administered at a concentration of 1 μg / ml to about 50 μg / ml. In an embodiment, insulin, an insulin analogue, an insulin agonist, and / or an insulin partial agonist are administered at a concentration of 2 μg / ml to about 50 μg / ml. In an embodiment, insulin, an insulin analogue, an insulin agonist, and / or an insulin partial agonist are administered at a concentration of 3 μg / ml to about 50 μg / ml. In an embodiment, insulin, an insulin analogue, an insulin agonist, and / or an insulin partial agonist are administered at a concentration of 4 μg / ml to about 50 μg / ml. In an embodiment, insulin, an insulin analogue, an insulin agonist, and / or an insulin partial agonist are administered at a concentration of 5 μg / ml to about 50 μg / ml. In an embodiment, insulin, an insulin analogue, an insulin agonist, and / or an insulin partial agonist are administered at a concentration of 6 μg / ml to about 50 μg / ml. In an embodiment, insulin, an insulin analogue, an insulin agonist, and / or an insulin partial agonist are administered at a concentration of 7 μg / ml to about 50 μg / ml. In an embodiment, insulin, an insulin analogue, an insulin agonist, and / or an insulin partial agonist are administered at a concentration of 8 μg / ml to about 50 μg / ml. In an embodiment, insulin, an insulin analogue, an insulin agonist, and / or an insulin partial agonist are administered at a concentration of 9 μg / ml to about 50 μg / ml. In an embodiment, insulin, an insulin analogue, an insulin agonist, and / or an insulin partial agonist are administered at a concentration of 10 μg / ml to about 50 μg / ml. In an embodiment, insulin, an insulin analogue, an insulin agonist, and / or an insulin partial agonist are administered at a concentration of 15 μg / ml to about 50 μg / ml. In an embodiment, insulin, an insulin analogue, an insulin agonist, and / or an insulin partial agonist are administered at a concentration of 20 μg / ml to about 50 μg / ml. In an embodiment, insulin, an insulin analogue, an insulin agonist, and / or an insulin partial agonist are administered at a concentration of 25 μg / ml to about 50 μg / ml. In an embodiment, insulin, an insulin analogue, an insulin agonist, and / or an insulin partial agonist are administered at a concentration of 30 μg / ml to about 50 μg / ml. In an embodiment, insulin, an insulin analogue, an insulin agonist, and / or an insulin partial agonist are administered at a concentration of 35 μg / ml to about 50 μg / ml. In an embodiment, insulin, an insulin analogue, an insulin agonist, and / or an insulin partial agonist are administered at a concentration of 40 μg / ml to about 50 μg / ml. In an embodiment, insulin, an insulin analogue, an insulin agonist, and / or an insulin partial agonist are administered at a concentration of 45 μg / ml to about 50 μg / ml.

[0161] In embodiments, insulin, an insulin analogue, an insulin agonist, and / or an insulin partial agonist are administered at a concentration of from 1 μg / ml to about 45 μg / ml. In embodiments, insulin, an insulin analogue, an insulin agonist, and / or an insulin partial agonist are administered at a concentration of from 1 μg / ml to about 40 μg / ml. In embodiments, insulin, an insulin analogue, an insulin agonist, and / or an insulin partial agonist are administered at a concentration of from 1 μg / ml to about 35 μg / ml. In embodiments, insulin, an insulin analogue, an insulin agonist, and / or an insulin partial agonist are administered at a concentration of from 1 μg / ml to about 30 μg / ml. In embodiments, insulin, an insulin analogue, an insulin agonist, and / or an insulin partial agonist are administered at a concentration of from 1 μg / ml to about 25 μg / ml. In embodiments, insulin, an insulin analogue, an insulin agonist, and / or an insulin partial agonist are administered at a concentration of from 1 μg / ml to about 20 μg / ml. In embodiments, insulin, an insulin analogue, an insulin agonist, and / or an insulin partial agonist are administered at a concentration of from 1 μg / ml to about 15 μg / ml. In embodiments, insulin, an insulin analogue, an insulin agonist, and / or an insulin partial agonist are administered at a concentration of from 1 μg / ml to about 10 μg / ml. In embodiments, insulin, an insulin analogue, an insulin agonist, and / or an insulin partial agonist are administered at a concentration of from 1 μg / ml to about 9 μg / ml. In embodiments, insulin, an insulin analogue, an insulin agonist, and / or an insulin partial agonist are administered at a concentration of from 1 μg / ml to about 8 μg / ml. In embodiments, insulin, an insulin analogue, an insulin agonist, and / or an insulin partial agonist are administered at a concentration of from 1 μg / ml to about 7 μg / ml. In embodiments, insulin, an insulin analogue, an insulin agonist, and / or an insulin partial agonist are administered at a concentration of from 1 μg / ml to about 6 μg / ml. In embodiments, insulin, an insulin analogue, an insulin agonist, and / or an insulin partial agonist are administered at a concentration of from 1 μg / ml to about 5 μg / ml. In embodiments, insulin, an insulin analogue, an insulin agonist, and / or an insulin partial agonist are administered at a concentration of from 1 μg / ml to about 4 μg / ml. In embodiments, insulin, an insulin analogue, an insulin agonist, and / or an insulin partial agonist are administered at a concentration of from 1 μg / ml to about 3 μg / ml. In embodiments, insulin, an insulin analogue, an insulin agonist, and / or an insulin partial agonist are administered at a concentration of from 1 μg / ml to about 2 μg / ml. The concentration can be any value or sub-range within the recited range including the endpoints.

[0162] In an embodiment, a T cell population is contacted with a polynucleotide and insulin, an insulin analog, an insulin agonist, and / or an insulin partial agonist simultaneously. In an embodiment, a T cell population is contacted with a polynucleotide and insulin, an insulin analog, an insulin agonist, and / or an insulin partial agonist sequentially. In an embodiment, a T cell population is contacted with an insulin inhibitor prior to the polynucleotide.

[0163] In an embodiment, the gene editing efficiency of the engineered T cell population is increased by at least about 0.1-fold to at least about 5.0-fold relative to an engineered T cell population not cultured in the presence of insulin, an insulin analogue, an insulin agonist, or an insulin partial agonist. In an embodiment, the gene editing efficiency of the engineered T cell population is increased by at least about 0.2-fold to at least about 5.0-fold relative to an engineered T cell population not cultured in the presence of insulin, an insulin analogue, an insulin agonist, or an insulin partial agonist. In an embodiment, the gene editing efficiency of the engineered T cell population is increased by at least about 0.3-fold to at least about 5.0-fold relative to an engineered T cell population not cultured in the presence of insulin, an insulin analogue, an insulin agonist, or an insulin partial agonist. In an embodiment, the gene editing efficiency of the engineered T cell population is increased by at least about 0.4-fold to at least about 5.0-fold relative to an engineered T cell population not cultured in the presence of insulin, an insulin analogue, an insulin agonist, or an insulin partial agonist. In an embodiment, the gene editing efficiency of the engineered T cell population is increased by at least about 0.5-fold to at least about 5.0-fold relative to an engineered T cell population not cultured in the presence of insulin, an insulin analogue, an insulin agonist, or an insulin partial agonist. In an embodiment, the gene editing efficiency of the engineered T cell population is increased by at least about 0.6-fold to at least about 5.0-fold relative to an engineered T cell population not cultured in the presence of insulin, an insulin analogue, an insulin agonist, or an insulin partial agonist. In an embodiment, the gene editing efficiency of the engineered T cell population is increased by at least about 0.7-fold to at least about 5.0-fold relative to an engineered T cell population not cultured in the presence of insulin, an insulin analogue, an insulin agonist, or an insulin partial agonist. In an embodiment, the gene editing efficiency of the engineered T cell population is increased by at least about 0.8-fold to at least about 5.0-fold relative to an engineered T cell population not cultured in the presence of insulin, an insulin analogue, an insulin agonist, or an insulin partial agonist. In an embodiment, the gene editing efficiency of the engineered T cell population is increased by at least about 0.9-fold to at least about 5.0-fold relative to an engineered T cell population not cultured in the presence of insulin, an insulin analogue, an insulin agonist, or an insulin partial agonist. In an embodiment, the gene editing efficiency of the engineered T cell population is increased by at least about 1.0-fold to at least about 5.0-fold relative to an engineered T cell population not cultured in the presence of insulin, an insulin analogue, an insulin agonist, or an insulin partial agonist. In an embodiment, the gene editing efficiency of the engineered T cell population is increased by at least about 1.5-fold to at least about 5.0-fold relative to an engineered T cell population not cultured in the presence of insulin, an insulin analogue, an insulin agonist, or an insulin partial agonist.In embodiments, the gene editing efficiency of the engineered T cell population is increased by at least about 2.0-fold to at least about 5.0-fold relative to an engineered T cell population not cultured in the presence of insulin, an insulin analog, an insulin agonist, or an insulin partial agonist. In embodiments, the gene editing efficiency of the engineered T cell population is increased by at least about 2.5-fold to at least about 5.0-fold relative to an engineered T cell population not cultured in the presence of insulin, an insulin analog, an insulin agonist, or an insulin partial agonist. In embodiments, the gene editing efficiency of the engineered T cell population is increased by at least about 3.0-fold to at least about 5.0-fold relative to an engineered T cell population not cultured in the presence of insulin, an insulin analog, an insulin agonist, or an insulin partial agonist. In embodiments, the gene editing efficiency of the engineered T cell population is increased by at least about 3.5-fold to at least about 5.0-fold relative to an engineered T cell population not cultured in the presence of insulin, an insulin analog, an insulin agonist, or an insulin partial agonist. In embodiments, the gene editing efficiency of the engineered T cell population is increased by at least about 4.0-fold to at least about 5.0-fold relative to an engineered T cell population not cultured in the presence of insulin, an insulin analog, an insulin agonist, or an insulin partial agonist. In embodiments, the gene editing efficiency of the engineered T cell population is increased by at least about 4.5-fold to at least about 5.0-fold relative to an engineered T cell population not cultured in the presence of insulin, an insulin analog, an insulin agonist, or an insulin partial agonist.

[0164] In an embodiment, the gene editing efficiency of the engineered T cell population is increased by at least about 0.1-fold to at least about 4.5-fold relative to an engineered T cell population not cultured in the presence of insulin, an insulin analog, an insulin agonist, or an insulin partial agonist. In an embodiment, the gene editing efficiency of the engineered T cell population is increased by at least about 0.1-fold to at least about 4.0-fold relative to an engineered T cell population not cultured in the presence of insulin, an insulin analog, an insulin agonist, or an insulin partial agonist. In an embodiment, the gene editing efficiency of the engineered T cell population is increased by at least about 0.1-fold to at least about 3.5-fold relative to an engineered T cell population not cultured in the presence of insulin, an insulin analog, an insulin agonist, or an insulin partial agonist. In an embodiment, the gene editing efficiency of the engineered T cell population is increased by at least about 0.1-fold to at least about 3.0-fold relative to an engineered T cell population not cultured in the presence of insulin, an insulin analog, an insulin agonist, or an insulin partial agonist. In an embodiment, the gene editing efficiency of the engineered T cell population is increased by at least about 0.1-fold to at least about 2.5-fold relative to an engineered T cell population not cultured in the presence of insulin, an insulin analog, an insulin agonist, or an insulin partial agonist. In an embodiment, the gene editing efficiency of the engineered T cell population is increased by at least about 0.1-fold to at least about 2.0-fold relative to an engineered T cell population not cultured in the presence of insulin, an insulin analog, an insulin agonist, or an insulin partial agonist. In an embodiment, the gene editing efficiency of the engineered T cell population is increased by at least about 0.1-fold to at least about 1.5-fold relative to an engineered T cell population not cultured in the presence of insulin, an insulin analog, an insulin agonist, or an insulin partial agonist. In an embodiment, the gene editing efficiency of the engineered T cell population is increased by at least about 0.1-fold to at least about 1.0-fold relative to an engineered T cell population not cultured in the presence of insulin, an insulin analog, an insulin agonist, or an insulin partial agonist. In an embodiment, the gene editing efficiency of the engineered T cell population is increased by at least about 0.1-fold to at least about 0.9-fold relative to an engineered T cell population not cultured in the presence of insulin, an insulin analog, an insulin agonist, or an insulin partial agonist. In an embodiment, the gene editing efficiency of the engineered T cell population is increased by at least about 0.1-fold to at least about 0.8-fold relative to an engineered T cell population not cultured in the presence of insulin, an insulin analog, an insulin agonist, or an insulin partial agonist. In an embodiment, the gene editing efficiency of the engineered T cell population is increased by at least about 0.1-fold to at least about 0.7-fold relative to an engineered T cell population not cultured in the presence of insulin, an insulin analog, an insulin agonist, or an insulin partial agonist.In an embodiment, the gene editing efficiency of the engineered T cell population is increased by at least about 0.1-fold to at least about 0.6-fold relative to an engineered T cell population not cultured in the presence of insulin, an insulin analog, an insulin agonist, or an insulin partial agonist. In an embodiment, the gene editing efficiency of the engineered T cell population is increased by at least about 0.1-fold to at least about 0.5-fold relative to an engineered T cell population not cultured in the presence of insulin, an insulin analog, an insulin agonist, or an insulin partial agonist. In an embodiment, the gene editing efficiency of the engineered T cell population is increased by at least about 0.1-fold to at least about 0.4-fold relative to an engineered T cell population not cultured in the presence of insulin, an insulin analog, an insulin agonist, or an insulin partial agonist. In an embodiment, the gene editing efficiency of the engineered T cell population is increased by at least about 0.1-fold to at least about 0.3-fold relative to an engineered T cell population not cultured in the presence of insulin, an insulin analog, an insulin agonist, or an insulin partial agonist. In an embodiment, the gene editing efficiency of the engineered T cell population is increased by at least about 0.1-fold to at least about 0.2-fold relative to an engineered T cell population not cultured in the presence of insulin, an insulin analog, an insulin agonist, or an insulin partial agonist.

[0165] In an embodiment, the gene editing efficiency of the engineered T cell population is increased by at least 0.1-fold to at least 5.0-fold relative to an engineered T cell population not cultured in the presence of insulin, an insulin analog, an insulin agonist, or an insulin partial agonist. In an embodiment, the gene editing efficiency of the engineered T cell population is increased by at least 0.2-fold to at least 5.0-fold relative to an engineered T cell population not cultured in the presence of insulin, an insulin analog, an insulin agonist, or an insulin partial agonist. In an embodiment, the gene editing efficiency of the engineered T cell population is increased by at least 0.3-fold to at least 5.0-fold relative to an engineered T cell population not cultured in the presence of insulin, an insulin analog, an insulin agonist, or an insulin partial agonist. In an embodiment, the gene editing efficiency of the engineered T cell population is increased by at least 0.4-fold to at least 5.0-fold relative to an engineered T cell population not cultured in the presence of insulin, an insulin analog, an insulin agonist, or an insulin partial agonist. In an embodiment, the gene editing efficiency of the engineered T cell population is increased by at least 0.5-fold to at least 5.0-fold relative to an engineered T cell population not cultured in the presence of insulin, an insulin analog, an insulin agonist, or an insulin partial agonist. In an embodiment, the gene editing efficiency of the engineered T cell population is increased by at least 0.6-fold to at least 5.0-fold relative to an engineered T cell population not cultured in the presence of insulin, an insulin analog, an insulin agonist, or an insulin partial agonist. In an embodiment, the gene editing efficiency of the engineered T cell population is increased by at least 0.7-fold to at least 5.0-fold relative to an engineered T cell population not cultured in the presence of insulin, an insulin analog, an insulin agonist, or an insulin partial agonist. In an embodiment, the gene editing efficiency of the engineered T cell population is increased by at least 0.8-fold to at least 5.0-fold relative to an engineered T cell population not cultured in the presence of insulin, an insulin analog, an insulin agonist, or an insulin partial agonist. In an embodiment, the gene editing efficiency of the engineered T cell population is increased by at least 0.9-fold to at least 5.0-fold relative to an engineered T cell population not cultured in the presence of insulin, an insulin analog, an insulin agonist, or an insulin partial agonist. In an embodiment, the gene editing efficiency of the engineered T cell population is increased by at least 1.0-fold to at least 5.0-fold relative to an engineered T cell population not cultured in the presence of insulin, an insulin analog, an insulin agonist, or an insulin partial agonist. In an embodiment, the gene editing efficiency of the engineered T cell population is increased by at least 1.5-fold to at least 5.0-fold relative to an engineered T cell population not cultured in the presence of insulin, an insulin analog, an insulin agonist, or an insulin partial agonist.In an embodiment, the gene editing efficiency of the engineered T cell population is increased by at least 2.0-fold to at least 5.0-fold relative to an engineered T cell population not cultured in the presence of insulin, an insulin analogue, an insulin agonist, or an insulin partial agonist. In an embodiment, the gene editing efficiency of the engineered T cell population is increased by at least 2.5-fold to at least 5.0-fold relative to an engineered T cell population not cultured in the presence of insulin, an insulin analogue, an insulin agonist, or an insulin partial agonist. In an embodiment, the gene editing efficiency of the engineered T cell population is increased by at least 3.0-fold to at least 5.0-fold relative to an engineered T cell population not cultured in the presence of insulin, an insulin analogue, an insulin agonist, or an insulin partial agonist. In an embodiment, the gene editing efficiency of the engineered T cell population is increased by at least 3.5-fold to at least 5.0-fold relative to an engineered T cell population not cultured in the presence of insulin, an insulin analogue, an insulin agonist, or an insulin partial agonist. In an embodiment, the gene editing efficiency of the engineered T cell population is increased by at least 4.0-fold to at least 5.0-fold relative to an engineered T cell population not cultured in the presence of insulin, an insulin analogue, an insulin agonist, or an insulin partial agonist. In an embodiment, the gene editing efficiency of the engineered T cell population is increased by at least 4.5-fold to at least 5.0-fold relative to an engineered T cell population not cultured in the presence of insulin, an insulin analogue, an insulin agonist, or an insulin partial agonist.

[0166] In an embodiment, the gene editing efficiency of the engineered T cell population is increased by at least 0.1-fold to at least 4.5-fold relative to an engineered T cell population not cultured in the presence of insulin, an insulin analog, an insulin agonist, or an insulin partial agonist. In an embodiment, the gene editing efficiency of the engineered T cell population is increased by at least 0.1-fold to at least 4.0-fold relative to an engineered T cell population not cultured in the presence of insulin, an insulin analog, an insulin agonist, or an insulin partial agonist. In an embodiment, the gene editing efficiency of the engineered T cell population is increased by at least 0.1-fold to at least 3.5-fold relative to an engineered T cell population not cultured in the presence of insulin, an insulin analog, an insulin agonist, or an insulin partial agonist. In an embodiment, the gene editing efficiency of the engineered T cell population is increased by at least 0.1-fold to at least 3.0-fold relative to an engineered T cell population not cultured in the presence of insulin, an insulin analog, an insulin agonist, or an insulin partial agonist. In an embodiment, the gene editing efficiency of the engineered T cell population is increased by at least 0.1-fold to at least 2.5-fold relative to an engineered T cell population not cultured in the presence of insulin, an insulin analog, an insulin agonist, or an insulin partial agonist. In an embodiment, the gene editing efficiency of the engineered T cell population is increased by at least 0.1-fold to at least 2.0-fold relative to an engineered T cell population not cultured in the presence of insulin, an insulin analog, an insulin agonist, or an insulin partial agonist. In an embodiment, the gene editing efficiency of the engineered T cell population is increased by at least 0.1-fold to at least 1.5-fold relative to an engineered T cell population not cultured in the presence of insulin, an insulin analog, an insulin agonist, or an insulin partial agonist. In an embodiment, the gene editing efficiency of the engineered T cell population is increased by at least 0.1-fold to at least 1.0-fold relative to an engineered T cell population not cultured in the presence of insulin, an insulin analog, an insulin agonist, or an insulin partial agonist. In an embodiment, the gene editing efficiency of the engineered T cell population is increased by at least 0.1-fold to at least 0.9-fold relative to an engineered T cell population not cultured in the presence of insulin, an insulin analog, an insulin agonist, or an insulin partial agonist. In an embodiment, the gene editing efficiency of the engineered T cell population is increased by at least 0.1-fold to at least 0.8-fold relative to an engineered T cell population not cultured in the presence of insulin, an insulin analog, an insulin agonist, or an insulin partial agonist. In an embodiment, the gene editing efficiency of the engineered T cell population is increased by at least 0.1-fold to at least 0.7-fold relative to an engineered T cell population not cultured in the presence of insulin, an insulin analog, an insulin agonist, or an insulin partial agonist.In an embodiment, the gene editing efficiency of the engineered T cell population is increased by at least 0.1-fold to at least 0.6-fold relative to an engineered T cell population not cultured in the presence of insulin, an insulin analog, an insulin agonist, or an insulin partial agonist. In an embodiment, the gene editing efficiency of the engineered T cell population is increased by at least 0.1-fold to at least 0.5-fold relative to an engineered T cell population not cultured in the presence of insulin, an insulin analog, an insulin agonist, or an insulin partial agonist. In an embodiment, the gene editing efficiency of the engineered T cell population is increased by at least 0.1-fold to at least 0.4-fold relative to an engineered T cell population not cultured in the presence of insulin, an insulin analog, an insulin agonist, or an insulin partial agonist. In an embodiment, the gene editing efficiency of the engineered T cell population is increased by at least 0.1-fold to at least 0.3-fold relative to an engineered T cell population not cultured in the presence of insulin, an insulin analog, an insulin agonist, or an insulin partial agonist. In an embodiment, the gene editing efficiency of the engineered T cell population is increased by at least 0.1-fold to at least 0.2-fold relative to an engineered T cell population not cultured in the presence of insulin, an insulin analog, an insulin agonist, or an insulin partial agonist. The fold increase can be any value or sub-range within the recited range including the endpoints.

[0167] In an embodiment, the gene editing efficiency of the engineered T cell population is increased by about 2-fold to about 3-fold. In an embodiment, the gene editing efficiency of the engineered T cell population is increased 2-fold to 3-fold. The fold increase can be any value or sub-range within the recited range including the endpoints.

[0168] In embodiments, the gene editing efficiency of the engineered T cell population is from about 1% to about 99%. In embodiments, the gene editing efficiency of the engineered T cell population is from about 5% to about 99%. In embodiments, the gene editing efficiency of the engineered T cell population is from about 10% to about 99%. In embodiments, the gene editing efficiency of the engineered T cell population is from about 20% to about 99%. In embodiments, the gene editing efficiency of the engineered T cell population is from about 30% to about 99%. In embodiments, the gene editing efficiency of the engineered T cell population is from about 40% to about 99%. In embodiments, the gene editing efficiency of the engineered T cell population is from about 50% to about 99%. In embodiments, the gene editing efficiency of the engineered T cell population is from about 60% to about 99%. In embodiments, the gene editing efficiency of the engineered T cell population is from about 70% to about 99%. In embodiments, the gene editing efficiency of the engineered T cell population is from about 80% to about 99%. In embodiments, the gene editing efficiency of the engineered T cell population is from about 90% to about 99%. In embodiments, the gene editing efficiency of the engineered T cell population is from about 91% to about 99%. In embodiments, the gene editing efficiency of the engineered T cell population is from about 92% to about 99%. In embodiments, the gene editing efficiency of the engineered T cell population is from about 93% to about 99%. In embodiments, the gene editing efficiency of the engineered T cell population is from about 94% to about 99%. In embodiments, the gene editing efficiency of the engineered T cell population is from about 95% to about 99%. In embodiments, the gene editing efficiency of the engineered T cell population is from about 96% to about 99%. In embodiments, the gene editing efficiency of the engineered T cell population is from about 97% to about 99%. In embodiments, the gene editing efficiency of the engineered T cell population is from about 98% to about 99%.

[0169] In embodiments, the gene editing efficiency of the engineered T cell population is from about 1% to about 98%. In embodiments, the gene editing efficiency of the engineered T cell population is from about 1% to about 97%. In embodiments, the gene editing efficiency of the engineered T cell population is from about 1% to about 96%. In embodiments, the gene editing efficiency of the engineered T cell population is from about 1% to about 95%. In embodiments, the gene editing efficiency of the engineered T cell population is from about 1% to about 94%. In embodiments, the gene editing efficiency of the engineered T cell population is from about 1% to about 93%. In embodiments, the gene editing efficiency of the engineered T cell population is from about 1% to about 92%. In embodiments, the gene editing efficiency of the engineered T cell population is from about 1% to about 91%. In embodiments, the gene editing efficiency of the engineered T cell population is from about 1% to about 90%. In embodiments, the gene editing efficiency of the engineered T cell population is from about 1% to about 80%. In embodiments, the gene editing efficiency of the engineered T cell population is from about 1% to about 70%. In embodiments, the gene editing efficiency of the engineered T cell population is from about 1% to about 60%. In embodiments, the gene editing efficiency of the engineered T cell population is from about 1% to about 50%. In embodiments, the gene editing efficiency of the engineered T cell population is from about 1% to about 40%. In embodiments, the gene editing efficiency of the engineered T cell population is from about 1% to about 30%. In embodiments, the gene editing efficiency of the engineered T cell population is from about 1% to about 20%. In embodiments, the gene editing efficiency of the engineered T cell population is from about 1% to about 10%. In embodiments, the gene editing efficiency of the engineered T cell population is from about 1% to about 5%.

[0170] In embodiments, the gene editing efficiency of the engineered T cell population is from 1% to 99%. In embodiments, the gene editing efficiency of the engineered T cell population is from 5% to 99%. In embodiments, the gene editing efficiency of the engineered T cell population is from 10% to 99%. In embodiments, the gene editing efficiency of the engineered T cell population is from 20% to 99%. In embodiments, the gene editing efficiency of the engineered T cell population is from 30% to 99%. In embodiments, the gene editing efficiency of the engineered T cell population is from 40% to 99%. In embodiments, the gene editing efficiency of the engineered T cell population is from 50% to 99%. In embodiments, the gene editing efficiency of the engineered T cell population is from 60% to 99%. In embodiments, the gene editing efficiency of the engineered T cell population is from 70% to 99%. In embodiments, the gene editing efficiency of the engineered T cell population is from 80% to 99%. In embodiments, the gene editing efficiency of the engineered T cell population is from 90% to 99%. In embodiments, the gene editing efficiency of the engineered T cell population is from 91% to 99%. In embodiments, the gene editing efficiency of the engineered T cell population is from 92% to 99%. In embodiments, the gene editing efficiency of the engineered T cell population is from 93% to 99%. In embodiments, the gene editing efficiency of the engineered T cell population is from 94% to 99%. In embodiments, the gene editing efficiency of the engineered T cell population is from 95% to 99%. In embodiments, the gene editing efficiency of the engineered T cell population is from 96% to 99%. In embodiments, the gene editing efficiency of the engineered T cell population is from 97% to 99%. In embodiments, the gene editing efficiency of the engineered T cell population is from 98% to 99%.

[0171] In an embodiment, the gene editing efficiency of the engineered T cell population is 1% to 98%. In an embodiment, the gene editing efficiency of the engineered T cell population is 1% to 97%. In an embodiment, the gene editing efficiency of the engineered T cell population is 1% to 96%. In an embodiment, the gene editing efficiency of the engineered T cell population is 1% to 95%. In an embodiment, the gene editing efficiency of the engineered T cell population is 1% to 94%. In an embodiment, the gene editing efficiency of the engineered T cell population is 1% to 93%. In an embodiment, the gene editing efficiency of the engineered T cell population is 1% to 92%. In an embodiment, the gene editing efficiency of the engineered T cell population is 1% to 91%. In an embodiment, the gene editing efficiency of the engineered T cell population is 1% to 90%. In an embodiment, the gene editing efficiency of the engineered T cell population is 1% to 80%. In an embodiment, the gene editing efficiency of the engineered T cell population is 1% to 70%. In an embodiment, the gene editing efficiency of the engineered T cell population is 1% to 60%. In an embodiment, the gene editing efficiency of the engineered T cell population is 1% to 50%. In an embodiment, the gene editing efficiency of the engineered T cell population is 1% to 40%. In an embodiment, the gene editing efficiency of the engineered T cell population is 1% to 30%. In an embodiment, the gene editing efficiency of the engineered T cell population is 1% to 20%. In an embodiment, the gene editing efficiency of the engineered T cell population is 1% to 10%. In an embodiment, the gene editing efficiency of the engineered T cell population is 1% to 5%. The percentage increase can be any value or sub-range within the recited range including the endpoints.

[0172] 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 75% 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 85% 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 91% 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 93% 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 95% 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 97% to about 99%. In an embodiment, the knockout efficiency of the engineered T cell population is about 98% to about 99%.

[0173] In embodiments, the knockout efficiency of the engineered T cell population is from about 70% to about 98%. In embodiments, the knockout efficiency of the engineered T cell population is from about 70% to about 97%. In embodiments, the knockout efficiency of the engineered T cell population is from about 70% to about 96%. In embodiments, the knockout efficiency of the engineered T cell population is from about 70% to about 95%. In embodiments, the knockout efficiency of the engineered T cell population is from about 70% to about 94%. In embodiments, the knockout efficiency of the engineered T cell population is from about 70% to about 93%. In embodiments, the knockout efficiency of the engineered T cell population is from about 70% to about 92%. In embodiments, the knockout efficiency of the engineered T cell population is from about 70% to about 91%. In embodiments, the knockout efficiency of the engineered T cell population is from about 70% to about 90%. In embodiments, the knockout efficiency of the engineered T cell population is from about 70% to about 85%. In embodiments, the knockout efficiency of the engineered T cell population is from about 70% to about 80%. In embodiments, the knockout efficiency of the engineered T cell population is from about 70% to about 75%.

[0174] In embodiments, the knockout efficiency of the engineered T cell population is from 70% to 99%. In embodiments, the knockout efficiency of the engineered T cell population is from 75% to 99%. In embodiments, the knockout efficiency of the engineered T cell population is from 80% to 99%. In embodiments, the knockout efficiency of the engineered T cell population is from 85% to 99%. In embodiments, the knockout efficiency of the engineered T cell population is from 90% to 99%. In embodiments, the knockout efficiency of the engineered T cell population is from 91% to 99%. In embodiments, the knockout efficiency of the engineered T cell population is from 92% to 99%. In embodiments, the knockout efficiency of the engineered T cell population is from 93% to 99%. In embodiments, the knockout efficiency of the engineered T cell population is from 94% to 99%. In embodiments, the knockout efficiency of the engineered T cell population is from 95% to 99%. In embodiments, the knockout efficiency of the engineered T cell population is from 96% to 99%. In embodiments, the knockout efficiency of the engineered T cell population is from 97% to 99%. In embodiments, the knockout efficiency of the engineered T cell population is from 98% to 99%.

[0175] In embodiments, the knockout efficiency of the engineered T cell population is from 70% to 98%. In embodiments, the knockout efficiency of the engineered T cell population is from 70% to 97%. In embodiments, the knockout efficiency of the engineered T cell population is from 70% to 96%. In embodiments, the knockout efficiency of the engineered T cell population is from 70% to 95%. In embodiments, the knockout efficiency of the engineered T cell population is from 70% to 94%. In embodiments, the knockout efficiency of the engineered T cell population is from 70% to 93%. In embodiments, the knockout efficiency of the engineered T cell population is from 70% to 92%. In embodiments, the knockout efficiency of the engineered T cell population is from 70% to 91%. In embodiments, the knockout efficiency of the engineered T cell population is from 70% to 90%. In embodiments, the knockout efficiency of the engineered T cell population is from 70% to 85%. In embodiments, the knockout efficiency of the engineered T cell population is from 70% to 80%. In embodiments, the knockout efficiency of the engineered T cell population is from 70% to 75%. The percentage knockout efficiency can be any value or sub-range within the recited ranges including the endpoints. For the methods provided herein, in embodiments, the knockout efficiency is about 90%. In embodiments, the knockout efficiency is 90%.

[0176] In embodiments, the knock-in efficiency of the engineered T cell population is from about 1% to about 99%. In embodiments, the knock-in efficiency of the engineered T cell population is from about 5% to about 99%. In embodiments, the knock-in efficiency of the engineered T cell population is from about 10% to about 99%. In embodiments, the knock-in efficiency of the engineered T cell population is from about 20% to about 99%. In embodiments, the knock-in efficiency of the engineered T cell population is from about 30% to about 99%. In embodiments, the knock-in efficiency of the engineered T cell population is from about 40% to about 99%. In embodiments, the knock-in efficiency of the engineered T cell population is from about 50% to about 99%. In embodiments, the knock-in efficiency of the engineered T cell population is from about 60% to about 99%. In embodiments, the knock-in efficiency of the engineered T cell population is from about 70% to about 99%. In embodiments, the knock-in efficiency of the engineered T cell population is from about 80% to about 99%. In embodiments, the knock-in efficiency of the engineered T cell population is from about 90% to about 99%. In embodiments, the knock-in efficiency of the engineered T cell population is from about 91% to about 99%. In embodiments, the knock-in efficiency of the engineered T cell population is from about 92% to about 99%. In embodiments, the knock-in efficiency of the engineered T cell population is from about 93% to about 99%. In embodiments, the knock-in efficiency of the engineered T cell population is from about 94% to about 99%. In embodiments, the knock-in efficiency of the engineered T cell population is from about 95% to about 99%. In embodiments, the knock-in efficiency of the engineered T cell population is from about 96% to about 99%. In embodiments, the knock-in efficiency of the engineered T cell population is from about 97% to about 99%. In embodiments, the knock-in efficiency of the engineered T cell population is from about 98% to about 99%.

[0177] In embodiments, the knock-in efficiency of the engineered T cell population is from about 1% to about 98%. In embodiments, the knock-in efficiency of the engineered T cell population is from about 1% to about 97%. In embodiments, the knock-in efficiency of the engineered T cell population is from about 1% to about 96%. In embodiments, the knock-in efficiency of the engineered T cell population is from about 1% to about 95%. In embodiments, the knock-in efficiency of the engineered T cell population is from about 1% to about 94%. In embodiments, the knock-in efficiency of the engineered T cell population is from about 1% to about 93%. In embodiments, the knock-in efficiency of the engineered T cell population is from about 1% to about 92%. In embodiments, the knock-in efficiency of the engineered T cell population is from about 1% to about 91%. In embodiments, the knock-in efficiency of the engineered T cell population is from about 1% to about 90%. In embodiments, the knock-in efficiency of the engineered T cell population is from about 1% to about 80%. In embodiments, the knock-in efficiency of the engineered T cell population is from about 1% to about 70%. In embodiments, the knock-in efficiency of the engineered T cell population is from about 1% to about 60%. In embodiments, the knock-in efficiency of the engineered T cell population is from about 1% to about 50%. In embodiments, the knock-in efficiency of the engineered T cell population is from about 1% to about 40%. In embodiments, the knock-in efficiency of the engineered T cell population is from about 1% to about 30%. In embodiments, the knock-in efficiency of the engineered T cell population is from about 1% to about 20%. In embodiments, the knock-in efficiency of the engineered T cell population is from about 1% to about 10%. In embodiments, the knock-in efficiency of the engineered T cell population is from about 1% to about 5%.

[0178] In an embodiment, the knock-in efficiency of the engineered T cell population is from 1% to 99%. In an embodiment, the knock-in efficiency of the engineered T cell population is from 5% to 99%. In an embodiment, the knock-in efficiency of the engineered T cell population is from 10% to 99%. In an embodiment, the knock-in efficiency of the engineered T cell population is from 20% to 99%. In an embodiment, the knock-in efficiency of the engineered T cell population is from 30% to 99%. In an embodiment, the knock-in efficiency of the engineered T cell population is from 40% to 99%. In an embodiment, the knock-in efficiency of the engineered T cell population is from 50% to 99%. In an embodiment, the knock-in efficiency of the engineered T cell population is from 60% to 99%. In an embodiment, the knock-in efficiency of the engineered T cell population is from 70% to 99%. In an embodiment, the knock-in efficiency of the engineered T cell population is from 80% to 99%. In an embodiment, the knock-in efficiency of the engineered T cell population is from 90% to 99%. In an embodiment, the knock-in efficiency of the engineered T cell population is from 91% to 99%. In an embodiment, the knock-in efficiency of the engineered T cell population is from 92% to 99%. In an embodiment, the knock-in efficiency of the engineered T cell population is from 93% to 99%. In an embodiment, the knock-in efficiency of the engineered T cell population is from 94% to 99%. In an embodiment, the knock-in efficiency of the engineered T cell population is from 95% to 99%. In an embodiment, the knock-in efficiency of the engineered T cell population is from 96% to 99%. In an embodiment, the knock-in efficiency of the engineered T cell population is from 97% to 99%. In an embodiment, the knock-in efficiency of the engineered T cell population is from 98% to 99%.

[0179] In embodiments, the knock-in efficiency of the engineered T cell population is from 1% to 98%. In embodiments, the knock-in efficiency of the engineered T cell population is from 1% to 97%. In embodiments, the knock-in efficiency of the engineered T cell population is from 1% to 96%. In embodiments, the knock-in efficiency of the engineered T cell population is from 1% to 95%. In embodiments, the knock-in efficiency of the engineered T cell population is from 1% to 94%. In embodiments, the knock-in efficiency of the engineered T cell population is from 1% to 93%. In embodiments, the knock-in efficiency of the engineered T cell population is from 1% to 92%. In embodiments, the knock-in efficiency of the engineered T cell population is from 1% to 91%. In embodiments, the knock-in efficiency of the engineered T cell population is from 1% to 90%. In embodiments, the knock-in efficiency of the engineered T cell population is from 1% to 80%. In embodiments, the knock-in efficiency of the engineered T cell population is from 1% to 70%. In embodiments, the knock-in efficiency of the engineered T cell population is from 1% to 60%. In embodiments, the knock-in efficiency of the engineered T cell population is from 1% to 50%. In embodiments, the knock-in efficiency of the engineered T cell population is from 1% to 40%. In embodiments, the knock-in efficiency of the engineered T cell population is from 1% to 30%. In embodiments, the knock-in efficiency of the engineered T cell population is from 1% to 20%. In embodiments, the knock-in efficiency of the engineered T cell population is from 1% to 10%. In embodiments, the knock-in efficiency of the engineered T cell population is from 1% to 5%. The percent knock-in efficiency can be any value or sub-range within the recited range including the endpoints. For the methods provided herein, in embodiments, the knock-in efficiency is about 60%. In embodiments, the knock-in efficiency is 60%.

[0180] Provided herein are methods for increasing the expansion of an engineered T cell population, comprising: (i) contacting a T cell population with insulin, an insulin analog, an insulin agonist, an insulin partial agonist, and a polynucleotide to form the engineered T cell population, and (ii) expanding the engineered T cell population to form an expanded engineered T cell population, wherein the insulin, insulin analog, insulin agonist, and / or insulin partial agonist increases the expanded engineered T cell population relative to an engineered T cell population not contacted with insulin, an insulin analog, an insulin agonist, and / or an insulin partial agonist.

[0181] In an embodiment, contacting a T cell population with a polynucleotide comprises transfecting the T cell population with the polynucleotide. In an embodiment, the polynucleotide is donor DNA. In an embodiment, the polynucleotide comprises: single-stranded DNA, double-stranded DNA, linear DNA strand, plasmid, nanoplasmid or minicircle. In an embodiment, the polynucleotide comprises single-stranded DNA. In an embodiment, the polynucleotide comprises double-stranded DNA. In an embodiment, the polynucleotide comprises a linear DNA strand. In an embodiment, the polynucleotide comprises a plasmid. In an embodiment, the polynucleotide comprises a nanoplasmid. In an embodiment, the polynucleotide comprises a minicircle.

[0182] In an embodiment, 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 transfecting the T cell population with the gene editing reagent or a polynucleotide encoding the gene editing reagent. In an embodiment, contacting the T cell population with a gene editing reagent comprises transfecting the T cell population with the gene editing reagent. In an embodiment, contacting the T cell population with a gene editing reagent comprises transfecting the T cell population with a polynucleotide encoding the gene editing reagent. In an embodiment, the T cell population is transfected simultaneously with the polynucleotide and the gene editing reagent or a polynucleotide encoding the gene editing reagent. In an embodiment, the T cell population is transfected simultaneously with the polynucleotide and the gene editing reagent. In an embodiment, the T cell population is transfected simultaneously with the polynucleotide and a polynucleotide encoding the gene editing reagent.

[0183] In an embodiment, a T cell population is contacted with insulin, an insulin analogue, an insulin agonist, and / or an insulin partial agonist at about 1 μg / ml to about 50 μg / ml. In an embodiment, a T cell population is contacted with insulin, an insulin analogue, an insulin agonist, and / or an insulin partial agonist at about 2 μg / ml to about 50 μg / ml. In an embodiment, a T cell population is contacted with insulin, an insulin analogue, an insulin agonist, and / or an insulin partial agonist at about 3 μg / ml to about 50 μg / ml. In an embodiment, a T cell population is contacted with insulin, an insulin analogue, an insulin agonist, and / or an insulin partial agonist at about 4 μg / ml to about 50 μg / ml. In an embodiment, a T cell population is contacted with insulin, an insulin analogue, an insulin agonist, and / or an insulin partial agonist at about 5 μg / ml to about 50 μg / ml. In an embodiment, a T cell population is contacted with insulin, an insulin analogue, an insulin agonist, and / or an insulin partial agonist at about 6 μg / ml to about 50 μg / ml. In an embodiment, a T cell population is contacted with insulin, an insulin analogue, an insulin agonist, and / or an insulin partial agonist at about 7 μg / ml to about 50 μg / ml. In an embodiment, a T cell population is contacted with insulin, an insulin analogue, an insulin agonist, and / or an insulin partial agonist at about 8 μg / ml to about 50 μg / ml. In an embodiment, a T cell population is contacted with insulin, an insulin analogue, an insulin agonist, and / or an insulin partial agonist at about 9 μg / ml to about 50 μg / ml. In an embodiment, a T cell population is contacted with insulin, an insulin analogue, an insulin agonist, and / or an insulin partial agonist at about 10 μg / ml to about 50 μg / ml. In an embodiment, a T cell population is contacted with insulin, an insulin analogue, an insulin agonist, and / or an insulin partial agonist at about 15 μg / ml to about 50 μg / ml. In an embodiment, a T cell population is contacted with insulin, an insulin analogue, an insulin agonist, and / or an insulin partial agonist at about 20 μg / ml to about 50 μg / ml. In an embodiment, a T cell population is contacted with insulin, an insulin analogue, an insulin agonist, and / or an insulin partial agonist at about 25 μg / ml to about 50 μg / ml. In an embodiment, a T cell population is contacted with insulin, an insulin analogue, an insulin agonist, and / or an insulin partial agonist at about 30 μg / ml to about 50 μg / ml. In an embodiment, a T cell population is contacted with insulin, an insulin analogue, an insulin agonist, and / or an insulin partial agonist at about 35 μg / ml to about 50 μg / ml. In an embodiment, a T cell population is contacted with insulin, an insulin analogue, an insulin agonist, and / or an insulin partial agonist at about 40 μg / ml to about 50 μg / ml.In an embodiment, a T cell population is contacted with insulin, an insulin analog, an insulin agonist, and / or an insulin partial agonist at about 45 μg / ml to about 50 μg / ml.

[0184] In an embodiment, a T cell population is contacted with insulin, an insulin analog, an insulin agonist, and / or an insulin partial agonist at about 1 μg / ml to about 45 μg / ml. In an embodiment, a T cell population is contacted with insulin, an insulin analog, an insulin agonist, and / or an insulin partial agonist at about 1 μg / ml to about 40 μg / ml. In an embodiment, a T cell population is contacted with insulin, an insulin analog, an insulin agonist, and / or an insulin partial agonist at about 1 μg / ml to about 35 μg / ml. In an embodiment, a T cell population is contacted with insulin, an insulin analog, an insulin agonist, and / or an insulin partial agonist at about 1 μg / ml to about 30 μg / ml. In an embodiment, a T cell population is contacted with insulin, an insulin analog, an insulin agonist, and / or an insulin partial agonist at about 1 μg / ml to about 25 μg / ml. In an embodiment, a T cell population is contacted with insulin, an insulin analog, an insulin agonist, and / or an insulin partial agonist at about 1 μg / ml to about 20 μg / ml. In an embodiment, a T cell population is contacted with insulin, an insulin analog, an insulin agonist, and / or an insulin partial agonist at about 1 μg / ml to about 15 μg / ml. In an embodiment, a T cell population is contacted with insulin, an insulin analog, an insulin agonist, and / or an insulin partial agonist at about 1 μg / ml to about 10 μg / ml. In an embodiment, a T cell population is contacted with insulin, an insulin analog, an insulin agonist, and / or an insulin partial agonist at about 1 μg / ml to about 9 μg / ml. In an embodiment, a T cell population is contacted with insulin, an insulin analog, an insulin agonist, and / or an insulin partial agonist at about 1 μg / ml to about 8 μg / ml. In an embodiment, a T cell population is contacted with insulin, an insulin analog, an insulin agonist, and / or an insulin partial agonist at about 1 μg / ml to about 7 μg / ml. In an embodiment, a T cell population is contacted with insulin, an insulin analog, an insulin agonist, and / or an insulin partial agonist at about 1 μg / ml to about 6 μg / ml. In an embodiment, a T cell population is contacted with insulin, an insulin analog, an insulin agonist, and / or an insulin partial agonist at about 1 μg / ml to about 5 μg / ml. In an embodiment, a T cell population is contacted with insulin, an insulin analog, an insulin agonist, and / or an insulin partial agonist at about 1 μg / ml to about 4 μg / ml. In an embodiment, a T cell population is contacted with insulin, an insulin analog, an insulin agonist, and / or an insulin partial agonist at about 1 μg / ml to about 3 μg / ml. In an embodiment, a T cell population is contacted with insulin, an insulin analog, an insulin agonist, and / or an insulin partial agonist at about 1 μg / ml to about 2 μg / ml.

[0185] In an embodiment, a T cell population is contacted with insulin, an insulin analog, an insulin agonist, and / or an insulin partial agonist at 1 μg / ml to 50 μg / ml. In an embodiment, a T cell population is contacted with insulin, an insulin analog, an insulin agonist, and / or an insulin partial agonist at 2 μg / ml to 50 μg / ml. In an embodiment, a T cell population is contacted with insulin, an insulin analog, an insulin agonist, and / or an insulin partial agonist at 3 μg / ml to 50 μg / ml. In an embodiment, a T cell population is contacted with insulin, an insulin analog, an insulin agonist, and / or an insulin partial agonist at 4 μg / ml to 50 μg / ml. In an embodiment, a T cell population is contacted with insulin, an insulin analog, an insulin agonist, and / or an insulin partial agonist at 5 μg / ml to 50 μg / ml. In an embodiment, a T cell population is contacted with insulin, an insulin analog, an insulin agonist, and / or an insulin partial agonist at 6 μg / ml to 50 μg / ml. In an embodiment, a T cell population is contacted with insulin, an insulin analog, an insulin agonist, and / or an insulin partial agonist at 7 μg / ml to 50 μg / ml. In an embodiment, a T cell population is contacted with insulin, an insulin analog, an insulin agonist, and / or an insulin partial agonist at 8 μg / ml to 50 μg / ml. In an embodiment, a T cell population is contacted with insulin, an insulin analog, an insulin agonist, and / or an insulin partial agonist at 9 μg / ml to 50 μg / ml. In an embodiment, a T cell population is contacted with insulin, an insulin analog, an insulin agonist, and / or an insulin partial agonist at 10 μg / ml to 50 μg / ml. In an embodiment, a T cell population is contacted with insulin, an insulin analog, an insulin agonist, and / or an insulin partial agonist at 15 μg / ml to 50 μg / ml. In an embodiment, a T cell population is contacted with insulin, an insulin analog, an insulin agonist, and / or an insulin partial agonist at 20 μg / ml to 50 μg / ml. In an embodiment, a T cell population is contacted with insulin, an insulin analog, an insulin agonist, and / or an insulin partial agonist at 25 μg / ml to 50 μg / ml. In an embodiment, a T cell population is contacted with insulin, an insulin analog, an insulin agonist, and / or an insulin partial agonist at 30 μg / ml to 50 μg / ml. In an embodiment, a T cell population is contacted with insulin, an insulin analog, an insulin agonist, and / or an insulin partial agonist at 35 μg / ml to 50 μg / ml. In an embodiment, a T cell population is contacted with insulin, an insulin analog, an insulin agonist, and / or an insulin partial agonist at 40 μg / ml to 50 μg / ml.In an embodiment, a T cell population is contacted with insulin, an insulin analogue, an insulin agonist, and / or an insulin partial agonist at 45 μg / ml to 50 μg / ml.

[0186] In embodiments, a T cell population is contacted with insulin, an insulin analog, an insulin agonist, and / or an insulin partial agonist at 1 μg / ml to 45 μg / ml. In embodiments, a T cell population is contacted with insulin, an insulin analog, an insulin agonist, and / or an insulin partial agonist at 1 μg / ml to 40 μg / ml. In embodiments, a T cell population is contacted with insulin, an insulin analog, an insulin agonist, and / or an insulin partial agonist at 1 μg / ml to 35 μg / ml. In embodiments, a T cell population is contacted with insulin, an insulin analog, an insulin agonist, and / or an insulin partial agonist at 1 μg / ml to 30 μg / ml. In embodiments, a T cell population is contacted with insulin, an insulin analog, an insulin agonist, and / or an insulin partial agonist at 1 μg / ml to 25 μg / ml. In embodiments, a T cell population is contacted with insulin, an insulin analog, an insulin agonist, and / or an insulin partial agonist at 1 μg / ml to 20 μg / ml. In embodiments, a T cell population is contacted with insulin, an insulin analog, an insulin agonist, and / or an insulin partial agonist at 1 μg / ml to 15 μg / ml. In embodiments, a T cell population is contacted with insulin, an insulin analog, an insulin agonist, and / or an insulin partial agonist at 1 μg / ml to 10 μg / ml. In embodiments, a T cell population is contacted with insulin, an insulin analog, an insulin agonist, and / or an insulin partial agonist at 1 μg / ml to 9 μg / ml. In embodiments, a T cell population is contacted with insulin, an insulin analog, an insulin agonist, and / or an insulin partial agonist at 1 μg / ml to 8 μg / ml. In embodiments, a T cell population is contacted with insulin, an insulin analog, an insulin agonist, and / or an insulin partial agonist at 1 μg / ml to 7 μg / ml. In embodiments, a T cell population is contacted with insulin, an insulin analog, an insulin agonist, and / or an insulin partial agonist at 1 μg / ml to 6 μg / ml. In embodiments, a T cell population is contacted with insulin, an insulin analog, an insulin agonist, and / or an insulin partial agonist at 1 μg / ml to 5 μg / ml. In embodiments, a T cell population is contacted with insulin, an insulin analog, an insulin agonist, and / or an insulin partial agonist at 1 μg / ml to 4 μg / ml. In embodiments, a T cell population is contacted with insulin, an insulin analog, an insulin agonist, and / or an insulin partial agonist at 1 μg / ml to 3 μg / ml. In embodiments, a T cell population is contacted with insulin, an insulin analog, an insulin agonist, and / or an insulin partial agonist at 1 μg / ml to 2 μg / ml. The concentration can be any value or sub-range within the recited range including the endpoints.

[0187] In embodiments, a T cell population is contacted with a polynucleotide and insulin, an insulin analogue, an insulin agonist, and / or an insulin partial agonist simultaneously. In embodiments, a T cell population is contacted with a polynucleotide and insulin, an insulin analogue, an insulin agonist, and / or an insulin partial agonist sequentially. In embodiments, a T cell population is contacted with insulin, an insulin analogue, an insulin agonist, and / or an insulin partial agonist before being contacted with the polynucleotide. In embodiments, a T cell population is contacted with insulin, an insulin analogue, an insulin agonist, and / or an insulin partial agonist after being contacted with the polynucleotide. In embodiments, a T cell population is contacted with insulin, an insulin analogue, an insulin agonist, and / or an insulin partial agonist before and after being contacted with the polynucleotide.

[0188] In an embodiment, the amplified engineered T cell population is increased by at least about 0.1-fold to at least about 5.0-fold relative to an engineered T cell population that has not been contacted with insulin, an insulin analogue, an insulin agonist, and / or an insulin partial agonist. In an embodiment, the amplified engineered T cell population is increased by at least about 0.2-fold to at least about 5.0-fold relative to an engineered T cell population that has not been contacted with insulin, an insulin analogue, an insulin agonist, and / or an insulin partial agonist. In an embodiment, the amplified engineered T cell population is increased by at least about 0.3-fold to at least about 5.0-fold relative to an engineered T cell population that has not been contacted with insulin, an insulin analogue, an insulin agonist, and / or an insulin partial agonist. In an embodiment, the amplified engineered T cell population is increased by at least about 0.4-fold to at least about 5.0-fold relative to an engineered T cell population that has not been contacted with insulin, an insulin analogue, an insulin agonist, and / or an insulin partial agonist. In an embodiment, the amplified engineered T cell population is increased by at least about 0.5-fold to at least about 5.0-fold relative to an engineered T cell population that has not been contacted with insulin, an insulin analogue, an insulin agonist, and / or an insulin partial agonist. In an embodiment, the amplified engineered T cell population is increased by at least about 0.6-fold to at least about 5.0-fold relative to an engineered T cell population that has not been contacted with insulin, an insulin analogue, an insulin agonist, and / or an insulin partial agonist. In an embodiment, the amplified engineered T cell population is increased by at least about 0.7-fold to at least about 5.0-fold relative to an engineered T cell population that has not been contacted with insulin, an insulin analogue, an insulin agonist, and / or an insulin partial agonist. In an embodiment, the amplified engineered T cell population is increased by at least about 0.8-fold to at least about 5.0-fold relative to an engineered T cell population that has not been contacted with insulin, an insulin analogue, an insulin agonist, and / or an insulin partial agonist. In an embodiment, the amplified engineered T cell population is increased by at least about 0.9-fold to at least about 5.0-fold relative to an engineered T cell population that has not been contacted with insulin, an insulin analogue, an insulin agonist, and / or an insulin partial agonist. In an embodiment, the amplified engineered T cell population is increased by at least about 1.0-fold to at least about 5.0-fold relative to an engineered T cell population that has not been contacted with insulin, an insulin analogue, an insulin agonist, and / or an insulin partial agonist. In an embodiment, the amplified engineered T cell population is increased by at least about 1.5-fold to at least about 5.0-fold relative to an engineered T cell population that has not been contacted with insulin, an insulin analogue, an insulin agonist, and / or an insulin partial agonist. In an embodiment, the amplified engineered T cell population is increased by at least about 2.0-fold to at least about 5.0-fold relative to an engineered T cell population that has not been contacted with insulin, an insulin analogue, an insulin agonist, and / or an insulin partial agonist.In an embodiment, the expanded engineered T cell population is increased by at least about 2.5-fold to at least about 5.0-fold relative to an engineered T cell population that has not been contacted with insulin, an insulin analog, an insulin agonist, and / or an insulin partial agonist. In an embodiment, the expanded engineered T cell population is increased by at least about 3.0-fold to at least about 5.0-fold relative to an engineered T cell population that has not been contacted with insulin, an insulin analog, an insulin agonist, and / or an insulin partial agonist. In an embodiment, the expanded engineered T cell population is increased by at least about 3.5-fold to at least about 5.0-fold relative to an engineered T cell population that has not been contacted with insulin, an insulin analog, an insulin agonist, and / or an insulin partial agonist. In an embodiment, the expanded engineered T cell population is increased by at least about 4.0-fold to at least about 5.0-fold relative to an engineered T cell population that has not been contacted with insulin, an insulin analog, an insulin agonist, and / or an insulin partial agonist. In an embodiment, the expanded engineered T cell population is increased by at least about 4.5-fold to at least about 5.0-fold relative to an engineered T cell population that has not been contacted with insulin, an insulin analog, an insulin agonist, and / or an insulin partial agonist.

[0189] In an embodiment, the expanded engineered T cell population is increased by at least about 0.1-fold to at least about 4.5-fold relative to an engineered T cell population that has not been contacted with insulin, an insulin analogue, an insulin agonist, and / or an insulin partial agonist. In an embodiment, the expanded engineered T cell population is increased by at least about 0.1-fold to at least about 4.0-fold relative to an engineered T cell population that has not been contacted with insulin, an insulin analogue, an insulin agonist, and / or an insulin partial agonist. In an embodiment, the expanded engineered T cell population is increased by at least about 0.1-fold to at least about 3.5-fold relative to an engineered T cell population that has not been contacted with insulin, an insulin analogue, an insulin agonist, and / or an insulin partial agonist. In an embodiment, the expanded engineered T cell population is increased by at least about 0.1-fold to at least about 3.0-fold relative to an engineered T cell population that has not been contacted with insulin, an insulin analogue, an insulin agonist, and / or an insulin partial agonist. In an embodiment, the expanded engineered T cell population is increased by at least about 0.1-fold to at least about 2.5-fold relative to an engineered T cell population that has not been contacted with insulin, an insulin analogue, an insulin agonist, and / or an insulin partial agonist. In an embodiment, the expanded engineered T cell population is increased by at least about 0.1-fold to at least about 2.0-fold relative to an engineered T cell population that has not been contacted with insulin, an insulin analogue, an insulin agonist, and / or an insulin partial agonist. In an embodiment, the expanded engineered T cell population is increased by at least about 0.1-fold to at least about 1.5-fold relative to an engineered T cell population that has not been contacted with insulin, an insulin analogue, an insulin agonist, and / or an insulin partial agonist. In an embodiment, the expanded engineered T cell population is increased by at least about 0.1-fold to at least about 1.0-fold relative to an engineered T cell population that has not been contacted with insulin, an insulin analogue, an insulin agonist, and / or an insulin partial agonist. In an embodiment, the expanded engineered T cell population is increased by at least about 0.1-fold to at least about 0.9-fold relative to an engineered T cell population that has not been contacted with insulin, an insulin analogue, an insulin agonist, and / or an insulin partial agonist. In an embodiment, the expanded engineered T cell population is increased by at least about 0.1-fold to at least about 0.8-fold relative to an engineered T cell population that has not been contacted with insulin, an insulin analogue, an insulin agonist, and / or an insulin partial agonist. In an embodiment, the expanded engineered T cell population is increased by at least about 0.1-fold to at least about 0.7-fold relative to an engineered T cell population that has not been contacted with insulin, an insulin analogue, an insulin agonist, and / or an insulin partial agonist. In an embodiment, the expanded engineered T cell population is increased by at least about 0.1-fold to at least about 0.6-fold relative to an engineered T cell population that has not been contacted with insulin, an insulin analogue, an insulin agonist, and / or an insulin partial agonist.In an embodiment, the expanded engineered T cell population is increased by at least about 0.1-fold to at least about 0.5-fold relative to an engineered T cell population that has not been contacted with insulin, an insulin analog, an insulin agonist, and / or an insulin partial agonist. In an embodiment, the expanded engineered T cell population is increased by at least about 0.1-fold to at least about 0.4-fold relative to an engineered T cell population that has not been contacted with insulin, an insulin analog, an insulin agonist, and / or an insulin partial agonist. In an embodiment, the expanded engineered T cell population is increased by at least about 0.1-fold to at least about 0.3-fold relative to an engineered T cell population that has not been contacted with insulin, an insulin analog, an insulin agonist, and / or an insulin partial agonist. In an embodiment, the expanded engineered T cell population is increased by at least about 0.1-fold to at least about 0.2-fold relative to an engineered T cell population that has not been contacted with insulin, an insulin analog, an insulin agonist, and / or an insulin partial agonist.

[0190] In embodiments, the expanded engineered T cell population is increased by at least 0.1-fold to at least 5.0-fold relative to an engineered T cell population that has not been contacted with insulin, an insulin analogue, an insulin agonist, and / or an insulin partial agonist. In embodiments, the expanded engineered T cell population is increased by at least 0.2-fold to at least 5.0-fold relative to an engineered T cell population that has not been contacted with insulin, an insulin analogue, an insulin agonist, and / or an insulin partial agonist. In embodiments, the expanded engineered T cell population is increased by at least 0.3-fold to at least 5.0-fold relative to an engineered T cell population that has not been contacted with insulin, an insulin analogue, an insulin agonist, and / or an insulin partial agonist. In embodiments, the expanded engineered T cell population is increased by at least 0.4-fold to at least 5.0-fold relative to an engineered T cell population that has not been contacted with insulin, an insulin analogue, an insulin agonist, and / or an insulin partial agonist. In embodiments, the expanded engineered T cell population is increased by at least 0.5-fold to at least 5.0-fold relative to an engineered T cell population that has not been contacted with insulin, an insulin analogue, an insulin agonist, and / or an insulin partial agonist. In embodiments, the expanded engineered T cell population is increased by at least 0.6-fold to at least 5.0-fold relative to an engineered T cell population that has not been contacted with insulin, an insulin analogue, an insulin agonist, and / or an insulin partial agonist. In embodiments, the expanded engineered T cell population is increased by at least 0.7-fold to at least 5.0-fold relative to an engineered T cell population that has not been contacted with insulin, an insulin analogue, an insulin agonist, and / or an insulin partial agonist. In embodiments, the expanded engineered T cell population is increased by at least 0.8-fold to at least 5.0-fold relative to an engineered T cell population that has not been contacted with insulin, an insulin analogue, an insulin agonist, and / or an insulin partial agonist. In embodiments, the expanded engineered T cell population is increased by at least 0.9-fold to at least 5.0-fold relative to an engineered T cell population that has not been contacted with insulin, an insulin analogue, an insulin agonist, and / or an insulin partial agonist. In embodiments, the expanded engineered T cell population is increased by at least 1.0-fold to at least 5.0-fold relative to an engineered T cell population that has not been contacted with insulin, an insulin analogue, an insulin agonist, and / or an insulin partial agonist. In embodiments, the expanded engineered T cell population is increased by at least 1.5-fold to at least 5.0-fold relative to an engineered T cell population that has not been contacted with insulin, an insulin analogue, an insulin agonist, and / or an insulin partial agonist. In embodiments, the expanded engineered T cell population is increased by at least 2.0-fold to at least 5.0-fold relative to an engineered T cell population that has not been contacted with insulin, an insulin analogue, an insulin agonist, and / or an insulin partial agonist.In an embodiment, the expanded engineered T cell population is increased by at least 2.5-fold to at least 5.0-fold relative to an engineered T cell population that has not been contacted with insulin, an insulin analogue, an insulin agonist, and / or an insulin partial agonist. In an embodiment, the expanded engineered T cell population is increased by at least 3.0-fold to at least 5.0-fold relative to an engineered T cell population that has not been contacted with insulin, an insulin analogue, an insulin agonist, and / or an insulin partial agonist. In an embodiment, the expanded engineered T cell population is increased by at least 3.5-fold to at least 5.0-fold relative to an engineered T cell population that has not been contacted with insulin, an insulin analogue, an insulin agonist, and / or an insulin partial agonist. In an embodiment, the expanded engineered T cell population is increased by at least 4.0-fold to at least 5.0-fold relative to an engineered T cell population that has not been contacted with insulin, an insulin analogue, an insulin agonist, and / or an insulin partial agonist. In an embodiment, the expanded engineered T cell population is increased by at least 4.5-fold to at least 5.0-fold relative to an engineered T cell population that has not been contacted with insulin, an insulin analogue, an insulin agonist, and / or an insulin partial agonist.

[0191] In an embodiment, the expanded engineered T cell population is increased by at least 0.1-fold to at least 4.5-fold relative to an engineered T cell population not contacted with insulin, an insulin analogue, an insulin agonist, and / or an insulin partial agonist. In an embodiment, the expanded engineered T cell population is increased by at least 0.1-fold to at least 4.0-fold relative to an engineered T cell population not contacted with insulin, an insulin analogue, an insulin agonist, and / or an insulin partial agonist. In an embodiment, the expanded engineered T cell population is increased by at least 0.1-fold to at least 3.5-fold relative to an engineered T cell population not contacted with insulin, an insulin analogue, an insulin agonist, and / or an insulin partial agonist. In an embodiment, the expanded engineered T cell population is increased by at least 0.1-fold to at least 3.0-fold relative to an engineered T cell population not contacted with insulin, an insulin analogue, an insulin agonist, and / or an insulin partial agonist. In an embodiment, the expanded engineered T cell population is increased by at least 0.1-fold to at least 2.5-fold relative to an engineered T cell population not contacted with insulin, an insulin analogue, an insulin agonist, and / or an insulin partial agonist. In an embodiment, the expanded engineered T cell population is increased by at least 0.1-fold to at least 2.0-fold relative to an engineered T cell population not contacted with insulin, an insulin analogue, an insulin agonist, and / or an insulin partial agonist. In an embodiment, the expanded engineered T cell population is increased by at least 0.1-fold to at least 1.5-fold relative to an engineered T cell population not contacted with insulin, an insulin analogue, an insulin agonist, and / or an insulin partial agonist. In an embodiment, the expanded engineered T cell population is increased by at least 0.1-fold to at least 1.0-fold relative to an engineered T cell population not contacted with insulin, an insulin analogue, an insulin agonist, and / or an insulin partial agonist. In an embodiment, the expanded engineered T cell population is increased by at least 0.1-fold to at least 0.9-fold relative to an engineered T cell population not contacted with insulin, an insulin analogue, an insulin agonist, and / or an insulin partial agonist. In an embodiment, the expanded engineered T cell population is increased by at least 0.1-fold to at least 0.8-fold relative to an engineered T cell population not contacted with insulin, an insulin analogue, an insulin agonist, and / or an insulin partial agonist. In an embodiment, the expanded engineered T cell population is increased by at least 0.1-fold to at least 0.7-fold relative to an engineered T cell population not contacted with insulin, an insulin analogue, an insulin agonist, and / or an insulin partial agonist. In an embodiment, the expanded engineered T cell population is increased by at least 0.1-fold to at least 0.6-fold relative to an engineered T cell population not contacted with insulin, an insulin analogue, an insulin agonist, and / or an insulin partial agonist.In embodiments, the amplified engineered T cell population is increased by at least 0.1-fold to at least 0.5-fold relative to an engineered T cell population that has not been contacted with insulin, an insulin analogue, an insulin agonist, and / or an insulin partial agonist. In embodiments, the amplified engineered T cell population is increased by at least 0.1-fold to at least 0.4-fold relative to an engineered T cell population that has not been contacted with insulin, an insulin analogue, an insulin agonist, and / or an insulin partial agonist. In embodiments, the amplified engineered T cell population is increased by at least 0.1-fold to at least 0.3-fold relative to an engineered T cell population that has not been contacted with insulin, an insulin analogue, an insulin agonist, and / or an insulin partial agonist. In embodiments, the amplified engineered T cell population is increased by at least 0.1-fold to at least 0.2-fold relative to an engineered T cell population that has not been contacted with insulin, an insulin analogue, an insulin agonist, and / or an insulin partial agonist. For the methods provided herein, in embodiments, the amplified engineered T cell population is increased by about 2.0-fold to about 3.0-fold. In embodiments, the amplified engineered T cell population is increased by 2.0-fold to 3.0-fold. The fold increase can be any value or subrange within the recited range including the endpoints.

[0192] In embodiments, the engineered T cell population expands by at least about 0.1-fold to at least about 1000-fold. In embodiments, the engineered T cell population expands by at least about 0.5-fold to at least about 1000-fold. In embodiments, the engineered T cell population expands by at least about 1.0-fold to at least about 1000-fold. In embodiments, the engineered T cell population expands by at least about 5.0-fold to at least about 1000-fold. In embodiments, the engineered T cell population expands by at least about 10-fold to at least about 1000-fold. In embodiments, the engineered T cell population expands by at least about 20-fold to at least about 1000-fold. In embodiments, the engineered T cell population expands by at least about 30-fold to at least about 1000-fold. In embodiments, the engineered T cell population expands by at least about 40-fold to at least about 1000-fold. In embodiments, the engineered T cell population expands by at least about 50-fold to at least about 1000-fold. In embodiments, the engineered T cell population expands by at least about 100-fold to at least about 1000-fold. In embodiments, the engineered T cell population expands by at least about 200-fold to at least about 1000-fold. In embodiments, the engineered T cell population expands by at least about 300-fold to at least about 1000-fold. In embodiments, the engineered T cell population expands by at least about 400-fold to at least about 1000-fold. In embodiments, the engineered T cell population expands by at least about 500-fold to at least about 1000-fold. In embodiments, the engineered T cell population expands by at least about 600-fold to at least about 1000-fold. In embodiments, the engineered T cell population expands by at least about 700-fold to at least about 1000-fold. In embodiments, the engineered T cell population expands by at least about 800-fold to at least about 1000-fold. In embodiments, the engineered T cell population expands by at least about 900-fold to at least about 1000-fold.

[0193] In embodiments, the engineered T cell population expands by at least about 0.1-fold to at least about 900-fold. In embodiments, the engineered T cell population expands by at least about 0.1-fold to at least about 800-fold. In embodiments, the engineered T cell population expands by at least about 0.1-fold to at least about 700-fold. In embodiments, the engineered T cell population expands by at least about 0.1-fold to at least about 600-fold. In embodiments, the engineered T cell population expands by at least about 0.1-fold to at least about 500-fold. In embodiments, the engineered T cell population expands by at least about 0.1-fold to at least about 400-fold. In embodiments, the engineered T cell population expands by at least about 0.1-fold to at least about 300-fold. In embodiments, the engineered T cell population expands by at least about 0.1-fold to at least about 200-fold. In embodiments, the engineered T cell population expands by at least about 0.1-fold to at least about 100-fold. In embodiments, the engineered T cell population expands by at least about 0.1-fold to at least about 50-fold. In embodiments, the engineered T cell population expands by at least about 0.1-fold to at least about 40-fold. In embodiments, the engineered T cell population expands by at least about 0.1-fold to at least about 30-fold. In embodiments, the engineered T cell population expands by at least about 0.1-fold to at least about 20-fold. In embodiments, the engineered T cell population expands by at least about 0.1-fold to at least about 10-fold. In embodiments, the engineered T cell population expands by at least about 0.1-fold to at least about 5.0-fold. In embodiments, the engineered T cell population expands by at least about 0.1-fold to at least about 1.0-fold. In embodiments, the engineered T cell population expands by at least about 0.1-fold to at least about 0.5-fold.

[0194] In embodiments, the engineered T cell population is expanded by at least 0.1-fold to at least 1000-fold. In embodiments, the engineered T cell population is expanded by at least 0.5-fold to at least 1000-fold. In embodiments, the engineered T cell population is expanded by at least 1.0-fold to at least 1000-fold. In embodiments, the engineered T cell population is expanded by at least 5.0-fold to at least 1000-fold. In embodiments, the engineered T cell population is expanded by at least 10-fold to at least 1000-fold. In embodiments, the engineered T cell population is expanded by at least 20-fold to at least 1000-fold. In embodiments, the engineered T cell population is expanded by at least 30-fold to at least 1000-fold. In embodiments, the engineered T cell population is expanded by at least 40-fold to at least 1000-fold. In embodiments, the engineered T cell population is expanded by at least 50-fold to at least 1000-fold. In embodiments, the engineered T cell population is expanded by at least 100-fold to at least 1000-fold. In embodiments, the engineered T cell population is expanded by at least 200-fold to at least 1000-fold. In embodiments, the engineered T cell population is expanded by at least 300-fold to at least 1000-fold. In embodiments, the engineered T cell population is expanded by at least 400-fold to at least 1000-fold. In embodiments, the engineered T cell population is expanded by at least 500-fold to at least 1000-fold. In embodiments, the engineered T cell population is expanded by at least 600-fold to at least 1000-fold. In embodiments, the engineered T cell population is expanded by at least 700-fold to at least 1000-fold. In embodiments, the engineered T cell population is expanded by at least 800-fold to at least 1000-fold. In embodiments, the engineered T cell population is expanded by at least 900-fold to at least 1000-fold.

[0195] In embodiments, the engineered T cell population expands by at least 0.1-fold to at least 900-fold. In embodiments, the engineered T cell population expands by at least 0.1-fold to at least 800-fold. In embodiments, the engineered T cell population expands by at least 0.1-fold to at least 700-fold. In embodiments, the engineered T cell population expands by at least 0.1-fold to at least 600-fold. In embodiments, the engineered T cell population expands by at least 0.1-fold to at least 500-fold. In embodiments, the engineered T cell population expands by at least 0.1-fold to at least 400-fold. In embodiments, the engineered T cell population expands by at least 0.1-fold to at least 300-fold. In embodiments, the engineered T cell population expands by at least 0.1-fold to at least 200-fold. In embodiments, the engineered T cell population expands by at least 0.1-fold to at least 100-fold. In embodiments, the engineered T cell population expands by 0.1-fold to at least 50-fold. In embodiments, the engineered T cell population expands by at least 0.1-fold to at least 40-fold. In embodiments, the engineered T cell population expands by at least 0.1-fold to at least 30-fold. In embodiments, the engineered T cell population expands by at least 0.1-fold to at least 20-fold. In embodiments, the engineered T cell population expands by at least 0.1-fold to at least 10-fold. In embodiments, the engineered T cell population expands by at least 0.1-fold to at least 5.0-fold. In embodiments, the engineered T cell population expands by at least 0.1-fold to at least 1.0-fold. In embodiments, the engineered T cell population expands by at least 0.1-fold to at least 0.5-fold. The fold increase can be any value or sub-range within the recited range including the endpoints. For the methods provided herein, in embodiments, the engineered T cells expand by about 20-fold. In embodiments, the engineered T cells expand by 20-fold.

[0196] In embodiments, the methods disclosed herein (including their embodiments) are carried out under Good Manufacturing Practice (GMP).

[0197] Engineered T cell composition

[0198] Compositions are provided herein that particularly include an engineered T cell population prepared by the methods provided herein (including their embodiments). The engineered T cell population can have increased viability and / or expansion compared to an engineered T cell population prepared by a method in which the T cell population is not contacted with insulin, insulin analogs, insulin agonists, and / or insulin partial agonists prior to generating the engineered T cell population. Accordingly, in one aspect, an engineered T cell population is provided that is prepared by contacting a T cell population with a polynucleotide and insulin, insulin analogs, insulin agonists, and / or insulin partial agonists.

[0199] In an embodiment, a T cell population is contacted with a polynucleotide in the presence of insulin, an insulin analog, an insulin agonist, and / or an insulin partial agonist. In an embodiment, a T cell population is sequentially contacted with a polynucleotide and insulin, an insulin analog, an insulin agonist, and / or an insulin partial agonist. In an embodiment, a T cell population is contacted with insulin, an insulin analog, an insulin agonist, and / or an insulin partial agonist prior to the polynucleotide. In an embodiment, a T cell population is contacted with a polynucleotide and insulin, an insulin analog, an insulin agonist, and / or an insulin partial agonist simultaneously. In an embodiment, a T cell population is contacted with insulin, an insulin analog, an insulin agonist, and / or an insulin partial agonist after the polynucleotide.

[0200] In an embodiment, a T cell population is cultured with insulin, an insulin analog, an insulin agonist, and / or an insulin partial agonist for from about 30 minutes to about 48 hours. In an embodiment, a T cell population is cultured with insulin, an insulin analog, an insulin agonist, and / or an insulin partial agonist for from about 1 hour to about 48 hours. In an embodiment, a T cell population is cultured with insulin, an insulin analog, an insulin agonist, and / or an insulin partial agonist for from about 2 hours to about 48 hours. In an embodiment, a T cell population is cultured with insulin, an insulin analog, an insulin agonist, and / or an insulin partial agonist for from about 4 hours to about 48 hours. In an embodiment, a T cell population is cultured with insulin, an insulin analog, an insulin agonist, and / or an insulin partial agonist for from about 6 hours to about 48 hours. In an embodiment, a T cell population is cultured with insulin, an insulin analog, an insulin agonist, and / or an insulin partial agonist for from about 12 hours to about 48 hours. In an embodiment, a T cell population is cultured with insulin, an insulin analog, an insulin agonist, and / or an insulin partial agonist for from about 24 hours to about 48 hours.

[0201] In an embodiment, the T cell population is cultured with insulin, an insulin analog, an insulin agonist, and / or an insulin partial agonist for about 30 minutes to about 24 hours. In an embodiment, the T cell population is cultured with insulin, an insulin analog, an insulin agonist, and / or an insulin partial agonist for about 30 minutes to about 12 hours. In an embodiment, the T cell population is cultured with insulin, an insulin analog, an insulin agonist, and / or an insulin partial agonist for about 30 minutes to about 6 hours. In an embodiment, the T cell population is cultured with insulin, an insulin analog, an insulin agonist, and / or an insulin partial agonist for about 30 minutes to about 4 hours. In an embodiment, the T cell population is cultured with insulin, an insulin analog, an insulin agonist, and / or an insulin partial agonist for about 30 minutes to about 2 hours. In an embodiment, the T cell population is cultured with insulin, an insulin analog, an insulin agonist, and / or an insulin partial agonist for about 30 minutes to about 1 hour.

[0202] In an embodiment, the T cell population is cultured with insulin, an insulin analog, an insulin agonist, and / or an insulin partial agonist for about 30 minutes. In an embodiment, the T cell population is cultured with insulin, an insulin analog, an insulin agonist, and / or an insulin partial agonist for about 1 hour. In an embodiment, the T cell population is cultured with insulin, an insulin analog, an insulin agonist, and / or an insulin partial agonist for about 2 hours. In an embodiment, the T cell population is cultured with insulin, an insulin analog, an insulin agonist, and / or an insulin partial agonist for about 4 hours. In an embodiment, the T cell population is cultured with insulin, an insulin analog, an insulin agonist, and / or an insulin partial agonist for about 6 hours. In an embodiment, the T cell population is cultured with insulin, an insulin analog, an insulin agonist, and / or an insulin partial agonist for about 12 hours. In an embodiment, the T cell population is cultured with insulin, an insulin analog, an insulin agonist, and / or an insulin partial agonist for about 24 hours. In an embodiment, the T cell population is cultured with insulin, an insulin analog, an insulin agonist, and / or an insulin partial agonist for about 48 hours.

[0203] T cell composition

[0204] The present disclosure provides compositions comprising a T cell population and insulin, an insulin analogue, an insulin agonist, and / or an insulin partial agonist, wherein the compositions can be used to generate engineered T cell populations. The Applicant has demonstrated that insulin enhances gene editing efficiency in T cells. The Applicant has further demonstrated that the compositions provided herein (including their examples) generate engineered T cell populations with increased cell viability and expansion compared to compositions that do not include insulin. Accordingly, in one aspect, there is provided a composition comprising a T cell population, a polynucleotide, and insulin, an insulin analogue, an insulin agonist, and / or an insulin partial agonist. In an example, the T cell population comprises engineered T cells. In an example, the engineered T cells are engineered as described herein.

[0205] In an example, the composition further comprises a gene editing reagent.

[0206] Pharmaceutical composition

[0207] It is contemplated that the compositions provided herein (including T cell compositions and engineered T cell compositions) may be effective in treating diseases (e.g., cancer). For example, the engineered T cells provided herein can comprise exogenous T cell receptors specific for cancer cell antigens. Accordingly, in one aspect, there is provided a pharmaceutical composition comprising the engineered T cells provided herein (including their examples). In an example, the pharmaceutical composition further comprises a pharmaceutically acceptable excipient.

[0208] It should be understood that the examples and embodiments described herein are for illustrative purposes only, and that various modifications or alterations thereof will occur to those skilled in the art and will be included within the spirit and scope of the present application and the scope of the appended claims. All publications, patents, and patent applications cited herein are hereby incorporated by reference in their entirety for all purposes.

[0209] Method of treatment

[0210] It is contemplated that the engineered T cells provided herein (including their examples) are specific for disease-related antigens (e.g., cancer cell antigens), thereby allowing for effective targeting of cancer cells. The engineered T cells can comprise, for example, one or more exogenous T cell receptors that are engineered to be specific for an individual's cancer cells, thereby allowing for personalized and specific targeting of cancer cells. Accordingly, in one aspect, there is provided 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 provided herein (including their examples) or the pharmaceutical composition provided herein (including their examples). In an example, the method comprises administering a therapeutically effective amount of the engineered T cells provided herein (including their examples). In an example, the method comprises administering a therapeutically effective amount of the pharmaceutical composition provided herein (including their examples).

[0211] For the methods provided herein, in embodiments, engineered T cells can be generated from a subject. For example, T cells can be extracted from a subject and contacted ex vivo with a polynucleotide (e.g., a donor nucleic acid) and insulin to generate engineered T cells, which are then administered back to the subject. Thus, in embodiments, the engineered T cells are autologous T cells. In embodiments, the engineered T cells can be generated from T cells that are not taken from the subject. For example, the engineered T cells can be generated from a healthy subject (e.g., a subject without cancer). Thus, in embodiments, the engineered T cells are allogeneic T cells.

[0212] 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.

[0213] In embodiments, the cancer is leukemia, lymphoma, carcinoma, sarcoma, brain cancer, glioma, glioblastoma, neuroblastoma, prostate cancer, colorectal cancer, pancreatic cancer, medulloblastoma, melanoma, cervical cancer, gastric cancer, ovarian cancer, lung cancer, head and neck cancer, breast cancer, liver cancer, or uterine cancer. In embodiments, the cancer is carcinoma. In embodiments, the cancer is sarcoma. In embodiments, the cancer is brain cancer. In embodiments, the cancer is glioma. In embodiments, the cancer is glioblastoma. In embodiments, the cancer is neuroblastoma. In embodiments, the cancer is prostate cancer. In embodiments, the cancer is colorectal cancer. In embodiments, the cancer is pancreatic cancer. In embodiments, the cancer is medulloblastoma. In embodiments, the cancer is cervical cancer. In embodiments, the cancer is gastric cancer. In embodiments, the cancer is ovarian cancer. In embodiments, the cancer is lung cancer. In embodiments, the cancer is head and neck cancer. In embodiments, the cancer is breast cancer. In embodiments, the cancer is liver cancer. In embodiments, the cancer is uterine cancer.

[0214] Examples

[0215] Example 1: Insulin treatment significantly improves the efficiency of engineering and expansion of T cells

[0216] Transgenic delivery using electroporation allows for a virus-free, convenient, efficient, and safe method for engineering cells. Compared to the use of recombinant viral vectors that have been widely used in the field of cell therapy, genome editing using electroporation is time-saving and easy to manufacture, and enables a more precise genome targeting method when using CRISPR / Cas9 for gene editing. However, electroporation has a great impact on cells, resulting in poor cell viability and low recovery rate after transfection. Therefore, an effective, safe, and easy-to-manufacture method for efficiently improving T cell engineering, cell expansion, and the number of all edited T cells (TEC) during the engineering of T cell receptors (TCR) via electroporation is needed. Here, Wilms' tumor gene 1 (WT1) peptide-associated T cell receptor (TCR) (which provides a clinically relevant system) was used as a DNA template to test the effect of our engineering process on the derived T cells as the final drug product (FDP). Our findings revealed that the Akt and ERK signaling pathways were activated during the engineering of T cells, and their activation was associated with the viability and expansion of T cell cultures. Further activation of these signaling pathways by adding insulin to the cultured T cells effectively enhanced T cell expansion, the number of all edited cells (TEC), and the efficiency of engineering the TCR of the FDP, contributing to a cell expansion and TEC improvement up to 4-fold and a 2-fold enhancement in knock-in efficiency. Without being bound by theory, these findings suggest that adding insulin to the T cell culture during the engineering of T cells improves the mitochondrial membrane potential and thus mitochondrial function, enhances T cell metabolism, and / or attenuates apoptosis. All these enhancements may be due to the activation of both the Akt and ERK signaling pathways, which was observed when T cells were pretreated with insulin before transfection. Notably, in our study, the T cell editing efficiency in the FDP was also improved after pretreatment of T cells with insulin before transfection compared to the control group. In addition, no significant differences were observed in the phenotype or T cell function between the insulin pretreatment group and the control group, indicating that insulin pretreatment of T cells is a safe and translationally relevant method for the engineering of T cells.

[0217] Insulin treatment stimulates the Akt signaling pathway: Recently, it has been reported that the insulin receptor plays a crucial role in T cell immunity in vivo. In numerous studies, insulin has been shown to stimulate the cell growth of many different cell types. However, its potential during the engineering and manufacturing of T cells was previously unknown. Our preliminary analysis of the activated cell signaling pathways during the engineering of T cells indicated that both the Akt and Erk signaling pathways could be activated during the engineering of T cells ( Figure 1 ).

[0218] To confirm that adding insulin to T cell cultures during the engineering process can have a positive effect on T cell growth, naive human CD8+ T cells were isolated and stimulated with anti-CD3 / CD28 antibodies for 2 days, followed by starving them for 4 hours (h) by culturing them in phosphate-buffered saline (PBS). Cells were then stimulated by adding insulin to the cultures, and samples were collected as indicated ( Figure 1 ). Western blot data showed that insulin induced phosphorylation of AKT in human CD8+ T cells as early as 15 minutes after insulin treatment ( Figure 1 ), and the level of AKT phosphorylation began to decline 3 hours after treatment ( Figure 1 ). Additionally, the level of cleaved caspase 3 (C-Cas3) was significantly reduced after adding insulin, indicating that insulin treatment can also attenuate the T cell apoptosis process, as Figure 1 shown. No significant increase in ERK1 / 2 or STAT5 phosphorylation was observed after stimulating starved cells with insulin, indicating that adding insulin to the medium of activated CD8+ T cells initially triggers AKT signaling. However, the potential effect of insulin treatment at later time points on the Ras-Raf-MEK-Erk signaling pathway in cultured T cells has not been determined.

[0219] The Akt and Erk signaling pathways are activated during the engineering of T cells, and their activation is associated with the growth and viability of the culture: To achieve more efficient gene editing and lower toxicity during the engineering of the TCR, we used the Lonza Nucleofector system to co-electroporate human primary CD8+ T cells with CRISPR-Cas9 / sgRNA ribonucleoprotein (RNP) and nanoplasmid DNA donors. The DNA plasmid design and targeted insertion location have been reported previously. Briefly, a 1572 bp WT1 peptide-specific TCR template was targeted to TRAC exon 1 using homology-directed repair ( Figure 7A ). A schematic of the optimized 15-day process (including T cell activation, electroporation, expansion, and harvest) is shown in Figure 7B . To identify signaling pathways that may affect the growth and expansion of T cells isolated from different donors, T cells isolated from two representative donors were cultured according to the same protocol. The Lonza EH115 pulse code was used with a 100 μl cuvette in the Lonza Nucleofector instrument according to the manufacturer's protocol. T cell growth rate and viability were measured during this process, and cell samples were collected at designated time points for Western blot analysis.

[0220] Our data show that under the same culture conditions, compared with the higher-performing donor B, the lower-performing donor cells (donor A) with greater viability loss and lower expansion rate exhibited stronger and more persistent Akt and Erk phosphorylation( Figure 8A and 8B ). Akt phosphorylation was measured in donor A on day 3 (24 hours after transfection), and this Akt phosphorylation remained strong until day 8 and beyond, while Akt phosphorylation in donor B began to gradually weaken from day 4 to day 6( Figure 8A , top). Similarly, in donor B cells with higher performance compared to donor A, Erk phosphorylation decreased from day 3 to day 4, while in donor A it persisted until day 8( Figure 8A , bottom). Our findings suggest that the activation of the Akt and Erk signaling pathways may play a role in T cell growth / expansion after electroporation. Therefore, our aim was to investigate whether in vitro stimulation of the Akt and Erk signaling pathways could enhance the growth and expansion rate of engineered T cells.

[0221] Insulin pretreatment of cultured T cells before electroporation improves the viability, expansion, and all edited cells during the engineering of the TCR process: To further investigate the effect of the Akt and Erk signaling pathways on T cell growth, T cells obtained from several independent donors underwent the process of engineering T cells with or without insulin treatment before electroporation, as insulin is a known activator of both the Akt and Erk signaling pathways. We first tested whether adding insulin to the T cell culture either before or after transfection (before or after TFX) or both before and after transfection ([before + after] TFX) had a more significant effect on T cell growth. As previously reported, chemically defined and serum-free medium was used in all experiments. Although treating the cultures with insulin improved T cell expansion under all tested conditions (before, after, or before + after TFX treatment), adding insulin before TFX showed the greatest improvement in both cell expansion and TEC levels( Figure 10A and 10B ). Treating the T cell cultures with insulin after TFX showed a moderate effect on cell expansion and TEC levels, as the cultures treated with insulin before TFX or before + after TFX had comparable culture growth and TEC levels compared to the control (CTR) and after TFX, respectively.

[0222] We also tested different insulin concentrations and treatment time points before TFX and their effects on T cell growth, expansion, and TEC levels. T cells were treated with the specified concentration of insulin for a specific duration only before transfection. Our data show that pre-treating T cells with insulin 24 hours before electroporation significantly enhanced T cell expansion during the 15-day culture Figures 2A to 2C) and TEC levels ( Figures 2D to 2F ). Notably, this enhancement was observed at all tested concentrations of insulin from low (1 μg / ml) to high (25 μg / ml) and in all tested donors. On average, pre - treating T cells with insulin prior to TFX improved cell culture expansion and TEC levels in all donors by approximately 4 - fold. Treating T cell cultures with 25 μg / ml (high) insulin for different lengths of time (6 hours, 24 hours, 48 hours) prior to TFX successfully improved T cell expansion and TEC levels in all tested donors, resulting in an improvement in expansion ( Figures 9A to 9C ) and TEC levels ( Figures 9D to 9F ) of approximately 3 - to 4 - fold. Interestingly, shorter insulin treatment durations prior to TFX (6 hours and 24 hours) showed the greatest enhancement in T cell growth and TEC levels. Therefore, high - concentration insulin treatment and treatment durations of 6 hours and 24 hours were used in subsequent experimental designs.

[0223] Insulin pretreatment improves the T cell expansion rate and TEC levels by enhancing the expression / activation of the Akt and Erk signaling pathways: To further investigate the status of Akt and Erk activation during insulin treatment, T cells were treated with insulin (25 μg / ml) 24 hours prior to electroporation, and cell pellets were isolated at designated time points during the process of engineering the T cells. Western blot analysis of these samples showed that pretreatment with insulin increased the levels of total Akt and Erk in cultured T cells ( Figure 3A 、 3B 、3D), and although the ratios of phosphorylated Akt (P - Akt) / Akt and phosphorylated Erk (P - Erk) / Erk remained comparable in untreated T cells and T cells pretreated with insulin prior to TFX ( Figure 3C 、 3E ), the absolute levels of pAkt and pErk were higher in insulin - pretreated T cells ( Figure 3A ).

[0224] Insulin treatment enhances the survival of edited T cells and thus enhances the level of T cell editing in the final drug product:Higher levels of Akt and Erk signaling pathway expression / activation may be the root cause of the higher T cell growth observed during the engineering process. However, the level of T cell growth is also affected by variability between donors. In theory, for donors with poor cell health, treating cultured cells with insulin may improve the TCR knock-in rate by improving T cell survival and expansion. Adding insulin to cultured T cells should not negatively impact Cas9-mediated cleavage of the gene of interest, nor should it affect the homologous directed repair process in our final T cell product. However, we observed that in two independent donors, the total knock-in percentage of T cells treated with insulin before or after TFX was significantly higher than that of the control group( Figures 4A to 4D ). In donor 1, the knock-in efficiency of T cells treated with insulin before TFX reached 35.5% (low insulin concentration), 29.8% (medium insulin concentration), and 31.3% (high insulin concentration), while the control group had only a KI efficiency of 3.25%( Figure 4A ). Consistent with this trend, in donor 2, the knock-in efficiency of T cells treated with insulin before TFX also reached 29.8% (low), 34.1% (medium), and 45.6% (high), which was significantly higher than that of the control group (7.88% KI)( Figure 4B ). In donor 1, the knock-in efficiency of T cells treated with insulin after TFX showed 18.4% (medium) and 13.8% (high), and in donor 2, it showed 11.6% (low), 8.98% (medium), and 10.1% (high).

[0225] Although under the post-TFX conditions, the improvement in KI was not as robust as in the pre-TFX conditions, insulin treatment still showed higher TCR editing values compared to the control group( Figure 4C and 4D ). The improvement in the knock-in rate observed in the final T cell product may be due to the survival and growth support of edited T cells. In T cell cultures pretreated with insulin, the observed improvement in TEC levels tended to have a higher amplification fold. Without being bound by theory, the increase in editing efficiency in the observed FDP( Figure 4E ) may be due to improved metabolic support in the treated cells.

[0226] Insulin pretreatment enhances T cell metabolism and mitochondrial function:To further understand how insulin pretreatment enhances T cell growth and TCR editing efficiency, we investigated the effects of insulin pretreatment on T cell metabolism and mitochondrial function. Glucose uptake in T cells was measured using the cell-permeable fluorescent glucose analog 2-NBDG. 2-NBDG has been widely used to evaluate cell metabolism and proliferation. T cells were treated with 25 μg / mL insulin for 24 h. 2-NBDG uptake rates were measured in untreated (control) or insulin-pretreated T cells before transfection ( Figure 5A ). Compared with the control group, T cells pretreated with insulin showed significantly higher 2-NBDG uptake and accumulation rates ( Figure 5A ). The same trend was maintained 48 h after transfection ( Figure 5B ), although insulin was thoroughly washed out by electroporation before transfection, indicating a much higher metabolic rate of T cells before and after the transfection process. Notably, insulin-treated T cells had a significantly higher metabolic rate compared with untreated and untransfected / untreated control groups ( Figure 5B ). This implies that the higher T cell metabolic rate conferred by insulin treatment is maintained in a donor-independent manner during and after the electroporation process, as a similar trend was observed for different independent donors ( Figures 5A to 5B , Figure 11A and 11B ).

[0227] We also investigated the potential causes of enhanced metabolism after insulin pretreatment. Since mitochondrial function and integrity may be impaired during the electroporation process, which may also trigger apoptosis, we tested the effects of insulin pretreatment on mitochondrial function. During the engineering of T cells, T cells were treated with JC-10 to measure the mitochondrial membrane potential (MMP) level. Before transfection, we observed comparable MMP levels between the insulin-pretreated group and the untreated control group ( Figure 5C and 11C ), and when tested 48 h after electroporation, insulin-pretreated T cells showed statistically significantly higher MMP levels in a donor-independent manner ( Figure 5D and 11D ). In addition, our data confirmed that the electroporation process had a negative impact on the MMP level in T cells even 48 h after transfection compared with the untransfected control group ( Figure 5D and 11D ).

[0228] Mitochondrial mass is another indicator of mitochondrial health in cells, and electroporation-mediated mitochondrial damage can lead to mitochondrial mass loss and weakened mitochondrial function. We measured mitochondrial mass in insulin-pretreated or untreated T cells using nonyl acridine orange (NAO) and observed a significant and donor-independent decrease in mitochondrial mass in untreated T cells after electroporation compared to insulin-pretreated T cells ( Figure 5F and 11F ). Note that before electroporation, insulin-pretreated and untreated T cells had comparable mitochondrial mass ( Figure 5E and 11E ), indicating that mitochondrial mass loss is mainly due to the electroporation process.

[0229] We further explored the mRNA transcription levels of some members of the Bcl-2 protein family, which can enhance mitochondrial membrane integrity by blocking Bax / Bak-mediated mitochondrial membrane permeabilization, thereby attenuating apoptosis. qPCR data showed a significant increase in the level of Bcl-2L1 mRNA in insulin-pretreated (24 hours) T cells ( Figure 5G ). Bcl-2L1 is one of the crucial markers and is reported to play an important role in maintaining mitochondrial health and function. The higher level of Bcl-2L1 expression in insulin-pretreated T cells can effectively counteract the negative effects of mitochondrial membrane permeabilization during electroporation.

[0230] Insulin pretreatment does not affect the FDP characteristics and T cell function: Higher levels of the T cell memory phenotype in FDP are closely related to clinical efficacy during T cell therapy. Memory T cells, specifically including central memory T cells (TCM) and stem cell memory T cells (TSCM), have the potential to proliferate and survive long-term once reintroduced into patients. Our protocol for engineering T cells generates more than 90% TCM and TSCM in FDP ( Figures 6A to 6B , Figures 12A to 12B ). A high percentage of the memory T cell phenotype can promote a higher proliferation rate and enhance the tumor-killing ability of T cells. Insulin treatment of T cells before or after TFX, tested in different donors, does not affect the FDP T cell phenotype compared to the untreated control group ( Figures 6A to 6B , Figures 12A to 12F ). Insulin pretreatment of T cells led to a significant increase in the cell expansion rate, cell editing efficiency, and thus a significant increase in the total number of edited cells in engineered T cells.

[0231] To test the impact of such changes on T cell function, CD8+ T cells derived from insulin-pretreated or untreated conditions were compared in T cell activation and target cell killing assays ( Figures 6C to 6D)。FDPs from multiple donors were cryopreserved and then thawed prior to use. Cells from untreated controls (CTR) or insulin-pretreated groups were cultured with a specific peptide (WT1) at the indicated concentration for up to 24 hours, which binds to the engineered TCR. Flow cytometry analysis revealed comparable expression profiles of CD137, a T cell activation marker, between T cells derived from insulin-pretreated or untreated conditions ( Figure 6C ), indicating that insulin treatment does not affect T cell activation.

[0232] T cell function was tested using a T cell-mediated target cell killing assay. Target cells T2 (T) were labeled with CFSE-Far Red, pulsed with 20 μM WT1 peptide, and then co-incubated with T cell FDPs (E) at the indicated E:T ratios. These data confirmed that, at all different E:T ratios, T cells pretreated with insulin had comparable target cell killing capabilities to untreated control groups ( Figure 6D ), indicating that insulin treatment does not affect the killing of antigen-specific tumor cells by engineered T cells.

[0233] In summary, our data demonstrate the safety of using insulin pretreatment during the engineering of T cells, outlining the broad impact of growth factor stimulation in the field of T cell adoptive transfer, specifically in improving the efficiency of T cell engineering, increasing the expansion rate, and increasing the total number of edited cells. By improving T cell growth and editing efficiency after electroporation, T cells with three- to four-fold higher TECs compared to untreated control groups and comparable activation, proliferation, and killing capabilities were obtained. Notably, the effects of insulin pretreatment were not donor- or antigen-restricted, and all independent donors tested showed a consistent trend of enhanced growth and improved editing after insulin pretreatment.

[0234] Materials and methods: Ethical statement: All experimental methods were conducted in accordance with approved guidelines. All donor materials were purchased from commercial suppliers, and signed informed consent forms were stored at the supplier's site. All cell culture procedures and processes were documented according to the guidelines approved by Genentech.

[0235] RNA ribonucleoprotein and DNA plasmid:The single guide RNA sequences for both TRAC and TRBC were derived as described by Oh, Senger, et al. and ordered from Synthego (Menlo Park, CA, USA). The SpyFi Cas9 protein was purchased from Aldevron (Fargo, ND, USA), and preformed RNPs (60 pmol total), HDR templates (≤8 μg), and T cells were resuspended in P3 buffer as we previously published. The nanoplasmid with the WT1 TCR sequence was ordered from Nature Technologies (Lincoln, NE, USA) and used at a working concentration of 150 μg / ml for electroporation.

[0236] Cell isolation and activation: Peripheral blood mononuclear cells (PBMCs) were isolated from human cryopreserved leukocyte concentrates (leukopak) using Ficoll gradient centrifugation (400 rpm, 25 minutes). All samples were obtained from healthy donors carrying the HLA A*02:01 MHC class I complex. CD8+ T cells were isolated using a Miltenyi AutoMACS cell separator according to the manufacturer's protocol. The isolated CD8+ T cells were then mixed with Miltenyi T cell TransACT reagent (1:100) (catalog number 130-111-160), 25 ng / mL IL-7 (catalog number 130-095-367), and 50 ng / mL IL-15 (catalog number 130-095-760) in FUJIPrime-XV medium (Irvine Scientific catalog number 91154) for 48 hours for activation.

[0237] Electroporation: According to the manufacturer's protocol, cells were electroporated using a 4D-Nucleofector system (Lonza). After activation and insulin treatment, 1x10 7 (10 million) CD8+ T cells were washed and resuspended in Lonza P3 primary cell nucleofector solution (catalog number V4XP-3024), then mixed with the pre-mixed RNPs and DNA plasmids, and then transferred to a Lonza 100-μL cuvette. After electroporation, 400 μl of FUJIPrime-XV medium was added to the cuvette and incubated for 15 minutes, then the cells were seeded into culture flasks. The pulse code used for all experiments in this manus...

Claims

1. A method for editing an endogenous gene in a T cell population, the method comprising: Under conditions that permit entry of a polynucleotide encoding a gene editing reagent or the gene editing reagent into cells, contacting the T cell population with the gene editing reagent or the polynucleotide, and culturing the T cell population in the presence of one or more of the following before and / or during and / or after the contacting step to obtain an engineered T cell population: insulin, insulin analogs, insulin agonists, and / or insulin partial agonists.

2. The method according to claim 1, further comprising contacting the T cell population with donor DNA.

3. The method according to claim 1 or 2, wherein the polynucleotide encoding the gene editing reagent comprises: Single-stranded DNA, double-stranded DNA, linear DNA strands, plasmids, nanoplasmids, or minicircles.

4. The method according to claim 3, wherein the polynucleotide encoding the gene editing reagent comprises a plasmid, the plasmid comprising a plasmid backbone and a polynucleotide sequence encoding the gene editing reagent.

5. The method according to claim 4, wherein the plasmid further comprises the donor DNA.

6. The method according to any one of claims 2 to 5, wherein the donor DNA sequence comprises a polynucleotide encoding a gene product.

7. The method according to claim 6, wherein the T cell population is obtained from a subject.

8. The method according to claim 7, wherein the gene product sequence comprises a chimeric antigen receptor (CAR), a T cell receptor (TCR), a human leukocyte antigen (HLA), or an allogeneic immune defense receptor (ADR) or a subunit thereof.

9. The method according to claim 8, wherein the TCR sequence comprises an exogenous TCR-β subunit or a fragment thereof and / or an exogenous TCR-α subunit or a fragment thereof, or a chimeric antigen receptor and / or a subunit thereof.

10. The method according to claim 9, wherein the TCR sequence comprises an exogenous TCR-β subunit or a fragment thereof and an exogenous TCR-α subunit or a fragment thereof.

11. The method according to claim 10, wherein the TCR sequence is inserted into the TRAC or TRBC locus.

12. The method according to claim 10, wherein the TCR sequence is inserted into the TRAC locus.

13. The method according to claim 10, wherein the TCR sequence is inserted into the TRBC locus.

14. The method according to claim 1 or 2, wherein contacting the T cell population with the gene editing reagent or the polynucleotide encoding the gene editing reagent comprises transfecting the T cell population with the gene editing reagent or the polynucleotide encoding the gene editing reagent.

15. The method according to claim 14, wherein the transfection comprises electroporation.

16. The method according to claim 14, wherein the transfection comprises nucleofection.

17. The method according to claim 14, wherein the transfection comprises liposomal transfection.

18. The method according to claim 14, wherein the transfection comprises microfluidic transfection.

19. The method according to any one of claims 1 to 18, wherein prior to the contacting step, the T cell population is cultured in the presence of insulin, an insulin analogue, an insulin agonist, and / or an insulin partial agonist.

20. The method according to claim 19, which comprises culturing the T cell population in the presence of insulin, an insulin analogue, an insulin agonist, and / or an insulin partial agonist for 48 hours, 24 hours, 12 hours, 6 hours, 4 hours, 2 hours, 1 hour, or 30 minutes prior to the contacting step.

21. The method according to any one of claims 1 to 18, wherein after the contacting step, the T cell population is cultured in the presence of insulin, an insulin analogue, an insulin agonist, and / or an insulin partial agonist.

22. The method according to claim 21, which comprises culturing the T cell population in the presence of insulin, an insulin analogue, an insulin agonist, and / or an insulin partial agonist for 48 hours, 24 hours, 12 hours, 6 hours, 4 hours, 2 hours, 1 hour, or 30 minutes after the contacting step.

23. The method according to any one of claims 1 to 18, wherein prior to and after the contacting step, the T cell population is cultured in the presence of insulin, an insulin analogue, an insulin agonist, and / or an insulin partial agonist.

24. The method according to claim 23, which comprises culturing the T cell population in the presence of insulin, an insulin analogue, an insulin agonist, and / or an insulin partial agonist for 48 hours, 24 hours, 12 hours, 6 hours, 4 hours, 2 hours, 1 hour, or 30 minutes prior to the contacting step.

25. The method according to claim 23 or 24, which comprises culturing the T cell population in the presence of insulin, an insulin analogue, an insulin agonist, and / or an insulin partial agonist for 48 hours, 24 hours, 12 hours, 6 hours, 4 hours, 2 hours, 1 hour, or 30 minutes after the contacting step.

26. The method according to any one of claims 1 to 25, wherein the insulin, insulin analogue, insulin agonist, and / or insulin partial agonist is administered at a concentration of about 1 μg / ml to about 50 μg / ml.

27. The method according to claim 26, wherein the insulin, insulin analogue, insulin agonist, and / or insulin partial agonist is administered at a concentration of about 1 μg / ml, about 5 μg / ml, or about 25 μg / ml.

28. The method according to claim 27, wherein the T cell population is transfected with the donor DNA and the gene editing reagent or the polynucleotide encoding the gene editing reagent simultaneously.

29. The method according to any one of claims 1 to 28, wherein the gene editing reagent comprises an RNA-guided nuclease.

30. The method according to claim 29, wherein the RNA-guided nuclease is a CRISPR-Cas system.

31. The method according to claim 30, wherein the CRISPR-Cas system comprises Cas9 or a Cas9 variant.

32. The method according to any one of claims 1 to 31, wherein the gene editing reagent comprises a CRISPR-Cas system, the CRISPR-Cas system comprising a Cas protein and a guide RNA.

33. The method according to any one of claims 1 to 32, wherein at least 80% of the engineered T cells are central memory T cells (TCM) and / or stem cell memory T cells (TSCM).

34. A method of monitoring the cell viability of a population of engineered T cells, the method comprising measuring mitochondrial function and cell metabolism over time.

35. The method according to claim 34, wherein the mitochondrial membrane potential is measured.

36. The method according to claim 35, wherein a dye-based assay is used to measure the mitochondrial membrane potential.

37. The method according to claim 36, wherein the dye is JC-1 or JC-10.

38. A method of monitoring the cell viability of a population of engineered T cells, the method comprising measuring cell metabolism markers over time.

39. The method according to claim 38, wherein the method comprises measuring changes in glucose metabolism, Bcl-2 expression, Bcl-XL expression, Bax expression or Bad expression over time.

40. The method according to claim 39, wherein a glucose analog is used to monitor glucose metabolism of the population of engineered T cells.

41. The method according to claim 40, wherein the analog is 2-NBDG.

42. A method of increasing the cell viability of a population of engineered T cells, the method comprising contacting a population of T cells in the presence of insulin, an insulin analog, an insulin agonist, an insulin partial agonist, a gene editing reagent or a polynucleotide encoding a gene editing reagent to form the population of engineered T cells, wherein the population of engineered T cells has increased cell viability, growth and / or gene editing efficiency relative to a population of engineered T cells not contacted with insulin, an insulin analog, an insulin agonist or an insulin partial agonist, and wherein the population of engineered T cells is administered to a subject in need thereof.

43. The method according to claim 42, further comprising contacting the population of T cells with donor DNA.

44. The method according to claim 42 or 43, wherein the polynucleotide comprises: Single-stranded DNA, double-stranded DNA, linear DNA strands, plasmids, nanoplasmids or minicircles.

45. The method according to claim 44, wherein the polynucleotide comprises a plasmid, the plasmid comprising a plasmid backbone and a polynucleotide sequence encoding the gene editing reagent.

46. The method according to claim 45, wherein the plasmid further comprises the donor DNA.

47. The method according to any one of claims 43 to 46, wherein the donor DNA sequence comprises a polynucleotide encoding a gene product.

48. The method according to claim 47, wherein the gene product is autologous or allogeneic to the subject.

49. The method according to claim 47, wherein the gene product sequence comprises a chimeric antigen receptor (CAR), a T cell receptor (TCR), a human leukocyte antigen (HLA), or an allogeneic immune defense receptor (ADR) or a subunit thereof.

50. The method according to claim 49, wherein the TCR sequence comprises an exogenous TCR-β subunit or a fragment thereof and / or an exogenous TCR-α subunit or a fragment thereof, or a chimeric antigen receptor and / or a subunit thereof.

51. The method according to claim 50, wherein the TCR sequence comprises an exogenous TCR-β subunit or a fragment thereof and an exogenous TCR-α subunit or a fragment thereof.

52. The method according to claim 51, wherein the TCR sequence is inserted into the TRAC or TRBC locus.

53. The method according to claim 51, wherein the TCR sequence is inserted into the TRAC locus.

54. The method according to claim 51, wherein the TCR sequence is inserted into the TRBC locus.

55. The method according to claim 42 or 43, wherein contacting the T cell population with the gene editing reagent or the polynucleotide encoding the gene editing reagent comprises transfecting the T cell population with the gene editing reagent or the polynucleotide encoding the gene editing reagent.

56. The method according to claim 55, wherein the transfection comprises electroporation.

57. The method according to claim 55, wherein the transfection comprises nucleofection.

58. The method according to claim 55, wherein the transfection comprises liposomal transfection.

59. The method according to claim 55, wherein the transfection comprises microfluidic transfection.

60. The method according to any one of claims 42 to 59, wherein the T cell population is cultured in the presence of insulin, an insulin analogue, an insulin agonist, and / or an insulin partial agonist before the contacting step.

61. The method according to claim 60, which comprises culturing the T cell population in the presence of insulin, an insulin analogue, an insulin agonist, and / or an insulin partial agonist for 48 hours, 24 hours, 12 hours, 6 hours, 4 hours, 2 hours, 1 hour, or 30 minutes before the contacting step.

62. The method according to any one of claims 42 to 59, wherein the T cell population is cultured in the presence of insulin, an insulin analogue, an insulin agonist, and / or an insulin partial agonist after the contacting step.

63. The method according to claim 62, which comprises culturing the T cell population in the presence of insulin, an insulin analogue, an insulin agonist, and / or an insulin partial agonist for 48 hours, 24 hours, 12 hours, 6 hours, 4 hours, 2 hours, 1 hour, or 30 minutes after the contacting step.

64. The method according to any one of claims 42 to 59, wherein the T cell population is cultured in the presence of insulin, an insulin analogue, an insulin agonist, and / or an insulin partial agonist before and after the contacting step.

65. The method according to claim 64, which includes culturing the T cell population in the presence of insulin, an insulin analogue, an insulin agonist, and / or an insulin partial agonist for 48 hours, 24 hours, 12 hours, 6 hours, 4 hours, 2 hours, 1 hour, or 30 minutes before the contacting step.

66. The method according to claim 64 or 65, which includes culturing the T cell population in the presence of insulin, an insulin analogue, an insulin agonist, and / or an insulin partial agonist for 48 hours, 24 hours, 12 hours, 6 hours, 4 hours, 2 hours, 1 hour, or 30 minutes after the contacting step.

67. The method according to any one of claims 42 to 66, wherein the insulin, insulin analogue, insulin agonist, and / or insulin partial agonist is administered at a concentration of about 1 μg / ml to about 50 μg / ml.

68. The method according to claim 67, wherein the insulin, insulin analogue, insulin agonist, and / or insulin partial agonist is administered at a concentration of about 1 μg / ml, about 5 μg / ml, or about 25 μg / ml.

69. The method according to claim 68, wherein the T cell population is transfected with the donor DNA and the gene editing reagent or the polynucleotide encoding the gene editing reagent simultaneously.

70. The method according to any one of claims 42 to 69, wherein the gene editing reagent includes an RNA-guided nuclease.

71. The method according to claim 70, wherein the RNA-guided nuclease is a CRISPR-Cas system.

72. The method according to claim 71, wherein the CRISPR-Cas system includes Cas9 or a Cas9 variant.

73. The method according to any one of claims 42 to 72, wherein the gene editing reagent includes a CRISPR-Cas system, and the CRISPR-Cas system comprises a Cas protein and a guide RNA.

74. The method according to any one of claims 42 to 73, wherein at least 80% of the engineered T cells are TCM and / or TSCM.

75. The method according to any one of claims 42 to 74, wherein the cell viability and culture performance of the engineered T cell population are monitored, and the method includes measuring mitochondrial function and cell metabolism over time.

76. The method according to claim 75, wherein the mitochondrial membrane potential is measured.

77. The method according to claim 76, wherein a dye-based assay is used to measure the mitochondrial membrane potential.

78. The method according to claim 77, wherein the dye is JC-1 or JC-10.

79. The method according to any one of claims 42 to 74, wherein the cell viability and culture performance of the T cell population are monitored, and the method includes measuring cell metabolism markers over time.

80. The method according to claim 79, wherein the method includes measuring the change in glucose metabolism over time.

81. The method according to claim 80, wherein a glucose analog is used to monitor the glucose metabolism of the engineered T cell population.

82. The method according to claim 81, wherein the analog is 2-NBDG.

83. The method according to any one of claims 75 to 82, wherein the cell viability of the engineered T cell population is increased by at least about 0.1-fold to at least about 5.0-fold relative to an engineered T cell population not cultured in the presence of insulin, an insulin analog, an insulin agonist, or an insulin partial agonist.

84. The method according to claim 83, wherein the cell viability is increased by about 2.0-fold.

85. The method according to any one of claims 75 to 83, wherein the cell viability of the engineered T cell population is from about 30% to about 95%.

86. A method of increasing the gene editing efficiency of an engineered T cell population, the method comprising contacting a T cell population with insulin, an insulin analog, an insulin agonist, an insulin partial agonist, a gene editing reagent, and a polynucleotide to form the engineered T cell population, wherein the engineered T cell population has an increased gene editing efficiency relative to an engineered T cell population not contacted with insulin, an insulin analog, an insulin agonist, or an insulin partial agonist.

87. The method according to claim 86, wherein contacting the T cell population with the polynucleotide comprises transfecting the T cell population with the polynucleotide.

88. The method according to claim 86 or 87, wherein the polynucleotide is donor DNA.

89. The method according to any one of claims 86 to 88, wherein the polynucleotide comprises: Single-stranded DNA, double-stranded DNA, linear DNA strand, plasmid, nanoplasmid, or minicircle.

90. The method according to any one of claims 86 to 89, further comprising contacting the T cell population with a gene editing reagent.

91. The method according to claim 90, wherein contacting the T cell population with the gene editing reagent comprises transfecting the T cell population with the gene editing reagent or a polynucleotide encoding the gene editing reagent.

92. The method according to claim 91, wherein the T cell population is transfected with the polynucleotide and the gene editing reagent or the polynucleotide encoding the gene editing reagent simultaneously.

93. The method according to any one of claims 86 to 92, wherein the T cell population is contacted with insulin, an insulin analog, an insulin agonist, and / or an insulin partial agonist at a concentration of about 1 μg / ml to about 50 μg / ml.

94. The method according to any one of claims 86 to 93, wherein the T cell population is contacted with the polynucleotide and insulin, an insulin analog, an insulin agonist, and / or an insulin partial agonist simultaneously.

95. The method according to any one of claims 86 to 94, wherein the T cell population is contacted with the polynucleotide and insulin, an insulin analog, an insulin agonist, and / or an insulin partial agonist sequentially.

96. The method according to claim 95, wherein the T cell population is contacted with an insulin inhibitor prior to the polynucleotide.

97. The method according to any one of claims 86 to 96, wherein the gene editing efficiency of the engineered T cell population is increased by at least about 0.1-fold to at least about 5-fold relative to an engineered T cell population that has not been contacted with insulin, an insulin analog, an insulin agonist, and / or an insulin partial agonist.

98. The method according to claim 97, wherein the gene editing efficiency of the engineered T cell population is increased by about 2.0-fold to about 3.0-fold.

99. The method according to any one of claims 86 to 98, wherein the gene editing efficiency of the engineered T cell population is from about 1% to about 99%.

100. The method according to any one of claims 86 to 99, wherein the knockout efficiency of the engineered T cell population is from about 70% to about 99%.

101. The method according to claim 100, wherein the knockout efficiency is about 90%.

102. The method according to any one of claims 86 to 101, wherein the knock-in efficiency of the engineered T cell population is from about 20% to about 99%.

103. The method according to claim 102, wherein the knock-in efficiency is about 60%.

104. A method for increasing the expansion of an engineered T cell population, the method comprising: (i) contacting a T cell population with insulin, an insulin analog, an insulin agonist, and / or an insulin partial agonist and a polynucleotide to form the engineered T cell population, and (ii) expanding the engineered T cell population to form an expanded engineered T cell population, wherein the insulin, insulin analog, insulin agonist, and / or insulin partial agonist increases the expanded engineered T cell population relative to an engineered T cell population that has not been contacted with insulin, an insulin analog, an insulin agonist, and / or an insulin partial agonist.

105. The method according to claim 104, wherein contacting the T cell population with the polynucleotide comprises transfecting the T cell population with the polynucleotide.

106. The method according to claim 104 or 105, wherein the polynucleotide is donor DNA.

107. The method according to any one of claims 104 to 106, wherein the polynucleotide comprises: Single-stranded DNA, double-stranded DNA, linear DNA strand, plasmid, nanoplasmid, or minicircle.

108. The method according to any one of claims 104 to 107, further comprising contacting the T cell population with a gene editing reagent.

109. The method according to claim 108, wherein contacting the T cell population with the gene editing reagent comprises transfecting the T cell population with the gene editing reagent or a polynucleotide encoding the gene editing reagent.

110. The method according to claim 109, wherein the T cell population is transfected simultaneously with the polynucleotide and the gene editing reagent or the polynucleotide encoding the gene editing reagent.

111. The method according to any one of claims 104 to 110, wherein the T cell population is contacted with insulin, an insulin analogue, an insulin agonist, and / or an insulin partial agonist at about 1 μg / ml to about 50 μg / ml.

112. The method according to any one of claims 104 to 111, wherein the T cell population is contacted with the polynucleotide and insulin, an insulin analogue, an insulin agonist, and / or an insulin partial agonist simultaneously.

113. The method according to any one of claims 104 to 111, wherein the T cell population is contacted with the polynucleotide and insulin, an insulin analogue, an insulin agonist, and / or an insulin partial agonist sequentially.

114. The method according to claim 113, wherein the T cell population is contacted with insulin, an insulin analogue, an insulin agonist, and / or an insulin partial agonist before being contacted with the polynucleotide.

115. The method according to claim 113, wherein the T cell population is contacted with insulin, an insulin analogue, an insulin agonist, and / or an insulin partial agonist after being contacted with the polynucleotide.

116. The method according to claim 113, wherein the T cell population is contacted with insulin, an insulin analogue, an insulin agonist, and / or an insulin partial agonist before and after being contacted with the polynucleotide.

117. The method according to any one of claims 104 to 116, wherein the expanded engineered T cell population is increased by at least about 0.1-fold to at least about 5.0-fold relative to an engineered T cell population not contacted with insulin, an insulin analogue, an insulin agonist, and / or an insulin partial agonist.

118. The method according to claim 117, wherein the expanded engineered T cell population is increased by about 2.0-fold to about 3.0-fold.

119. The method according to any one of claims 104 to 118, wherein the engineered T cell population is expanded by at least about 0.1-fold to at least about 1000-fold.

120. The method according to claim 119, wherein the engineered T cells are expanded by about 20-fold.

121. An engineered T cell population prepared by the method according to any one of claims 1 to 120.

122. A pharmaceutical composition comprising the engineered T cells according to claim 121.

123. 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 according to claim 96 or the pharmaceutical composition according to claim 97.

124. The method according to claim 123, wherein the disease is cancer.

125. The method according to claim 123 or 124, wherein the cancer is leukemia, lymphoma, carcinoma, sarcoma, brain cancer, glioma, glioblastoma, neuroblastoma, prostate cancer, colorectal cancer, pancreatic cancer, medulloblastoma, melanoma, cervical cancer, gastric cancer, ovarian cancer, lung cancer, head and neck cancer, breast cancer, liver cancer or uterine cancer.

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