Efficient TCR gene editing in T lymphocytes

Abstract

The present disclosure relates to engineered T cells, methods for making and using same, and reagents for making engineered T cells.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Application No. 63 / 165,509, filed March 24, 2021, and U.S. Provisional Application No. 63 / 323,065, filed March 24, 2022, each of which is incorporated by reference herein in its entirety for all purposes.

[0002] Sequence Listing This application contains a Sequence Listing that has been submitted electronically in ASCII format and is incorporated by reference in its entirety. The ASCII copy is written to a file named 048893-532001WO_ST25.txt, created on Mar. 24, 2022, and is 170,857 bytes in size. [Background technology]

[0003] background T cell therapy has attracted increasing interest for the treatment of many different diseases, including cancer, infectious diseases, and inflammatory diseases. However, the preparation of therapeutic T cells using current viral-based engineering approaches is time-consuming, costly, and poses certain safety concerns. Furthermore, previous methods of producing engineered T cells using non-viral methods can have low editing efficiency and limit cell yields. Summary of the Invention

[0004] overview The present technology relates generally to engineered T cells and related systems, methods and kits. The technology addresses the low efficiency and low numbers of engineered T cells that are typically produced using known non-viral methods.

[0005] In one aspect, an engineered T cell is provided. The engineered T cell comprises a nucleic acid sequence encoding a polypeptide comprising an exogenous T cell receptor (TCR) or a portion thereof. In an embodiment, the exogenous TCR or a portion thereof is an exogenous T cell receptor beta (TCR-beta) and / or an exogenous T cell receptor alpha (TCR-alpha), or a portion thereof. In an embodiment, the nucleic acid sequence is inserted into a TCR locus. In an embodiment, the TCR locus is a TCR-alpha locus of the engineered T cell. In an embodiment, the TCR locus is a TCR-beta locus of the engineered T cell.

[0006] In embodiments, the exogenous TCR-alpha comprises at least a portion of an endogenous TCR-alpha. In embodiments, the exogenous TCR-alpha comprises an exogenous TCR-alpha (VJ) domain and an endogenous TCR-alpha constant domain.

[0007] In an embodiment, the nucleic acid sequence further comprises a polyadenylation (polyA) sequence. In an embodiment, the nucleic acid sequence further comprises a stop codon. In an embodiment, the stop codon is 3' to the exogenous TCR and 5' to the polyA sequence. In an embodiment, the TCR knock-in (KI) construct further comprises a polyA sequence. In an embodiment, SEQ ID NO:1 comprises a polyA sequence.

[0008] In another related aspect, a composition is provided comprising isolated T cells, wherein at least 5% of the cells are engineered T cells. In an embodiment, each engineered T cell comprises a nucleic acid sequence encoding a polypeptide comprising an exogenous TCR-beta and an exogenous TCR-alpha. In an embodiment, each engineered T cell comprises a nucleic acid sequence encoding a polypeptide comprising an exogenous TCR-beta and an exogenous TCR-alpha. In an embodiment, each engineered T cell comprises a nucleic acid sequence encoding a polypeptide comprising at least a portion of an exogenous TCR-beta and at least a portion of an exogenous TCR-alpha. In an embodiment, the portion of the exogenous TCR-alpha is a TCR-alpha VJ region. In an embodiment, a nucleic acid sequence is inserted into the TCR-alpha locus of the engineered T cell.

[0009] In embodiments, the engineered T cells do not express a functional endogenous TCR-beta protein. In embodiments, the engineered T cells do not express a functional endogenous TCR-alpha protein. In embodiments, the exogenous TCR-alpha (VJ) domain forms part of a heterologous TCR-alpha that includes at least a portion of the T cell's endogenous TCR-alpha. In embodiments, the TCR-alpha locus is a TCR-alpha constant region. In embodiments, the exogenous TCR-beta and the heterologous TCR-alpha are expressed from a nucleic acid to form a functional TCR. In embodiments, the engineered T cells are bound to an antigen. In embodiments, the engineered T cells are bound to a cancer cell. In embodiments, the TCR binds to an antigen presented on a major histocompatibility complex class I (MHCI) molecule.

[0010] In embodiments, the antigen is a neo-antigen or a tumor associated antigen (TAA). In embodiments, the antigen is a neo-antigen. In embodiments, the antigen is a TAA. In embodiments, the neo-antigen or TAA is selected from WT1, JAK2, NY-ESO1, PRAME, KRAS, or an antigen from Table 1 or Table 2. In embodiments, the antigen is WT1. The MHCI allele can be any known or discovered allele. In an embodiment, the MHCI is selected from the group consisting of HLA-A02:01, A*02:03, A*02:06, A*02:07, A*023:01, A*26:01, A*29:02, A*30:01, A*30:02, A*31:01, A*32:01, A*68:01, A*68:02, B*18:01, B*35:03, B*40:01, B*40:02 , B*40:06, B*46:01, B*51:01, B*53:01, B*57:01, B*58:01, C*01:02, C*02:02, C*03:02, C*03:03, C*05:01, C*06:02, C*07:01, C*08:01, C*08:02, C*12:03, C*14:02, or C*15:02. In embodiments, the antigen is specific to a cancer in the subject receiving the engineered T cells.

[0011] Related methods, compositions and reagents can be found in the following patent applications: U.S. Patent Application Publication No. 20180355429 A1 "DETERMINING ANTIGEN-SPECIFIC T-CELLS", U.S. Patent Application Publication No. 20180087109 A1 "DETERMINING ANTIGEN-SPECIFIC T-CELLS", WO 2017112944 A1 "HIGH AFFINITY T CELL RECEPTORS AND USES THEREOF", WO 2016161273 A1 "METHOD OF IDENTIFYING HUMAN COMPATIBLE T CELL RECEPTORS SPECIFIC FOR AN ANTIGENIC TARGET", U.S. Patent Application Publication No. 20160138011 A1 "MULTIPLEXED DIGITAL QUANTITATION OF REARRANGED LYMPHOID RECEPTORS IN A COMPLEX MIXTURE", International Publication No. WO 2016069886 A1 "HIGHLY-MULTIPLEXED SIMULTANEOUS DETECTION OF NUCLEIC ACIDS ENCODING PAIRED ADAPTIVE IMMUNE RECEPTOR HETERODIMERS FROM MANY SAMPLES", U.S. Patent Application Publication No. 20150275296 A1 "DETERMINING ANTIGEN-SPECIFIC T-CELLS", International Publication No. WO 2010053587 A2 "METHODS OF MONITORING CONDITIONS BY SEQUENCE ANALYSIS", International Publication No. WO 2011139371 A1 "MONITORING HEALTH AND DISEASE STATUS USING CLONOTYPE PROFILES", International Publication No. WO 2011139372 A1 "SEQUENCE ANALYSIS OF COMPLEX AMPLICONS", International Publication No. 2013059725 A1 "QUANTIFICATION OF ADAPTIVE IMMUNE CELL GENOMES IN A COMPLEX MIXTURE OF CELLS", U.S. Patent Application Publication No. 20130196328 A1 "RARE"CLONOTYPES AND USES THEREOF", U.S. Patent Application Publication No. 20130236895 A1 "METHOD OF SEQUENCE DETERMINATION USING SEQUENCE TAGS", U.S. Patent Application Publication No. 20130065768 A1 "RANDOM ARRAY SEQUENCING OF LOW-COMPLEXITY LIBRARIES", U.S. Patent Application Publication No. 20130150252 "DETECTION AND MEASUREMENT OF TISSUE-INFILTRATING LYMPHOCYTES", WO 2013134162 A2 "DETERMINING PAIRED IMMUNE RECEPTOR CHAINS FROM FREQUENCY MATCHED SUBUNITS", WO 2013169957 A1 "COMPOSITIONS AND METHOD FOR MEASURING AND CALIBRATING AMPLIFICATION BIAS IN "MULTIPLEXED PCR REACTIONS", International Publication No. 2013188471A2 "METHOD OF SEQUENCE DETERMINATION USING SEQUENCE TAGS", International Publication No. 2013188831A1 "UNIQUELY TAGGED REARRANGED ADAPTIVE IMMUNE RECEPTOR GENES IN A COMPLEX GENE SET", International Publication No. 2014055561A1 "IMMUNOCOMPETENCE ASSESSMENT BY ADAPTIVE IMMUNE RECEPTOR DIVERSITY AND CLONALITY CHARACTERIZATION", International Publication No. 2014130685A1 "RARE CLONOTYPES AND USES THEREOF", International Publication No. 2014145992A1 "UNIQUELY TAGGED REARRANGED ADAPTIVE IMMUNE RECEPTOR GENES IN A COMPLEX GENE SET" COMPLEX GENE SET”, International Publication No. 2015002908 A1 “LARGE-SCALE BIOMOLECULAR ANALYSIS WITH SEQUENCETAGS", International Publication No. 2015058159A1 "PREDICTING PATIENT RESPONSIVENESS TO IMMUNE CHECKPOINT INHIBITORS", International Publication No. 2015106161A1 "METHODS FOR DEFINING AND PREDICTING IMMUNE RESPONSE TO ALLOGRAFT", International Publication No. 2015160439A2 "QUANTIFICATION OF ADAPTIVE IMMUNE CELL GENOMES IN A COMPLEX MIXTURE OF CELLS", International Publication No. 2016086029A1 "CHARACTERIZATION OF ADAPTIVE IMMUNE RESPONSE TO VACCINATION OR INFECTION USING IMMUNE REPERTOIRE SEQUENCING", U.S. Patent Application Publication No. 20180282808A1 "DETERMINING WT-1 SPECIFIC T CELLS AND and WT-1 SPECIFIC T CELL RECEPTORS (TCRS) (each of which is incorporated by reference in its entirety for all that is taught therein, including, but not limited to, all methods of making, methods of use, reagents, cells, proteins, nucleic acids, compositions, etc.).

[0012] In an embodiment, the expression of endogenous TCR-beta gene is disrupted by gene editing. In an embodiment, the expression of endogenous TCR-beta gene is disrupted in more than about 80% of cells. In an embodiment, the expression of endogenous TCR-beta gene is disrupted in more than about 90% of cells. In an embodiment, the expression of endogenous TCR-beta gene is disrupted in more than about 95% of cells.

[0013] In an embodiment, the expression of the endogenous TCR-alpha gene is disrupted by gene editing. In an embodiment, the endogenous TCR-alpha gene is disrupted in more than about 80% of the cells. In an embodiment, the endogenous TCR-alpha gene is disrupted in more than about 90% of the cells. In an embodiment, the endogenous TCR-alpha gene is disrupted in more than about 95% of the cells.

[0014] In an embodiment, the nucleic acid sequence further encodes a self-cleaving peptide. In an embodiment, the self-cleaving peptide is a self-cleaving viral peptide. In an embodiment, the self-cleaving viral peptide is T2A (SEQ ID NO: 49), P2A (SEQ ID NO: 50), E2A (SEQ ID NO: 51), F2A (SEQ ID NO: 52). In an embodiment, the unique self-cleaving viral peptide sequence is labeled.

[0015] In an embodiment, the engineered T cells express CD45RO, CC chemokine receptor type 7 (CCR7) and L-selectin (CD62L). In an embodiment, the engineered T cells have a central memory (CM) T cell phenotype. In an embodiment, the engineered T cells have a naive T cell phenotype. In an embodiment, the engineered T cells have a naive T cell phenotype are CD45RA+CD45RO-CD27+CD95-. In an embodiment, the engineered T cells have a stem cell memory T cell phenotype. In an embodiment, the engineered T cells have a stem cell memory T cell phenotype are CD45RA+CD45RO-CD27+CD95+CD58+CCR7-Hi TCF1+. In an embodiment, the engineered T cells have a central memory T cell phenotype. In an embodiment, the engineered T cells have a central memory T cell phenotype are CD45RO+CD45RA-CD27+CD95+CD58+. In embodiments, the engineered T cells have a precursor exhausted T cell phenotype. In embodiments, the engineered T cells with precursor exhausted T cell phenotype are PD-1+SLAMF6+TCF1+TIM3-CD39-. In embodiments, the engineered T cells with precursor exhausted T cell phenotype express PD-1 at low or intermediate levels compared to PD-1 high exhausted T cells. In embodiments, the engineered T cells with precursor exhausted T cell phenotype express PD-1 at low or intermediate levels compared to recently activated T cells. In embodiments, the T cells are autologous to a subject in need thereof.

[0016] In another related aspect, there is provided a pharmaceutical composition comprising a population of engineered T cells as described herein, including embodiments, and a pharma- ceutical acceptable excipient.

[0017] In embodiments, at least 10% of the cells in the composition comprising isolated T cells are engineered T cells. In embodiments, at least 20% of the cells are engineered T cells. In embodiments, at least 30% of the cells are engineered T cells. In embodiments, at least 40% of the cells are engineered T cells. In embodiments, at least 50% of the cells are engineered T cells. In embodiments, at least 60% of the cells are engineered T cells. In embodiments, at least 70% of the cells are engineered T cells. In embodiments, at least 80% of the cells are engineered T cells.

[0018] In an embodiment, the crude product is about 0.1×10 5 ~Approx. 1×10 11 In some embodiments, the composition comprises at least 1×10 engineered T cells. 8 In some embodiments, the composition comprises about 1×10 engineered T cells. 8 pieces~approx. 1×10 11 In some embodiments, the composition comprises at least 1×10 engineered T cells. 9 In some embodiments, the composition comprises at least 1×10 engineered T cells. 10 In some embodiments, the composition comprises at least 1×10 engineered T cells. 11 In an embodiment, the composition further comprises a pharma- ceutically acceptable excipient.

[0019] In another related embodiment, there is provided a T cell comprising an RNA transcript having an mRNA transcript transcribed from a TCR transgene inserted into the TCR-alpha or TCR-beta locus.

[0020] In another interrelated aspect, a non-viral method for generating engineered T cells is provided. In an embodiment, the method uses clustered regularly interspaced short palindromic repeats (CRISPR)-based gene editing. In an embodiment, the method uses Cas9. In an embodiment, the method uses high-fidelity Cas9. In an embodiment, the method uses SpyFi Cas9. In an embodiment, the gene editing reagents are provided as Cas9 ribonucleoprotein particles (RNPs). The method includes a) contacting a T cell with a first CRISPR / Cas9 RNP and donor DNA under conditions that allow the first CRISPR / Cas9 RNP and donor DNA to enter the T cell, where the first Cas9 / RNP comprises a first guide RNA that targets an endogenous TCR locus, and the donor DNA comprises a nucleic acid sequence encoding a TCR in a format specified herein; b) incubating the T cell for a period of time; and c) culturing the cell in a medium for a period of time to allow the donor DNA to recombine into the endogenous TCR locus, thereby forming an engineered T cell. In an embodiment, the TCR locus targeted by the first guide RNA is the TCR-alpha locus. In an embodiment, the TCR locus targeted by the second guide RNA is the TCR-beta locus. In an embodiment, the first RNP comprises a first gene editing protein and the first guide RNA in a molar excess of the guide RNA. In an embodiment, the first RNP comprises a first gene editing protein, and the ratio of the first guide RNA to the first gene editing protein is between 1:1 and 100:1.

[0021] In an embodiment, the TCR locus targeted by the first guide RNA is the TCR-alpha locus, and the T cell is further contacted with a second RNP comprising a second guide RNA targeting an endogenous TCR-beta locus. In an embodiment, the T cell is also contacted with a second donor DNA, the second donor DNA comprising a nucleic acid sequence encoding a TCR in a format as specified herein. The contacting of the cells can be performed in any order. In an embodiment, the T cell is contacted with the second RNP and optionally the second donor DNA in step a). In an embodiment, the T cell is contacted with the second RNP and the second donor DNA between steps a) and b). In an embodiment, the T cell is contacted with the second RNP and the second donor DNA before step a). In an embodiment, the T cell is contacted with the second RNP and the second donor DNA after step c). In an embodiment, the second RNP comprises the second gene editing protein and the second guide RNA in a molar excess of the guide RNA. In an embodiment, the second RNP comprises a second gene editing protein, and the ratio of the second guide RNA to the second gene editing protein is 1:1 to 100:1. In an embodiment, less than 10% of the engineered T cells express functional endogenous TCR-beta compared to a control. In an embodiment, less than 1% of the engineered T cells express functional endogenous TCR-beta compared to a control. In an embodiment, the engineered T cells do not express functional endogenous TCR-beta.

[0022] In embodiments, the first guide RNA is discovered by methods known to those of skill in the art to target the exon 1, exon 2, or exon 3 region of the TCR constant alpha region locus (TRAC). In embodiments, the first guide RNA comprises one or more of TRAC1 (SEQ ID NO: 7), TRAC2 (SEQ ID NO: 8), TRAC3 (SEQ ID NO: 9), TRAC4 (SEQ ID NO: 10), TRAC5 (SEQ ID NO: 11), TRAC6 (SEQ ID NO: 12), TRAC7 (SEQ ID NO: 13), TRAC8 (SEQ ID NO: 14), TRAC9 (SEQ ID NO: 15), TRAC10 (SEQ ID NO: 16), TRAC11 (SEQ ID NO: 17), TRAC12 (SEQ ID NO: 18), TRAC13 (SEQ ID NO: 19), TRAC14 (SEQ ID NO: 20), TRAC15 (SEQ ID NO: 21), or TRAC16 (SEQ ID NO: 22). In embodiments, the first guide RNA comprises one of TRAC1, TRAC3, TRAC4, TRAC5, TRAC7, TRAC12, or TRAC15. In embodiments, the first guide RNA targets TRAC1. In embodiments, the first guide RNA comprises TRAC3. In embodiments, the first guide RNA comprises a nucleic acid sequence in Table 10.

[0023] In an embodiment, the second guide RNA is discovered by methods known to those skilled in the art to target the exon 1 region of both TCR constant beta region loci (TRBC) in T cells. In an embodiment, the second guide RNA is selected from the group consisting of TRBC1 (SEQ ID NO: 23), TRBC2 (SEQ ID NO: 24), TRBC3 (SEQ ID NO: 25), TRBC4 (SEQ ID NO: 26), TRBC5 (SEQ ID NO: 27), TRBC6 (SEQ ID NO: 28), TRBC7 (SEQ ID NO: 29), TRBC8 (SEQ ID NO: 30), TRBC9 (SEQ ID NO: 31), TRBC10 (SEQ ID NO: 32), TRBC11 (SEQ ID NO: 33), TRBC12 (SEQ ID NO: 34), TRBC13 (SEQ ID NO: 35), T The second guide RNA comprises one or more of RBC14 (SEQ ID NO:36), TRBC15 (SEQ ID NO:37), TRBC16 (SEQ ID NO:38), TRBC17 (SEQ ID NO:39), TRBC18 (SEQ ID NO:40), TRBC19 (SEQ ID NO:41), TRBC20 (SEQ ID NO:42), TRBC21 (SEQ ID NO:43), TRBC22 (SEQ ID NO:44), TRBC23 (SEQ ID NO:45), TRBC24 (SEQ ID NO:46), TRBC25 (SEQ ID NO:47), or TRBC26 (SEQ ID NO:48). In an embodiment, the second guide RNA comprises one or more of TRBC4, TRBC8, TRBC13, TRBC19, TRBC20, TRBC21, TRBC22, TRBC23, or TRBC26. In an embodiment, the second guide RNA comprises a nucleic acid sequence of Table 11.

[0024] In embodiments, the conditions that allow the RNP and donor DNA to enter the cells comprise electroporation. In embodiments, the electroporation method comprises a commercially available electroporation kit (e.g., NUCLEOFECTION (Lonza)). In embodiments, method step b) comprises incubating the T cells for at least 10 minutes. In embodiments, the T cells are incubated at about 37° C. In embodiments, the T cells are incubated at less than about 37° C. In embodiments, the medium comprises a cytokine. In embodiments, the cytokine comprises interleukin-2 (IL-2), interleukin-7 (IL-7) and / or interleukin-15 (IL-15). In embodiments, the cytokine comprises IL-2. In embodiments, the cytokine comprises IL-7. In embodiments, the cytokine comprises IL-15. In embodiments, the cytokine comprises IL-7 and IL-15.

[0025] In some embodiments, method step a) is carried out in the presence of a negatively charged polymer. In embodiments, the polymer is poly(glutamic acid) (PGA) or a variant thereof, poly(aspartic acid), heparin, or poly(acrylic acid). In embodiments, the PGA is poly(L-glutamic acid) or a variant thereof. In embodiments, the PGA is poly(D-glutamic acid) or a variant thereof. In embodiments, the PGA or a variant thereof has an average molecular weight between 15 kilodaltons (kDa) and 50 kDa. In some embodiments, about 2 μg / μL to about 15 μg / μL of polymer is added.

[0026] In embodiments, the amount of the first and / or second RNP is about 0.2 pmol / μL to about 10 pmol / μL. In embodiments, the amount of donor DNA is about 0.5 fmol / μL to about 0.5 pmol / μL. In embodiments, the amount of RNP is about 5 pmol to about 200 pmol. In embodiments, the amount of donor DNA is about 0.01 pmol to about 10 pmol. In embodiments, a portion of the donor DNA recombines into an endogenous TCR-alpha locus. In embodiments, the TCR-alpha locus is a TCR-alpha constant region locus. In embodiments, the exogenous TCR-alpha (VJ) domain forms a portion of a heterologous TCR-alpha that comprises at least a portion of the endogenous TCR-alpha of the engineered T cell. In embodiments, the donor DNA comprises a left homology arm and a right homology arm.

[0027] In an embodiment, the left homology arm and the right homology arm are homologous to an endogenous TCR-alpha locus. In an embodiment, the left homology arm is about 50 bases to about 2000 bases in length. In an embodiment, the left homology arm is about 100 bases to about 1000 bases in length. In an embodiment, the left homology arm is about 200 bases to about 800 bases in length. In an embodiment, the right homology arm is about 200 bases to about 2000 bases in length. In an embodiment, the right homology arm is about 100 bases to about 1000 bases in length. In an embodiment, the left homology arm and the right homology arm are homologous to an endogenous TCR-beta locus. In an embodiment, the left homology arm is about 200 bases to about 800 bases in length. In an embodiment, the left homology arm is about 250 bases to about 700 bases in length. In an embodiment, the right homology arm is about 200 bases to about 800 bases in length. In an embodiment, the right homology arm is about 250 bases to about 700 bases in length.

[0028] In embodiments, the donor DNA comprises double stranded DNA (dsDNA). In embodiments, the donor DNA is on a plasmid, nanoplasmid, or minicircle. In embodiments, the donor DNA is contained within a plasmid. In embodiments, the donor DNA is on a nanoplasmid. In embodiments, the donor DNA is on a minicircle. In embodiments, the donor DNA is linear. In embodiments, the donor DNA is a PCR product. In embodiments, the donor DNA comprises single stranded DNA (ssDNA). In embodiments, the donor DNA is not chemically modified. In embodiments, the donor DNA comprises a chemical modification. In embodiments, the modification comprises a 5' phosphate or a 5' phosphorothioate.

[0029] In an embodiment, the donor DNA and the RNP are incubated together prior to method step a), In an embodiment, the gene editing protein comprises at least one nuclear localization signal (NLS).

[0030] In an embodiment, the T cells are activated prior to method step a). In an embodiment, the T cells are activated for 24 hours to 96 hours. In an embodiment, the T cells are activated in the presence of a cytokine. In an embodiment, the cytokine comprises IL-2, IL-7 and / or IL-15. In an embodiment, the T cells are activated in the presence of about 1 ng / mL to about 200 ng / mL of IL-2. In an embodiment, the T cells are activated in the presence of about 0 ng / mL to about 50 ng / mL of IL-2. In an embodiment, the T cells are activated in the presence of about 10 ng / mL of IL-2. In an embodiment, the T cells are activated in the presence of about 1 ng / mL to about 100 ng / mL of IL-7. In an embodiment, the T cells are activated in the presence of about 5 ng / mL to about 500 ng / mL of IL-15. In an embodiment, the T cells are activated in the presence of an anti-CD3 antibody and / or an anti-CD28 antibody. In an embodiment, the T cells are activated in the presence of a CD3 agonist and / or a CD28 agonist. In an embodiment, the anti-CD3 antibody and / or the anti-CD28 antibody are conjugated to a substrate. In an embodiment, the CD3 agonist and / or the CD28 agonist are conjugated to a substrate. In an embodiment, the method step)a is performed within about 24 hours after activation. In an embodiment, the method step)a is performed about 24 hours to about 72 hours after activation. In an embodiment, the method step)a is performed about 36 hours to about 60 hours after activation.

[0031] In another interrelated aspect, there is provided a method for generating a population of engineered T cells comprising performing a method as described herein, including embodiments, on a population of T cells. In embodiments, at least about 5% of the population of T cells are engineered T cells. In embodiments, between about 5% and about 100% of the population of T cells are engineered T cells. In embodiments, at least about 50% of the population of T cells are engineered T cells. In embodiments, at least about 60% of the population of T cells are engineered T cells. In embodiments, at least about 25% of the T cell population is viable after method step c). In embodiments, at least about 50% of the T cell population is viable after method step c). In embodiments, at least about 75% of the T cell population is viable after method step c). The amount may be any value or subrange within the recited range, including the endpoints.

[0032] In an embodiment, the method further comprises contacting the T cells with a second RNP comprising a guide RNA targeting an endogenous TCR-beta locus. In an embodiment, at least about 5% of the population of T cells are engineered T cells, and less than about 20% of the engineered T cells express endogenous TCR-beta. In an embodiment, about 10% to about 100% of the population of T cells are engineered T cells. Contacting the T cells with the second RNP can occur before, during, or after contact with the first RNP. In an embodiment, the first RNP comprises a guide RNA targeting an endogenous TCR-alpha locus.

[0033] In an embodiment, method step a) comprises the following steps, in any order: (i) adding donor DNA to the chamber, (ii) adding RNP to the chamber, and (iii) adding T cell(s) to the chamber. In an embodiment, method step a) comprises the following steps, in this order: (i) adding RNP to the chamber, (ii) adding donor DNA to the chamber, and (iii) adding T cells to the chamber. In an embodiment, method step a) comprises the following steps, in this order: (i) adding RNP to the chamber, (ii) adding T cells to the chamber, and (iii) adding donor DNA to the chamber. In an embodiment, method step a) comprises the following steps, in any order: (i) adding donor DNA to the chamber, (ii) adding RNP to the chamber, (iii) adding a negatively charged polymer to the chamber, and (iv) adding T cells to the chamber. In an embodiment, method step a) comprises the following steps, in this order: (i) adding donor DNA to the chamber, (ii) adding RNP to the chamber, (iii) adding a negatively charged polymer to the chamber, and (iv) adding T cells to the chamber. In an embodiment, method step a) comprises the following steps, in this order: (i) combining RNP and a negatively charged polymer to form an RNP-polymer mixture, (ii) adding donor DNA to the chamber, (iii) adding the RNP-polymer mixture to the chamber, and (iv) adding T cells to the chamber. In an embodiment, method step a) comprises the following steps, in this order: (i) adding RNP to the chamber, (ii) adding donor DNA to the chamber, (iii) adding a negatively charged polymer to the chamber, and (iv) adding T cells to the chamber. In an embodiment, T cells are not pipetted during method steps a) and b). In some embodiments, the negatively charged polymer comprises PGA. When more than one RNP and / or more than one donor DNA are used, they can be added simultaneously or in any order. In embodiments, no negatively charged polymer is added to the chamber.

[0034] In another related aspect, an engineered T cell is provided. The engineered T cell is produced by the methods described herein, including the embodiments. In another related aspect, an engineered T cell population is provided. The engineered T cell population is produced by the methods described herein, including the embodiments.

[0035] In another interrelated embodiment, a method is provided for treating a subject with cancer, the method comprising: a) providing a population of T cells, b) engineering at least a subset of the population of T cells to express an exogenous T cell receptor (TCR) and knock out endogenous TCR-beta, thereby forming an engineered population of T cells, where the exogenous TCR binds to an antigen expressed by the cancer, c) expanding the population of engineered T cells, and d) administering the expanded population of engineered T cells to the subject.

[0036] In embodiments, the antigen is a neo-antigen or tumor associated antigen (TAA). In embodiments, at least a portion of the genome and / or transcriptome of the cancer has been sequenced to determine the presence of the antigen. In embodiments, the engineered T cells are generated using the methods described herein, including embodiments. In embodiments, the antigen is Wilms tumor gene 1 (WT1), Janus kinase 2 (JAK2), New York esophageal squamous cell carcinoma-1 (NY-ESO1), PRAME nuclear receptor transcription regulator (PRAME) or mutant Kirsten rat sarcoma virus (KRAS). In embodiments, the antigen is specific for the cancer. In embodiments, the TCR binds to an antigen presented on a major histocompatibility complex class I (MHCI) molecule. In embodiments, the MHCI comprises an MHCI allele expressed by the subject. In embodiments, the expanded population of engineered T cells comprises 1×10 5 ~1×10 9 In embodiments, the expanded population of engineered T cells comprises at least 1 x 108 In embodiments, the expanded population of engineered T cells comprises about 1×10 8 pieces~approx. 1×10 11 In embodiments, the expanded population of engineered T cells comprises at least 1 x 10 9 In embodiments, the expanded population of engineered T cells comprises at least 1 x 10 10 In embodiments, the expanded population of engineered T cells comprises at least 1 x 10 11 In embodiments, the T cells are autologous to the subject.

[0037] In another interrelated embodiment, a method is provided for treating a subject with cancer, the method comprising: a) providing a first population of T cells isolated from the subject; b) engineering at least a subset of the first population of T cells to express a first exogenous T cell receptor (TCR) and knock out endogenous TCR-beta, thereby forming a first population of engineered T cells, where the exogenous TCR binds to a first antigen expressed by the cancer; c) expanding the first population of engineered T cells; and d) administering the expanded first population of engineered T cells to the subject. e) providing a second population of T cells isolated from the subject; f) engineering at least a subset of the second population of T cells to express a second exogenous TCR and knock out endogenous TCR-beta, thereby forming a second population of engineered T cells, wherein the exogenous TCR binds to a second antigen expressed by the cancer; g) expanding the second population of engineered T cells; and h) administering the expanded second population of engineered T cells to the subject.

[0038] In an embodiment, at least a portion of the genome or transcriptome of the cancer is sequenced to determine the presence of a first antigen and a second antigen. In an embodiment, the first antigen is WT1, JAK2, NY-ESO1, PRAME, mutant KRAS, or HPV. In an embodiment, the first TCR and / or the second TCR binds to an antigen presented on an MHCl molecule. In an embodiment, the antigen is a neoantigen or a TAA. In an embodiment, the MHCI comprises an MHCI allele expressed by the subject.

[0039] In an embodiment, the expanded population of first engineered T cells is greater than or equal to 1×10 5 ~1×10 9 In an embodiment, the expanded population of first engineered T cells comprises at least 1 x 10 8 In an embodiment, the expanded population of first engineered T cells comprises at least 1 x 10 9 In an embodiment, the expanded population of first engineered T cells comprises about 1×10 8 pieces~approx. 1×10 11 In an embodiment, the expanded population of second engineered T cells comprises 1×10 5 ~1×10 9 In an embodiment, the expanded population of second engineered T cells comprises at least 1 x 10 8 In an embodiment, the expanded population of second engineered T cells comprises at least 1 x 10 9 In an embodiment, the expanded population of second engineered T cells comprises about 1 x 10 8 pieces~approx. 1×10 11 The engineered T cells include

[0040] In an embodiment, the T cells are autologous to the subject. In an embodiment, an additional population of engineered T cells is administered to the patient, where the T cells in the additional population of engineered T cells express a third exogenous TCR that binds to a third antigen expressed by the cancer and do not express endogenous TCR-beta. In an embodiment, an additional population of engineered T cells is administered to the patient, where the T cells in the additional population of engineered T cells express up to five exogenous TCRs that bind to an antigen expressed by the cancer (one at a time) and do not express endogenous TCR-beta. In an embodiment, an additional population of engineered T cells is administered to the patient, where the T cells in the additional population of engineered T cells express up to ten exogenous TCRs that bind to an antigen expressed by the cancer (one at a time) and do not express endogenous TCR-beta. In embodiments, further additional plurality of engineered T cells are administered to the patient, where the T cells in these additional plurality of engineered T cells express additional exogenous TCRs that bind (one at a time) to additional antigens expressed by the cancer, and do not express endogenous TCR-beta.

[0041] In another related aspect, a method of treating cancer is provided. The method comprises administering a T cell, composition, or pharmaceutical composition described herein, including embodiments, to a patient with cancer. In embodiments, the method further comprises administering an anti-cancer therapy to the subject. In embodiments, the anti-cancer therapy comprises immunotherapy, chemotherapy, and / or radiation.

[0042] In another interrelated aspect, a guide RNA is provided. The first guide RNA is discovered by methods known to those skilled in the art to target the exon 1, exon 2 or exon 3 region of the TCR constant alpha region locus (TRAC). In an embodiment, the guide RNA targets the endogenous TCR-alpha locus. In an embodiment, the guide RNA targets one or more of the following sequences from the TCR-alpha locus: TRAC1 (SEQ ID NO: 7), TRAC2 (SEQ ID NO: 8), TRAC3 (SEQ ID NO: 9), TRAC4 (SEQ ID NO: 10), TRAC5 (SEQ ID NO: 11), TRAC6 (SEQ ID NO: 12), TRAC7 (SEQ ID NO: 13), TRAC8 (SEQ ID NO: 14), TRAC9 (SEQ ID NO: 15), TRAC10 (SEQ ID NO: 16), TRAC11 (SEQ ID NO: 17), TRAC12 (SEQ ID NO: 18), TRAC13 (SEQ ID NO: 19), TRAC14 (SEQ ID NO: 20), TRAC15 (SEQ ID NO: 21), or TRAC16 (SEQ ID NO: 22). In an embodiment, the guide RNA targeting the endogenous TCR-alpha locus comprises a nucleic acid sequence of Table 10. In an embodiment, the endogenous TCR-alpha locus is an endogenous TCR-alpha constant region.

[0043] In another interrelated embodiment, a guide RNA is provided. The second guide RNA is discovered by methods known to those skilled in the art to target the exon 1 region of both TCR constant beta region loci (TRBC). In an embodiment, the guide RNA targets the endogenous TCR-beta locus. In an embodiment, the guide RNA targets the endogenous TCR-beta locus. In an embodiment, the guide RNA targets the exon 1 region of both TCR-beta loci, TRBC1 (SEQ ID NO: 23), TRBC2 (SEQ ID NO: 24), TRBC3 (SEQ ID NO: 25), TRBC4 (SEQ ID NO: 26), TRBC5 (SEQ ID NO: 27), TRBC6 (SEQ ID NO: 28), TRBC7 (SEQ ID NO: 29), TRBC8 (SEQ ID NO: 30), TRBC9 (SEQ ID NO: 31), TRBC10 (SEQ ID NO: 32), TRBC11 (SEQ ID NO: 33), TRBC12 (SEQ ID NO: 34), TRBC13 (SEQ ID NO: 35), TRBC14 (SEQ ID NO: 36), TRBC15 (SEQ ID NO: 37), TRBC16 (SEQ ID NO: 38), TRBC17 (SEQ ID NO: 39), TRBC18 (SEQ ID NO: 40), TRBC19 (SEQ ID NO: 41), TRBC20 (SEQ ID NO: 42), TRBC21 (SEQ ID NO: 43), TRBC22 (SEQ ID NO: 44), TRBC23 (SEQ ID NO: 45), TRBC24 (SEQ ID NO: 46), TRBC25 (SEQ ID NO: 47), TRBC26 (SEQ ID NO: 48), TRBC27 (SEQ ID NO: 49), TRBC28 (SEQ ID NO: 50), TRBC29 (SEQ ID NO: 51), TR 35), TRBC14 (SEQ ID NO: 36), TRBC15 (SEQ ID NO: 37), TRBC16 (SEQ ID NO: 38), TRBC17 (SEQ ID NO: 39), TRBC18 (SEQ ID NO: 40), TRBC19 (SEQ ID NO: 41), TRBC20 (SEQ ID NO: 42), TRBC21 (SEQ ID NO: 43), TRBC22 (SEQ ID NO: 44), TRBC23 (SEQ ID NO: 45), TRBC24 (SEQ ID NO: 46), TRBC25 (SEQ ID NO: 47), or TRBC26 (SEQ ID NO: 48). In an embodiment, the guide RNA targeting the endogenous TCR-beta locus comprises a nucleic acid sequence of Table 11.

[0044] In another interrelated aspect, a nucleic acid is provided. The nucleic acid comprises a nucleic acid sequence comprising an exogenous TCR-beta coding sequence and an exogenous TCR-alpha coding sequence, the nucleic acid sequence further comprising a first self-cleaving peptide coding sequence. In an embodiment, the nucleic acid further comprises a first homology arm and a second homology arm. In an embodiment, the nucleic acid further comprises a second self-cleaving peptide coding sequence. In an embodiment, the nucleic acid comprises, in 5' to 3' order: (i) a first homology arm, (ii) a first self-cleaving viral peptide coding sequence, (iii) an exogenous TCR-beta coding sequence, (iv) a second self-cleaving viral peptide coding sequence, (v) an exogenous TCR-alpha coding sequence, and (vi) a second homology arm. In an embodiment, the nucleic acid further comprises an enzymatic cleavage site. In an embodiment, the enzymatic cleavage site is located between the TCR-beta coding sequence and the second self-cleaving viral peptide coding sequence. In an embodiment, the enzyme cleavage site is a furin enzyme cleavage site. In an embodiment, the nucleic acid further comprises a GSG amino acid sequence. In an embodiment, GSG precedes one or both of the first self-cleaving viral peptide coding sequence and the second self-cleaving viral peptide coding sequence.

[0045] In embodiments, the nucleic acid further comprises a polyadenylation (polyA) sequence. In embodiments, the polyadenylation sequence may be located immediately 3' to the TCR coding sequence. In embodiments, the polyadenylation sequence may be located immediately 5' to the second homology arm. In embodiments, the polyadenylation sequence is the bovine growth hormone polyadenylation sequence (bgh-polyA).

[0046] In an embodiment, the first homology arm is homologous to an endogenous TCR-alpha locus in a human T cell. In an embodiment, the second homology arm is homologous to an endogenous TCR-alpha locus in a human T cell. In an embodiment, the endogenous TCR-alpha locus is a TCR-alpha constant region. In an embodiment, the first self-cleaving viral peptide is T2A, P2A, E2A or F2A. In an embodiment, the second self-cleaving viral peptide is T2A, P2A, E2A or F2A. In an embodiment, the first self-cleaving viral peptide and the second self-cleaving viral peptide are different. In an embodiment, the first self-cleaving peptide coding sequence is 5' of an exogenous TCR-beta coding sequence. In an embodiment, the second self-cleaving peptide coding sequence is 5' of an exogenous TCR-alpha coding sequence. In an embodiment, the second self-cleaving peptide coding sequence is 5' to the exogenous TCR-alpha coding sequence.In an embodiment, the nucleic acid is a plasmid, nanoplasmid or minicircle.

[0047] In another interrelated aspect, a kit for producing engineered T cells is provided. The kit comprises a TCR-alpha targeted guide RNA as described herein, including embodiments. In an embodiment, the kit further comprises a TCR-beta targeted guide RNA as described herein, including embodiments. In an embodiment, the kit further comprises a gene editing reagent or a nucleic acid encoding a gene editing reagent. In an embodiment, the gene editing reagent is a CRISPR system. In an embodiment, the kit further comprises donor DNA. In an embodiment, the donor DNA comprises a nucleic acid sequence encoding a polypeptide comprising an exogenous TCR-beta domain and an exogenous TCR-alpha domain. In an embodiment, the donor DNA comprises a nucleic acid sequence encoding a polypeptide comprising an exogenous TCR-beta domain and an exogenous TCR-alpha (VJ) domain. In an embodiment, the exogenous TCR-beta and the heterologous TCR-alpha form a TCR capable of binding to an antigen. In an embodiment, the TCR binds to an antigen presented on a major histocompatibility complex class I (MHCI) molecule. In embodiments, the antigen is WT1, JAK2, NY-ESO1, PRAME, mutant KRAS, or an antigen of Table 1 or Table 2. In embodiments, the kit further comprises poly(glutamic acid) (PGA) or a variant thereof. In embodiments, the kit further comprises a nucleic acid as described herein, including embodiments. [Brief description of the drawings]

[0048] [Figure 1A]1A and 1B are bar graphs showing knock-in efficiency of TRAC1-mNeon template in response to varying electroporation conditions. FIG. 1A shows the frequency of knock-in positive mNeon+TCRab+ cells, and FIG. 1B shows the frequency of residual TCRab expressing cells. Cells were electroporated with TRAC1 RNP only (RNP), NTC (non-targeted guide control without TCR knockout) RNP + 4 μg of TRAC1-mNeon (4 μg), or 6 μg / 4 μg / 2 μg / 1 μg of TRAC1-mNeon + TRAC1 RNP (6 μg, 4 μg, 2 μg and 1 μg, respectively). All cells were electroporated in P2 buffer with EH100 pulse code, except for the rightmost group of the two bars, which represents the group of cells electroporated in P3 buffer with EH115 pulse code. After electroporation, cells were incubated at either 37° C. or 32° C. [Figure 1B] Same as above.

[0049] [Figure 2A] Figures 2A and 2B are bar graphs showing the recovery of total CD8+ cells (Figure 2A) and knock-in positive CD8+ T cells (Figure 2B) depending on the amount of template used for electroporation. Groups of cells electroporated with RNP only (RNP) or NTC + 4 μg template (NTC + 4 μg) served as controls. After electroporation, cells were incubated at either 37°C or 32°C. [Figure 2B] Same as above.

[0050] [Diagram 3] FIG. 3 is a bar graph showing cell viability after electroporation for the data in FIGS. 2A-2B.

[0051] [Figure 4]Figure 4 is a flow cytometry analysis showing knock-in of the TRAC1-mNeon template. The top row shows an analysis of cells electroporated in P2 buffer using the EH100 pulse code, and the bottom row shows an analysis of cells electroporated in P3 buffer using the EH115 pulse code. The green (top) box in each plot indicates detection of TRAC1-mNeon knock-in cells, and the red (bottom) box indicates detection of residual TCRab expressing cells. T cells from three different donors were evaluated.

[0052] [Diagram 5] Figure 5 is a flow cytometry dot plot showing knock-in of TRAC1-mNeon template. The top and bottom rows are dot plots representing cell populations electroporated with 6 μg or 4 μg of template, respectively. The green (top) box in each plot indicates detection of TRAC1-mNeon knock-in cells, and the red (bottom) box indicates detection of residual TCRab expressing cells. T cells from three different donors were evaluated.

[0053] [Figure 6] Figure 6 is a flow cytometry analysis showing knock-in of TRAC1-mNeon template. The top and bottom rows are dot plots representing cells electroporated with 2 μg or 1 μg of template, respectively. The green (top) boxes in each plot indicate detection of TRAC1-mNeon knock-in cells, and the red (bottom) boxes indicate detection of residual TCRab expressing cells. T cells from three different donors were evaluated.

[0054] [Figure 7]Figure 7 is a flow cytometry analysis showing no significant off-target integration. The top and bottom rows show dot plots representing cell populations electroporated with RNP alone or NTC RNP + 4 μg TRAC 1-mNeon, respectively. The green (top) box in each plot indicates detection of TRAC1-mNeon knock-in cells, and the red (bottom) box indicates detection of TCRab expressing cells. T cells from three different donors were evaluated.

[0055] [Figure 8-1] Figures 8A and 8B show knock-in of TRAC1-mNeon (Figure 8A) and Rab11A-YFP (Figure 8B) templates in T cells. The left panel shows flow cytometry analysis to detect knock-in positive cells, and the right panel is a graph showing the percentage of knock-in depending on the amount of template used for electroporation. T cells from three different donors were evaluated. [Figure 8-2] Same as above.

[0056] [Figure 8-3] (C) Flow cytometry plots showing no significant off-target integration by NTC RNP. Various donor templates and Cas9 enzymes were tested. T cells from three different donors were evaluated. [Figure 8-4] Same as above. [Figure 8-5] Same as above. [Figure 8-6] Same as above.

[0057] [Figure 8-7] FIG. 8D is a flow cytometry analysis of T cells electroporated with TRAC1 RNP+4 μg TRAC1-mNeon template (right) and NTC RNP+4 μg TRAC1-mNeon template (left). [Figure 8-8] Same as above. [Figure 8-9] Same as above.

[0058] [Figure 9A] Figures 9A and 9B are bar graphs showing knock-in efficiency as a function of the TRAC locus used for knockout and the amount of template DNA used for electroporation. Figure 9A shows the frequency of knock-in positive mNeon+TCRab+ cells generated using the TRAC1-mNeon template, and Figure 9B shows the frequency of knock-in positive mNeon+TCRab+ cells generated using the TRAC3-mNeon template. [Figure 9B] Same as above.

[0059] [Figure 10A] 10A-10C are bar graphs showing TCRab knockout efficiency. The graphs show the frequency of TCR+Neon- cells after electroporation with various amounts of TRAC1-mNeon template (FIG. 10A), TRAC3-mNeon template (FIG. 10B), or no template (FIG. 10C). [Figure 10B] Same as above. [Figure 10C] Same as above.

[0060] [Figure 11A] 11A-11D are bar graphs showing the recovery of knock-in positive mNeon+TCRab+ T cells as a function of the amount of template used. FIG. 11A and FIG. 11B show the recovery of mNeon+TCRab+ cells from T cells electroporated with various amounts of TRAC1-mNeon template and TRAC3-mNeon template, respectively. FIG. 11C and FIG. 11D show the recovery of total CD8+ cells from cells electroporated with various amounts of TRAC1-mNeon template and TRAC3-m template, respectively. [Figure 11B] Same as above. [Figure 11C] Same as above. [Figure 11D] Same as above.

[0061] [Figure 12A]12A and 12B are bar graphs showing cell viability (percentage of live cells) following electroporation with various amounts of TRAC1-mNeon (FIG. 14A) or TRAC3-mNeon (FIG. 14B) templates. Cell viability was assessed by the absence of 7AAD staining. [Figure 12B] Same as above.

[0062] [Figure 13] Figure 13: Flow cytometry analysis showing knock-in of TRAC1-mNeon (top) and TRAC3-mNeon (bottom) templates. 6 μg of template was used for electroporation. Green (top) boxes indicate detection of knock-in cells, and red (bottom) boxes indicate detection of residual TCRab expressing cells.

[0063] [Figure 14] Figure 14 is a flow cytometry dot plot showing knock-in of TRAC1-mNeon (top) and TRAC3-mNeon (bottom) templates. 4 μg of each template was used for electroporation. Green (top) boxes indicate detection of knock-in cells, and red (bottom) boxes indicate detection of residual TCRab expressing cells.

[0064] [Figure 15] Figure 15 is a flow cytometry dot plot showing knock-in of TRAC1-mNeon (top) and TRAC3-mNeon (bottom) templates. 2 μg of each template was used for electroporation. Green (top) boxes indicate detection of knock-in cells, and red (bottom) boxes indicate detection of residual TCRab expressing cells.

[0065] [Figure 16]Figure 16 is a flow cytometry dot plot showing knock-in of TRAC1-mNeon (top) and TRAC3-mNeon (bottom) templates. 1 μg of each template was used for electroporation. Green (top) boxes indicate detection of knock-in cells, and red (bottom) boxes indicate detection of residual TCRab expressing cells.

[0066] [Figure 17] Figure 17 is a graph showing knock-in of TRAC3-mNeon template in T cells (top) and cell viability after electroporation (bottom) as a function of the amount of RNP used for electroporation. T cells were electroporated with various amounts of TRAC3 RNP and a fixed amount of TRAC3-mNeon template. Cells electroporated without template RNP (NT RNP) served as a negative control.

[0067] [Figure 18-1] FIG. 18 is a flow cytometry dot plot showing knock-in of TRAC3-mNeon template in T cells as a function of the amount of TRAC3-mNeon template and TRAC3 RNP used in electroporation for samples from the first donor. [Figure 18-2] Same as above. [Figure 18-3] Same as above. [Figure 18-4] Same as above.

[0068] [Figure 19-1] FIG. 19 is a flow cytometry dot plot showing knock-in of TRAC3-mNeon template in T cells as a function of the amount of TRAC3-mNeon template and TRAC3 RNP used in electroporation for samples from a second donor. [Figure 19-2] Same as above. [Figure 19-3] Same as above. [Figure 19-4] Same as above.

[0069] [Figure 20-1] FIG. 20 is a flow cytometry dot plot showing knock-in of TRAC3-mNeon template in T cells as a function of the amount of TRAC3-mNeon template and TRAC3 RNP used in electroporation for samples from a third donor. [Figure 20-2] Same as above. [Figure 20-3] Same as above. [Figure 20-4] Same as above.

[0070] [Figure 21] FIG. 21 shows flow cytometry histograms showing TCRab knockout using various amounts of TRAC3 RNP (KO RNP).

[0071] [Figure 22] Figure 22 is an image of an agarose gel showing the forward (pFW) and reverse (pRV) ssDNA products generated from a Strandase reaction using a dsDNA template. The strandase reaction was driven to completion using 20 μg of double-stranded TRAC3-mNeon and TRAC3-NY-ESO1. The leftmost lane shows molecular weight markers. The ssDNA yield from the reaction is shown on the right.

[0072] [Figure 23-1]Figures 23A-C show knock-in efficiency and cell viability following electroporation with either ss or ds TRAC 3-mNeon templates. Figure 23A is a flow cytometry dot plot showing TRAC3-mNeon knock-in in T cells. Cells were subjected to electroporation with TRAC3 NRP or control NT RNP and various amounts of TRAC3-mNEON template. Either dsDNA, forward ssDNA strand (ssDNA FW) or reverse ssDNA (ssDNA RV) templates were used. Figure 23B is a graph showing cell viability and Figure 23C is a graph showing knock-in percentage under the indicated conditions. [Figure 23-2] Same as above. [Figure 23-3] Same as above. [Figure 23-4] Same as above. [Figure 23-5] Same as above. [Figure 23-6] Same as above. [Figure 23-7] Same as above.

[0073] [Figure 24A] Figures 24A and 24B show knock-in efficiency depending on the media conditions used for various TRANSACT™ titrations and T cell activation. Bar graphs show the frequency of knock-in positive T cells 4 days (Figure 24A) or 5 days (Figure 24B) after electroporation with the TRAC3-mNeon template. Media and cytokine conditions tested were RPMI and X-VIVO (XV) media containing either IL-2 (10 ng / mL), IL-7 (25 ng / mL) and IL-15 (50 ng / mL) (2 / 7 / 15), or IL-7 (25 ng / mL) and IL-15 (50 ng / mL) (7 / 15). [Figure 24B] Same as above.

[0074] [Figure 25A]Figures 25A and 25B are graphs showing the recovery and proliferation of knock-in positive mNeon+TCRab+ cells after electroporation with TRAC3-mNeon. Figure 25A shows the recovery of CD8+ cells after electroporation of cells activated with various TRANSACT™ titrations. Media and cytokine conditions tested are as shown in Figure 24. Figure 25B shows the proliferation of mNeon+TCRab+ cells from day 4 to day 5 after electroporation with cells activated with various media and TRANSACT™ titration conditions. TA20, TA50, TA100 and TA200 represent 1:20, 1:50, 1:100 and 1:200 TRANSACT™ titrations, respectively. RPMI and XV represent RPMI medium and X-VIVO medium, respectively. [Figure 25B] Same as above.

[0075] [Figure 26A] Figures 26A and 26B are graphs showing CD8+ cell recovery and proliferation after electroporation with TRAC3-mNeon. Figure 26A shows CD8+ cell recovery from cells activated with various TRANSACT™ titrations. Media and cytokine conditions tested are as shown in Figure 24. Figure 26B shows CD8+ cell proliferation from day 4 to day 5 after electroporation using cells activated with various media and TRANSACT™ titration conditions. TA20, TA50, TA100 and TA200 represent 1:20, 1:50, 1:100 and 1:200 TRANSACT™ titrations, respectively. Cells were grown in RPMI medium or X-VIVO™ (XV) medium. [Figure 26B] Same as above.

[0076] [Figure 27A]Figures 27A and 27B are bar graphs showing the knock-in efficiency of TRAC3-mNeon for electroporation performed 48 or 72 hours after activation using various T cell activation conditions. Figures 27A and 27B show the percentage of knock-in positive mNeon+TCRab+ cells 4 or 5 days after electroporation, respectively. Media and cytokine conditions tested are as shown in Figure 24. [Figure 27B] Same as above.

[0077] [Figure 28] FIG. 28 shows graphs demonstrating proliferation of knock-in positive (mNeon+TCRab+) CD8+ T cells after electroporation at 72 hours post-activation (left) and a comparison of cell proliferation after electroporation at 48 hours versus 72 hours post-activation (right). Various media conditions and TRANSACT™ titrations were tested for T cell activation. 48 hours and 72 hours indicate data points at 48 hours and 72 hours post-activation, respectively; TA20, TA50, TA100 and TA200 indicate 1:20, 1:50, 1:100 and 1:200 TRANSACT™ titrations, respectively; RPMI and XV indicate RPMI medium and X-VIVO medium, respectively.

[0078] [Figure 29-1] Figures 29A and 29B are flow cytometry dot plots showing detection of mNeon+TCRab+ T cells activated in RPMI medium prior to electroporation. Figure 29A shows analysis of cells activated with 1:50 TRANSACT™ and 1:200 TRANSACT™, and Figure 29B shows analysis of cells activated with 1:20 and 1:100 TRANSACT™ titrations. [Figure 29-2] Same as above. [Figure 29-3] Same as above. [Figure 29-4] Same as above.

[0079] [Figure 30-1]Figures 30A and 30B are flow cytometry dot plots showing detection of mNeon+TCRab+T cells activated with X-VIVO™ medium prior to electroporation. Figure 30A shows analysis of cells activated with 1:50 TRANSACT™ and 1:200 TRANSACT™, and Figure 30B shows analysis of cells activated with 1:20 and 1:100 TRANSACT™ titrations. [Figure 30-2] Same as above. [Figure 30-3] Same as above. [Figure 30-4] Same as above.

[0080] [Figure 31-1] Figures 31A and 31B are flow cytometry dot plots showing detection of knock-in positive mNeon+TCRab+ cells activated in RPMI or X-VIVO™ medium prior to electroporation. Figure 31A shows analysis of cells activated with 1:50 TRANSACT™, and Figure 31B shows analysis of cells activated with 1:100 TRANSACT™ titration. [Figure 31-2] Same as above. [Figure 31-3] Same as above. [Figure 31-4] Same as above.

[0081] [Diagram 32] Figure 32 is an image of an agarose gel showing the forward (pFW) and reverse (pRV) ssDNA products generated from a Strandase reaction using a double-stranded TRAC3-mNeon template. The left-most lane of the agarose gel is a molecular weight marker. The ssDNA yield from the reaction is shown on the right.

[0082] [Figure 33A]Figures 33A and 33B are flow cytometry analyses showing knock-in efficiency of dsDNA, forward single stranded DNA (ssDNA FW) and reverse ssDNA (ssDNA RV) TRAC-mNeon template DNA by detection of mNeon+TCRab+T cells. Figure 33A shows an analysis of cells from donor 1 and Figure 33B shows an analysis of cells from donor 2. [Figure 33B] Same as above.

[0083] [Figure 34A] FIG. 34A and FIG. 34B are graphs showing the frequency of mNeon+TCRab+ T cells (FIG. 34A) and % viable cells (FIG. 34B) in response to various amounts of either double-stranded DNA (ds), forward ssDNA (ssDNA-FW) or reverse ssDNA (ssDNA-RV) TRAC-mNeon template used for electroporation. [Figure 34B] Same as above.

[0084] [Figure 35A] Figures 35A and 35B are graphs showing TCRab knockout due to interference with template knockout efficiency. Figure 35A shows percent TCRab expression, and Figure 35B shows percent TCRab knockout. ds indicates dsDNA, ssFW indicates ssDNA forward strand, and ssRV indicates ssDNA reverse strand. [Figure 35B] Same as above.

[0085] [Figure 36-1]Figures 36A-C show TRAC-mNeon knock-in efficiency as a function of different knock-in sites and different amounts of TRACsg RNP included in electroporation. Figure 36A is a schematic of the TRAC locus with different sgRNA sites. Figure 36B is a flow cytometry analysis showing knock-in efficiency using TRACsg1, TRACsg3, TRACsg4 and TRACsg5 knock-in sites, and Figure 36C shows knock-in efficiency using TRACsg7, TRACsg12 and TRACsg15 knock-in sites. [Figure 36-2] Same as above. [Figure 36-3] Same as above. [Figure 36-4] Same as above. [Figure 36-5] Same as above.

[0086] [Figure 37] FIG. 37 is a graph showing mNeon knock-in efficiency by detection of mNeon+TCR+ cells (top) and cell viability after electroporation using various amounts of each indicated template (bottom).

[0087] [Figure 38A] Figures 38A-C show the effect of media and supplements on mNeon+ knock-in in T cells after activation and electroporation. Figure 38A is a bar graph showing cell recovery 5 days after initial T cell activation, and Figure 38B is a bar graph showing cell recovery 7 days after initial T cell activation. Figure 38C is a graph showing T cell proliferation 5-7 days for knock-in+ cells. Cells were cultured and activated in RPMI, X-Vivo (XV), supplemented X-Vivo (sXV or Sup.XV) supplemented with either IL-2+IL-7+IL-15 (2 / 7 / 15) or IL-7+IL-15 (7 / 15). TA30, TA100 and TA200 represent TRANSACT™ titrations of 1:30, 1:100 and 1:200, respectively. [Figure 38B] Same as above. [Figure 38C] Same as above.

[0088] [Figure 39A] Figures 39A and 39B show the effect of media and supplements on mNeon knock-in after T cell activation and electroporation. Figure 39A is a bar graph showing the frequency of knock-in positive mNeon+TCRab+ cells 5 days after initial T cell activation, and Figure 39B is a bar graph showing the frequency of knock-in positive mNeon+TCRab+ cells 7 days after initial T cell activation. Cells were cultured and activated under the conditions shown in Figure 38. [Figure 39B] Same as above.

[0089] [Figure 40A] Figures 40A and 40B show the effect of activation media and supplements on CD8+ T cell recovery after electroporation. Figure 40A is a bar graph showing cell recovery 5 days after initial T cell activation, and Figure 40B is a bar graph showing cell recovery 7 days after initial T cell activation. Cells were cultured and activated under the conditions shown in Figure 38. [Figure 40B] Same as above.

[0090] [Diagram 41] 41 is a graph showing total CD8+ cell proliferation between days 5 and 7 after T cell activation. Cells were cultured and activated under the conditions shown in FIG.

[0091] [Diagram 42] Figure 42 is a bar graph showing the fold expansion of total CD8+ T cells (black bars) and knock-in positive Neon+TCRab+ cells (red bars) from the time of electroporation to day 5 post-electroporation. Cells were cultured and activated under the conditions shown in Figure 40.

[0092] [Figure 43-1] Figure 43 shows flow cytometry dot plots showing the effect of TRANSACT™ titration in RPMI medium on mNeon knock-in efficiency. Medium contained IL-2, IL-7 and IL-15 cytokines. [Figure 43-2] Same as above.

[0093] [Figure 44-1] Figure 44 shows flow cytometry dot plots showing the effect of TRANSACT™ titration in X-VIVO™ medium on mNeon knock-in efficiency of a representative TRAC locus donor DNA. The medium contained IL-2, IL-7 and IL-15 cytokines. [Figure 44-2] Same as above.

[0094] [Figure 45-1] Figure 45 shows flow cytometry dot plots showing the effect of TRANSACT™ titration in X-VIVO™ medium on mNeon knock-in efficiency. The medium contained IL-7 and IL-15 cytokines. [Figure 45-2] Same as above.

[0095] [Figure 46-1] Figure 46 shows flow cytometry dot plots demonstrating the effect of TRANSACT™ addition in supplemented X-VIVO™ medium on mNeon knock-in efficiency. Medium contained IL-2, IL-7 and IL-15 cytokines. [Figure 46-2] Same as above.

[0096] [Figure 47-1] Figure 47 shows flow cytometry analysis demonstrating the effect of TRANSACT™ titration in supplemented X-VIVO™ medium on mNeon knock-in efficiency. Medium contained IL-7 and IL-15 cytokines. [Figure 47-2] Same as above.

[0097] [Figure 48-1]Figures 48A and 48B are flow cytometry dot plots showing the effect of IL-2 on knock-in efficiency and frequency of knock-in positive mNeon+TCR+ cells in T cells (Figure 48A) using RAB11A-GFP (left) or TRAC3 (right) templates (Figure 48B). [Figure 48-2] Same as above. [Figure 48-3] Same as above.

[0098] [Figure 49-1] Figure 49A and Figure 49B show the effect of resting cells after electroporation on knock-in efficiency. Figure 49A shows flow cytometry analysis of cells electroporated with mNeon-TRAC3 template 3 days after electroporation, and Figure 49B shows flow cytometry analysis of cells electroporated with Rab11A-YFP template 3 days after electroporation. After electroporation, cells were subjected to the following conditions (from left to right): 100 μL of buffer was added and immediately transferred; 20 μL of buffer was added and incubated at 37°C for 15 minutes; 100 μL of buffer was added and incubated at 37°C for 15 minutes; 5 minutes of incubation followed by 20 μL of medium and then incubated at 37°C for 15 minutes; or 5 minutes of incubation followed by 100 μL of medium and then incubated at 37°C for 15 minutes. [Figure 49-2] Same as above. [Figure 49-3] Same as above. [Figure 49-4] Same as above. [Figure 49-5] Same as above. [Figure 49-6] Same as above.

[0099] [Figure 50] FIG. 50 is a graph showing the frequency of knock-in positive mNeon+TCR+ cells under each of the conditions tested in FIG. 49A and FIG. 49B.

[0100] [Figure 51-1]Figures 51A-C show knock-in efficiency as a function of varying amounts of either unmodified dsDNA template or 5'-phosphate modified dsDNA PCR template used for electroporation. Figure 51A is a flow cytometry dot plot showing detection of knock-in positive mNeon+TCRab+ cells, Figure 51B is a graph showing the frequency of mNeon+TCRab+ cells, and Figure 51C is a graph showing cell viability by analysis of PI negative cells after electroporation with dsDNA template with (w / 5'-P) or without (w / o 5'-P) 5'-phosphate. [Figure 51-2] Same as above. [Figure 51-3] Same as above. [Figure 51-4] Same as above.

[0101] [Figure 52-1] Figures 52A-C show knock-in efficiency using various amounts of either unmodified or 5'-phosphorothioate modified dsDNA templates for electroporation. Figure 52A is a flow cytometry analysis showing detection of knock-in positive mNeon+TCRab+ cells, Figure 52B is a graph showing the frequency of mNeon+TCRab+ cells, and Figure 52C is a graph showing cell viability by analysis of PI negative cells after electroporation using dsDNA templates with (w / 5'-Pthio) or without (w / o5'-Pthio) 5'-phosphorothioate. [Figure 52-2] Same as above. [Figure 52-3] Same as above. [Figure 52-4] Same as above.

[0102] [Figure 53-1]Figures 53A-C show knock-in efficiency using various amounts of unmodified reverse strand (RV) ssDNA template or 5'-phosphorothioate modified reverse strand (RV) ssDNA template compared to unmodified ds DNA template for electroporation. Figure 53A is a flow cytometry analysis showing detection of knock-in positive mNeon+TCRab+ cells, Figure 53B is a graph showing the frequency of mNeon+TCRab+ cells, and Figure 53C is a graph showing cell viability by analysis of PI negative cells after electroporation using ssDNA template with (w / 5'-Pthio) or without (w / o5'-Pthio) 5'-phosphorothioate. [Figure 53-2] Same as above. [Figure 53-3] Same as above. [Figure 53-4] Same as above. [Figure 53-5] Same as above. [Figure 53-6] Same as above.

[0103] [Figure 54] FIG. 54 is a graph comparing the frequency of knock-in positive mNeon+TCRab+ (top) and live cells (bottom) using various amounts of dsDNA template, 5'-phosphate modified dsDNA template (w / 5'-P), 5'-phosphorothioate modified dsDNA template (w / 5'-Pthio), ssDNA template, or 5'-phosphorothioate modified ssDNA template (w / 5'-Pthio) for electroporation.

[0104] [Figure 55] Figure 55 is a flow cytometry analysis showing the effect of electroporation time point on template knock-in efficiency. Electroporation was performed 60 hours (top), 48 hours (middle), or 36 hours (bottom) after activation.

[0105] [Figure 56]Figure 56 is a flow cytometry analysis showing the effect of PGA on knock-in efficiency by detection of knock-in positive mNeon+TCRab+ cells. Cells were electroporated with template DNA and 60 pmol of RNP (1:1 sgRNA:Cas9) in either the presence (bottom) or absence (top) of PGA.

[0106] [Figure 57] Figure 57 is a flow cytometry analysis showing the effect of PGA on knock-in efficiency by detection of knock-in positive mNeon+TCRab+ cells. Cells were electroporated with template DNA and 80 pmol of RNP at 3:1 sgRNA:Cas9 in either the presence (bottom) or absence (top) of PGA.

[0107] [Figure 58A] Figures 58A and 58B are bar graphs showing cell viability of CD8+ T cells electroporated with or without PGA. Cell viability was assessed 3 (Figure 58A) and 5 (Figure 58B) days after electroporation (days 5 and 7 of cell culture) for samples electroporated with and without template DNA (control) and with and without PGA. The amount of RNP used for electroporation (pmol) is indicated on the x-axis. [Figure 58B] Same as above.

[0108] [Figure 59A] Figures 59A and 59B are bar graphs showing the frequency of mNeon+ cells for T cells electroporated with or without PGA. The frequency of knock-in positive cells was assessed 5 days (Figure 59A) and 7 days (Figure 59B) after electroporation. Cells were electroporated with and without template DNA and with or without PGA. The amount of RNP used for electroporation (pmol) is shown on the x-axis. [Figure 59B] Same as above.

[0109] [Figure 60A] Figures 60A and 60B are bar graphs showing mNeon+ cell recovery for T cells electroporated with or without PGA. Cell recovery was assessed 5 days (Figure 60A) and 7 days (Figure 60B) after electroporation. Cells were electroporated with and without template DNA and with or without PGA. The amount of RNP used for electroporation (pmol) is shown on the x-axis. [Figure 60B] Same as above.

[0110] [Figure 61A] Figures 61A and 61B are bar graphs showing the effect of electroporation time on mNeon knock-in efficiency. Cells were electroporated 36, 48, or 60 hours after T cell activation, and in controls, cells did not receive template during electroporation. mNeon+ cell frequency (Figure 61A) and recovery (Figure 61B) were assessed 3 days after electroporation. [Figure 61B] Same as above.

[0111] [Figure 62A] Figures 62A and 62B are bar graphs showing the effect of electroporation time on the efficiency of mNeon knock-in into the TRAC locus. mNeon+ cell frequency (Figure 62A) and recovery (Figure 62B) were assessed 5 days after electroporation. Cells were electroporated 36, 48, or 60 hours after T cell activation with TransAct, and in controls, cells did not receive template during electroporation. [Figure 62B] Same as above.

[0112] [Figure 63-1]Figures 63A-D show the effect of reagent addition order on knock-in efficiency. Figure 63A is a flow cytometry analysis (bottom) of cells electroporated with various orders of addition of template, RNP and PGA to T cells, designated and numbered 1-6 (top). Knock-in efficiency was assessed by detection of mNeon+TCRab+ cells. Figure 63B is a graph showing the frequency of mNeon+TCR+ cells for samples of cells electroporated with different orders of addition as indicated on the x-axis of the graph, Figure 63C is a graph showing the frequency of TCR knockout cells, and Figure 63D is a graph showing the frequency of live cells. [Figure 63-2] Same as above. [Figure 63-3] Same as above. [Figure 63-4] Same as above. [Figure 63-5] Same as above. [Figure 63-6] Same as above.

[0113] [Figure 64-1] Figures 64A-D show knock-in efficiency using linear PCR product dsDNA template compared to plasmid template for electroporation. Figure 64A shows flow cytometry dot plots detecting mNeon+TCRab+ cells from cells electroporated with various amounts of PCR product dsDNA or pUC57 plasmid. Figure 64B shows the frequency of mNeon+TCR+ cells for samples of cells electroporated with various amounts of PCR product or pUC57 plasmid template as indicated on the x-axis of the graph, Figure 64C shows the frequency of live cells, and Figure 64D shows the frequency of TCR knockout cells. [Figure 64-2] Same as above. [Figure 64-3] Same as above. [Figure 64-4] Same as above. [Figure 64-5] Same as above. [Figure 64-6] Same as above.

[0114] [Figure 65A] Figures 65A-C show the effect of cell density and type of donor template used for electroporation on knock-in efficiency. T cells were electroporated with 1 μg or 2 μg of linear PCR product dsDNA (L) or plasmid (P) and either 5×106 (5e6), 2×106 (2e6) or 1×106 (1e6) cells. Figure 65A is a graph showing the frequency of mNeon+TCRab+ cells 5 days after activation, Figure 65B is a graph showing the frequency of mNeon+TCRab+ cells 7 days after activation, and Figure 65C is a graph showing the expansion of mNeon+TCRab+ cells from day 5 to day 7. [Figure 65B] Same as above. [Figure 65C] Same as above.

[0115] [Figure 66A] Figure 66A and Figure 66B show mNeon+TCRab+ cell recovery in response to electroporation at various cell densities with different amounts and types of donor templates. T cells were electroporated with 1 μg or 2 μg of linear PCR product dsDNA (L) or plasmid (P) and either 5×106 (5e6), 2×106 (2e6) or 1×106 (1e6) cells. Figure 66A is a graph showing the recovery of mNeon+TCRab+ cells at day 7 after activation, and Figure 66B is a graph showing the proliferation of mNeon+TCRab+ cells from day 5 to day 7. The 5e6 cell density condition was omitted to better visualize proliferation at lower cell densities. [Figure 66B] Same as above.

[0116] [Figure 67-1] Figure 67 is a flow cytometry analysis showing detection of knock-in positive mNeon+TCRab+ T cells from cells electroporated with linear dsDNA template. Various amounts of template and cell densities were used for electroporation. [Figure 67-2]Same as above. [Figure 67-3] Same as above. [Figure 67-4] Same as above.

[0117] [Figure 68-1] Figure 68 is a flow cytometry analysis of knock-in positive mNeon+TCRab+ T cells from cells electroporated with linear dsDNA template (top) compared to cells electroporated with plasmid template (bottom). Either 1 μg or 2 μg of template was used. [Figure 68-2] Same as above. [Figure 68-3] Same as above.

[0118] [Figure 69-1] FIG. 69 is a flow cytometry dot plot showing detection of total CD8+ cells, CD45RA+ cells, and CD45RO+ cells on day 0 of T cell activation for three donors. [Figure 69-2] Same as above. [Figure 69-3] Same as above.

[0119] [Figure 70] FIG. 70 is a schematic diagram of T cell differentiation showing that TSCM are less differentiated cells (top) and a table showing various cytokine combinations that can promote the generation of T cell subsets (bottom).

[0120] [Figure 71A]Figures 71A-C show knock-in efficiency using different TRAC loci and either linear dsDNA or plasmid DNA as template. Figure 71A is a graph showing the frequency of knock-in positive mNeon+TCRab+ cells at day 4 compared to day 7 after T cell activation, Figure 71B shows mNeon+TCRab+ cell recovery at days 4 and 7 after activation, and Figure 71C shows fold expansion of mNeon+TCRab+ cells. TRAC3, TRAC4, TRAC5, TRAC7, TRAC12 and TRAC15 templates in linear dsDNA (L) or plasmid DNA (P) form were used for electroporation. [Figure 71B] Same as above. [Figure 71C] Same as above.

[0121] [Figure 72-1] Figure 72 is a flow cytometry analysis showing detection of knock-in positive mNeon+TCRab+ T cells electroporated with various templates. Cells were electroporated with RNP only control (top row, left three panels), TRAC3 linear dsDNA template (top row, right three panels), TRAC3 plasmid (bottom row, left three panels) or TRAC4 plasmid (bottom row, right three panels). [Figure 72-2] Same as above. [Figure 72-3] Same as above.

[0122] [Figure 73-1] Figure 73 is a flow cytometry analysis showing detection of knock-in positive mNeon+TCRab+ T cells electroporated with various templates. Cells were electroporated with TRAC5 plasmid (top row, left three panels), TRAC7 plasmid (top row, right three panels), TRAC12 plasmid (bottom row, left three panels) or TRAC15 plasmid (bottom row, right three panels). [Figure 73-2] Same as above. [Figure 73-3] Same as above.

[0123] [Figure 74-1] FIG. 74 is a flow cytometry analysis profiling total CD8+ (left column), CD45RA+ (middle column), and CD45RO+ (right column) T cells from donor samples used in the experiments shown in FIG. 72 and FIG. 73. [Figure 74-2] Same as above. [Figure 74-3] Same as above.

[0124] [Figure 75] Figure 75 is a flow cytometry dot plot showing knock-in efficiency of NY-ESO1 TCR with either gp100 TCR, MART3 TCR, WT1C TCR, or MAGEAB3 TCR, as indicated. Flow cytometry data was collected 7 days after T cell activation, when plasmid templates encoding NY-ESO1 TCR were electroporated simultaneously with templates encoding the other TCRs. Cells were stained with Immudex peptide-MHC dextramer WB3247-APC (NY-ESO1 TCR) along with WB3469-PE (WT1 TCR), WB2162-PE (MART TCR), WB3415_PE (MAGEA3 TCR), or WB2156_PE (gp100 TCR).

[0125] [Figure 76A]Figures 76A-C are graphs showing the effect of linear versus plasmid template, amount of template used, and cell density on knock-in efficiency. Figure 76A is a graph showing frequency and Figure 76B is a graph showing recovery of knock-in positive mNeon+TCRab+ cells at 5 and 7 days post-activation. Figure 76C is a graph showing recovery of CD8+ T cells at 5 and 7 days post-activation. Either 2 million (2M), 5 million (5M), or 10 million (10M) cells were used as indicated on the x-axis of the graph. Either 1 μg or 2 μg of linear dsDNA PCR product (L) or plasmid (P) template was used for electroporation for each group of cells. [Figure 76B] Same as above. [Figure 76C] Same as above.

[0126] [Figure 77-1] Figure 77 is a flow cytometry analysis showing detection of knock-in positive mNeon+TCRab+ T cells. Either 2 million (2M, top row) or 5 million cells (5M, bottom row) with 1 μg of linear dsDNA or 2 μg of plasmid DNA template were used for electroporation. [Figure 77-2] Same as above.

[0127] [Figure 78-1] Figure 78 is a flow cytometry dot plot detecting knock-in positive mNeon+TCRab+ T cells. Ten million cells were electroporated with either 1 μg of linear dsDNA TRAC3-mNeon (top) or 2 μg of TRAC3-mNeon plasmid (bottom) template. [Figure 78-2] Same as above.

[0128] [Figure 79A]Figures 79A-D are bar graphs showing the frequency of knock-in positive mNeon+TCRab+ cells at 5 days (Figure 79A) and 7 days (Figure 79C) after activation, and residual TCR-expressing mNeon-TCRab+ cells at 5 days (Figure 79B) and 7 days (Figure 79D) after activation. All cell populations were electroporated in P3 buffer and different pulse codes were tested, as indicated on the x-axis of the graphs. [Figure 79B] Same as above. [Figure 79C] Same as above. [Fig. 79D] Same as above.

[0129] [Figure 80A] Figures 80A-D are bar graphs showing the recovery of knock-in positive mNeon+TCRab+ cells at days 5 (Figure 80A) and 7 (Figure 80C) post-activation, and total CD8+ cells at days 5 (Figure 80B) and 7 (Figure 80D) post-activation. All cell populations were electroporated in P3 buffer and different pulse codes were tested, as indicated on the x-axis of the graphs. [Figure 80B] Same as above. [Fig. 80C] Same as above. [Fig. 80D] Same as above.

[0130] [Figure 81-1] Figures 81A and 81B are flow cytometry analyses showing detection of knock-in positive mNeon+TCRab+ T cells 7 days after activation using samples from donor 1. Figure 81A shows data for cells electroporated using the following pulse programs: for the top row, left to right: no program, DS150, DS120, EH100; for the bottom row, left to right: CA137, CM138, CM137, and CM150. Figure 81B shows data for cells electroporated using the following pulse programs: for the top row, left to right: EO100, EN138, EN150, EN113; for the bottom row, left to right: DN100, DS138, DS137, DS130. [Figure 81-2] Same as above. [Figure 81-3] Same as above. [Figure 81-4] Same as above.

[0131] [Figure 82-1] Figures 82A and 82B are flow cytometry analyses showing detection of knock-in positive mNeon+TCRab+ T cells 7 days after activation using samples from donor 2. Figure 82A shows data for cells electroporated using the following pulse programs: for the top row, left to right: no program, DS150, DS120, EH100; for the bottom row, left to right: CA137, CM138, CM137, and CM150. Figure 82B shows data for cells electroporated using the following pulse programs: for the top row, left to right: EO100, EN138, EN150, EN113; for the bottom row, left to right: DN100, DS138, DS137, DS130. [Figure 82-2] Same as above. [Figure 82-3] Same as above. [Figure 82-4] Same as above.

[0132] [Figure 83-1] Figures 83A and 83B are flow cytometry analyses showing detection of knock-in positive mNeon+TCRab+ T cells 7 days after activation using samples from donor 3. Figure 83A shows data for cells electroporated using the following pulse programs: for the top row, left to right: no program, DS150, DS120, EH100; for the bottom row, left to right: CA137, CM138, CM137, and CM150. Figure 83B shows data for cells electroporated using the following pulse programs: for the top row, left to right: EO100, EN138, EN150, EN113; for the bottom row, left to right: DN100, DS138, DS137, DS130. [Figure 83-2] Same as above. [Figure 83-3] Same as above. [Figure 83-4] Same as above.

[0133] [Figure 84-1] Figure 84 shows flow cytometry dot plots profiling the donor sample used in the experiments shown in Figures 81-83. Cells were analyzed on day 0 of activation. Plots showing total CD8+ cells, CD45RA+ cells, CD45RO+ cells, and CD45RA-CD45RO- (RA-RO-) cells are shown as indicated. [Figure 84-2] Same as above. [Figure 84-3] Same as above.

[0134] [Figure 85-1] Figures 85A-D are flow cytometry analyses showing knock-in efficiency of various amounts of linear dsDNA TRAC templates mNeon-TRACsg3 (Figure 85A), mNeon-TRACsg4 (Figure 85B), mNeon-TRACsg5 (Figure 85C) and mNeon-TRACsg12 (Figure 85D) in T cells. The rightmost column is a dot plot of cells electroporated with the addition of TRBC21 RNP. [Figure 85-2] Same as above. [Figure 85-3] Same as above. [Figure 85-4] Same as above. [Figure 85-5] Same as above. [Figure 85-6] Same as above. [Figure 85-7] Same as above. [Figure 85-8] Same as above.

[0135] [Figure 86A]Figure 86A and Figure 86B are graphs showing knock-in positive mNeon+TCR+ cells. Figure 86A is a graph showing the frequency of mNeon+TCRab+ cells using various amounts of mNeon-TRAC3, mNeon-TRAC4, mNeon-TRAC5 and mNeon-TRAC12 templates for electroporation. Figure 86B is a graph showing the frequency of mNeon+TCRab+ cells using 1 μg of mNeon-TRAC3, mNeon-TRAC4, mNeon-TRAC5 or mNeon-TRAC12 templates for electroporation with or without TRBC21 RNP. [Figure 86B] Same as above.

[0136] [Figure 87-1] Figure 87 is a flow cytometry analysis showing detection of knock-in positive mNeon+TCRab+ T cells for cell populations electroporated with either the CI unit electroporator (top) or the ACE electroporator (bottom). Cells were analyzed 5 days after activation and different pulse codes were tested. [Figure 87-2] Same as above.

[0137] [Figure 88-1] Figure 88 shows flow cytometry analysis detecting knock-in positive mNeon+TCRab+ T cells in cell populations electroporated with different pulse codes. Cells were analyzed 12 days after activation. [Figure 88-2] Same as above.

[0138] [Figure 89A]Figure 89A and Figure 89B are flow cytometry analysis showing the effect of various concentrations of PGA on knock-in efficiency. Figure 89A is a flow cytometry dot plot of cells electroporated in the presence of 0 μg, 12.5 μg, or 254 μg of PGA. Cells electroporated with NT (non-targeted guide control, i.e., no TCR knockout) RNP are the control group. Figure 89B is a flow cytometry dot plot of cells electroporated in the presence of 50 μg, 100 μg, 200 μg, or 400 μg of PGA. [Fig. 89B] Same as above.

[0139] [Figure 90A] Figures 90A and 90B are graphs showing the frequency of knock-in positive mNeon+TCRab+ cells (Figure 90A) and the recovery rate of mNeon+ cells (Figure 90B) depending on the concentration of PGA used during electroporation. [Figure 90B] Same as above.

[0140] [Figure 91] Figure 91 shows the effect of PGA concentration and PGA variants on knock-in efficiency. Graphs showing the frequency of knock-in positive mNeon+TCRab+ cells (top) and the recovery rate of mNeon+ cells (bottom). PGA concentrations and variants are shown on the x-axis.

[0141] [Figure 92A] Figures 92A-D are bar graphs showing the effect of electroporation pulse codes on the knock-in efficiency of TRAC3-mNeon in T cells. The frequency of knock-in positive mNeon+TCRab+ cells at 5 days (Figure 92A) and 7 days (Figure 92C) after activation, and the frequency of TCRab+mNeon- cells at 5 days (Figure 92B) and 7 days (Figure 92D) after activation are shown. [Fig. 92B] Same as above. [Fig. 92C] Same as above. [Fig. 92D]Same as above.

[0142] [Figure 93A] Figures 93A and 93B are flow cytometry dot plots showing the effect of electroporation pulse codes on TRAC3-mNeon knock-in efficiency in T cells. Figure 93A shows flow cytometry analysis of cells electroporated with EH115, EN138 and EN158 pulse codes. Figure 93B shows flow cytometry analysis of cells electroporated with EW113, EH111 and EO100 pulse codes. The percentage of mNeon+TCRab+ cells is as indicated in the upper right corner of each plot. [Figure 93B] Same as above.

[0143] [Figure 94A] Figures 94A and 94B are flow cytometry dot plots showing the effect of electroporation pulse codes on TRAC3-mNeon knock-in efficiency in T cells. Figure 94A shows flow cytometry analysis of cells electroporated with EO115, EO128, and EO151 pulse codes. Figure 94B shows flow cytometry analysis of cells electroporated with DS130, DS137, and DS138 pulse codes. The percentage of mNeon+TCRab+ cells is as indicated in the upper right corner of each plot. [Figure 94B] Same as above.

[0144] [Figure 95A] Figures 95A-D are bar graphs showing knock-in efficiency of TRAC3-mNeon in T cells in response to varying electroporation pulse codes as indicated on the x-axis of the graph. The frequency of knock-in positive mNeon+TCRab+ cells at 5 days post-activation (Figure 95A) and 7 days post-activation (Figure 95C) and the recovery of mNeon+TCRab+ cells at 5 days post-activation (Figure 95B) and 7 days post-activation (Figure 95D) are shown. [Figure 95B]Same as above. [Figure 95C] Same as above. [Fig. 95D] Same as above.

[0145] [Figure 96-1] Figure 96 is a flow cytometry analysis showing knock-in efficiency of TRAC3-NY-ESO-4 TCR plasmid template in T cells. Cells electroporated with RNP only (no template) are the control group (leftmost three columns). From left to right for each of the three rows of plots, the flow cytometry analysis shows detection of NY-ESO-4TCR+ cells among total CD8+ T cells, TCRab+ cells among total CD8+ T cells, and NY-ESO-4TCR+ cells among CD8+TCRab+ cells. [Figure 96-2] Same as above. [Figure 96-3] Same as above.

[0146] [Figure 97A] Figure 97A and Figure 97B are flow cytometric analyses showing the effectiveness of various cell staining methods. Figure 97A is flow cytometric detection of NY-ESO-4TCRab+ cells using dextramer (Dex) staining (left) or dextramer staining (right) in the presence of other antibodies. Figure 97B is flow cytometric detection of NY-ESO-4TCRab+ using GNE tetramer. Dot plots of cells electroporated with RNP control are shown in the left column. [Figure 97B] Same as above.

[0147] [Figure 98-1] Figure 98 is a flow cytometry analysis showing knock-in efficiency of various TCRs using TRAC3 plasmid templates in HLA-A0201 CD8+ T cells. TRAC3-NY-ESO-4, TRAC3-WTIC-13, TRAC3-MART2 and TRAC3-MART3 templates were used for electroporation. [Figure 98-2] Same as above.

[0148] [Figure 99] Figure 99 is a flow cytometry analysis showing knock-in efficiency of TRAC3-mNeon plasmid template using two different electroporators. In each dot plot, the upper box represents knock-in positive mNeon+TCRab+ cells and the lower box represents residual TCRab expressing cells.

[0149] [Figure 100A] FIG. 100A and FIG. 100B are graphs comparing the frequency of knock-in positive mNeon+TCRab+ T cells (FIG. 100A) and the number of mNeon+ T cells (FIG. 100B) at day 6 post-activation depending on the pulse code used for electroporation. The pulse codes used are as indicated on the x-axis, with the rightmost four pulse codes being the formation codes. The square and circle data markers represent data for cells electroporated with two different electroporators. [Figure 100B] Same as above.

[0150] [Figure 101A] FIG. 101A and FIG. 101B are graphs comparing the frequency of knock-in positive mNeon+TCRab+ T cells (FIG. 101A) and the number of mNeon+ T cells (FIG. 101B) at day 8 post-activation depending on the pulse code used for electroporation. The pulse codes used are as indicated on the x-axis, with the rightmost four pulse codes being the formation codes. The square and circle data markers represent data for cells electroporated with two different electroporators. [Figure 101B] Same as above.

[0151] [Figure 102-1]FIG. 102A-C show the effect of TRBC RNP on knock-in efficiency of NY-ESO1 TCR into the TRAC locus using the TRAC3-1G4(NY-ESO1) plasmid. FIG. 102A is a flow cytometry analysis of knock-in positive pMHC(NY-ESO1)+TCRab+ cells using various RNPs as indicated above each dot plot. FIG. 102B is a graph showing the frequency of TCRab+pMHC+ cells, and FIG. 102C is a graph showing the frequency of total pMHC+ cells. The RNPs used for electroporation are as indicated below the graphs. [Figure 102-2] Same as above. [Figure 102-3] Same as above. [Figure 102-4] Same as above. [Figure 102-5] Same as above.

[0152] [Figure 103-1] Figure 103A and Figure 103B show the effect of TRBC separate TBBC RNP on the knock-in efficiency of NY-ESO1 TCR to TRAC locus combined with TRAC3 RNP using TRAC3-1G4 (NY-ESO1) plasmid. Figure 103A is a flow cytometry analysis showing the detection of double positive pMHC (NY-ESO1) + cells using various RNPs as shown above each dot plot. Figure 103B is a graph showing the frequency of double positive pMHC + cells. The RNPs used for electroporation are as shown below the graph. [Figure 103-2] Same as above. [Figure 103-3] Same as above. [Figure 103-4] Same as above.

[0153] [Figure 104-1]Figures 104A and 104B show the effect of different homology arm lengths on knock-in efficiency of TRACsg3-mNeon, TRACsg4-mNeon, TRACsg5-mNeon and TRACsg12-mNeon templates in T cells. Flow cytometry analysis showing knock-in efficiency as a function of homology arm length (HA length) for left / right arms 300 / 300, 400 / 400 and 500 / 500 (Figure 104A), as well as longest / longest (varying for each template), longest / 300 and 300 / longest (Figure 104B). [Figure 104-2] Same as above. [Figure 104-3] Same as above. [Figure 104-4] Same as above.

[0154] [Figure 105A] Figures 105A-H are graphs showing the effect of different homology arm lengths on knock-in efficiency of TRACsg3-mNeon, TRACsg4-mNeon, TRACsg5-mNeon and TRACsg12-mNeon templates in T cells. The frequency of knock-in positive mNeon+TCRab+ cells using TRAC3-mNeon (Figure 105A), TRAC4-mNeon (Figure 105C), TRAC5-mNeon (Figure 105E) or TRAC12-mNeon (Figure 105G) templates for electroporation is shown. The number of mNeon+ cells using TRAC3-mNeon (Figure 105B) and TRAC4-mNeon (Figure 105D), TRAC5-mNeon (Figure 105F) or TRAC12-mNeon (Figure 105H) templates for electroporation is shown. The change in length of the homology arm (HA) of the left / right arm is indicated on the x-axis. [Figure 105B] Same as above. [Figure 105C] Same as above. [Fig. 105D] Same as above. [Figure 105E] Same as above. [Fig. 105F] Same as above. [Figure 105G] Same as above. [Fig. 105H]Same as above.

[0155] [Fig. 106] 106 is a graph showing T2 cell killing by WT1 and gp100B TCR knock-in cells. T2 cells were electroporated with various concentrations of target peptide.

[0156] [Figure 107A] 107A-107D are graphs showing the overall effect of TRBC22 RNP titration. FIG. 107A shows the percentage of Neon positive TRAC+ / -TRBC samples 8 days after electroporation under the indicated conditions. FIG. 107B shows the percentage of Neon positive samples 8 days after electroporation under the indicated conditions. FIG. 107C shows the percentage of Neon positive TRAC3 / TRBC22 samples 7 days after electroporation under the indicated conditions. FIG. 107D shows the percentage of Neon positive TRAC3 / TRBC22 samples 7 days after electroporation under the indicated conditions. The dashed box in FIG. 107D shows the second electroporation with TRBC22 RNP after 24 hours, comparing EW113 to EO100. 24w vs. 48w indicates whether the cells were cultured in 24-well or 48-well plates after the initial electroporation (2×10 6 cells). [Figure 107B] Same as above. [Figure 107C] Same as above. [Fig. 107D] Same as above.

[0157] [Figure 108A] Figures 108A and 108B are graphs of combined data from all experiments showing that the use of TRBC22 RNP appears to have minimal effect on neon knock-in. Figure 108A shows the percentage of Neon positive samples. Figure 108B shows the number of Neon positive samples. [Figure 108B] Same as above.

[0158] [Figure 109A] 109A-109C are graphs showing the results of a series of sequential electroporations performed on TRAC3 RNP+ donor templates and TRBC22 RNPs 24 hours later. FIG. 109A shows the percentage (or frequency) of Neon positive samples. FIG. 109B shows the number of Neon positive samples. FIG. 109C shows the frequency or percentage of TCRab+Neon negative samples. [Figure 109B] Same as above. [Figure 109C] Same as above.

[0159] [Figure 110A] Figures 110A-110D are graphs showing the results of testing knock-in frequencies for various templates. Figures 110A and 110B show the frequency of Neon positive samples for two single experiments. Figures 110C-110D show the combined data for both experiments, with Figure 110C showing the frequency of Neon positive samples and Figure 110D showing the number of Neon positive samples. [Figure 110B] Same as above. [Figure 110C] Same as above. [Figure 110D] Same as above.

[0160] [Figure 111-1] FIG. 111 is flow cytometry data at day 7 of TRAC3 RNP alone (30 pmol or 60 pmol) with different donor templates using plasmid or nanoplasmid. [Figure 111-2] Same as above.

[0161] [Figure 112-1] FIG. 112 is flow cytometry data on day 7 from a titration experiment using 30 pmol or 60 pmol of TRAC3 RNP and 10 pmol, 20 pmol, or 30 pmol of TRB22 RNP as shown in FIG. [Figure 112-2] Same as above. [Figure 112-3] Same as above.

[0162] [Figure 113-1] FIG. 113 shows flow cytometry data 7 days after sequential electroporations, all using 30 pmol of TRAC3 RNP and 30 pmol of TRBC22 RNP, with the electroporation codes shown. [Figure 113-2] Same as above.

[0163] [Fig. 114A] Figure 114A and Figure 114B are graphs showing data for pUC57 vs. Nanoplasmid vs. Minicircle DNA 6 days after electroporation. Figure 114A shows the percentage (top graph) and absolute number (bottom graph) of mNeon+TCR+ samples. Figure 114B shows the amount of DNA used in the experiment. [Fig. 114B] Same as above.

[0164] [Fig. 115A] Figure 115A and Figure 115B are graphs showing data for pUC57 vs. Nanoplasmid vs. Minicircle DNA 8 days after electroporation. Figure 115A shows the percentage (top graph) and absolute number (bottom graph) of mNeon+TCR+ samples. Figure 115B shows the amount of DNA used in the experiment. [Fig. 115B] Same as above.

[0165] [Fig. 116A] Figure 116A and Figure 116B are graphs showing the effect on cell viability by equivalent molecules of each template on pUC57, nanoplasmid or minicircle. Figure 116A shows the percentage of cell viability 6 days after electroporation, and Figure 116B shows the percentage of cell viability 8 days after electroporation. [Fig. 116B] Same as above.

[0166] [Figure 117]FIG. 117 is a graph of the change in knock-in cell number over time from days 6 to 8 after electroporation based on the data from FIG. 116A and FIG. 116B.

[0167] [Fig. 118A] Figure 118A and Figure 118B are graphs summarizing cytokine readout monitoring: Figure 118A shows the concentration of TNF-alpha and Figure 118B shows the concentration of IL-6 after electroporation with pUC57, nanoplasmid or minicircle. [Fig. 118B] Same as above.

[0168] [Figure 119] FIG. 119 shows the concentrations of IFN-γ (top graph), IL-5 (middle graph) and IL-13 (bottom graph) following electroporation with pUC57, nanoplasmid or minicircle.

[0169] [Fig. 120A] Figures 120A-B are graphs showing data for pUC57 vs. nanoplasmid vs. minicircle DNA on day 6. Figure 120A shows the percentage (top graph) and absolute number (bottom graph) of mNeon+TCR+ samples on day 6. Figure 120B shows the amount of DNA used in this experiment. [Fig. 120B] Same as above.

[0170] [Fig. 121A] Figures 121A-B are graphs showing data for pUC57 vs. nanoplasmid vs. minicircle DNA on day 8. Figure 121A shows the percentage (top graph) and absolute number (bottom graph) of mNeon+TCR+ samples on day 8. Figure 121B shows the amount of DNA used in this experiment. [Fig. 121B] Same as above.

[0171] [Figure 122-1]FIG. 122 shows flow cytometry data on day 8 of electroporation of T cells electroporated with pUC57, nanoplasmid, minicircle and PCR samples. [Figure 122-2] Same as above. [Figure 122-3] Same as above.

[0172] [Figure 123] FIG. 123 is a pair of graphs showing the effect on cell viability following electroporation with equivalent molecules of the indicated templates, with data from 6 days post-electroporation shown in the top graph and data from 8 days post-electroporation shown in the bottom graph.

[0173] [Figure 124] FIG. 124 is a graph showing the change in the number of knock-in cells over time from day 6 to day 8, based on the data in FIG.

[0174] [Fig. 125] Figure 125 is a graph showing that the addition of TRBC RNP does not reduce knock-in efficiency. The left graph shows the percentage of Neon positive samples after electroporation with templates on plasmid or nanoplasmid, and the right graph shows the number of Neon positive samples.

[0175] [Fig. 126A] Figures 126A-B are graphs showing that knock-in efficiency increases with higher amounts of nanoplasmid. Figure 126A shows the knock-in efficiency at 5 days (left graph) and 7 days (right graph) after electroporation. Figure 126B shows the change in knock-in cell number at 5 days and 7 days after electroporation. [Fig. 126B] Same as above.

[0176] [Figure 127-1]FIG. 127 is a graph of flow cytometry data on day 7 of electroporation for TRBC19 or TRBC22 plasmid titrations (10 pmol or 30 pmol) with 60 pmol of TRAC3. [Figure 127-2] Same as above. [Figure 127-3] Same as above.

[0177] [Figure 128-1] Figure 128 is a graph of flow cytometry data 7 days after electroporation for 30 pmol of TRAC3 alone (top row), or TRBC22 (30 pmol, middle row) or TRBC19 (30 pmol, bottom row) on plasmid (left three rows) or nanoplasmid (right three rows). [Figure 128-2] Same as above. [Figure 128-3] Same as above.

[0178] [Figure 129-1] FIG. 129 is a graph of flow cytometry data 7 days after electroporation for 2 μg, 4 μg, 6 μg, or 8 μg of GenScript plasmid. [Figure 129-2] Same as above.

[0179] [Figure 130-1] FIG. 130 is a graph of flow cytometry data 7 days after electroporation for Nanoplasmid titration. [Figure 130-2] Same as above. [Figure 130-3] Same as above.

[0180] [Fig. 131A] Figure 131A and Figure 131B are graphs showing the results of Neon knock-in 7 days after electroporation using templates on plasmid or nanoplasmid with or without different addition orders of PGA and template. Figure 131A shows the percentage of Neon positive cells and Figure 131B shows the number of Neon positive cells. [Fig. 131B] Same as above.

[0181] [Fig. 132A] Figure 132A-C are graphs showing that codon optimization can dramatically reduce the effect of template addition order on TCR knock-in efficiency and knock-in efficiency. Figure 132A shows the percentage of WT1+ (total CD8+) samples 7 days after electroporation. Figure 132B shows the percentage of CD3+ samples 7 days after electroporation. Figure 132C shows the percentage of WT1+ (total CD3+) samples 7 days after electroporation. [Fig. 132B] Same as above. [Fig. 132C] Same as above.

[0182] [Fig. 133] Figure 133 is a graph showing the number of WT1 TCR+ cells 7 days after electroporation based on the data in Figures 132A to 132C. The conditions graphed, from left to right, are template added first, template added last, and no PGA.

[0183] [Figure 134-1] FIG. 134 is a graph of flow cytometry data 7 days after electroporation of Neon-templated knock-in with or without different orders of addition of PGA and template. [Figure 134-2] Same as above. [Figure 134-3] Same as above. [Figure 134-4] Same as above.

[0184] [Figure 135-1] FIG. 135 is a graph of flow cytometry data 7 days after electroporation of WT1_5213 with or without different orders of addition of PGA and template. [Figure 135-2] Same as above. [Figure 135-3]Same as above. [Fig. 135-4] Same as above. [Fig. 135-5] Same as above. [Fig. 135-6] Same as above.

[0185] [Figure 136-1] FIG. 136 is a graph of flow cytometry data 7 days after electroporation for WT1_Ref. [Figure 136-2] Same as above. [Figure 136-3] Same as above. [Figure 136-4] Same as above. [Figure 136-5] Same as above. [Figure 136-6] Same as above.

[0186] [Figure 137-1] FIG. 137 is a graph showing flow cytometry data 7 days after electroporation for WT1 64_9. [Figure 137-2] Same as above. [Figure 137-3] Same as above. [Figure 137-4] Same as above. [Figure 137-5] Same as above. [Figure 137-6] Same as above.

[0187] [Figure 138] FIG. 138 is a cartoon representation of target cell killing (top) and a graph of percent killing (bottom) for neo-TCR and non-TCR compared to control.

[0188] [Figure 139]Figure 139 is a cartoon representation of the insertion of a DNA template into a TCR-alpha locus as described herein. The DNA template (top) includes (5' to 3'): a left (5') homology arm, a NEO-TCR (a first self-cleaving peptide (e.g., T2A), a TCR-beta V segment, a TCR-beta D segment, a TCR-beta J segment, a TCR-beta constant region, a second self-cleaving peptide (e.g., P2A), a TCR-alpha V segment, a TCR-alpha J segment, and a portion of the TCR-alpha constant region), and a right (3') homology arm. A nuclease or gene editing agent (e.g., CRISPR / Cas9) cleaves the T cell's endogenous TCR-alpha constant region (middle) and promotes homologous recombination of the DNA template with the endogenous TCR-alpha locus within the constant region. The resulting edited TCR-alpha locus (bottom) contains the NEO-TCR in frame with a portion of the endogenous TCR-alpha constant region.

[0189] [Fig. 140A]Figure 140A-I. Plasmid-based donor templates enable efficient non-viral gene editing of the TRAC locus in primary T cells. (Figure 140A-C) Titration of linear dsDNA donor templates. (Figure 140A) Diagram of linear dsDNA knock-in construct TRAC-mNG. (Figure 140B) Bar graphs (n=4) showing knock-in efficiency, cell viability, total cell recovery, and edited cell recovery (mNG positive cells) 3 days after electroporation with 1 μg, 2 μg, 4 μg, 6 μg, or 8 μg of linear dsDNA donor template with Cas9-RNP targeting the TRAC locus. Circles represent individual donors and bars represent median with range. (Figure 140C) Representative contour plot showing frequency of CD8 T cells expressing mNG. (Figure 140D-F) Titration of pUC57 plasmid donor template (Figure 140D) Diagram of pUC57 knock-in construct TRAC-mNG. (Figure 140E) Bar graphs showing frequency of CD8 T cells expressing mNG, cell viability, total cell recovery and edited cell recovery (mNG positive cells) 3 days after electroporation with 1 μg, 2 μg, 4 μg, 6 μg or 8 μg of pUC57 plasmid donor template with Cas9-RNP targeting the TRAC locus (n=4). Circles represent individual donors and bars represent median with range. (Figure 140F) Representative contour plot showing frequency of CD8 T cells expressing mNG. (Figure 140G-I) Titration of nanoplasmid donor template (Figure 140G) Diagram of nanoplasmid knock-in construct TRAC-mNG. (FIG. 140H) Bar graphs showing the frequency of mNG-expressing CD8 T cells, total cell recovery, and edited cell recovery (mNG positive cells) 3 days after electroporation with 1 μg, 2 μg, 4 μg, 6 μg, or 8 μg of nanoplasmid donor template with Cas9-RNP targeting the TRAC locus (n=4). Circles represent individual donors and bars represent median values ​​with range. (FIG. 140I) Representative contour plot showing the frequency of mNG-expressing CD8 T cells. [Fig. 140B] Same as above. [Fig. 140C] Same as above. [Fig. 140D]Same as above. [Figure 140E] Same as above. [Fig. 140F] Same as above. [Fig. 140G] Same as above. [Fig. 140H] Same as above. [Fig. 140I] Same as above.

[0190] [Figure 141-1]Figures 141A-F. Optimization of CRISPR / Cas9-mediated gene knock-in with plasmid-based donor DNA in CD4 and CD8 T cells. (Figure 141A,B) Homology arm optimization of plasmid-based donor templates. (Figure 141A) Representative contour plots showing the frequency of CD8 T cells expressing mNG and (Figure 141B) bar graphs (n=2) showing knock-in efficiency, cell viability, total cell recovery and edited cell recovery (mNG positive cells) 3 days after electroporation with pUC57 plasmid or nanoplasmid donor templates with homology arm lengths ranging from 100bp to 2000bp (equimolar amount to 4μg of 2000bp construct) with Cas9-RNP targeting the TRAC locus. Circles represent individual donors and bars represent median with range. (FIG. 141C) Frequency of mNG-expressing CD8 T cells, cell viability, total cell recovery, and edited cell recovery (mNG positive cells) 3 days after electroporation at 24, 36, 48, or 72 hours of culture using nanoplasmid donor template with Cas9-RNP targeting the TRAC locus (n=4). Circles represent individual donors and bars represent median with range. (FIG. 141D) Optimization of nucleofection pulse codes in CD8 T cells electroporated with nanoplasmid donor template and Cas9-RNP targeting the TRAC locus. Graph shows frequency of mNG-expressing cells and edited cell recovery (mNG positive cells) 3 days after electroporation. Each circle represents a separate pulse code. Data is representative of three independent CD8 T cell donors. (Fig. 141E,F) Gene editing targeting the TRAC locus in CD4 T cells. (Fig. 141E) Representative contour plots showing the frequency of CD4 T cells expressing mNG, and (Fig. 141F) bar graphs (n=3) showing knock-in efficiency, cell viability, total cell recovery, and edited cell recovery (mNG positive cells) 5 days after electroporation of CD4 T cells with TRAC-mNG nanoplasmid donor template along with Cas9-RNP targeting the TRAC locus. Circles represent individual donors and bars represent median with range. [Figure 141-2] Same as above. [Figure 141-3] Same as above. [Figure 141-4] Same as above. [Figure 141-5] Same as above. [Figure 141-6] Same as above. [Figure 141-7] Same as above.

[0191] [Fig. 142A]Figure 142A-J. Non-viral T cell receptor editing using plasmid DNA donors (Figure 142A) Diagram of TCR alpha and beta genomic loci. V gene (purple), D gene (red) and J gene (blue) and constant region (green) segments. sgTRAC and sgTRBC targeting sites are indicated. (Figure 142B) Diagram of nanoplasmid knock-in constructs TRAC-1G4TCR, TRAC-TCR6-2 and TRAC-CD19CAR. (Fig. 142C,E,G) Representative contour plots (left) and bar graphs (right) showing the frequency of CD8 T cells expressing (Fig. 142C) NY-ESO1-specific 1G4 TCR, (Fig. 142E) CMV-specific pp65 6-2 TCR, (Fig. 142G) CD19-CAR 5 days after electroporation using nanoplasmid donor templates with Cas9-RNP targeting the TRAC locus (n=3). Circles represent individual donors and bars represent median with range. (Fig. 142D,F,H) Bar graphs showing cell viability, total cell recovery, and edited cell recovery 5 days after electroporation using nanoplasmid donor templates encoding (Fig. 142D) NY-ESO1-specific 1G4 TCR, (Fig. 142F) CMV-specific pp65 6-2 TCR, (Fig. 142H) CD19-CAR, along with Cas9-RNP targeting the TRAC locus (n=3). Circles represent individual donors and bars represent median with range. (FIG. 142I) Lactate levels in culture supernatants, analyzed by luminescence using the Lactate-Glo Assay, were measured 1, 3, 5, and 7 days after transfection of CD8 T cells with sgTRAC / sgTRBC Cas9-RNP (RNP only) or sgTRAC / sgTRBC Cas9-RNP and a nanoplasmid donor template targeting the TRAC locus (RNP + nanoplasmid) compared to untransfected control T cells (no RNP).(Figure 142J) The number of cells recovered from culture 7 days after transfection of CD8 T cells with sgTRAC / sgTRBC Cas9-RNP (RNP only) or sgTRAC / sgTRBC Cas9-RNP with a nanoplasmid donor template targeting the TRAC locus (RNP + nanoplasmid) was compared to untransfected control T cells (no RNP). [Fig. 142B] Same as above. [Fig. 142C] Same as above. [Fig. 142D] Same as above. [Fig. 142E] Same as above. [Fig. 142F] Same as above. [Fig. 142G] Same as above. [Fig. 142H] Same as above. [Fig. 142I] Same as above. [Fig. 142J] Same as above.

[0192] [Fig. 143A]Figures 143A-I: TCR engineered T cells recognize and kill antigen-expressing target cells. Representative (Figure 143A) histograms and (Figure 143B) bar graphs (n=4) showing CD137 frequency of IG4 TCR knock-in CD8+ T cells activated with the indicated doses of NY-ESO 157-165 peptide. Circles represent individual donors and bars represent medians with range. Representative (Figure 143C) histograms and (Figure 143D) bar graphs (n=3) showing CD137 frequency of pp65 TCR knock-in CD8+ T cells activated with the indicated doses of pp65495-503 peptide. Circles represent individual donors and bars represent medians with range. (Figure 143E) Representative histograms showing frequency of CFSE positive target cells relative to CFSE negative reference cells in co-cultures with IG4 TCR knock-in CD8+ T cells in the absence or presence of cognate peptide. (Fig. 143F) Graph showing specific lysis calculated without peptide or in the presence of 0.1 μM NY-ESO157-165 peptide. Circles represent individual donors and bars represent median values ​​with range (n=4). This experiment was performed twice. (Fig. 143G) Bar graph showing IFN-β, TNF-α and Granzyme B (GzmB) production by TCR knockout or 1G4 TCR knock-in CD8+ T cells from three donors co-cultured with A-375 cells expressing the NY-ESO-1 antigen. Circles represent technical replicates and bars represent median values ​​with range (n=3). This experiment was performed twice. (FIG. 143H) Representative images of A-375 cells expressing NY-ESO-1 antigen, labeled with a cytoplasmic dye, and co-cultured with TCR knockout cells (left panel) or 1G4 TCR knock-in cells (right panel) 2 and 18 hours after culture seeding in the presence of Caspase 3 / 7-green apoptosis reagent. (FIG. 143I) Target cell killing over time as measured by the number of Cas3 / 7 positive objects in co-cultures of A-375 cells expressing NY-ESO-1 antigen, labeled with a cytoplasmic dye, and co-cultured with TCR knockout cells (open circles) or 1G4 TCR knock-in cells (closed circles). Mean ± standard deviation of six technical replicates. This experiment was performed twice.RM one-way ANOVA with Geisser-Greenhouse correction (Figure 143B-D); paired t-test (Figure 143F); *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001 in one-way ANOVA (G) or Tukey's multiple comparison test, two-way ANOVA (Figure 143I). [Fig. 143B] Same as above. [Fig. 143C] Same as above. [Fig. 143D] Same as above. [Fig. 143E] Same as above. [Fig. 143F] Same as above. [Fig. 143G] Same as above. [Fig. 143H] Same as above. [Fig. 143I] Same as above.

[0193] [Fig. 144A]Figures 144A-M. Generation of reporters of gene expression. (Figure 144A) Diagram of nanoplasmid knock-in construct RAB11A-YFP. (Figure 144B) Histogram overlay of YFP expression of CD8 T cells transfected with RAB11A-YFP nanoplasmid with or without RAB11A targeting Cas9-RNP 10 days after electroporation. (Figure 144C) Bar graphs showing frequency of YFP expression (left) and cell viability (right) of CD8 T cells transfected with RAB11A-YFP nanoplasmid with or without RAB11A targeting Cas9-RNP 10 days after electroporation (n=3). Circles represent individual donors and bars represent median with range. (Figure 144D) Diagram of nanoplasmid knock-in construct AAVS1-mNG. (Figure 144E) Histogram overlay of mNG expression in CD8 T cells transfected with AAVS1-mNG nanoplasmid with or without AAVS1 targeting Cas9-RNP 10 days after electroporation. (Figure 144F) Bar graphs (n=4) showing frequency of mNG expression (left) and cell viability (right) in CD8 T cells transfected with AAVS1-mNG nanoplasmid with or without AAVS1 targeting Cas9-RNP 10 days after electroporation. Circles represent individual donors and bars represent median with range. (Figure 144G) Diagram of nanoplasmid knock-in construct CD4-mNG. (Figure 144H) Representative contour plots showing frequency of CD4 and CD8 T cells expressing mNG 10 days after electroporation of nanoplasmid donor template and Cas9 / RNP targeting CD4 locus. (FIG. 144I) Bar graphs showing the frequency (top) and cell viability (bottom) of mNG-expressing CD4 and CD8 T cells 10 days after electroporation of nanoplasmid donor templates and Cas9 / RNP targeting the CD4 locus (n=4 for CD4 T cells, n=3 for CD8 T cells). Circles represent individual donors and bars represent medians with range.(Figure 144J) Histogram overlay of CD4 expression in CD4 T cells transfected with CD4-mNG nanoplasmid and either a non-targeting control Cas9-RNP (sgNTC) or a Cas9-RNP targeting the CD4 locus (sgCD4) 10 days after electroporation. (Figure 144K) Diagram of nanoplasmid knock-in constructs TNFRSF9-mNG and RAB11A-YFP (left) and representative contour plots showing the frequency of CD137- and mNG-expressing CD8 T cells after electroporation with the nanoplasmid mNG reporter construct targeting the TNFRSF9 locus or the constitutive YFP expression construct targeting the RAB11A locus together with the respective Cas9-RNPs either without reactivation or 6 hours after reactivation with Transact (right). (Figure 144L) Bar graphs (n=4) showing the frequency of YFP-expressing CD8 T cells (left) and mNG-expressing CD8 T cells (right) over time after electroporation with nanoplasmid mNG reporter constructs targeting the TNFRSF9 locus or constitutive YFP expression constructs targeting the RAB11A locus with the respective Cas9-RNPs and reactivation with Transact at 0 hours. Circles represent individual donors and bars represent median values ​​with range. (Figure 144M) Bar graphs (n=4) showing the median fluorescence intensity (gMFI) of CD137 expression in CD8 T cells over time after electroporation with nanoplasmid mNG reporter constructs targeting the TNFRSF9 locus (right) or constitutive YFP expression constructs targeting the RAB11A locus (left) with the respective Cas9-RNPs and reactivation with Transact at 0 hours. Circles represent individual donors and bars represent median values ​​with range. [Fig. 144B] Same as above. [Fig. 144C] Same as above. [Fig. 144D] Same as above. [Fig. 144E] Same as above. [Fig. 144F] Same as above. [Fig. 144G] Same as above. [Fig. 144H]Same as above. [Fig. 144I] Same as above. [Fig. 144J] Same as above. [Figure 144K] Same as above. [Fig. 144L] Same as above. [Figure 144M] Same as above.

[0194] [Fig. 145A]Figures 145A-145K. Multiple gene knock-in in human T cells. (Figures 145A-145C) Diagrams of nanoplasmid knock-in constructs are shown above. Representative contour plots (left) and bar graphs (right) showing the frequency of CD8 T cells expressing mNG (n=3) 10 days after electroporation with a nanoplasmid TRAC-mNG donor template and Cas9 / RNP targeting the TRAC locus (Figure 145A), mCherry (n=3) 10 days after electroporation with a nanoplasmid TRAC-mCherry donor template and Cas9 / RNP targeting the TRAC locus (Figure 145B), or either mNG or mCherry (n=3) 10 days after electroporation with two nanoplasmid donor templates (TRAC-mNG and TRAC-mCherry) and Cas9 / RNP targeting the TRAC locus (Figure 145C). The graphs on the right in (Figure 145C) show the percentage of transgene-expressing cells expressing either mNG (bottom right of the histogram), mCherry (top left) or both (top right). In the bar graphs, circles represent individual donors and bars represent medians with range. (Figure 145D) Diagram of the nanoplasmids, RAB11A-YFP and TRAC-mCherry used in the dual targeting study. (Figure 145E) Representative contour plots showing the frequency of CD8 T cells expressing either YFP (bottom right), mCherry (top left) or both (top right) 10 days after electroporation with nanoplasmid donors RAB11A-YFP and TRAC-mCherry and Cas9 / RNP targeting the RAB11A and TRAC loci. (FIG. 145F) Bar graphs (n=4) showing CD8 T cell knock-in efficiency (left), cell viability (center), and total cell recovery (right) 10 days after electroporation with nanoplasmid donors RAB11A-YFP and TRAC-mCherry targeting the RAB11A and TRAC loci and Cas9 / RNP. Circles represent individual donors and bars represent median values ​​with range.(Figure 145G) Percentage of transgene-expressing cells co-transfected with nanoplasmid donors RAB11A-YFP and TRAC-mCherry and Cas9 / RNP targeting the RAB11A and TRAC loci expressing YFP, mCherry, or both (n=4). Circles represent individual donors and bars represent medians with range. (Figure 145H) Diagram of the nanoplasmids, AAVS1-mNG and TRAC-mCherry used in the dual targeting study. (Figure 145I) Representative contour plot showing the frequency of CD8 T cells expressing mNG, mCherry, or both 10 days after electroporation with nanoplasmid donors AAVS1-mNG and TRAC-mCherry and Cas9 / RNP targeting the AAVS1 and TRAC loci. (FIG. 145J) Bar graphs (n=4) showing CD8 T cell knock-in efficiency (left), cell viability (center), and total cell recovery (right) 10 days after electroporation with nanoplasmid donors AAVS1-mNG and TRAC-mCherry and Cas9 / RNP targeting the AAVS1 and TRAC loci. Circles represent individual donors and bars represent medians with range. (FIG. 145K) Percentage of transgene-expressing cells co-transfected with nanoplasmid donors AAVS1-mNG and TRAC-mCherry and Cas9 / RNP targeting the AAVS1 and TRAC loci expressing mNG, mCherry, or both (n=4). Circles represent individual donors and bars represent medians with range. [Fig. 145B] Same as above. [Fig. 145C] Same as above. [Fig. 145D] Same as above. [Fig. 145E] Same as above. [Fig. 145F] Same as above. [Fig. 145G] Same as above. [Fig. 145H] Same as above. [Fig. 145I] Same as above. [Fig. 145J] Same as above. [Figure 145K] Same as above.

[0195] [Fig. 146A] Figures 146A-G. Non-viral CRISPR gene editing with large payloads. (Figure 146A) Diagram of nanoplasmid knock-in constructs TRAC_NotchICD_mNG, TRAC_NotchICD_1G4 and TRAC_THEMIS_1G4. (Fig. 146B,D,F) Representative contour plots showing the frequency of CD8 T cells expressing (Fig. 146B) mNG or (Fig. 146D,F) 1G4 TCR 5 days after electroporation with (Fig. 146B) NotchICD_mNG, (Fig. 146D) NotchICD_1G4 or (Fig. 146F) THEMIS_1G4 nanoplasmid donor templates with Cas9 / RNP targeting the TRAC locus. (Fig. 146C,E,G) Representative contour plots showing the frequency of CD8 T cells expressing (Fig. 146C) mNG or (Fig. 146E,G) 1G4 TCR 5 days after electroporation with (Fig. 146C) NotchICD_mNG, (Fig. 146E) NotchICD_1G4 or (Fig. 146G) THEMIS_1G4 nanoplasmid donor templates with Cas9 / RNP targeting the TRAC locus. Bar graphs showing T cell frequency and cell viability (n=3). Circles represent individual donors and bars represent median values ​​with range. [Fig. 146B] Same as above. [Figure 146C] Same as above. [Fig. 146D] Same as above. [Figure 146E] Same as above. [Fig. 146F] Same as above. [Fig. 146G] Same as above.

[0196] [Fig. 147A]Figure 147A-H. Optimization of non-viral gene editing in primary T cells using plasmid-based donor templates. (Figure 147A-F) Titration of linear dsDNA and nanoplasmid donor templates in RPMI / FBS medium. (Figure 147A) Diagram of linear dsDNA knock-in construct TRAC-mNG. (Figure 147B) Bar graphs (n=4) showing knock-in efficiency, cell viability, total cell recovery, and edited cell recovery (mNG positive cells) of CD8 T cells cultured in RPMI / 10% FBS 3 days after electroporation with 1 μg, 2 μg, 4 μg linear dsDNA donor template with Cas9 / RNP targeting the TRAC locus. (Figure 147C) Representative contour plots showing frequency (left), viability (second from left), total cell recovery (second from right), and edited cell recovery (right) of CD8 T cells expressing mNG. Circles represent individual donors and bars represent medians with range. (Fig. 147D) Diagram of nanoplasmid knock-in construct TRAC-mNG. (Fig. 147E) Bar graphs (n=4) showing knock-in efficiency, cell viability, total cell recovery and edited cell recovery (mNG positive cells) of CD8 T cells cultured in RPMI / 10% FBS 3 days after electroporation with 1 μg, 2 μg, 4 μg of nanoplasmid donor template with Cas9 / RNP targeting the TRAC locus. Circles represent individual donors and bars represent medians with range. (Fig. 147F) Representative contour plots showing frequency (left), viability (second from left), total cell recovery (second from right) and edited cell recovery (right) of CD8 T cells expressing mNG. (FIG. 147G) Bar graphs showing knock-in efficiency (left), cell viability (second from left), total cell recovery (second from right), and edited cell recovery (right) 3 days after electroporation with either 2 μg of linear dsDNA or nanoplasmid donor template with Cas9-RNP targeting the TRAC locus in the absence of poly-L-glutamic acid (PGA) (n=3). Circles represent individual donors and bars represent median values ​​with range.(FIG. 147H) Bar graphs (n=3) showing knock-in efficiency (left), cell viability (second from left), total cell recovery (second from right), and edited cell recovery (right) 3 days after electroporation with 2 μg of either linear dsDNA or nanoplasmid donor template with (tCTS) or without (w / o tCTS) truncated Cas9 target sequence along with Cas9-RNP targeting the TRAC locus. Circles represent individual donors and bars represent median values ​​with range. [Fig. 147B] Same as above. [Fig. 147C] Same as above. [Fig. 147D] Same as above. [Fig. 147E] Same as above. [Fig. 147F] Same as above. [Fig. 147G] Same as above. [Fig. 147H] Same as above.

[0197] [Fig. 148A]Figure 148A-H. Cytokine production and stress responses induced in T cells after exposure to dsDNA donor templates. (Figure 148A) IFN-α and IFN-γ measured by SIMOA, TNF-α and IL-2 measured by Luminex from CD8 T cells 18 hours after transfection with Cas9 / RNP targeting the TRAC locus alone or with nanoplasmid donor template compared to untransfected control T cells (no RNP). Circles represent individual donors; bars represent median with range (n-3). (Figure 148B) GSEA enrichment analysis results from CD8 T cells after transfection with Cas9 / RNP targeting TRAC with nanoplasmid donor template compared to Cas9 / RNP alone. Gene sets for interferon-γ response, interferon-α response, TNFα signaling, and inflammatory response were significantly enriched. (Fig. 148C) GSEA enrichment analysis results from CD8 T cells after transfection with Cas9 / RNP targeting TRAC with linear dsDNA donor template compared to Cas9 / RNP alone. Gene sets for interferon-gamma response, interferon-alpha response, TNFalpha signaling, and inflammatory response were significantly enriched. (Fig. 148B,C) The y-axis represents enrichment score and the x-axis is genes represented in the gene set (vertical black line). The colored bands at the bottom represent the degree of differentially expressed genes (red for upregulation, blue for downregulation). (Fig. 148D) GSEA of all 375 upregulated genes in both nanoplasmid / Cas9-RNP and linear dsDNA / Cas9-RNP versus Cas9-RNP alone using GSEA MSigDB Hallmark 2020. (Figure 148E-H) Heatmap showing genes upregulated in nanoplasmid / Cas9-RNP and linear dsDNA / Cas9-RNP versus Cas9-RNP alone, most of which contributed to (Figure 148E) interferon-α response, (Figure 148F) TNFα response, (Figure 148G) apoptosis, or (Figure 148H) inflammatory response (all MSigDB hallmarks).Color coded by RNA-seq count data normalized using variance stabilizing transformation (VST). [Fig. 148B] Same as above. [Fig. 148C] Same as above. [Fig. 148D] Same as above. [Fig. 148E] Same as above. [Fig. 148F] Same as above. [Figure 148G] Same as above. [Fig. 148H] Same as above.

[0198] [Fig. 149A]Figures 149A-149O. Non-viral T cell receptor editing in CD4+ and CD8+ T cells using plasmid DNA donors. (Figure 149A) TCR expression on cell surface by flow cytometry in CD8 T cells 48 hours after transfection with Cas9-RNP targeting the TRAC (sgTRAC) or TRBC (sgTRBC) locus (Figures 149B-G) TCR editing in CD4 T cells. Representative contours showing the frequency of CD8 T cells expressing (Figure 149B) NY-ESO1-specific 1G4 TCR, (Figure 149D) CMV-specific pp65 6-2 TCR, (Figure 149F) CD19-CAR 5 days after electroporation with nanoplasmid donor template with Cas9-RNP targeting the TRAC locus. Bar graphs (n=4) showing knock-in efficiency and cell viability 5 days after electroporation using nanoplasmid donor templates encoding (Fig. 149C) NY-ESO1-specific 1G4 TCR, (Fig. 149E) CMV-specific pp65-6-2 TCR, (Fig. 149G) CD19-CAR, together with Cas9-RNP targeting the TRAC locus. Circles represent individual donors; bars represent median values ​​with range (n-3). (Fig. 149H) Diagram showing all possible translocation events between TRAC, TRBC1 and TRBC2 genomic loci (Fig. 149I). Bar graphs (n=4) showing the frequency of individual translocation events between TRAC, TRBC1 and TRBC2 genomic loci quantified by ddPCR in CD8 T cells co-transfected with Cas9-RNP targeting the TRAC and TRBC loci or in non-transfected control T cells. Circles represent individual donors; bars represent median values ​​with range (n-3). This experiment was performed twice. (FIG. 149J) Representative histograms and (FIG. 149K) bar graphs showing CD137 frequency of pp65 TCR knock-in CD8+ T cells stimulated with the indicated concentrations of pp65495-503 peptide. Circles represent individual donors and bars represent median with range (n=3). This experiment was performed twice. (FIG. 149L) Bar graphs showing IFN-β (left) and TNF-α (right) production by pp65 TCR knock-in CD8+ T cells stimulated with the indicated concentrations of pp65495-503 peptide.Circles represent individual donors and bars represent medians with ranges (n=3). This experiment was performed twice. (Figure 149M) Representative histogram showing the frequency of CFSE-positive target cells and CFSE-negative reference cells in co-culture with pp65 TCR knock-in CD8+ T cells in the absence or presence of cognate peptide. (Figure 149N) Graph showing specific lysis calculated in the absence of peptide or with 0.1 μM pp65495-503 peptide. Circles represent individual donors and bars represent medians with ranges (n=3). This experiment was performed twice. (Figure 149O) Bar graph showing IFN-β and TNF-α production by TCR6-2 (irrelevant TCR) or CD19-CAR knock-in CD4+ T cells from two donors (D1, D2) in co-culture with CD19-expressing B cells. Circles represent technical replicates and bars represent medians with ranges (n=9). This experiment was performed twice. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001 in RM one-way ANOVA (Figure 149A,K), paired t-test (Figure 149N) one-way ANOVA (Figure 149O) with Geisser-Greenhouse correction. [Fig. 149B] Same as above. [Fig. 149C] Same as above. [Fig. 149D] Same as above. [Fig. 149E] Same as above. [Fig. 149F] Same as above. [Figure 149G] Same as above. [Fig. 149H] Same as above. [Fig. 149I] Same as above. [Fig. 149J] Same as above. [Figure 149K] Same as above. [Figure 149L] Same as above. [Figure 149M] Same as above. [Fig. 149N] Same as above. [Fig. 149O] Same as above.

[0199] [Fig. 150A]Figures 150A-I. Kinetics of gene expression following transient transfection of dsDNA, plasmids and nanoplasmids. (Figure 150A) Diagram of the nanoplasmid knock-in construct RAB11A-YFP. (Figure 150B) Representative histograms showing the frequency of CD8 T cells expressing YFP 3, 5 or 7 days after electroporation with promoter-containing nanoplasmid donor templates with (paired red / right bars) or without (paired blue / left bars) Cas9 / RNP targeting the RAB11A locus. (Figure 150C) Graphs showing YFP expression, cell viability, total cell recovery, and edited cell recovery 3, 5, or 7 days after electroporation with a promoter-containing nanoplasmid donor template with (red / right bars) or without (blue / left bars) Cas9 / RNP targeting the RAB11A locus (n=3). (Figure 150D) Diagram of linear dsDNA knock-in construct RAB11A-YFP. (Figure 150E) Representative histograms showing the frequency of CD8 T cells expressing YFP 3, 5, or 7 days after electroporation with a promoter-containing linear dsDNA donor template with (top) or without (bottom) Cas9 / RNP targeting the RAB11A locus. (FIG. 150F) Graphs showing YFP expression frequency, cell viability, total cell recovery, and edited cell recovery 3, 5, or 7 days after electroporation with a promoter-containing linear dsDNA donor template with (paired red / right bars) or without (paired blue / left bars) Cas9 / RNP targeting the RAB11A locus (n=3). (FIG. 150G) Diagram of pUC57 plasmid knock-in construct RAB11A-YFP. (FIG. 150H) Representative histograms showing the frequency of CD8 T cells expressing YFP 3, 5, or 7 days after electroporation with a promoter-containing pUC57 plasmid donor template with (top) or without (bottom) Cas9 / RNP targeting the RAB11A locus.(Figure 150I) Graphs showing YFP expression frequency, cell viability, total cell recovery, and edited cell recovery 3, 5, or 7 days after electroporation with a promoter-containing pUC57 plasmid donor template with (paired red / right bars) or without (paired blue / left bars) Cas9 / RNP targeting the RAB11A locus (n=3). [Fig. 150B] Same as above. [Fig. 150C] Same as above. [Fig. 150D] Same as above. [Figure 150E] Same as above. [Fig. 150F] Same as above. [Figure 150G] Same as above. [Fig. 150H] Same as above. [Fig. 150I] Same as above.

[0200] [Fig. 151A]Figure 151A-G. Multiple gene knock-in in human T cells. (Figure 151A-C) Diagrams of pUC57 plasmid knock-in constructs are shown at the top. (Figure 151A) Representative contour plots (left) and bar graphs (right) showing the frequency of CD8 T cells expressing mNG (n=3) 10 days after electroporation with a pUC57 plasmid TRAC-mNG donor template and Cas9 / RNP targeting the TRAC locus, (Figure 151B) mCherry (n=3) 10 days after electroporation with a pUC57 plasmid TRAC-mCherry donor template and Cas9 / RNP targeting the TRAC locus, or (Figure 151C) either mNG or mCherry (n=3) 10 days after electroporation with two pUC57 plasmid donor templates (TRAC-mNG and TRAC-mCherry) and Cas9 / RNP targeting the TRAC locus. The graphs on the right in (Figure 151C) show the percentage of transgene-expressing cells expressing either mNG (green / bottom right of histogram), mCherry (red / top left) or both (blue / top right). Circles represent individual donors and bars represent medians with range. (Figure 151D) Diagram of pUC57 plasmids, RAB11A-YFP and TRAC-mCherry, used in dual targeting studies. (Figure 151E) Representative contour plots showing the frequency of CD8 T cells expressing either YFP, mCherry or both 10 days after electroporation with RAB11A-YFP and TRAC-mCherry and Cas9 / RNP of pUC57 donors targeting the RAB11A and TRAC loci. (FIG. 151F) Bar graphs (n=4) showing CD8 T cell knock-in efficiency, cell viability, and total cell recovery 10 days after electroporation with pUC57 donor RAB11A-YFP and TRAC-mCherry and Cas9 / RNP targeting the RAB11A and TRAC loci. Circles represent individual donors and bars represent median values ​​with range.(FIG. 151G) Percentage of transgene-expressing cells co-transfected with pUC57 donor templates RAB11A-YFP and TRAC-mCherry and Cas9 / RNP targeting the RAB11A and TRAC loci expressing YFP, mCherry, or both (n=4). Circles represent individual donors and bars represent median values ​​with range. This experiment was performed in triplicate. *p<0.05 **p<0.01 in RM one-way ANOVA with Geisser-Greenhouse correction. [Fig. 151B] Same as above. [Fig. 151C] Same as above. [Fig. 151D] Same as above. [Fig. 151E] Same as above. [Fig. 151F] Same as above. [Fig. 151G] Same as above.

[0201] [Fig. 152] Figure 152. Two TCR knock-in constructs with and without polyadenylation (bGHpA) sequence. The TRACg3_WT1C13_pUC57 construct contains a truncated TCR-alpha sequence consisting of the entire variable region and a small segment of the constant region before the right homology arm. When copied into the TRAC3 cleavage site in the genome, the truncated TCR-alpha in the construct is made entirely by in-frame fusion with genomic DNA that provides the remaining part of the TCR-alpha constant chain sequence. The construct named Schober_TRACg3_WT1C13_pUC57 differs from TRACg3_WT1C13_pUC57 in that it contains the full-length TCR-alpha sequence followed by a stop codon and a polyadenylation sequence from the bovine growth hormone gene (bGHpA) before the right homology arm. It does not require an in-frame fusion with the remaining TCR-alpha constant chain sequence in the genome for TCR-alpha expression.

[0202] [Fig. 153]Figure 153. Flow cytometry scatter plot of donor primary cells electroporated with two TCR knock-in construct RNPs with and without polyadenylation (bGHpA) sequence (Schober_TRACg3_WT1C13_pUC57 and TRACg3_WT1C13_pUC57).

[0203] [Fig. 154] Figure 154. Bar graph comparison (% pMHC+ and TCR+pMHC+) of donor primary cells electroporated with two TCR knock-in construct RNPs with and without polyadenylation (bGHpA) sequence (Schober_TRACg3_WT1C13_pUC57 and TRACg3_WT1C13_pUC57).

[0204] [Fig. 155] Figure 155. Schematic of knock-in at TCRB1 and TCRB2 locus nanoplasmid constructs.

[0205] [Fig. 156] Figure 156. Flow cytometry scatter plot of donor primary cells electroporated with two knock-ins at the TCRB1 and TCRB2 loci nanoplasmid constructs without the TCR alpha sequence.

[0206] [Fig. 157] Figure 157. Bar graph comparison of donor primary cells electroporated with two knock-ins at the TCRB1 and TCRB2 locus nanoplasmid constructs without the TCR alpha sequence (% pMHC+ and TCR+pMHC+).

[0207] [Fig. 158] Figure 158. Scatter plot of cell proliferation rates at days 5 and 7 of donor primary cells electroporated with two knock-ins at the TCRB1 and TCRB2 loci nanoplasmid constructs without the TCR alpha sequence.

[0208] [Fig. 159] Figure 159. Flow cytometry scatter plots of non-viral TCR knock-in using Jurkat cells at day 7 with and without IL-2 activation.

[0209] [Fig. 160] Figure 160. Flow cytometry scatter plot of non-viral TCR knock-in using primary CD4+ cells not activated with IL-2.

[0210] [Figure 161-1] Figure 161. Schematic diagram of knockout of TCR-alpha and TCR-beta loci using TCRA1-TCRA16 and TCRB1-TCRB26 guide RNAs (including plots of % negative T cells for TCR-alpha or beta by flow cytometry). [Figure 161-2] Same as above.

[0211] [Figure 162-1] Figure 162. Schematic of knockout of TCR-alpha and TCR-beta loci using TCRA1-TCRA16 and TCRB1-TCRB26 guide RNAs, including plots of % negative T cells for TCR-alpha or beta by flow cytometry. Boxed sequences indicate guide RNA sequences with greater than 90% knockout efficiency. [Figure 162-2] Same as above.

[0212] [Fig. 163]Figure 163. Schematic diagram of a construct containing a self-cleaving peptide (SCP). The SCP upstream of the TCRb gene is T2A, and the second SCP sequence upstream of the TCRa gene is P2A. The two consecutive SCPs in this example are not 100% identical to prevent erroneous recombination (i.e., each of the upstream or downstream P2A coding sequences could recombine with each other, resulting in a non-functional recombination locus). By using different nucleic acid sequences to code for each P2A site, the only homologous regions of the TCR donor and acceptor sites are the 5' and 3' arms. In this example, different up and downstream SCP sequences are used (T2A and P2A, respectively, and are not limited to only T2A and P2A sequences). Alternatively, the nucleic acid sequences coding for each of the two SCPs can be codon divergent, but still code for the same SCP peptide sequence. A short neutral GSG sequence is optionally added upstream of each SCP sequence. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0213] Detailed Description After reading this description, it will be clear to a person skilled in the art how to implement the present disclosure in various alternative embodiments and alternative applications. However, not all of the various embodiments of the present invention are described herein. It will be understood that the embodiments presented herein are presented by way of example only and are not limiting. Therefore, this detailed description of various alternative embodiments should not be interpreted as limiting the scope or breadth of the present disclosure described herein.

[0214] Before the present technology is disclosed and described, it is to be understood that the embodiments described below are not limited to particular compositions, methods of preparing such compositions, or uses thereof, as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting.

[0215] The detailed description is divided into various sections solely for the convenience of the reader, and disclosures found in any section may be combined with those of another section. Titles or subtitles may be used herein for the convenience of the reader and are not intended to affect the scope of the disclosure.

[0216] I. Definition Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. In this specification and the claims that follow, reference will be made to certain terms that shall be defined to have the following meanings.

[0217] The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting. As used herein, the singular forms "a," "an," and "the" are intended to include the plural forms as well, unless the context clearly indicates otherwise.

[0218] "Optional" or "optionally" means that the subsequently described event or circumstance may or may not occur, and that the description includes instances when the event or circumstance occurs and instances when it does not occur.

[0219] The term "about" when used prior to a numerical designation, e.g., temperature, time, amount, concentration, etc., including a range, indicates an approximation that may vary by (+) or (-) 10%, 5%, 1%, or any subrange or subvalue therebetween. Preferably, the term "about" means that the amount may vary by + / - 10%.

[0220] "Comprising" or "comprises" is intended to mean that the compositions and methods include the recited elements but do not exclude others. "Consisting essentially of," when used to define compositions and methods, is intended to mean excluding other elements that are essential to the combination for the described purpose. Thus, a composition consisting essentially of the elements defined in the present invention does not exclude other materials or steps that do not materially affect the basic and novel characteristics of the claimed technology. "Consisting of" is intended to mean excluding more than trace amounts of other components and substantial method steps. Embodiments defined by each of these transition terms are within the scope of this disclosure.

[0221] "T cells" or "T lymphocytes", as used herein, are a type of lymphocyte (a subtype of white blood cell) that plays a central role in cell-mediated immunity. They can be distinguished from other lymphocytes, e.g., 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 helper T cells. Different types of T cells can be distinguished by the use of T cell detection agents.

[0222] "Memory T cells" are T cells that have previously encountered and responded to their cognate antigen during a previous infection, cancer encounter, or previous vaccination. Upon a second encounter, the cognate antigen memory T cells can regenerate (divide) to mount a faster and stronger immune response than when the immune system first responded to the pathogen.

[0223] "Regulatory T cells" or "suppressor T cells" are lymphocytes that modulate the immune system, maintain tolerance to self-antigens, and prevent autoimmune disease.

[0224] "Nucleic acid" refers to nucleotides (e.g., deoxyribonucleotides or ribonucleotides) and polymers thereof, or their complements, or nucleosides (e.g., deoxyribonucleosides or ribonucleosides), in either single-stranded, double-stranded, or multiple-stranded form. In embodiments, "nucleic acid" does not include nucleosides. The terms "polynucleotide," "oligonucleotide," "oligo," and the like, in their usual and customary sense, refer to a linear sequence of nucleotides. The term "nucleoside" in its usual and customary sense refers to a glycosylamine that contains a nucleobase and a five-carbon sugar (ribose or deoxyribose). Non-limiting examples of nucleosides include cytidine, uridine, adenosine, guanosine, thymidine, and inosine. The term "nucleotide" in its usual and customary sense refers to a single unit, i.e., monomer, of a polynucleotide or nucleic acid. A nucleotide can be a ribonucleotide, a deoxyribonucleotide, or a modified form thereof. Examples of nucleic acids contemplated herein include single-stranded and double-stranded DNA, single-stranded and double-stranded RNA, and hybrid molecules having a mixture of single-stranded and double-stranded DNA and RNA. Examples of nucleic acids contemplated herein, e.g., nucleic acids, include any type of RNA, e.g., mRNA, siRNA, miRNA, and guide RNA, as well as any type of DNA, genomic DNA, plasmid DNA, and minicircle DNA, and any fragments thereof. The term "duplex" in the context of nucleic acids refers to double-strandedness in the usual and customary sense. Nucleic acids can be linear or branched. For example, nucleic acids can be linear chains of nucleotides, or they can be branched, e.g., such that the nucleic acid comprises one or more arms or branches of nucleotides. Optionally, branched nucleic acids are repeatedly branched to form higher-order structures, such as dendrimers.

[0225] Nucleic acids, for example nucleic acids with phosphorothioate backbones, can contain one or more reactive moieties. As used herein, the term reactive moiety includes any group that can react with another molecule, for example a nucleic acid or a polypeptide, via covalent, non-covalent or other interaction. As an example, a nucleic acid can contain an amino acid reactive moiety that reacts with an amino acid on a protein or polypeptide via covalent, non-covalent or other interaction.

[0226] The term also encompasses nucleic acids containing known nucleotide analogs or modified backbone residues or linkages, which are synthetic, natural and non-natural, have similar binding properties as the reference nucleic acid, and are metabolized in a similar manner as the reference nucleotide. Examples of such analogs include, but are not limited to, phosphodiester derivatives, including, for example, phosphoramidates, phosphorodiamidates, phosphorothioates (also known as phosphothioates with a double bond sulfur replacing the oxygen in the phosphate), phosphorodithioates, phosphonocarboxylic acids, phosphonocarboxylates, phosphonoacetic acids, phosphonoformic acids, methylphosphonates, boronphosphonates, or O-methylphosphoramidite linkages (see Eckstein, OLIGONUCLEOTIDES AND ANALOGUES: A PRACTICAL APPROACH, Oxford University Press), as well as modifications to nucleotide bases, such as 5-methylcytidine or pseudouridine; and peptide nucleic acid backbones and linkages. Other analog nucleic acids include those with positive backbones; non-ionic backbones, modified sugars, and non-ribose backbones (e.g., phosphorodiamidate morpholino oligos or locked nucleic acids (LNAs) as known in the art), including those described in U.S. Pat. Nos. 5,235,033 and 5,034,506, and Chapters 6 and 7 of ASC Symposium Series 580, CARBOHYDRATE MODIFICATIONS IN ANTISENSE RESEARCH, Sanghui & Cook, eds. Nucleic acids containing one or more carbocyclic sugars are also included within one definition of nucleic acid. Modifications of the ribose-phosphate backbone may be made for a variety of reasons, such as to increase the stability and half-life of such molecules in physiological environments or as probes on biochips. Mixtures of natural nucleic acids and analogs can be made, or mixtures of different nucleic acid analogs, as well as mixtures of natural nucleic acids and analogs can be made. In embodiments, the internucleotide linkages in DNA are phosphodiester, phosphodiester derivatives, or a combination of both.

[0227] As used herein, the terms "left homology arm" and "right homology arm" are used to define the 5' and 3' genomic DNA fragments (genes, partial genes or other DNA segments to be introduced into the genome) flanking the DNA sequence of interest, respectively. For example, gene targeting by homologous recombination may typically involve transfecting cells with a targeting vector designed and constructed to contain two genomic DNA fragments, a gene, transgene, gene fragment or other DNA of interest flanked by a 5' or left homology arm and a 3' or right homology arm. After transfection, these two arms are used to promote homologous recombination between the DNA donor and the endogenous target locus.

[0228] As used herein, "unmodified donor DNA" is defined as DNA that is unchanged from its original state. As used herein, "chemically modified donor DNA" is defined as DNA that has undergone some form of chemical modification, such as the addition or subtraction of a bond or moiety. Examples of chemical modifications include, but are not limited to, phosphorothioation, phosphorylation, methylation, acetylation, etc.

[0229] As used herein, the term "ribonucleoprotein" or "RNP" is used to refer to a complex of a ribonucleic acid and an RNA-binding protein, such as Cas9.

[0230] As used herein, the term "CRISPR" or "clustered regularly interspaced short palindromic repeats" is used according to its simple ordinary meaning to refer to a genetic element that bacteria use as a type of acquired immunity to protect against viruses. CRISPRs contain short sequences that are derived from viral genomes and integrated into bacterial genomes. Cas (CRISPR-associated proteins) processes these sequences and cleaves matching viral DNA sequences. Thus, the CRISPR sequences act as a guide for Cas to recognize and cleave DNA that is at least partially complementary to the CRISPR sequences. By introducing a plasmid containing the Cas gene and a specifically constructed CRISPR into a eukaryotic cell, the eukaryotic genome can be cleaved at any desired location.

[0231] As used herein, the term "Cas9" or "CRISPR associated protein 9" is used according to its simple ordinary meaning to refer to an enzyme that uses a CRISPR sequence as a guide to recognize and cleave a specific strand of DNA that is at least partially complementary to the CRISPR sequence. The Cas9 enzyme, together with the CRISPR sequence, forms the basis of a technology known as CRISPR-Cas9 that can be used to edit genes in organisms. This editing process has a wide variety of applications, including basic biological research, development of biotechnology products, and treatment of disease.

[0232] As referred to herein, "CRISPR associated protein 9", "Cas9", "Csn1" or "Cas9 protein" includes either recombinant or native forms of Cas9 endonuclease or variants or homologues thereof that maintain Cas9 endonuclease enzymatic activity (e.g., within at least 50%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% activity compared to Cas9). In embodiments, the variant or homologue has at least 90%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity over the entire sequence or a portion of the sequence (e.g., a 50, 100, 150 or 200 contiguous amino acid portion) compared to a native Cas9 protein. In embodiments, the Cas9 protein is substantially identical to a protein identified by UniProt reference number Q99ZW2 or a variant or homologue having substantial identity thereto. In embodiments, the Cas9 protein has at least 75% sequence identity with the amino acid sequence of the protein identified by UniProt reference number Q99ZW2. In embodiments, the Cas9 protein has at least 80% sequence identity with the amino acid sequence of the protein identified by UniProt reference number Q99ZW2. In embodiments, the Cas9 protein has at least 85% sequence identity with the amino acid sequence of the protein identified by UniProt reference number Q99ZW2. In embodiments, the Cas9 protein has at least 90% sequence identity with the amino acid sequence of the protein identified by UniProt reference number Q99ZW2. In embodiments, the Cas9 protein has at least 95% sequence identity with the amino acid sequence of the protein identified by UniProt reference number Q99ZW2.

[0233] As referred to herein, "CRISPR-associated endonuclease Cas12a," "Cas12a," "Cas12," or "Cas12 protein" includes either recombinant or naturally occurring forms of Cas12 endonuclease or variants or homologs thereof that maintain Cas12 endonuclease enzymatic activity (e.g., within at least 50%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% activity compared to Cas12). In embodiments, the variants or homologs have at least 90%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity over the entire sequence or a portion of the sequence (e.g., a portion of 50, 100, 150, or 200 contiguous amino acids) compared to the naturally occurring Cas12 protein. In embodiments, the Cas12 protein is substantially identical to the protein identified by UniProt reference number A0Q7Q2, or a substantially identical variant or homologue.

[0234] A "guide RNA" or "gRNA" as provided herein refers to an RNA sequence that has sufficient complementarity with a target nucleic acid sequence to hybridize with the target sequence and direct sequence-specific binding of a CRISPR complex to the target sequence. For example, the gRNA can target Cas to a target nucleic acid. In an embodiment, the gRNA comprises a crRNA and a tracrRNA. For example, the gRNA can comprise a crRNA and a tracrRNA hybridized by base pairing. Thus, in an embodiment, the crRNA and the tracrRNA are two RNA molecules that form an RNA / RNA complex by complementary base pairing between the crRNA and the tracrRNA to form the gRNA. In an embodiment, the gRNA is a single gRNA (sgRNA), where both the crRNA and the tracrRNA are present in a single RNA molecule. In embodiments, the degree of complementarity between a guide RNA sequence and its corresponding target sequence is about 50%, 60%, 75%, 80%, 85%, 90%, 95%, 97.5%, 99% or more when optimally aligned using a suitable alignment algorithm. In embodiments, the degree of complementarity between a guide RNA sequence and its corresponding target sequence is at least about 50%, 60%, 75%, 80%, 85%, 90%, 95%, 97.5%, 99% when optimally aligned using a suitable alignment algorithm.

[0235] Non-limiting examples of CRISPR enzymes include Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9 (also known as Csn1 and Csx12), Cas10, Cas12, Cas13, Csy1, Csy2, Csy3, Cse1, Cse2, Csc1, Csc2, Csa5, Csn2, Csm2, Csm 3, Csm4, Csm5, Csm6, Cmr1, Cmr3, Cmr4, Cmr5, Cmr6, Csb1, Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, Csx1, Csx15, Csf1, Csf2, Csf3, Csf4, their homologs, their fusion proteins, or modified versions thereof. In an embodiment, the CRISPR enzyme is a Cas9 enzyme. In an embodiment, the Cas9 enzyme is S. pneumoniae, S. pyogenes, or S. thermophilus Cas9, or mutants derived therefrom in these organisms. In an embodiment, the CRISPR enzyme is codon-optimized for expression in eukaryotic cells. In embodiments, the CRISPR enzyme directs one or two strand cleavage at the target sequence, hi embodiments, the CRISPR enzyme lacks DNA strand cleavage activity.

[0236] As used herein, the term "nuclear localization signal," "nuclear localization sequence," or "NLS" is used to refer to an amino acid sequence that marks or tags a protein for introduction into the cell nucleus by nuclear transport. Typically, this signal consists of one or more short sequences of positively charged lysines or arginines exposed on the protein surface.

[0237] As used herein, the term "non-viral" or "non-viral gene therapy" refers to any nucleic acid sequence that does not include a virus, a viral vector, or a viral-mediated delivery of a nucleic acid. Examples of non-viral methods of nucleic acid delivery include, but are not limited to, needle injection, ballistic DNA, electroporation, sonoporation, photoporation, magnetofection, hydroporation, mechanical massage, chemical carriers, inorganic particles, cationic lipids, lipid emulsions, solid lipid nanoparticles, peptide-based complexes, polymer-based complexes, or mixtures thereof.

[0238] As used herein, the term "nanoplasmid" is used to refer to an engineered circular nucleic acid that contains at least a nucleic acid sequence of interest, a mini origin of replication (e.g., R6K), and a selection marker (e.g., a small RNA selection marker, RNA-OUT). Nanoplasmids contain less than 500 bp of prokaryotic DNA.

[0239] As used herein, the term "antigen" is used to describe a compound, composition, or chemical that, when administered to an immunocompetent subject, can induce an immune response, such as a cytotoxic T lymphocyte (CTL) response, a T helper cell response, a B cell response (e.g., the production of antibodies that specifically bind to an epitope), a NK cell response, or any combination thereof. Thus, an immunogenic or antigenic composition is a composition that can elicit an immune response in an immunocompetent subject.

[0240] As used herein, the term "neo-antigen" is used to describe newly formed antigens not previously recognized by the immune system. Neo-antigens can arise from altered tumor proteins formed as a result of tumor mutations or from viral proteins. Non-limiting examples of neo-antigens are listed in Table 2.

[0241] As used herein, the term "tumor associated antigen" or "TAA" is used to describe proteins that are significantly overexpressed in cancer compared to normal cells and are therefore also abundantly displayed on the surface of cancer cells. Non-limiting examples of TAAs are listed in Table 1.

[0242] [Table 1]

[0243] [Table 2]

[0244] As used herein, a "pharmaceutically acceptable carrier, diluent, or excipient" includes, but is not limited to, any adjuvant, carrier, additive, glidant, sweetener, diluent, preservative, dye / colorant, flavor enhancer, surfactant, wetting agent, dispersing agent, suspending agent, stabilizer, isotonic agent, solvent, surface active agent, or emulsifier approved by the U.S. Food and Drug Administration as acceptable for use in humans or veterinary medicine. Exemplary pharma- ceutically acceptable carriers include, but are not limited to, sugars such as lactose, glucose, and sucrose; starches such as corn starch and potato starch; cellulose and its derivatives such as sodium carboxymethylcellulose, ethylcellulose, and cellulose acetate; tragacanth; malt; gelatin; talc; cocoa butter, waxes, animal and vegetable fats, paraffin, silicones, bentonite, silicic acid, zinc oxide; oils such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil, soybean oil; glycols such as propylene glycol; polyols such as glycerin, sorbitol, mannitol, and polyethylene glycol; esters such as ethyl oleate, ethyl laurate; agar; buffers such as magnesium hydroxide, aluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline; Ringer's solution; ethyl alcohol; phosphate buffers; and any other compatible substance used in pharmaceutical formulations.

[0245] As used herein, the terms "population of cells" or "plurality of cells" can be used interchangeably and refer to two or more cells.

[0246] As used herein, the term "inflammatory disease" refers to a disease or condition characterized by abnormal inflammation (e.g., elevated levels of inflammation compared to a control, such as a healthy individual not afflicted with the disease). Examples of inflammatory diseases include autoimmune diseases, arthritis, rheumatoid arthritis, psoriatic arthritis, juvenile idiopathic arthritis, multiple sclerosis, systemic lupus erythematosus (SLE), myasthenia gravis, juvenile onset diabetes, type 1 diabetes, Guillain-Barré syndrome, Hashimoto's encephalitis, Hashimoto's thyroiditis, ankylosing spondylitis, psoriasis, Sjogren's syndrome, vasculitis, glomerulonephritis, autoimmune thyroiditis, Behcet's disease, Crohn's disease, and ulcers. In some cases, the conditions that may be affected include, but are not limited to, inflammatory bowel disease, bullous pemphigoid, sarcoidosis, ichthyosis, Graves' ophthalmopathy, inflammatory bowel disease, Addison's disease, vitiligo, asthma, allergic asthma, acne vulgaris, celiac disease, chronic prostatitis, inflammatory bowel disease, pelvic inflammatory disease, reperfusion injury, ischemia-reperfusion injury, stroke, sarcoidosis, transplant rejection, interstitial cystitis, atherosclerosis, scleroderma, and atopic dermatitis.

[0247] As used herein, the term "autoimmune disease" refers to a disease or condition in which the immune system of a subject has an abnormal immune response to a substance that does not normally induce an immune response in healthy subjects. Examples of autoimmune diseases that can be treated with the compounds, pharmaceutical compositions or methods described herein include acute disseminated encephalomyelitis (ADEM), acute necrotizing hemorrhagic leukoencephalitis, Addison's disease, agammaglobulinemia, alopecia areata, amyloidosis, ankylosing spondylitis, anti-GBM / anti-TBM nephritis, antiphospholipid syndrome (APS), autoimmune angioedema, autoimmune aplastic anemia, autoimmune autonomic neuropathy, autoimmune hepatitis, autoimmune hyperlipidemia, autoimmune immunodeficiency, autoimmune inner ear disease (AIED), autoimmune myocarditis, Autoimmune oophoritis, autoimmune pancreatitis, autoimmune retinopathy, autoimmune thrombocytopenic purpura (ATP), autoimmune thyroid disease, autoimmune urticaria, axonal or neuronal neuropathy, Baro's disease, Behçet's disease, bullous pemphigoid, cardiomyopathy, Castleman's disease, celiac disease, Chagas disease, chronic fatigue syndrome, chronic inflammatory demyelinating polyneuropathy (CIDP), chronic relapsing multifocal stomatitis (CRMO), Churg-Strauss syndrome, cicatricial pemphigoid / benign mucous membrane pemphigoid, Crohn's disease, Cogan's syndrome, Cold agglutinin disease, congenital heart block, Coxsackie myocarditis, CREST disease, essential mixed cryoglobulinemia, demyelinating neuropathy, dermatitis herpetiformis, dermatomyositis, Devic's disease (neuromyelitis optica), discoid lupus, Dressler's syndrome, endometriosis, eosinophilic esophagitis, eosinophilic fasciitis, erythema nodosum, experimental allergic myelitis, Evans syndrome, fibromyalgia, fibrosing alveolitis, giant cell arteritis (temporal arteritis), giant cell myocarditis, glomerulonephritis, Goodpasture's syndrome, granulomatosis with polyangiitis (GPA) (formerly Wegener's granulomatosis) encephalopathy), Graves' disease, Guillain-Barré syndrome, Hashimoto's encephalitis, Hashimoto's thyroiditis, hemolytic anemia, Henoch-Schönlein purpura, herpes gestationis, hypogammaglobulinemia, idiopathic thrombocytopenic purpura (ITP), IgA nephropathy, IgG4-related sclerosing disease, immunoregulatory lipoproteins, inclusion body myositis, interstitial cystitis, juvenile arthritis, juvenile diabetic (type 1 diabetes), juvenile dermatomyositis, Kawasaki syndrome, Lambert-Eaton syndrome, leukocytoclastic vasculitis, lichen planus, lichen sclerosus, acneic conjunctivitis,Linear immunoglobulin A disease (LAD), lupus (SLE), Lyme disease, chronic, Meniere's disease, microscopic polyangiitis, mixed connective tissue disease (MCTD), Mooren's ulcer, Mucha-Habermann disease, multiple sclerosis, myasthenia gravis, myositis, narcolepsy, neuromyelitis optica (Devic), neutropenia, ocular cicatricial pemphigoid, optic neuritis, relapsing rheumatism, PANDAS (pediatric autoimmune neuropsychiatric disorders associated with streptococcus), paraneoplastic Cerebellar degeneration, paroxysmal nocturnal hemoglobinuria (PNH), Parry-Romberg syndrome, Parsonase-Turner syndrome, pars planitis (peripheral uveitis), pemphigus, peripheral neuropathy, perivenous encephalomyelitis, pernicious anemia, POEMS syndrome, polyarteritis nodosa, autoimmune polyglandular syndrome types I, II and III, polymyalgia rheumatica, polymyositis, post-myocardial infarction syndrome, post-pericardiotomy syndrome, progesterone dermatitis, primary Primary biliary cirrhosis, primary sclerosing cholangitis, psoriasis, psoriatic arthritis, idiopathic pulmonary fibrosis, pyoderma gangrenosum, erythroblastic hypoplasia, Raynaud's phenomenon, reactive arthritis, reflex sympathetic dystrophy, Reiter's syndrome, relapsing polychondritis, restless legs syndrome, retroperitoneal fibrosis, rheumatic fever, rheumatoid arthritis, sarcoidosis, Schmidt's syndrome, gangrene, Sjogren's syndrome, sperm and testicular autoimmunity, stiff-pace These include: Johns Hopkins syndrome, subacute bacterial endocarditis (SBE), Susac syndrome, sympathetic ophthalmia, Takayasu arteritis, temporal arteritis / giant cell arteritis, thrombocytopenic purpura (TTP), Tolosa-Hunt syndrome, transverse myelitis, type 1 diabetes, ulcerative colitis, undifferentiated connective tissue disease (UCTD), uveitis, vasculitis, vesicular dermatosis, vitiligo, or Wegener's granulomatosis (i.e., granulomatosis with polyangiitis (GPA)).

[0248] As used herein, the term "inflammatory disease" refers to a disease or condition characterized by abnormal inflammation (e.g., elevated levels of inflammation compared to a control, such as a healthy individual not afflicted with the disease). Examples of inflammatory diseases include traumatic brain injury, arthritis, rheumatoid arthritis, psoriatic arthritis, juvenile idiopathic arthritis, multiple sclerosis, systemic lupus erythematosus (SLE), myasthenia gravis, juvenile onset diabetes, type 1 diabetes, Guillain-Barré syndrome, Hashimoto's encephalitis, Hashimoto's thyroiditis, ankylosing spondylitis, psoriasis, Sjogren's syndrome, vasculitis, glomerulonephritis, autoimmune thyroiditis, Behcet's disease, Crohn's disease, ulcerative colitis, bullous pemphigoid, sarcoidosis, ichthyosis, Graves' ophthalmopathy, inflammatory bowel disease, Addison's disease, vitiligo, asthma, allergic asthma, acne vulgaris, celiac disease, chronic prostatitis, inflammatory bowel disease, pelvic inflammatory disease, reperfusion injury, sarcoidosis, transplant rejection, interstitial cystitis, atherosclerosis, and atopic dermatitis.

[0249] As used herein, the term "cancer" refers to all types of cancer, neoplasms, or malignant tumors found in mammals (e.g., humans), including leukemia, lymphoma, carcinoma, and sarcoma. Exemplary cancers that may 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 may be treated with the compounds or methods provided herein include thyroid, endocrine system, brain, breast, cervix, colon, head and neck, liver, kidney, lung, ovary, pancreas, rectum, stomach, and uterine cancer. Further examples include thyroid carcinoma, bile duct carcinoma, pancreatic adenocarcinoma, cutaneous melanoma, colon adenocarcinoma, rectal adenocarcinoma, gastric adenocarcinoma, esophageal carcinoma, head and neck squamous cell carcinoma, breast invasive carcinoma, lung adenocarcinoma, lung squamous cell carcinoma, non-small cell lung carcinoma, mesothelioma, multiple myeloma, neuroblastoma, glioma, glioblastoma multiforme, ovarian cancer, rhabdomyosarcoma, primary thrombocytosis, primary macroglobulinemia, primary brain tumor, malignant pancreatic insulinoma, malignant carcinoid, bladder cancer, premalignant skin lesions, testicular cancer, thyroid cancer, neuroblastoma, esophageal cancer, genitourinary tract cancer, malignant hypercalcemia, endometrial cancer, adrenal cortical carcinoma, endocrine or exocrine pancreatic neoplasms, medullary thyroid cancer, medullary thyroid carcinoma, melanoma, colorectal cancer, papillary thyroid cancer, hepatocellular carcinoma, or prostate cancer. II. Engineered T Cells

[0250] In one aspect, engineered T cells are provided. Each engineered T cell comprises a nucleic acid sequence encoding a polypeptide comprising an exogenous TCR-beta and an exogenous TCR-alpha. In an embodiment, the engineered T cell comprises a nucleic acid sequence encoding a polypeptide comprising an exogenous TCR-beta and an exogenous TCR-alpha (VJ) domain. In an embodiment, the nucleic acid sequence is inserted into the TCR-alpha locus of the engineered T cell.

[0251] In another related aspect, there is provided a composition comprising isolated T cells, wherein at least 5% of the cells are engineered T cells, each engineered T cell comprising a nucleic acid sequence encoding a polypeptide comprising an exogenous TCR-beta and an exogenous TCR-alpha. In an embodiment, the nucleic acid sequence is inserted into the TCR-alpha locus of the engineered T cell.

[0252] In embodiments, at least 10% of the cells are engineered T cells. In embodiments, at least 15% of the cells are engineered T cells. In embodiments, at least 20% of the cells are engineered T cells. In embodiments, at least 25% of the cells are engineered T cells. In embodiments, at least 30% of the cells are engineered T cells. In embodiments, at least 40% of the cells are engineered T cells. In embodiments, at least 50% of the cells are engineered T cells. In embodiments, at least 60% of the cells are engineered T cells. In embodiments, between about 5% and 100% of the cells are engineered T cells. In embodiments, between about 5% and about 50% of the cells are engineered T cells. In embodiments, between about 5% and about 25% of the cells are engineered T cells. In embodiments, between about 5% and about 20% of the cells are engineered T cells. In embodiments, between about 5% and about 15% of the cells are engineered T cells. The percentages may be any value or subrange within the recited range, including the endpoints.

[0253] In embodiments, the engineered T cells do not express functional endogenous TCR-beta protein. In embodiments, less than about 50% of the engineered T cells express functional endogenous TCR-beta protein. In embodiments, less than about 40% of the engineered T cells express functional endogenous TCR-beta protein. In embodiments, less than about 30% of the engineered T cells express functional endogenous TCR-beta protein. In embodiments, less than about 20% of the engineered T cells express functional endogenous TCR-beta protein. In embodiments, less than about 10% of the engineered T cells express functional endogenous TCR-beta protein. In embodiments, less than about 5% of the engineered T cells express functional endogenous TCR-beta protein. In embodiments, less than about 4% of the engineered T cells express functional endogenous TCR-beta protein. In embodiments, less than about 3% of the engineered T cells express functional endogenous TCR-beta protein. In embodiments, less than about 2% of the engineered T cells express functional endogenous TCR-beta protein. In embodiments, less than about 1% of the engineered T cells express functional endogenous TCR-beta protein. In embodiments, about 0% to about 50% of the engineered T cells express functional endogenous TCR-beta protein. In embodiments, about 0% to about 25% of the engineered T cells express functional endogenous TCR-beta protein. In embodiments, about 1% to about 25% of the engineered T cells express functional endogenous TCR-beta protein. The percentages may be any value or subrange within the recited range, including the endpoints.

[0254] In embodiments, at least about 50% of the engineered T cells have a non-functional TCR-beta locus that is disrupted across all alleles. In embodiments, at least about 40% of the engineered T cells have a non-functional TCR-beta locus that is disrupted across all alleles. In embodiments, at least about 60%, 70%, 80% or 90% of the engineered T cells have a non-functional TCR-beta locus that is disrupted across all alleles. In embodiments, at least about 95%, 96%, 97%, 98%, or 99% of the engineered T cells have a non-functional TCR-beta locus that is disrupted across all alleles. In embodiments, between about 50% and about 100% of the engineered T cells have a non-functional TCR-beta locus that is disrupted across all alleles. In embodiments, between about 60% and about 95% of the engineered T cells have a non-functional TCR-beta locus that is disrupted across all alleles. The percentage can be any value or subrange within the recited range, including the endpoints.

[0255] In embodiments, less than about 50% of the engineered T cells express functional endogenous TCR-alpha protein. In embodiments, less than about 40% of the engineered T cells express functional endogenous TCR-alpha protein. In embodiments, less than about 30% of the engineered T cells express functional endogenous TCR-alpha protein. In embodiments, less than about 20% of the engineered T cells express functional endogenous TCR-alpha protein. In embodiments, less than about 10% of the engineered T cells express functional endogenous TCR-alpha protein. In embodiments, less than about 5% of the engineered T cells express functional endogenous TCR-alpha protein. In embodiments, less than about 4% of the engineered T cells express functional endogenous TCR-alpha protein. In embodiments, less than about 3% of the engineered T cells express functional endogenous TCR-alpha protein. In embodiments, less than about 2% of the engineered T cells express functional endogenous TCR-alpha protein. In embodiments, less than about 1% of the engineered T cells express functional endogenous TCR-alpha protein. In embodiments, between about 0% and about 50% of the engineered T cells express functional endogenous TCR-alpha protein. In embodiments, between about 0% and about 25% of the engineered T cells express functional endogenous TCR-alpha protein. In embodiments, between about 1% and about 25% of the engineered T cells express functional endogenous TCR-alpha protein. The percentages may be any value or subrange within the recited range, including the endpoints.

[0256] In embodiments, at least about 50% of the engineered T cells have a non-functional TCR-alpha locus that is disrupted across all alleles. In embodiments, at least about 40% of the engineered T cells have a non-functional TCR-alpha locus that is disrupted across all alleles. In embodiments, at least about 60%, 70%, 80% or 90% of the engineered T cells have a non-functional TCR-alpha locus that is disrupted across all alleles. In embodiments, at least about 95%, 96%, 97%, 98%, or 99% of the engineered T cells have a non-functional TCR-alpha locus that is disrupted across all alleles. In embodiments, between about 50% and about 100% of the engineered T cells have a non-functional TCR-alpha locus that is disrupted across all alleles. In embodiments, between about 60% and about 95% of the engineered T cells have a non-functional TCR-alpha locus that is disrupted across all alleles. The percentage can be any value or subrange within the recited range, including the endpoints.

[0257] In an embodiment, the exogenous TCR-alpha (VJ) domain forms part of a heterologous TCR-alpha that comprises at least a portion of the T cell's endogenous TCR-alpha. In an embodiment, the TCR-alpha locus is a TCR-alpha constant region. In an embodiment, the exogenous TCR-beta and the heterologous TCR-alpha are expressed from a nucleic acid to form a functional TCR. In an embodiment, at least 40% of the engineered T cells express the heterologous TCR-alpha. In an embodiment, at least 50% of the engineered T cells express the heterologous TCR-alpha. In an embodiment, at least 60% of the engineered T cells express the heterologous TCR-alpha. In an embodiment, at least 70% of the engineered T cells express the heterologous TCR-alpha. In an embodiment, at least 80% of the engineered T cells express the heterologous TCR-alpha. In an embodiment, at least 90% of the engineered T cells express the heterologous TCR-alpha.

[0258] In embodiments, the engineered T cells are bound to an antigen. In embodiments, the engineered T cells are bound to a cancer cell. In embodiments, the TCR binds to an antigen presented on a major histocompatibility complex class I (MHCI) molecule.

[0259] In embodiments, the antigen is a neo-antigen or tumor associated antigen (TAA). In embodiments, the antigen is a neo-antigen. In embodiments, the antigen is a TAA. In embodiments, the neo-antigen or TAA is selected from WT1, JAK2, NY-ESO1, PRAME, KRAS, HPV, or an antigen from Table 1 or Table 2. In embodiments, the antigen is WT1. In embodiments, the antigen is specific to the cancer of the subject receiving the engineered T cells. In embodiments, the antigen is expressed by or associated with the cancer of the subject receiving the engineered T cells.

[0260] In an embodiment, the nucleic acid sequence further encodes a self-cleaving peptide. In an embodiment, the self-cleaving peptide is a self-cleaving viral peptide. In an embodiment, the self-cleaving viral peptide is T2A. In an embodiment, the self-cleaving viral peptide is P2A. In an embodiment, the self-cleaving viral peptide is E2A. In an embodiment, the self-cleaving viral peptide is F2A.

[0261] In an embodiment, the nucleic acid sequence further encodes a protease cleavage site. In an embodiment, the protease cleavage site is a furin cleavage site.

[0262] In embodiments, the nucleic acid further comprises a polyadenylation (polyA) sequence. In embodiments, the polyadenylation sequence may be located immediately 3' to the TCR coding sequence. In embodiments, the polyadenylation sequence may be located immediately 5' to the second homology arm. In embodiments, the polyadenylation sequence is the bovine growth hormone polyadenylation sequence (bgh-polyA).

[0263] In embodiments, the engineered T cells express CD45RO, CC chemokine receptor type 7 (CCR7) and L-selectin (CD62L). In embodiments, the engineered T cells have a central memory (CM) T cell phenotype. In embodiments, the engineered T cells have a naive T cell phenotype. In embodiments, the engineered T cells have a naive T cell phenotype are CD45RA+CD45RO-CD27+CD95- (i.e., the cells express CD45RA and CD27 and do not express detectable levels of CD45RO and CD95). In embodiments, the engineered T cells have a stem cell memory T cell phenotype. In embodiments, the engineered T cells have a stem cell memory T cell phenotype are CD45RA+CD45RO-CD27+CD95+CD58+CCR7-Hi TCF1+. In embodiments, the engineered T cells have a central memory T cell phenotype. In embodiments, the engineered T cells with a central memory T cell phenotype are CD45RO+CD45RA-CD27+CD95+CD58+. In embodiments, the engineered T cells have a progenitor exhausted T cell phenotype. In embodiments, the engineered T cells with a progenitor exhausted T cell phenotype are PD-1+SLAMF6+TCF1+TIM3-CD39-. In embodiments, the engineered T cells with a progenitor exhausted T cell phenotype express PD-1 at low or intermediate levels compared to PD-1 high exhausted T cells, or to recently activated T cells. In embodiments, the engineered T cells with a progenitor exhausted T cell phenotype express PD-1 at low or intermediate levels compared to PD-1 levels expressed in recently activated T cells. In embodiments, the T cells are autologous to a subject in need thereof.

[0264] In another related aspect, there is provided a pharmaceutical composition comprising a population of engineered T cells as described herein, including embodiments, and a pharma- ceutical acceptable excipient.

[0265] In embodiments, at least 10% of the cells in the composition comprising isolated T cells are engineered T cells. In embodiments, at least 20% of the cells are engineered T cells. In embodiments, at least 30% of the cells are engineered T cells. In embodiments, at least 40% of the cells are engineered T cells. In embodiments, at least 50% of the cells are engineered T cells. In embodiments, at least 60% of the cells are engineered T cells. In embodiments, at least 70% of the cells are engineered T cells. In embodiments, at least 80% of the cells are engineered T cells. In embodiments, at least 90% of the cells are engineered T cells.

[0266] In embodiments, the composition comprises about 0.1×10 5 pieces~approx. 1×10 9 In some embodiments, the composition comprises at least 1×10 engineered T cells. 8 In some embodiments, the composition comprises at least 1×10 engineered T cells. 9 In an embodiment, the composition comprises 1×10 engineered T cells. 9 pieces~1×10 11 In some embodiments, the composition comprises at least 1×10 engineered T cells. 10 In some embodiments, the composition comprises at least 1×10 engineered T cells. 11 The number of cells can be any value or subrange between the recited ranges, including the endpoints.

[0267] In an embodiment, the composition further comprises a pharma- ceutically acceptable additive. Means for making such compositions or implants are described in the art (see, for example, Remington's Pharmaceutical Sciences, 16th Ed., Mack, ed. (1980)). Where appropriate, the engineered T cells may be formulated into semi-solid or liquid form preparations, such as capsules, solutions, injections, inhalants, or aerosols, in the usual manner for each route of administration. In an embodiment, the additive is a balanced salt solution, such as Hank's balanced salt solution, or saline. It will also be understood that, if desired, the compositions of the present invention may be administered in combination with other agents, such as cytokines, growth factors, hormones, small molecules, chemotherapeutic agents, prodrugs, drugs, antibodies, or various other pharma- ceutical active agents. There is virtually no limit to the other components that may be included in the composition, so long as the additional agents do not adversely affect the ability of the composition to deliver the intended therapy.

[0268] In another related aspect, there is provided a T cell comprising an RNA transcript having a structure as described herein, in embodiments, the T cell comprising the RNA transcript, wherein the mRNA transcript is transcribed from a TCR transgene inserted into the TCR-alpha and / or TCR-beta locus.

[0269] III. Methods for Preparation In another related embodiment, a method for generating an engineered T cell is provided, the method comprising: a) contacting a T cell with a first ribonucleoprotein particle (RNP) and donor DNA under conditions that allow the RNP and donor DNA to enter the T cell, where the first RNP comprises a first guide RNA that targets an endogenous TCR-alpha locus, and the donor DNA comprises a nucleic acid sequence that comprises a gene encoding a polypeptide comprising an exogenous TCR-beta and an exogenous TCR-alpha, b) incubating the T cell for a period of time, and c) culturing the cell in medium for a period of time to allow the donor DNA to insert into the endogenous TCR-alpha locus, thereby forming an engineered T cell.

[0270] In embodiments, the first RNP comprises a first gene editing protein and a first guide RNA in molar excess of the latter. In some embodiments, the first RNP comprises a first gene editing protein, and the ratio of the first guide RNA to the first gene editing protein is about 1:1 to about 100:1 molar ratio. In some embodiments, the ratio of the first guide RNA to the first gene editing protein is about 1:1 to about 75:1 molar ratio. In some embodiments, the ratio of the first guide RNA to the first gene editing protein is about 1:1 to about 50:1 molar ratio. In some embodiments, the ratio of the first guide RNA to the first gene editing protein is about 1:1 to about 25:1 molar ratio. In some embodiments, the ratio of the first guide RNA to the first gene editing protein is about 1:1 to about 10:1 molar ratio. In an embodiment, the ratio of the first guide RNA to the first gene editing protein is about 1:1 to about 5:1 molar ratio. In an embodiment, the ratio of the first guide RNA to the first gene editing protein is about 1:1 to about 4:1 molar ratio. In an embodiment, the ratio of the first guide RNA to the first gene editing protein is about 1:1 molar ratio. In an embodiment, the ratio of the first guide RNA to the first gene editing protein is about 2:1 molar ratio. In an embodiment, the ratio of the first guide RNA to the first gene editing protein is about 3:1 molar ratio. In an embodiment, the ratio of the first guide RNA to the first gene editing protein is about 4:1 molar ratio. In an embodiment, the ratio of the first guide RNA to the first gene editing protein is about 5:1 molar ratio. In an embodiment, the ratio of the first guide RNA to the first gene editing protein is about 6:1 molar ratio. In an embodiment, the ratio of the first guide RNA to the first gene editing protein is about 7:1 molar ratio. In an embodiment, the ratio of the first guide RNA to the first gene editing protein is about 8:1 molar ratio. In an embodiment, the ratio of the first guide RNA to the first gene editing protein is about 9:1 molar ratio. In an embodiment, the ratio of the first guide RNA to the first gene editing protein is about 10:1 molar ratio. In an embodiment, the ratio of the first guide RNA to the first gene editing protein is about 25:1 molar ratio.In an embodiment, the ratio of the first guide RNA to the first gene editing protein is about 50: 1 molar ratio. In an embodiment, the ratio of the first guide RNA to the first gene editing protein is about 75: 1 molar ratio. In an embodiment, the ratio of the first guide RNA to the first gene editing protein is about 100: 1 molar ratio. The molar ratio may be any value or subrange within the recited range, including the endpoints.

[0271] In an embodiment, the T cell is contacted with a second RNP comprising a second guide RNA targeting an endogenous TCR-beta locus. In an embodiment, the second RNP comprises a second gene editing protein and a molar excess of the second guide RNA of the latter. In an embodiment, the second RNP comprises a second gene editing protein, and the molar ratio of the second guide RNA to the second gene editing protein is 1:1 to 100:1. In an embodiment, the second RNP comprises a second gene editing protein, and the molar ratio of the second guide RNA to the second gene editing protein is 1:1 to 75:1. In an embodiment, the second RNP comprises a second gene editing protein, and the molar ratio of the second guide RNA to the second gene editing protein is 1:1 to 50:1. In an embodiment, the second RNP comprises a second gene editing protein, and the molar ratio of the second guide RNA to the second gene editing protein is 1:1 to 25:1. In an embodiment, the second RNP comprises a second gene editing protein, and the molar ratio of the second guide RNA to the second gene editing protein is 1:1 to 10:1. In an embodiment, the second RNP comprises a second gene editing protein, and the molar ratio of the second guide RNA to the second gene editing protein is 1:1 to 5:1. In an embodiment, the molar ratio of the second guide RNA to the second gene editing protein is 1:1 to 4:1. In an embodiment, the molar ratio of the second guide RNA to the second gene editing protein is about 1:1. In an embodiment, the molar ratio of the second guide RNA to the second gene editing protein is about 2:1. In an embodiment, the molar ratio of the second guide RNA to the second gene editing protein is about 3:1. In an embodiment, the molar ratio of the second guide RNA to the second gene editing protein is about 4:1. In an embodiment, the molar ratio of the second guide RNA to the second gene editing protein is about 5:1. In an embodiment, the ratio of the first guide RNA to the first gene editing protein is about 10: 1 molar ratio. In an embodiment, the ratio of the first guide RNA to the first gene editing protein is about 25: 1 molar ratio. In an embodiment, the ratio of the first guide RNA to the first gene editing protein is about 50: 1 molar ratio.In an embodiment, the ratio of the first guide RNA to the first gene editing protein is a molar ratio of about 75: 1. In an embodiment, the ratio of the first guide RNA to the first gene editing protein is a molar ratio of about 100: 1. The molar ratio may be any value or subrange within the recited ranges, including the endpoints.

[0272] In an embodiment, the T cell is contacted with the second RNP comprising the second guide RNA at the same time as the first RNP comprising the first guide RNA. In an embodiment, the T cell is contacted with the second RNP comprising the second guide RNA before the first RNP comprising the first guide RNA. In an embodiment, the T cell is contacted with the second RNP comprising the second guide RNA after the first RNP comprising the first guide RNA.

[0273] In embodiments, the first guide RNA is discovered by methods known to those of skill in the art to target the exon 1, exon 2, or exon 3 region of the TCR constant alpha region locus (TRAC). In embodiments, the first guide RNA targets one of the following sequences from the TCR-alpha locus: TRAC1 (SEQ ID NO: 7), TRAC2 (SEQ ID NO: 8), TRAC3 (SEQ ID NO: 9), TRAC4 (SEQ ID NO: 10), TRAC5 (SEQ ID NO: 11), TRAC6 (SEQ ID NO: 12), TRAC7 (SEQ ID NO: 13), TRAC8 (SEQ ID NO: 14), TRAC9 (SEQ ID NO: 15), TRAC10 (SEQ ID NO: 16), TRAC11 (SEQ ID NO: 17), TRAC12 (SEQ ID NO: 18), TRAC13 (SEQ ID NO: 19), TRAC14 (SEQ ID NO: 20), TRAC15 (SEQ ID NO: 21), or TRAC16 (SEQ ID NO: 22). In embodiments, the first guide RNA comprises one of TRAC1, TRAC3, TRAC4, TRAC5, TRAC7, TRAC12, or TRAC15. In embodiments, the first guide RNA targets the sequence TRAC1. In embodiments, the first guide RNA targets the sequence TRAC3. In embodiments, the first guide RNA comprises a nucleic acid sequence in Table 10. Any one or more of the guide RNAs may be explicitly excluded.

[0274] In an embodiment, the second guide RNA is discovered by methods known to those skilled in the art to target the exon 1 region of both TCR constant beta region loci (TRBC). In an embodiment, the second guide RNA is selected from the following TCR-beta loci: TRBC1 (SEQ ID NO: 23), TRBC2 (SEQ ID NO: 24), TRBC3 (SEQ ID NO: 25), TRBC4 (SEQ ID NO: 26), TRBC5 (SEQ ID NO: 27), TRBC6 (SEQ ID NO: 28), TRBC7 (SEQ ID NO: 29), TRBC8 (SEQ ID NO: 30), TRBC9 (SEQ ID NO: 31), TRBC10 (SEQ ID NO: 32), TRBC11 (SEQ ID NO: 33), TRBC12 (SEQ ID NO: 34), TRBC13 (SEQ ID NO: 35), TRBC14 (SEQ ID NO: 36), TRBC15 (SEQ ID NO: 37), TRBC16 (SEQ ID NO: 38), TRBC17 (SEQ ID NO: 39), TRBC18 (SEQ ID NO: 40), TRBC19 (SEQ ID NO: 41), TRBC20 (SEQ ID NO: 42), TRBC21 (SEQ ID NO: 43), TRBC22 (SEQ ID NO: 44), TRBC23 (SEQ ID NO: 45), TRBC24 (SEQ ID NO: 46), TRBC25 (SEQ ID NO: 47), TRBC26 (SEQ ID NO: 48), TRBC27 (SEQ ID NO: 49), TRBC28 (SEQ ID NO: 50), TRBC29 (SEQ ID NO: 51), TRBC30 (SEQ ID NO: 52), TRBC31 (SEQ ID NO: 53), TRBC32 (SEQ ID NO: 54), TRBC33 (SEQ ID NO: No. 35), TRBC14 (SEQ ID NO: 36), TRBC15 (SEQ ID NO: 37), TRBC16 (SEQ ID NO: 38), TRBC17 (SEQ ID NO: 39), TRBC18 (SEQ ID NO: 40), TRBC19 (SEQ ID NO: 41), TRBC20 (SEQ ID NO: 42), TRBC21 (SEQ ID NO: 43), TRBC22 (SEQ ID NO: 44), TRBC23 (SEQ ID NO: 45), TRBC24 (SEQ ID NO: 46), TRBC25 (SEQ ID NO: 47), or TRBC26 (SEQ ID NO: 48). In embodiments, the second guide RNA targets one or more of the following loci: TRBC4, TRBC8, TRBC13, TRBC19, TRBC20, TRBC21, TRBC22, TRBC23, or TRBC26. In embodiments, the second guide RNA comprises a nucleic acid sequence in Table 11. Any one or more of the guide RNAs may be explicitly excluded.

[0275] In some embodiments, the RNP comprises a CRISPR-associated (CAS) protein. In some embodiments, the heterodimeric protein is Cas9. In some embodiments, the Cas9 is Streptococcus pyogenes (Sp)Cas9. In some embodiments, the SpCas9 is wild-type SpCas9. In some embodiments, the SpCas9 is a mutant SpCas9.

[0276] In an embodiment, the RNP comprises a guide RNA. In an embodiment, the guide RNA is a synthetic nucleic acid. In an embodiment, the guide RNA comprises non-naturally occurring bases and / or backbone linkages known in the art. In an embodiment, the guide RNA is a single guide (sg) RNA. In an embodiment, the guide RNA comprises a tracer (tr) RNA and a crispr (cr) RNA. In an embodiment, the guide RNA comprises a phosphodiester derivative, including but not limited to phosphoramidate, phosphorodiamidate, phosphorothioate, phosphorodithioate, phosphonocarboxylic acid, phosphonocarboxylate, phosphonoacetic acid, phosphonoformic acid, methylphosphonate, boron phosphonate, or O-methyl phosphoramidite linkage.

[0277] In embodiments, the conditions that allow the RNP and donor DNA to enter the cells include electroporation. In embodiments, in method step b), the T cells are incubated for at least 10 minutes. In embodiments, the T cells are incubated at about 37° C. In embodiments, the T cells are incubated at less than about 37° C. In embodiments, the medium comprises a cytokine. In embodiments, the cytokine comprises IL-2, IL-7 and / or IL-15. In embodiments, the cytokine comprises IL-2. In embodiments, the cytokine comprises IL-7. In embodiments, the cytokine comprises IL-15. In embodiments, the cytokine comprises IL-7 and IL-15. Any one or more of the listed cytokines may be explicitly excluded.

[0278] In some embodiments, method step a) is carried out in the presence of a negatively charged polymer. In embodiments, the polymer is poly(glutamic acid) (PGA) or a variant thereof, poly(aspartic acid), heparin, or poly(acrylic acid). In embodiments, the PGA is poly(L-glutamic acid) or a variant thereof. In embodiments, the PGA is poly(D-glutamic acid) or a variant thereof. In embodiments, the PGA or a variant thereof has an average molecular weight between 15 kilodaltons (kDa) and 50 kDa. Any one or more of the polymers may be explicitly excluded.

[0279] In some embodiments, about 0.4 μg / μL to about 20 μg / μL of polymer is added. In some embodiments, about 2 μg / μL to about 12 μg / μL of polymer is added. In some embodiments, about 2 μg / μL to about 8 μg / μL of polymer is added. In some embodiments, about 4 μg / μL to about 8 μg / μL of polymer is added. In some embodiments, about 6 μg / μL to about 8 μg / μL of polymer is added. In some embodiments, about 6 μg / μL of polymer is added. In some embodiments, about 8 μg / μL of polymer is added. The amount may be any value or subrange within the recited range, including the endpoints. In embodiments, no polymer is added.

[0280] In an embodiment, the amount of RNP is about 0.04 pmol / μL to about 20 pmol / μL. In an embodiment, the amount of RNP is about 0.2 pmol / μL to about 8 pmol / μL. In an embodiment, the amount of RNP is about 0.6 pmol / μL to about 8 pmol / μL. In an embodiment, the amount of RNP is about 0.8 pmol / μL to about 8 pmol / μL. In an embodiment, the amount of RNP is about 1.2 pmol / μL to about 8 pmol / μL. In an embodiment, the amount of RNP is about 2 pmol / μL to about 8 pmol / μL. In an embodiment, the amount of RNP is about 3 pmol / μL to about 8 pmol / μL. In an embodiment, the amount of RNP is about 4 pmol / μL to about 8 pmol / μL. In an embodiment, the amount of RNP is about 0.2 pmol / μL to about 6 pmol / μL. In embodiments, the amount of RNP is from about 0.2 pmol / μL to about 4 pmol / μL. In embodiments, the amount of RNP is from about 0.2 pmol / μL to about 2 pmol / μL. In embodiments, the amount of RNP is from about 0.2 pmol / μL to about 1 pmol / μL. The amount can be any value or subrange within the recited range, including the endpoints.

[0281] In an embodiment, the amount of donor DNA is about 0.0002 pmol / μL to about 2 pmol / μL. In an embodiment, the amount of donor DNA is about 0.0004 pmol / μL to about 0.4 pmol / μL. In an embodiment, the amount of donor DNA is about 0.0004 pmol / μL to about 0.2 pmol / μL. In an embodiment, the amount of donor DNA is about 0.0004 pmol / μL to about 0.04 pmol / μL. In an embodiment, the amount of donor DNA is about 0.0004 pmol / μL to about 0.02 pmol / μL. In an embodiment, the amount of donor DNA is about 0.0004 pmol / μL to about 0.004 pmol / μL. In an embodiment, the amount of donor DNA is about 0.0002 pmol / μL to about 0.4 pmol / μL. In an embodiment, the amount of donor DNA is about 0.004 pmol / μL to about 0.4 pmol / μL. In an embodiment, the amount of donor DNA is about 0.02 pmol / μL to about 0.4 pmol / μL. In an embodiment, the amount of donor DNA is about 0.04 pmol / μL to about 0.4 pmol / μL. In an embodiment, the amount of donor DNA is about 0.08 pmol / μL to about 0.4 pmol / μL. In an embodiment, the amount of donor DNA is about 0.2 pmol / μL to about 0.4 pmol / μL. The amount may be any value or subrange within the recited range, including the endpoints.

[0282] The amounts of polymer, RNP, donor DNA, and any other components of the embodiments described herein can be scaled up as needed, e.g., to prepare engineered T cells for clinical use.

[0283] In an embodiment, the donor DNA recombines into an endogenous TCR-alpha locus. In an embodiment, the TCR-alpha locus is a TCR-alpha constant chain. In an embodiment, the donor DNA recombines into an endogenous TCR-beta locus.

[0284] In an embodiment, the donor DNA comprises a left homology arm and a right homology arm. In an embodiment, the left homology arm and the right homology arm are homologous to an endogenous TCR-alpha locus. In an embodiment, the left homology arm is about 50 bases to about 2000 bases in length. In an embodiment, the left homology arm is about 100 bases to about 1000 bases in length. In an embodiment, the left homology arm is about 200 bases to about 800 bases in length. In an embodiment, the right homology arm is about 200 bases to about 2000 bases in length. In an embodiment, the right homology arm is about 100 bases to about 1000. In an embodiment, the left homology arm and the right homology arm are homologous to an endogenous TCR-beta locus. In an embodiment, the left homology arm is about 200 bases to about 800 bases in length. In embodiments, the left homology arm is about 250 to about 700 bases in length. In embodiments, the right homology arm is about 200 to about 800 bases in length. In embodiments, the right homology arm is about 250 to about 700 bases in length. The lengths may be any value or subrange within the recited ranges, including the endpoints.

[0285] In embodiments, the donor DNA comprises double stranded DNA (dsDNA). In embodiments, the donor DNA is on a plasmid, nanoplasmid, or minicircle. In embodiments, the donor DNA is on a plasmid. In embodiments, the donor DNA is on a nanoplasmid. In embodiments, the donor DNA is on a minicircle. In embodiments, the donor DNA is linear. In embodiments, the donor DNA comprises single stranded DNA (ssDNA). In embodiments, the donor DNA is not chemically modified. In embodiments, the donor DNA comprises a chemical modification. In embodiments, the modification comprises a 5' phosphate or a 5' phosphorothioate.

[0286] In embodiments, the donor DNA and the RNP are incubated together prior to contacting the T cell with the first RNP and the donor DNA. In some embodiments, the gene editing protein of the RNP comprises a nuclear localization sequence (NLS).

[0287] In embodiments, the T cells are cultured in a medium. In embodiments, the medium is selected from RPMI, PRIME-XV, and / or X-VIVO.

[0288] In an embodiment, the T cells are activated prior to contacting the T cells with the first RNP and donor DNA. In an embodiment, the T cells are activated for 24 to 96 hours. In an embodiment, the T cells are activated in the presence of cytokines. In an embodiment, the cytokines include IL-2, IL-7 and / or IL-15.

[0289] In an embodiment, the T cells are activated with IL-2. In an embodiment, the T cells are activated in the presence of about 0 ng / mL to about 50 ng / mL of IL-2. In an embodiment, the T cells are activated in the presence of about 5 ng / mL to about 50 ng / mL of IL-2. In an embodiment, the T cells are activated in the presence of about 1 ng / mL to about 5 ng / mL of IL-2. In an embodiment, the T cells are activated in the presence of about 5 ng / mL to about 25 ng / mL of IL-2. In an embodiment, the T cells are activated in the presence of about 5 ng / mL to about 20 ng / mL of IL-2. In an embodiment, the T cells are activated in the presence of about 5 ng / mL to about 15 ng / mL of IL-2. In an embodiment, the T cells are activated in the presence of about 10 ng / mL of IL-2. The amount may be any value or subrange within the recited range, including the endpoint.

[0290] In an embodiment, the T cells are activated with IL-2. In an embodiment, the T cells are activated in the presence of about 0 units / mL to about 1000 units / mL of IL-2. In an embodiment, the T cells are activated in the presence of about 25 units / mL to about 500 units / mL of IL-2. In an embodiment, the T cells are activated in the presence of about 125 units / mL to about 500 units / mL of IL-2. In an embodiment, the T cells are activated in the presence of about 125 units / mL to about 400 units / mL of IL-2. In an embodiment, the T cells are activated in the presence of about 125 units / mL to about 300 units / mL of IL-2. In an embodiment, the T cells are activated in the presence of about 200 units / mL of IL-2. The amount may be any value or subrange within the recited range, including the endpoint.

[0291] In an embodiment, the T cells are activated with IL-7. In an embodiment, the T cells are activated in the presence of about 0 ng / mL to about 200 ng / mL of IL-7. In an embodiment, the T cells are activated in the presence of about 5 ng / mL to about 200 ng / mL of IL-7. In an embodiment, the T cells are activated in the presence of about 10 ng / mL to about 200 ng / mL of IL-7. In an embodiment, the T cells are activated in the presence of about 5 ng / mL to about 150 ng / mL of IL-7. In an embodiment, the T cells are activated in the presence of about 5 ng / mL to about 100 ng / mL of IL-7. In an embodiment, the T cells are activated in the presence of about 5 ng / mL to about 50 ng / mL of IL-7. In an embodiment, the T cells are activated in the presence of about 10 ng / mL to about 100 ng / mL of IL-7. In embodiments, T cells are activated in the presence of about 10 ng / mL to about 50 ng / mL IL-7. In embodiments, T cells are activated in the presence of about 25 ng / mL IL-7. The amount can be any value or subrange within the recited range, including the endpoints.

[0292] In an embodiment, the T cells are activated with IL-7. In an embodiment, the T cells are activated in the presence of about 0 units / mL to about 4000 units / mL of IL-7. In an embodiment, the T cells are activated in the presence of about 20 units / mL to about 2000 units / mL of IL-7. In an embodiment, the T cells are activated in the presence of about 20 units / mL to about 1000 units / mL of IL-7. In an embodiment, the T cells are activated in the presence of about 20 units / mL to about 500 units / mL of IL-7. In an embodiment, the T cells are activated in the presence of about 100 units / mL to about 500 units / mL of IL-7. In an embodiment, the T cells are activated in the presence of about 500 units / mL of IL-7. The amount may be any value or subrange within the recited range, including the endpoint.

[0293] In an embodiment, the T cells are activated with IL-15. In an embodiment, the T cells are activated in the presence of about 0 ng / mL to about 500 ng / mL of IL-15. In an embodiment, the T cells are activated in the presence of about 0 ng / mL to about 200 ng / mL of IL-15. In an embodiment, the T cells are activated in the presence of about 5 ng / mL to about 200 ng / mL of IL-15. In an embodiment, the T cells are activated in the presence of about 10 ng / mL to about 200 ng / mL of IL-15. In an embodiment, the T cells are activated in the presence of about 15 ng / mL to about 200 ng / mL of IL-15. In an embodiment, the T cells are activated in the presence of about 25 ng / mL to about 200 ng / mL of IL-15. In an embodiment, the T cells are activated in the presence of about 5 ng / mL to about 150 ng / mL of IL-15. In embodiments, T cells are activated in the presence of about 5 ng / mL to about 100 ng / mL of IL-15. In embodiments, T cells are activated in the presence of about 10 ng / mL to about 100 ng / mL of IL-15. In embodiments, T cells are activated in the presence of about 25 ng / mL to about 100 ng / mL of IL-15. In embodiments, T cells are activated in the presence of about 50 ng / mL of IL-15. The amount may be any value or subrange within the recited range, including the endpoint.

[0294] In an embodiment, the T cells are activated with IL-15. In an embodiment, the T cells are activated in the presence of about 0 units / mL to about 500 units / mL of IL-15. In an embodiment, the T cells are activated in the presence of about 25 units / mL to about 500 units / mL of IL-15. In an embodiment, the T cells are activated in the presence of about 125 units / mL to about 500 units / mL of IL-15. In an embodiment, the T cells are activated in the presence of about 125 units / mL to about 400 units / mL of IL-15. In an embodiment, the T cells are activated in the presence of about 125 units / mL to about 300 units / mL of IL-15. In an embodiment, the T cells are activated in the presence of about 200 units / mL of IL-15. The amount may be any value or subrange within the recited range, including the endpoint.

[0295] In an embodiment, the T cells are activated in the presence of an anti-CD3 antibody and / or an anti-CD28 antibody. In an embodiment, the T cells are activated in the presence of a CD3 agonist and / or a CD28 agonist. In an embodiment, the anti-CD3 antibody and / or the anti-CD28 antibody are conjugated to a substrate. In an embodiment, the CD3 agonist and / or the CD28 agonist are conjugated to a substrate. In an embodiment, the substrate is a colloidal polymer nanomatrix. In an embodiment, the substrate is a superparamagnetic particle. In an embodiment, the T cells are activated in the presence of T CELL TRANSACT™ (Miltenyi Biotech). In an embodiment, the T cells are activated in the presence of DYNABEADS® Human T-Activator CD3 / CD28 (Thermo Fisher Scientific). In an embodiment, the method step)a is performed within about 24 hours after activation. In an embodiment, the method step)a is performed within about 24 hours to about 72 hours after activation. In an embodiment, method step a) is carried out for about 36 hours to about 60 hours after activation.

[0296] In another interrelated aspect, there is provided a method for generating a population of engineered T cells comprising performing a method as described herein, including embodiments, on a population of T cells. In embodiments, at least about 5% of the population of T cells are recovered as engineered T cells. In embodiments, about 5% to about 100% of the population of T cells are recovered as engineered T cells. In embodiments, at least about 50% of the population of T cells are recovered as engineered T cells. In embodiments, at least about 60% of the population of T cells are recovered as engineered T cells. The percentages may be any value or subrange within the recited range, including the endpoints.

[0297] In an embodiment, the engineered T cells are expanded after genetic modification. In an embodiment, the engineered T cells are expanded at least about 2 times compared to the cell number on the first day after electroporation. In an embodiment, the engineered T cells are expanded at least about 5 times, at least about 10 times, at least about 15 times, at least about 20 times, at least about 50 times, at least about 100 times, at least about 200 times, at least about 500 times, at least about 1000 times compared to the cell number on the first day after electroporation.

[0298] In embodiments, about 5% to about 100% of the T cell population are viable after method step c). In embodiments, about 10% to about 100% of the T cell population are viable after method step c). In embodiments, about 15% to about 100% of the T cell population are viable after method step c). In embodiments, about 20% to about 100% of the T cell population are viable after method step c). In embodiments, about 25% to about 100% of the T cell population are viable after method step c). In embodiments, at least about 5% of the T cell population are viable after method step c). In embodiments, at least about 10% of the T cell population are viable after method step c). In embodiments, at least about 15% of the T cell population are viable after method step c). In embodiments, at least about 20% of the T cell population are viable after method step c). In embodiments, at least about 25% of the T cell population are viable after method step c). In embodiments, at least about 50% of the T cell population is viable after method step c). In embodiments, at least about 75% of the T cell population is viable after method step c). The percentages can be any value or subrange within the recited ranges, including the endpoints.

[0299] In an embodiment, the method further comprises contacting the cells with a second RNP comprising a guide RNA targeting an endogenous TCR-beta locus, and at least about 5% of the population of T cells are recovered as engineered T cells. In an embodiment, less than about 30% of the engineered T cells express functional endogenous TCR-beta protein. In an embodiment, less than about 20% of the engineered T cells express functional endogenous TCR-beta protein. In an embodiment, less than about 10% of the engineered T cells express functional endogenous TCR-beta protein. In an embodiment, less than about 5% of the engineered T cells express functional endogenous TCR-beta protein. The percentages may be any value or subrange within the recited range, including the endpoints.

[0300] In embodiments, about 10% to about 100% of the population of T cells are recovered as engineered T cells. In embodiments, at least about 20% of the population of T cells are recovered as engineered T cells. In embodiments, at least about 30% of the population of T cells are recovered as engineered T cells. In embodiments, at least about 40% of the population of T cells are recovered as engineered T cells. In embodiments, at least about 50% of the population of T cells are recovered as engineered T cells. In embodiments, at least about 60% of the population of T cells are recovered as engineered T cells. In embodiments, at least about 70% of the population of T cells are recovered as engineered T cells. In embodiments, at least about 80% of the population of T cells are recovered as engineered T cells. In embodiments, at least about 90% of the population of T cells are recovered as engineered T cells. The percentages may be any value or subrange within the recited range, including the endpoint. In embodiments, the percentage of engineered T cells is measured prior to expansion. In an embodiment, the percentage of engineered T cells is measured after expansion.

[0301] In an embodiment, method step a) comprises, in any order, the following steps: (i) adding donor DNA to the chamber, (ii) adding RNP to the chamber, (iii) adding a negatively charged polymer to the chamber, and (iv) adding T cells to the chamber. In an embodiment, method step a) comprises, in any order, the following steps: (i) combining RNP and a negatively charged polymer to form an RNP-PGA mixture, (ii) adding donor DNA to the chamber, (iii) adding the RNP-PGA mixture to the chamber, and (iv) adding T cells to the chamber. In an embodiment, method step a) comprises, in any order, the following steps: (i) adding RNP to the chamber, (ii) adding donor DNA to the chamber, and (iii) adding T cells to the chamber. In an embodiment, T cells are not pipetted during method steps a) and b). In some embodiments, the method includes adding a negatively charged polymer to the chamber. In embodiments, the method specifically excludes adding a negatively charged polymer to the chamber.

[0302] In another related aspect, an engineered T cell is provided. The engineered T cell is produced by the methods described herein, including the embodiments. In another related aspect, an engineered T cell population is provided. The engineered T cell population is produced by the methods described herein, including the embodiments.

[0303] IV.How to use The engineered T cells described herein can be used for any suitable purpose. For example, the engineered T cells can be administered to treat a disease in a subject in need thereof. The disease can be a neoplastic, infectious, or inflammatory disease.

[0304] In an embodiment, the neoplasm is a cancer. In an embodiment, the infection is a viral infection, a bacterial infection, a fungal infection, a protozoal infection, or a helminthic infection. In an embodiment, the viral infection is caused by human immunodeficiency virus, hepatitis C virus, hepatitis B virus, human cytomegalovirus, or a coronavirus. In embodiments, the inflammatory disease is an autoimmune disorder, allergy, arthritis, psoriasis, diabetes, Guillain-Barre syndrome, Hashimoto's encephalitis, Hashimoto's thyroiditis, ankylosing spondylitis, psoriasis, Sjogren's syndrome, vasculitis, glomerulonephritis, autoimmune thyroiditis, Behcet's disease, Crohn's disease, ulcerative colitis, bullous pemphigoid, sarcoidosis, ichthyosis, Graves' ophthalmopathy, inflammatory bowel disease, Addison's disease, vitiligo, asthma, allergic asthma, acne vulgaris, celiac disease, chronic prostatitis, inflammatory bowel disease, pelvic inflammatory disease, reperfusion injury, ischemia-reperfusion injury, stroke, sarcoidosis, transplant rejection, interstitial cystitis, atherosclerosis, scleroderma, and atopic dermatitis.

[0305] In another interrelated embodiment, a method is provided for treating a subject with cancer, the method comprising: a) providing a population of T cells, b) engineering at least a subset of the population of T cells to express an exogenous T cell receptor (TCR) and knock out endogenous TCR-beta, thereby forming an engineered population of T cells, where the exogenous TCR binds to an antigen expressed by the cancer, c) expanding the population of engineered T cells, and d) administering the expanded population of engineered T cells to the subject.

[0306] In another interrelated embodiment, a method is provided for treating a subject with cancer, the method comprising: a) providing a first population of T cells isolated from the subject; b) engineering at least a subset of the first population of T cells to express a first exogenous T cell receptor (TCR) and knock out endogenous TCR-beta, thereby forming a first population of engineered T cells, where the exogenous TCR binds to a first antigen expressed by the cancer; c) expanding the first population of engineered T cells; and d) administering the expanded first population of engineered T cells to the subject. and e) providing a second population of T cells isolated from the subject; f) engineering at least a subset of the second population of T cells to express a second exogenous TCR and knock out the endogenous TCR-beta, thereby forming a second population of engineered T cells, where the exogenous TCR binds to a second antigen expressed by the cancer; g) expanding the second population of engineered T cells; and h) administering the expanded second population of engineered T cells to the subject. In embodiments, any number of multiple engineered T cells, each containing a different TCR, can be prepared and administered to the subject. In embodiments, each TCR binds to a different antigen. In embodiments, two or more TCRs bind to the same antigen. The multiple T cells can be administered in any order, including simultaneously.

[0307] In another interrelated embodiment, a method is provided for treating a subject with cancer, the method comprising: a) providing a first population of T cells isolated from the subject; b) engineering at least a subset of the first population of T cells to express a first exogenous T cell receptor (TCR) and knock out endogenous TCR-alpha, thereby forming a first population of engineered T cells, where the exogenous TCR binds to a first antigen expressed by the cancer; c) expanding the first population of engineered T cells; and d) administering the expanded first population of engineered T cells to the subject. e) providing a second population of T cells isolated from the subject; f) engineering at least a subset of the second population of T cells to express a second exogenous TCR and knock out endogenous TCR-alpha, thereby forming a second population of engineered T cells, wherein the exogenous TCR binds to a second antigen expressed by the cancer; g) expanding the second population of engineered T cells; and h) administering the expanded second population of engineered T cells to the subject.

[0308] In an embodiment, a method for treating a subject having cancer, wherein the subject is treated with any one or a combination of populations of engineered T cells expressing exogenous TCR-beta and / or TCR-alpha.

[0309] In embodiments, the expanded population of engineered T cells is greater than 1×10 5 pieces~1×10 11 In embodiments, the expanded population of engineered T cells comprises at least 1 x 10 8 In embodiments, the expanded population of engineered T cells comprises at least 1 x 10 9 In embodiments, the expanded population of engineered T cells comprises at least 1 x 10 10In embodiments, the expanded population of engineered T cells comprises at least 1 x 10 11 engineered T cells. The number can be any value or subrange within the recited range, including the endpoints.

[0310] In embodiments, the T cells are autologous to the subject. In embodiments, the T cells are allogeneic to the subject.

[0311] In an embodiment, the antigen is a neo-antigen or TAA. In an embodiment, at least a portion of the genome and / or transcriptome of the cancer has been sequenced to determine the presence of the antigen. In an embodiment, the engineered T cells are generated using the methods described herein, including embodiments. In an embodiment, the antigen is WT1, JAK2, NY-ESO1, PRAME, or mutant KRAS, HPV. In an embodiment, the antigen is an antigen from Table 1 or Table 2. In an embodiment, the antigen is specific to the cancer. In an embodiment, the TCR binds to an antigen presented on a major histocompatibility complex class I (MHCI) molecule.

[0312] In an embodiment, the expanded population of first engineered T cells is greater than or equal to 1×10 5 ~1×10 11 In an embodiment, the expanded population of first engineered T cells comprises at least 1 x 10 8 In an embodiment, the expanded population of first engineered T cells comprises at least 1 x 10 9 In an embodiment, the expanded population of first engineered T cells comprises at least 1 x 10 10 In an embodiment, the expanded population of first engineered T cells comprises at least 1 x 10 11 In an embodiment, the expanded population of second engineered T cells comprises 1×10 5 ~1×10 11In an embodiment, the expanded population of second engineered T cells comprises at least 1 x 10 8 In an embodiment, the expanded population of second engineered T cells comprises at least 1 x 10 9 In an embodiment, the expanded population of second engineered T cells comprises at least 1 x 10 10 In an embodiment, the expanded population of second engineered T cells comprises at least 1 x 10 11 engineered T cells. The number can be any value or subrange within the recited range, including the endpoints.

[0313] In an embodiment, an additional population of engineered T cells is administered to the patient, where the T cells in the additional population of engineered T cells express a third exogenous TCR that binds to a third antigen expressed by the cancer. In an embodiment, an additional population of engineered T cells is administered to the patient, where the T cells in the additional population of engineered T cells express a fourth exogenous TCR that binds to a fourth antigen expressed by the cancer. In an embodiment, an additional population of engineered T cells is administered to the patient, where the T cells in the additional population of engineered T cells express a fifth exogenous TCR that binds to a fifth antigen expressed by the cancer. In an embodiment, an additional population of engineered T cells is administered to the patient, where the T cells in the additional population of engineered T cells express a sixth exogenous TCR that binds to a sixth antigen expressed by the cancer. In an embodiment, an additional population of engineered T cells is administered to the patient, where the T cells in the additional population of engineered T cells express a seventh exogenous TCR that binds to a seventh antigen expressed by the cancer. In embodiments, an additional population of engineered T cells is administered to the patient, where the T cells in the additional population of engineered T cells express an eighth exogenous TCR that binds to an eighth antigen expressed by the cancer. One of skill in the art will understand that any number of additional multiple T cells expressing any number of additional TCRs falls within the scope of the present disclosure.

[0314] In another related aspect, a method of treating cancer is provided. The method comprises administering a T cell, composition, or pharmaceutical composition described herein, including embodiments, to a patient having cancer. In embodiments, the method further comprises administering an anti-cancer therapy to the subject. In embodiments, the T cell, composition, or pharmaceutical composition described herein is administered to a patient undergoing anti-cancer therapy. In embodiments, a patient undergoing anti-cancer therapy is selected for administration of the T cell, composition, or pharmaceutical composition described herein. In embodiments, the anti-cancer therapy comprises immunotherapy, chemotherapy, and / or radiation.

[0315] In embodiments, the patient undergoes lymphodepletion prior to administration of the T cells, compositions, or pharmaceutical compositions described herein. In embodiments, the lymphodepleted patient is administered the T cells, compositions, or pharmaceutical compositions described herein. In embodiments, the lymphodepleted patient is selected for administration of the T cells, compositions, or pharmaceutical compositions described herein.

[0316] V. Nucleic Acids and Kits In another interrelated aspect, a guide RNA is provided. The first guide RNA is discovered by methods known to those skilled in the art to target the exon 1, exon 2 or exon 3 region of the TCR constant alpha region locus (TRAC). In an embodiment, the guide RNA targets the endogenous TCR-alpha locus in one or more of TRAC1 (SEQ ID NO: 7), TRAC2 (SEQ ID NO: 8), TRAC3 (SEQ ID NO: 9), TRAC4 (SEQ ID NO: 10), TRAC5 (SEQ ID NO: 11), TRAC6 (SEQ ID NO: 12), TRAC7 (SEQ ID NO: 13), TRAC8 (SEQ ID NO: 14), TRAC9 (SEQ ID NO: 15), TRAC10 (SEQ ID NO: 16), TRAC11 (SEQ ID NO: 17), TRAC12 (SEQ ID NO: 18), TRAC13 (SEQ ID NO: 19), TRAC14 (SEQ ID NO: 20), TRAC15 (SEQ ID NO: 21), or TRAC16 (SEQ ID NO: 22). In an embodiment, the guide RNA targeting the endogenous TCR-alpha locus comprises a nucleic acid sequence shown in Table 10. In an embodiment, the endogenous TCR-alpha locus is an endogenous TCR-alpha constant region.

[0317] In another interrelated aspect, a guide RNA is provided. The second guide RNA is discovered by methods known to those skilled in the art to target the exon 1 region of both TCR constant beta region loci (TRBC). In some embodiments, the guide RNA is selected from TRBC1 (SEQ ID NO: 23), TRBC2 (SEQ ID NO: 24), TRBC3 (SEQ ID NO: 25), TRBC4 (SEQ ID NO: 26), TRBC5 (SEQ ID NO: 27), TRBC6 (SEQ ID NO: 28), TRBC7 (SEQ ID NO: 29), TRBC8 (SEQ ID NO: 30), TRBC9 (SEQ ID NO: 31), TRBC10 (SEQ ID NO: 32), TRBC11 (SEQ ID NO: 33), TRBC12 (SEQ ID NO: 34), TRBC13 (SEQ ID NO: 35), TRBC14 (SEQ ID NO: 36), TRBC15 (SEQ ID NO: 37), TRBC16 (SEQ ID NO: 38), TRBC17 (SEQ ID NO: 39), TRBC18 (SEQ ID NO: 40), TRBC19 (SEQ ID NO: 41), TRBC20 (SEQ ID NO: 42), TRBC21 (SEQ ID NO: 43), TRBC22 (SEQ ID NO: 44), TRBC23 (SEQ ID NO: 45), TRBC24 (SEQ ID NO: 46), TRBC25 (SEQ ID NO: 47), TRBC26 (SEQ ID NO: 48), TRBC27 (SEQ ID NO: 49), TRBC28 (SEQ ID NO: 50), TRBC29 (SEQ ID NO: 51), TRBC30 (SEQ ID NO: 52), TRBC31 (SEQ ID NO: 53), TRBC32 (SEQ ID NO: 54), TRBC33 (SEQ ID NO: 55 No. 36), TRBC15 (SEQ ID NO: 37), TRBC16 (SEQ ID NO: 38), TRBC17 (SEQ ID NO: 39), TRBC18 (SEQ ID NO: 40), TRBC19 (SEQ ID NO: 41), TRBC20 (SEQ ID NO: 42), TRBC21 (SEQ ID NO: 43), TRBC22 (SEQ ID NO: 44), TRBC23 (SEQ ID NO: 45), TRBC24 (SEQ ID NO: 46), TRBC25 (SEQ ID NO: 47), or TRBC26 (SEQ ID NO: 48). In an embodiment, the guide RNA targeting the endogenous TCR-beta locus comprises a nucleic acid sequence shown in Table 11.

[0318] In another interrelated aspect, a nucleic acid is provided. The nucleic acid comprises a nucleic acid sequence comprising an exogenous TCR-beta coding sequence and an exogenous TCR-alpha coding sequence, the nucleic acid sequence further comprising a first self-cleaving peptide coding sequence. In an embodiment, the nucleic acid further comprises a first homology arm and a second homology arm. In an embodiment, the nucleic acid further comprises a second self-cleaving peptide coding sequence. In an embodiment, the nucleic acid comprises, in 5' to 3' order: (i) a first homology arm, (ii) a first self-cleaving viral peptide coding sequence, (iii) an exogenous TCR-beta coding sequence, (iv) a second self-cleaving viral peptide coding sequence, (v) an exogenous TCR-alpha coding sequence, (vi) optionally a polyA sequence, and (vii) a second homology arm. In an embodiment, the exogenous TCR-alpha coding sequence encodes an exogenous TCR-alpha VJ domain.

[0319] In an embodiment, the first homology arm is homologous to the endogenous TCR-alpha locus in human T cells. In an embodiment, the second homology arm is homologous to the endogenous TCR-alpha locus in human T cells. In an embodiment, the endogenous TCR-alpha locus is a TCR-alpha constant region. In an embodiment, the first homology arm is homologous to the endogenous TCR-beta locus in human T cells. In an embodiment, the second homology arm is homologous to the endogenous TCR-beta locus in human T cells.

[0320] In an embodiment, the first self-cleaving viral peptide is T2A, P2A, E2A or F2A. In an embodiment, the second self-cleaving viral peptide is T2A, P2A, E2A or F2A. In an embodiment, the first self-cleaving viral peptide and the second self-cleaving viral peptide are different. In an embodiment, the first self-cleaving viral peptide and the second self-cleaving viral peptide are the same. In an embodiment, the first self-cleaving peptide coding sequence is 5' of the exogenous TCR-alpha coding sequence (e.g., the exogenous TCR-alpha VJ domain coding sequence). In an embodiment, the second self-cleaving peptide coding sequence is 5' of the exogenous TCR-beta coding sequence. In an embodiment, the first self-cleaving peptide coding sequence is 5' of the exogenous TCR-beta coding sequence. In an embodiment, the second self-cleaving peptide coding sequence is 5' of the exogenous TCR-alpha coding sequence (e.g., the exogenous TCR-alpha VJ domain coding sequence). In an embodiment, the nucleic acid sequence comprises a polyA signal. In an embodiment, the polyA signal is 3' of the full-length TCR-alpha coding sequence. In an embodiment, the polyA signal is 3' of the full-length TCR-beta coding sequence.

[0321] In embodiments, the nucleic acid is a plasmid, a nanoplasmid or a minicircle. In embodiments, the nucleic acid is a plasmid. In embodiments, the nucleic acid is a nanoplasmid. In embodiments, the nucleic acid is a minicircle.

[0322] In another interrelated aspect, a kit for producing engineered T cells is provided. The kit comprises a TCR-alpha targeted guide RNA as described herein, including embodiments. In an embodiment, the kit further comprises a TCR-beta targeted guide RNA as described herein, including embodiments. In an embodiment, the kit further comprises a gene editing reagent or a nucleotide encoding the gene editing reagent. In an embodiment, the gene editing reagent is a CRISPR system. In an embodiment, the kit further comprises donor DNA. In an embodiment, the donor DNA comprises a nucleic acid sequence encoding a polypeptide comprising an exogenous TCR-beta domain and an exogenous TCR-alpha domain. In an embodiment, the exogenous TCR-beta and the heterologous TCR-alpha form a TCR capable of binding to an antigen. In an embodiment, the TCR binds to an antigen presented on a major histocompatibility complex class I (MHCI) molecule. In an embodiment, the antigen is WT1, JAK2, NY-ESO1, PRAME, mutant KRAS, or an antigen of Table 1 or Table 2. In an embodiment, the antigen is a neo-antigen. In an embodiment, the kit further comprises poly(glutamic acid) (PGA) or a variant thereof. In an embodiment, the kit further comprises a nucleic acid as described herein, including embodiments.

[0323] It should be understood that the examples and embodiments described herein are for illustrative purposes only, and that various modifications or changes taking into account thereof will be suggested to those skilled in the art and are to be included within the spirit and scope of this application and the scope of the appended claims. All publications, patents, and patent applications cited herein are hereby incorporated by reference in their entirety for all purposes. EXAMPLES

[0324] Those skilled in the art will appreciate that the descriptions of making and using the particles described herein are for illustrative purposes only, and that the disclosure is not limited by these examples.

[0325] Example 1. Purification and activation of T cells To purify T cells, the following reagents were used: STRAIGHTFROM® Buffy Coat CD8 MicroBeads (Miltenyi, Cat. No. 130-114-978), MACS Buffer (PBS / 0.5% BSA / 2 mM EDTA), QUADROMACS™ Separator (Miltenyi), X-VIVO without gentamicin and phenol red (Lonza, Cat. No. 04-744Q), and red blood cell lysis buffer.

[0326] Buffy coats from each donor were diluted to 80 mL with PBS / 0.5% BSA / 2 mM EDTA and 4 mL of STRAIGHTFROM® Buffy Coat CD8 MicroBeads were added per buffy coat (i.e., 4 mL of beads per 80 mL of blood). The tubes were mixed by inverting 5-8 times and incubated in the refrigerator (2-8 °C) for 15 min.

[0327] For magnetic separation, a whole blood column was placed in the magnetic field of a QUADROMACS® separator. The column was prepared by rinsing with 3 mL of separation (MACS) buffer, and the magnetically labeled cell suspension was applied to the prepared whole blood column. The flow-through contained non-labeled cells. The whole blood column was washed twice with 2 mL of separation buffer, then removed from the separator and placed in a new collection tube. The magnetically labeled cells were eluted by applying elution buffer, followed by firmly pushing the plunger into the column. The cells were pelleted by centrifugation at 300 g for 5 min, and the supernatant was removed. The cells were resuspended in 5 mL of red blood cell lysis buffer by pipetting up and down and incubated at room temperature for 5 min. The cells were washed with PBS and centrifuged again, after which the resulting cell pellet was resuspended by pipetting in 5 mL of X-VIVO™ medium containing 50 μM 2-mercaptoethanol and 10 μM N-acetyl-L-cysteine. Count the cells and the goal is to culture at 1 x 10 in X-VIVO™ 15 medium. 6 In cells / mL.

[0328] The following reagents were used to activate T cells: TRANSACT™ (Miltenyi, Catalog No. 130-111-160), 25 ng / mL research grade human IL-7 (Miltenyi, Catalog No. 130-095-367), 50 ng / mL research grade human IL-15 (Miltenyi, Catalog No. 130-095-760), 10 ng / mL research grade human IL-2 (Miltenyi, Catalog No. 130-097-743), X-VIVO™ 15 without gentamicin and phenol red (Lonza, Catalog No. 04-744Q), N-acetyl-L-cysteine ​​(Cat. No. A9165), and b-mercaptoethanol (Cat. No. 21985-023).

[0329] The tissue culture plates were plated according to the following protocol: cells were first plated in X-VIVO™ 15 containing 50 μM 2-mercaptoethanol and 10 μM N-acetyl-L-cysteine ​​at 1×10^6 cells / mL (4 mL per well) in 12-well plates or 5×10 in 6-well plates. 6 Cells were plated at a concentration of 1:100 (5 mL per well). T CellTRANSACT™ Reagent (1:100) was added to each well along with 10 ng / mL IL-2, 25 ng / mL IL-7, and 50 ng / mL IL-15. Cells were incubated for 36-48 hours at 37°C, 5% CO2, and humidified.

[0330] Example 2. T cell electroporation For electroporation of T cells, the following reagents and equipment were used: SpyFi™ Cas9 (Aldevron, catalog number 9214-5mg), sgRNA targeting the TCR (for examples, see Tables 3 and 4; and NTC), donor DNA template, Nuclease-Free Duplex Buffer (IDT, catalog number 1072570), poly-L-glutamic acid sodium salt 15,000-50,000 molecular weight (Sigma, catalog number P4761-100MG), PCR tubes, P3 Primary Cell Kit (Lonza V4SP-3096, 96 reactions), 4D-Nucleofector™ Core Unit (Lonza, catalog number AAF-1002B), and 4D-Nucleofector™ X unit (Lonza, catalog number AAF-1002X).

[0331] T cell electroporation. Unless otherwise stated, sgRNAs were reconstituted to 50 μM in nuclease-free duplex buffer, vortexed briefly, incubated at room temperature (RT) for 5 min, then vortexed again. If crRNA+tracrRNA were used, each was reconstituted at 100 μM and tracRNA (20 μL each) was added to the tube, vortexed briefly, and spun down. RNAs were annealed at 95°C for 5 min and slowly cooled to RT (~5 min).

[0332] X-VIVO™ 15 (900 μL) containing 50 μM 2-mercaptoethanol and 10 μM N-acetyl-L-cysteine, 10 ng / mL IL-2, 25 ng / mL IL-7, and 50 ng / mL IL-15 was dispensed into each well and incubated at 37° C. to warm the medium.

[0333] Separately, recovery medium (X-VIVO™ 15 medium containing 50 μM 2-mercaptoethanol and 10 μM N-acetyl-L-cysteine ​​without the addition of cytokines) was heated.

[0334] The RNP complex was prepared immediately before transfection as follows: 1. 3.5 μl (180 pmol) of sgRNA was added to an RNAse / DNAse free PCR tube. 2. 1 μl of high-fidelity Cas9 (60 pmol) was added and the solution was mixed gently by repeatedly pipetting (ratio 3:1). 3. Incubated at room temperature for 15 min to obtain 4.5 μl of RNP mixture ready for transfection.

[0335] Activated T cells were harvested and centrifuged at 300g. Activated T cells were washed with PBS and counted (by Vi-CELL). Cells were centrifuged again at 300g, then resuspended at 1x10^6 cells per 20μl of P3 nucleofection buffer with supplements and mixed by pipetting 2-3 times. Donor template (2μl, containing 1μg total) was added to a PCR tube. RNP (4.5μl) was added to the PCR tube and incubated for 1 minute at room temperature. If used, 2μl of PGA (resuspended at 100mg / mL) was added to the PCR tube and incubated for 1-2 minutes at room temperature. Cells (20μL) were added to the PCR tube and mixed by gently pipetting up and down. The mixture was transferred to a cuvette strip, a lid was added, and incubated in the cuvette strip for 3-5 minutes.

[0336] The cuvette strip was placed in a 4D-Nucleofector™ machine and electroporation was performed using the indicated program (e.g., Primary Cell P3 program and EH115 pulse code). 80 μl of pre-warmed X-VIVO™ 15 containing 50 μM 2-mercaptoethanol and 10 μM N-acetyl-L-cysteine ​​(no cytokines) was added per nucleofected cell and the cuvette was transferred to a 37° C. incubator for a minimum of 15 minutes. After the 15 minute incubation, the cells were transferred to the appropriate wells in a 48-well plate (X-VIVO™ 15 medium containing cytokines).

[0337] On day 3, cells were transferred to a 24-well plate and 1 mL of fresh X-VIVO™ 15 medium containing 50 μM 2-mercaptoethanol and 10 μM N-acetyl-L-cysteine ​​with 10 ng / mL IL-2, 25 ng / mL IL-7 and 50 ng / mL IL-15 was added. On day 5, cells were resuspended by pipetting. An aliquot (100 μL) was kept for analysis and the remainder was transferred to a 12-well plate and 2 mL of fresh X-VIVO™ 15 medium containing 50 μM 2-mercaptoethanol and 10 μM N-acetyl-L-cysteine ​​with 10 ng / mL IL-2, 25 ng / mL IL-7 and 50 ng / mL IL-15 was added. On day 7, cells were resuspended by pipetting and another 100 μL aliquot was taken for analysis.

[0338] Example 3. Flow cytometry analysis 100 μl of post-transfection cell culture was transferred to a 96-well V-bottom well. 100 μl of cold FACS buffer (0.5% bovine serum albumin (BSA) + sodium azide in PBS) was added and cells were spun down at 300×g for 5 min at 4° C. Cells were resuspended in 200 μl of FACS buffer and centrifuged at 300×g for 5 min at 4° C. Cells were then resuspended in 100 μl of working solution of fixable viability dye (e.g., eBioscience Fixable Viability Dye eFluor 506, Thermo Fisher catalog number 65-0866-14) made by diluting the dye 1:1000 in PBS (azide- and serum / protein-free). Cells were mixed well and incubated in the dark at 4° C. for 30 min. The cells were washed twice with cold FACS buffer, and 100 μL of FACS buffer was first added to the mixture of cells in the working solution of fixable viability dye, spun down at 300×g for 5 min at 4° C., then resuspended in 200 μL of FACS buffer, spun down at 300×g for 5 min at 4° C. The cells were then resuspended in the working solution of the appropriate fluorochrome-conjugated antibody. For most surface antibodies, the cells were incubated in the dark at 4° C. for 30 min. When the expression of exogenous T cell receptors was assessed by dextramer or tetramer staining, the cells were first incubated with 50 μL of the working solution of dextramer / tetramer reagent in the dark for 15 min at room temperature. After this incubation, 50 μL of the working solution of other fluorochrome-conjugated antibodies (e.g., CD8, CD3, etc.) was added to the cells, and the cells were incubated in the dark for another 15 min at 4° C. The cells were then resuspended in the working solution of the appropriate fluorochrome-conjugated antibody. For most surface antibodies, cells were incubated for 30 minutes at 4° C. in the dark. When expression of exogenous T cell receptors was assessed by dextramer or tetramer staining, cells were first incubated with 50 μL of a working solution of dextramer / tetramer reagent for 15 minutes at room temperature in the dark. After this incubation, 50 μL of a working solution of other fluorochrome-conjugated antibodies (e.g., CD8, CD3, etc.) was added to the cells and the cells were incubated for an additional 15 minutes at 4° C. in the dark.After incubation with antibodies, cells were washed twice with cold FACS buffer. After the second wash, cells were resuspended in 100 μL of FACS buffer and analyzed on the flow cytometer. To count cells, 10 μL of CountBright Absolute Counting Beads (Molecular Probes Cat. No. 402-ML-020) was added to the stained cells. A minimum of 1000 beads were collected on the flow cytometer to determine an accurate concentration count, and cell numbers were determined using calculations provided by the manufacturer.

[0339] Example 4. Donor DNA titration and electroporation conditions Experimental conditions were determined using donor DNA encoding the first 2A peptide, mNeon fluorescent protein, the second 2A peptide, and the TCRa VJ domain. T cells were activated using TRANSACT™ (1:30) in RPMI + 10% FBS, Glutamax, HEPES, non-essential amino acids, sodium pyruvate, beta-mercaptoethanol, and cytokines (10 ng / mL IL-2, 25 ng / mL IL-7, and 50 ng / mL IL-15). Cells were electroporated 48 hours after activation using Lonza P2 buffer and pulse code EH100 or P3 buffer and pulse code EH115.

[0340] RNP was prepared as follows: 1.6 μL PGA (15-50 kDa at 100 mg / mL in water) + 2 μL TRAC1 sgRNA (100 pmol) + 1 μL Cas9 (50 pmol); for RNP, 10 min incubation and additional 5 min incubation with TRAC1-mNeon construct (both incubations at room temperature). PCR product TRAC1-mNeon construct was titrated: 6 μg, 4 μg, 2 μg or 1 μg.

[0341] Electroporation conditions: TRAC1 RNP + increasing amounts of TRAC1-mNeon (6 μg, 4 μg, 2 μg, and 1 μg); NTC (non-targeted guide control, i.e., no TCR knockout) RNP + 4 μg TRAC1-mNeon; TRAC1 RNP + 2 μg TRAC1-mNeon + P3 buffer + EH115 pulse. After electroporation, cells were split in half: (1) at 37° C. for the entire culture period, and (2) a 24-hour "cold shock" at 32° C. was performed before being transferred to 37° C.

[0342] The results are shown in Figures 1A to 7.

[0343] Example 5. Targeting Different TRAC Loci and Titration of Donor DNA Donor DNA encoding the first 2A peptide, the mNeon fluorescent protein, the second 2A peptide and the TCRa VJ domain was used.

[0344] T cells were activated using TRANSACT™ (1:30) in RPMI + 10% FBS, Glutamax, HEPES, non-essential amino acids, sodium pyruvate, beta-mercaptoethanol, and cytokines (10 ng / mL IL-2, 25 ng / mL IL-7, and 50 ng / mL IL-15). Cells were electroporated 48 hours after activation using Lonza P3 buffer and EH115 pulse code. Cells were incubated at 37° C. immediately after electroporation.

[0345] RNPs were prepared as follows: 1.6 μL PGA (15-50 kDa at 100 mg / mL in water) + 2 μL TRAC1 or TRAC3 sgRNA + 1 μL Cas9. 10 min incubation for RNP and an additional 5 min incubation with the appropriate PCR product donor template (TRAC1-mNeon or TRAC3-mNeon); all incubations at room temperature. Cells were electroporated with TRAC1 or TRAC3 RNP + titrated amounts of the corresponding TRAC-Neon template (6 μg, 4 μg, 2 μg, and 1 μg), or TRAC1 RNP + titrated amounts of NY-ESO TCR template.

[0346] The results are shown in Figures 10A to 16.

[0347] Example 6. Titration of RNP Donor DNA encoding the first 2A peptide, the mNeon fluorescent protein, the second 2A peptide and the TCRa VJ domain was used.

[0348] T cells were activated using TRANSACT™ (1:30) in RPMI + 10% FBS, L-glutamine, non-essential amino acids, BME and cytokines (10 ng / mL IL-2, 25 ng / mL IL-7, 50 ng / mL IL-15). Cells were electroporated 48 hours after activation using P3 buffer and EH115 pulse code.

[0349] RNPs were prepared as follows: 1.6 μL PGA (15-50 kDa at 100 mg / mL in water) + RNP + IDT duplex buffer to equalize the total sample volume. 10 min incubation for RNP and additional 5 min incubation with template (TRAC 3 mNeon dsDNA (1 μg or 2 μg)) at room temperature. TRAC3 only (no TRBC) at 0 pmol, 10 pmol, 20 pmol, 40 pmol, 80 pmol, or 120 pmol of RNP. Four days after electroporation, cells were stained with TCRa / b-APC (BioLegend clone IP26) and flow cytometry analysis was performed. The results are shown in Figures 7-21.

[0350] Example 7. Single-stranded DNA template and double-stranded DNA donor template Donor DNA was used encoding the first 2A peptide, mNeon fluorescent protein, the second 2A peptide, and the TCRa VJ domain. T cells were activated using TRANSACT™ (1:30) in RPMI + 10% FBS, L-glutamine, non-essential amino acids, BME, and cytokines (10 ng / mL IL-2, 25 ng / mL IL-7, 50 ng / mL IL-15). Cells were electroporated 48 hours after activation using P3 buffer and EH115 pulse code.

[0351] RNPs were prepared as follows: Cas9+gRNA (10 min, room temperature), followed by 1.6 μL of PGA (15-50 kDa at 100 mg / mL in water), then template, incubated for 5 min at room temperature. Templates were TRAC3 mNeon dsDNA (0.125 μg, 0.25 μg, 0.50 μg) or TRAC3 ssDNA forward (FW) or reverse (RV). RNPs: NT or TRAC3 only (no TRBC) at 10 pmol. Four days after electroporation, cells were stained with TCRa / b-APC (BioLegend clone IP26) and flow cytometry analysis was performed. The results are shown in Figure 22-Figure 23C.

[0352] Example 8. Activation and electroporation conditions Donor DNA encoding the first 2A peptide, mNeon fluorescent protein, the second 2A peptide, and the TCRa VJ domain was used. T cells were activated with RPMI + 10% FBS, Glutamax, HEPES, non-essential amino acids, sodium pyruvate, beta-mercaptoethanol, and cytokines (10ng / mL IL-2, 25ng / mL IL-7, 50ng / mL IL-15); X-VIVO15 with 10ng / mL IL-2, 25ng / mL IL-7; and 50ng / mL IL-15; or X-VIVO15 with 25ng / mL IL-7 and 50ng / mL IL-15. TRANSACT (1:20, 1:50, 1:100, 1:200) was titrated for each set of media / cytokine conditions tested. Cells were electroporated 48 or 72 hours after activation.

[0353] RNPs were prepared as follows: 10 min incubation at room temperature for RNP (2 µL sgRNA + 1 µL Cas9, using 40 pmol per reaction); 1.6 µL of PGA (15–50 kDa at 100 mg / mL in water) + 2 µg of TRAC3-mNeon construct and an additional 5 min incubation at room temperature.

[0354] The results are shown in Figures 24A to 31B.

[0355] Example 9. Single-stranded DNA templates (forward and reverse) and double-stranded DNA Donor DNA was used encoding the first 2A peptide, mNeon fluorescent protein, the second 2A peptide, and the TCRa VJ domain. T cells were activated using TRANSACT™ (1:30) in X-VIVO + L-glutamine, non-essential amino acids, BME, and cytokines (10 ng / mL IL-2, 25 ng / mL IL-7, 50 ng / mL IL-15). Cells were electroporated 48 hours after activation using P3 buffer and EH115 pulse code.

[0356] RNPs were prepared as follows: SpyFi Cas9+sgRNA+PGA (1.6 μL / sample), 10 min incubation. Template was added (TRAC mNeon dsDNA (0.5 μg, 1.0 μg, 1.5 μg) and ssDNA (0.5 μg, 1.0 μg), see FIG. 34) and incubated for another 5 min at room temperature. Electroporation was performed with 40 pmols of RNP (no TRBCs). Four days after electroporation, cells were stained with TCRa / b-APC (BioLegend clone IP26) and flow cytometry analysis was performed. The results are shown in FIG. 33-FIG. 35B.

[0357] Example 10. Targeting additional TRAC loci for TCR knock-in Donor DNA was used encoding the first 2A peptide, mNeon fluorescent protein, the second 2A peptide, and the TCRa VJ domain. T cells were activated using TRANSACT™ (1:30) in X-VIVO + L-glutamine, non-essential amino acids, BME, and cytokines (10 ng / mL IL-2, 25 ng / mL IL-7, 50 ng / mL IL-15). Cells were electroporated 48 hours after activation using P3 buffer and EH115 pulse code.

[0358] RNPs were prepared as follows: SpyFi Cas9+sgRNA+PGA (1.6 μL / sample), 10 min incubation. Template was added (0.25 μg, 0.5 μg or 1.0 μg TRAC mNeon dsDNA) and incubated for another 5 min at room temperature. Electroporation was performed with 40 pmols of RNP (no TRBCs). Four days after electroporation, cells were stained with TCRa / b-APC (BioLegend clone IP26) and flow cytometry analysis was performed. The corresponding gRNA site in the TCR-alpha locus is shown in Figure 36A. See results in Figure 36B, Figure 36C, and Figure 37.

[0359] Example 11. Cytokine conditions and titration of TCR activation Donor DNA encoding the first 2A peptide, the mNeon fluorescent protein, the second 2A peptide and the TCRa VJ domain was used.

[0360] T cells were activated using TRANSACT™ (1:30, 1:100, or 1:200) in the following media conditions: [Table A]

[0361] Supplements added were Glutamax, non-essential amino acids, sodium pyruvate, and beta-mercaptoethanol. Cells were electroporated 48 hours after activation using P3 buffer and EH115 pulse code.

[0362] RNPs were prepared as follows: SpyFi Cas9+sgRNA+PGA (1.6 μL / sample) (3:1 sgRNA to Cas9 ratio), incubated for 10 min. Template was added (1 μg TRAC3-mNeon) and incubated for another 5 min at room temperature. Electroporation was performed with 40 pmols of RNP (no TRBCs). Four days after electroporation, cells were stained with TCRa / b-APC (BioLegend clone IP26) and flow cytometry analysis was performed. The results are shown in Figures 38A-47.

[0363] Example 12. Handling of cells after electroporation Donor DNA was used that encodes the first 2A peptide, mNeon fluorescent protein, the second 2A peptide, and the TCRa VJ domain. Activation was performed with X-VIVO medium containing 1:100 TRANSACT™, 10 ng / mL IL-2, 25 ng / mL IL-7, and 50 ng / mL IL-15. After 48 hours, electroporation was performed. Electroporation was performed according to the following steps: 1) 2:1 sgRNA:Cas9(SpyFi)+PGA for 15 min; 2) Add 0.5 μg mNeon template for 5 min; 3) Add cells; 4) P3 buffer, pulse code EH115; 5) Add cytokines to 100 μL of warmed X-VIVO and mix once with a pipette.

[0364] Three conditions were performed after electroporation: 1) Add 100 μL of X-VIVO containing cytokines and immediately transfer to 24-well plate; 2) Add 20 or 100 μL of X-VIVO containing cytokines without pipetting, wait 15 minutes at 37° C., then transfer to 24-well plate; 3) Wait 5 minutes at room temperature, add 20 μL or 100 μL of X-VIVO containing cytokines without pipetting, wait 15 minutes at 37° C., then transfer to 24-well plate. The results are shown in Figures 48A to 50.

[0365] Example 13. Double- and single-stranded DNA templates containing 5'-phosphate and phosphorothioate The PCR products used as templates in the above experiments were generated using primers lacking a 5' phosphate. Here, we used templates with 5' phosphate or phosphorothioate added to the ends of the primers. Donor DNA encoding the first 2A peptide, mNeon fluorescent protein, the second 2A peptide, and the TCRa VJ domain was used.

[0366] The experimental setup included a new order of addition and RNP formulation. Plating was performed using X-VIVO with 10ng / mL IL-2, 25ng / mL IL-7, 50ng / mL IL-15 and TRANSACT™ 1:100. Electroporation was performed 42 hours after activation using P3 buffer and an EH115 pulse. Templates used were 0.5μg, 1.0μg and 2.0μg of TRAC3 mNeon dsDNA and 40pmol of RNP. RNP formulations included SpyFi Cas 9 and TRACsg3 in a 2:1 ratio and were incubated at room temperature for 15 minutes.

[0367] The order of addition was as follows: 1) 2 μL template, 2) 2 μL RNP, 3) 1.6 μL of 10 mg / mL stock PGA, and 4) 20 μL cells. The time to pipette each component across all tubes was approximately 7-10 min. After electroporation, samples were left at room temperature for 5 min, followed by the addition of 20 μL of plain X-VIVO without pipetting, incubation at 37 °C for 15 min, and transfer to a 24-well plate containing 1 mL of X-VIVO medium with cytokines. Staining conditions on day 4 post-electroporation included TCRa / b-APC (BioLegend clone IP26) and PI. Results are shown in Figure 51A-Figure 54.

[0368] Example 14. Transfection timing and RNP titration. Donor DNA was used encoding the first 2A peptide, mNeon fluorescent protein, the second 2A peptide, and the TCRa VJ domain. Activation conditions included X-VIVO medium, 1:100 TRANSACT™, 10 ng / mL IL-2, 25 ng / mL IL-7, and 50 ng / mL IL15. Cells were electroporated 36, 48, or 72 hours after activation. Approximately 1×10 6Cells were used. For RNP generation, appropriate volumes of sgRNA and Cas9 were mixed and incubated at room temperature for 10 min. The Cas9 used was IDT HiFi Cas9. The order of reagent addition was as follows: 1) donor template, 2) RNP, 3) PGA, and 4) cells. 80 μL of pre-warmed X-VIVO medium without cytokines was added directly to the cuvette without pipetting the cells and incubated at 37 °C for 15 min before transferring to a larger plate.

[0369] [Table 3]

[0370] The results are shown in Figures 55 to 62B.

[0371] Example 15. Order of addition of reagents during transfection. Donor DNA encoding the first 2A peptide, mNeon fluorescent protein, the second 2A peptide and the TCRa VJ domain were used. Experimental set-up of the order of addition test. Plating included X-VIVO with gentamicin, 10 ng / mL IL-2, 25 ng / mL IL-7, 50 ng / mL IL-15 and TRANSACT™ 1:100. Electroporation was performed 48-52 hours after activation using P3 buffer and an EH115 pulse. The template used was 0.5 μg TRAC3 mNeon dsDNA, SEC purified. The RNP formulation and order of addition are shown in Figure 63A. After electroporation, samples were left at room temperature for 5 minutes, followed by the addition of 20 μL of plain X-VIVO without gentamicin without pipetting, incubated at 37 °C for 15 minutes and transferred to a 24-well plate containing 1 mL of X-VIVO medium with cytokines. Staining conditions included TCRa / b-APC (BioLegend clone IP26) and PI on day 4 post-electroporation. Results are shown in Figures 63B-D.

[0372] Example 16. dsPCR versus plasmid templates. Donor DNA encoding the first 2A peptide, the mNeon fluorescent protein, the second 2A peptide and the TCRa VJ domain was used.

[0373] Plating was performed using X-VIVO containing gentamicin, 10ng / mL IL-2, 25ng / mL IL-7, 50ng / mL IL-15, and TRANSACT™ 1:100. Electroporation was performed 48-52 hours after activation using P3 buffer and an EH115 pulse. Templates used were SEC purified TRAC3 mNeon dsDNA or GenScript pUC57 TRAC3 mNeon plasmid. RNP formulations were performed using SpyFi Cas 9+TRACsg3 at a 2:1 ratio followed by a 15 minute incubation at room temperature. The order of addition was: 1) 2μL template, 2) 2μL RNP, 3) 1.6μL of 10mg / mL stock PGA, and 4) 20μL cells. After electroporation, samples were left at room temperature for 5 min, followed by the addition of 20 μL of plain X-VIVO without gentamicin without pipetting, incubation at 37 °C for 15 min, and transfer to a 24-well plate containing 1 mL of X-VIVO medium with cytokines. Staining conditions 4 days after electroporation included TCRa / b-APC (BioLegend clone IP26) and PI. Results are shown in Figure 64A-D.

[0374] Example 17. Cell density and linear versus plasmid donor template. Donor DNA encoding the first 2A peptide, mNeon fluorescent protein, the second 2A peptide and the TCRa VJ domain was used. Parameters tested were: 1) cell density for electroporation, and 2) PCR product versus plasmid as donor template. Culture conditions included X-VIVO medium without gentamicin or phenol red supplemented with 50 μM 2-mercaptoethanol and 10 μM N-acetyl-L-cysteine, and 1:100 TRANSACT™ with 25 ng / mL IL-7 and 50 ng / mL IL-15 (no IL-2 added). Electroporation was performed 48 hours after activation using the TRAC3-Neon template, using P3 buffer and EH115 pulse code. The order of reagent addition was as follows: 1) donor template, 2) 60 pmol of RNP (3:1 pmol ratio of sgRNA:SpyFi Cas9, incubated at room temperature for 15 minutes), and 3) 2 μL of PGA (100 mg / mL). After electroporation, 80 μL of warmed medium without cytokines was added to the electroporation cuvette and the cells were incubated at 37 °C for 15 minutes before being transferred to a larger cell culture plate. The test conditions are shown in Table 4 below and are shown in Table 4. 6 Plasmid comparisons of cell density conditions were included.

[0375] [Table 4]

[0376] The results are shown in Figures 65A to 70.

[0377] Example 18. Targeting different TRAC loci using plasmid donor templates. Donor DNA encoding the first 2A peptide, mNeon fluorescent protein, second 2A peptide and TCRa VJ domain was used. Parameters tested were: 1) different TRAC loci for knock-in, and 2) PCR product versus plasmid as donor template. Culture conditions included X-VIVO medium without gentamicin or phenol red supplemented with 50 μM 2-mercaptoethanol and 10 μM N-acetyl-L-cysteine, and 1:100 TRANSACT™ with 25 ng / mL IL-7 and 50 ng / mL IL-15 (no IL-2 added). Electroporation was performed 48 hours after activation using the TRAC3-mNeon template using P3 buffer and EH115 pulse code. The order of reagent addition was as follows: 1) donor template, 2) 60 pmol of RNP (3:1 pmol ratio of sgRNA:SpyFi Cas9, incubated at room temperature for 15 min), and 3) 2 μL of PGA (100 mg / mL). After electroporation, 80 μL of warmed medium without cytokines was added to the electroporation cuvette and cells were incubated at 37 °C for 40 min before being transferred to a larger cell culture plate. Test conditions included 1 μg of the following templates: TRAC3L (TRAC3 PCR product), TRAC3 plasmid, TRAC4 plasmid, TRAC5 plasmid, TRAC7 plasmid, TRAC12 plasmid, and TRAC15 plasmid. Results are shown in Figures 71A-74.

[0378] Example 19. Co-transfection with two TCR constructs. The parameters tested were co-transfection of two different plasmid-based TCR templates. Culture conditions included X-VIVO medium without gentamicin or phenol red supplemented with 50 μM 2-mercaptoethanol and 10 μM N-acetyl-L-cysteine, and 1:100 TRANSACT™ containing 25 ng / mL IL-7 and 50 ng / mL IL-15 (no IL-2 added). Electroporation was performed 44 hours after activation with P3 buffer and an EH115 pulse using templates NYESO1_TRAC3 (SEQ ID NO:2), eRT80_gp100B_8_TRAC3 (SEQ ID NO:3), eRT76_MART_3_TRAC3 (SEQ ID NO:4), eJH52_WT1C_13_TRAC3 (SEQ ID NO:5) and eRT76_MAGEA3B_4_TRAC3 (SEQ ID NO:6).

[0379] The order of addition of reagents was as follows: 1) 0.5 µg of each donor template; 2) 40 pmol TRAC3 RNP (sgRNA:SpyFi Cas9 in a 3:1 pmol ratio that was preassembled for 15 min at room temperature before adding 1 µL of PGA at 100 mg / mL); 3) 40 pmol TRBCg21 RNP (sgRNA:SpyFi Cas9 in a 3:1 pmol ratio that was preassembled for 15 min at room temperature before adding 1 µL of PGA at 100 µg / µL); 4) 1 million cells resuspended in 20 µL of P3 buffer.

[0380] After electroporation, 80 μl of warmed cytokine-free medium was added to the electroporation cuvette and the cells were incubated at 37° C. for 15 minutes before being transferred to a larger cell culture plate. The results are shown in FIG.

[0381] Plasmids encoding the NY-ESO1 TCR were electroporated with several other TCRs and analyzed by flow cytometry 7 days after activation.

[0382] The results are shown in Figure 75.

[0383] Example 20. Cell density and electroporation conditions. Donor DNA encoding the first 2A peptide, the mNeon fluorescent protein, the second 2A peptide and the TCRa VJ domain was used.

[0384] Culture conditions included X-VIVO medium without gentamicin or phenol red supplemented with 50 μM 2-mercaptoethanol and 10 μM N-acetyl-L-cysteine, and 1:100 TRANSACT™ with 25 ng / mL IL-7 and 50 ng / mL IL-15. Electroporation was performed 48 hours after activation using the TRAC 3-mNeon template. The order of reagent addition was as follows: 1) donor template, 2) 60 pmol RNP (3:1 pmol ratio of sgRNA:Aldevron SpyFi Cas9, incubated at room temperature for 15 minutes), and 3) 2 μL PGA (100 mg / mL). After electroporation, 80 μL of warmed medium without cytokines was added to the electroporation cuvette and the cells were incubated at 37° C. for 40 minutes before being transferred to a larger cell culture plate. Cell densities were 2M, 5M, and 10M cells. To standardize for the number of molecules used per electroporation, knock-in efficiency tests were performed using 1 μg of linear PCR product template or 2 μg of plasmid template (TRAC3-mNeon). A pulse optimization program was performed using P3 buffer and 2 μg plasmid template, TRAC3-mNeon, research grade, using 1.5M cells per condition to allow testing of all conditions for each donor. Results are shown in Figures 76A-84.

[0385] Example 21. Knock-in at the TRAC locus with disruption of the endogenous TCR beta chain. Here, we compared the knock-in efficiency of various TRAC templates as PCR products and tested whether the addition of TRBCsg21 RNP had any adverse effect on the knock-in efficiency or staining pattern. Donor DNA encoding the first 2A peptide, mNeon fluorescent protein, second 2A peptide and TCRa VJ domain was used. The parameters tested were titration of four different PCR-based mNeon constructs targeting different regions of the TRAC locus, comparing TRAC RNP without TRBC RNP with TRBC RNP. Culture conditions included X-VIVO medium without gentamicin or phenol red supplemented with 50 μM 2-mercaptoethanol and 10 μM N-acetyl-L-cysteine, and 1:100 TRANSACT™ with 25 ng / mL IL-7 and 50 ng / mL IL-15 (no IL-2 added). Electroporation was performed 44 hours after activation using P3 buffer and EH115 pulse code. The templates were the following double-stranded PCR products: TRAC3_mNeon, TRAC4_mNeon, TRAC5_mNeon, and TRAC12_mNeon.

[0386] The order of reagent addition was as follows: 1) 0.25 μg, 0.5 μg or 1 μg of each donor template; 2) 60 pmol TRACsg3 RNP (pre-assembled for 15 min at room temperature before adding 1 μL of PGA at 100 mg / ml, sgRNA:SpyFi Cas9 at 2.5:1 pmol ratio); 3) Duplicate 1 μg template set only: 60 pmol TRBCsg21 RNP (pre-assembled for 15 min at room temperature before adding 1 μL of PGA at 100 μg / uL, sgRNA:SpyFi Cas9 at 2.5:1 pmol ratio); 4) Cells resuspended in 20 μL of P3 buffer. A duplicate set of the 1 μg template condition was created and only this set received both TRACsg3 and TRBCsg21 RNP. All other samples received only TRACsg3 RNP.

[0387] After electroporation, the cells were left in a 37° C. incubator for 10 min. Then, 80 μL of warmed medium without cytokines was added to the electroporation cuvette and the cells were incubated in a 37° C. incubator for an additional 10 min before being transferred to a larger cell culture plate.

[0388] The results are shown in Figures 85A to 86B.

[0389] Example 22. Poly(L-glutamic acid) and poly(D-glutamic acid) molecular weight variants. Donor DNA encoding the first 2A peptide, mNeon fluorescent protein, second 2A peptide and TCRa VJ domain was used. We used a gRNA:Cas9 ratio of 2.5:1 and we titrated the PGA from 15 to 50 kDa to better define its optimal range. Plating was performed using X-VIVO without gentamicin, containing 25 ng / mL IL-7, 50 ng / mL IL-15, b-Me, L-Cys and 1:100 TRANSACT™. Electroporation was performed 42 hours after activation in P3 buffer, EH115. The template used was 0.75 μg TRAC3 mNeon plasmid (GenScript industrial grade, endotoxin-free maxiprep). RNP used was 60 pmol with a sgRNA 3:Cas9 ratio of 2.5:1. After 1 hour, cells were added to the master mix and then electroporation was performed. The order of reagent addition was as follows: 1) RNP pre-incubated for 15 min, 2) template, 3) PGA, and 4) 1.5 million cells per cuvette. After electroporation, 80 μL of plain X-VIVO was added without pipetting, followed by incubation at 37°C for 1 h and transfer to a 48-well plate containing 1 mL of X-VIVO medium with cytokines. Staining conditions on days 6 and 8 post-activation included TCRa / b-APC (BioLegend clone IP26), 5 μL staining solution, and PI 1:200 per staining solution.

[0390] The results are shown in Figures 89 and 90.

[0391] The structure of poly(L-glutamic acid) is shown below. [ka]

[0392] Other uses of PGA and its derivatives include thermoplastic, fiber, film and membrane compositions, cryoprotectants, and superabsorbent polymers when crosslinked. PGA also has an affinity for binding various metal ions. Table 5 below lists the poly(L-glutamic acid) variants used in this study.

[0393] [Table 5]

[0394] Example 23. TCR knock-in. Culture conditions included X-VIVO medium without gentamicin or phenol red supplemented with 50 μM 2-mercaptoethanol and 10 μM N-acetyl-L-cysteine, and 1:100 TRANSACT™ with 25 ng / mL IL-7 and 50 ng / mL IL-15. Electroporation was performed 48 hours after activation using P3 buffer. The order of reagent addition was as follows: 1) TRAC3-Neon or TRAC3-TCR donor template, 2) 60 pmol RNP (3:1 pmol ratio of sgRNA:Aldevron SpyFi Cas9, incubated at room temperature for 15 minutes), and 3) 2 μL PGA 100 mg / mL. After electroporation, 80 μL of warmed medium without cytokines was added to the electroporation cuvette and the cells were incubated at 37° C. for 40 minutes before being transferred to a larger cell culture plate. The results are shown in Figures 92A-98.

[0395] Example 24. Electroporation conditions. Donor DNA was used encoding the first 2A peptide, mNeon fluorescent protein, the second 2A peptide, and the TCRa VJ domain. Plating was performed using X-VIVO medium without gentamicin, 25ng / mL IL-7, 50ng / mL IL-15, b-Me, L-Cys, and 1:100 TRANSACT™. Electroporation was performed 44 hours after activation in P3 buffer using 16 different pulse codes and two different machines. The template used was 0.75μg of TRAC3 mNeon plasmid (GenScript industrial grade, endotoxin-free maxiprep). The RNP used was 60pmol with a sgRNA 3:Cas9 ratio of 2.5:1. The order of addition of reagents was as follows: 1) RNP pre-incubated for 15 minutes, 2) template, 3) PGA, and 4) 1.5 million cells per cuvette. Enough master mix was made to cover all reactions. An aliquot of the mixture sufficient for 4 reactions was pipetted into each tube of a PCR 8 strip (n=3, or one strip per donor) per donor. Sufficient donor cells for 4 reactions were added to each tube. The cell mixture was transferred with a multichannel pipette directly from the PCR strip into two cuvettes for a total of 32 wells. Pairs of cuvettes were electroporated simultaneously on two different machines. After electroporation, 80 μL of plain X-VIVO was added without pipetting, followed by incubation at 37°C for 1 hour and transfer to a 48-well plate containing 1 mL of X-VIVO medium with cytokines. Staining conditions on days 6 and 8 post-activation included TCRa / b-APC (BioLegend clone IP26), 5 μL staining solution, and PI 1:200 per staining solution. Table 6 below shows the donor information.

[0396] [Table 6]

[0397] The results are shown in Figures 103-105B. Tables 7 and 8 below show the percentage of knock-in versus mNeon+ cell numbers on days 6 and 8.

[0398] Table 7. Comparison of knock-in % and mNeon+ cell numbers on day 6. [Table 7]

[0399] Table 8. Comparison of knock-in % and mNeon+ cell numbers on day 8. [Table 8]

[0400] Example 25. NY-ESO TCR knock-in with disruption of the endogenous TCR beta chain. Plating was performed using X-VIVO without gentamicin, 25ng / mL IL-7, 50ng / mL IL-15, b-Me, L-Cys and 1:100 TRANSACT™. Electroporation was performed 48 hours after activation in P3 buffer, code EH115. The template used was 0.75μg of TRAC3 1G4(NY-ESO1) plasmid (GenScript industrial grade endotoxin-free maxiprep) and the RNPs used were variable with a sgRNA3:Cas9 ratio of 2.5:1. The order of addition of reagents was as follows: 1) 0.75μg template, 2) 60pmol NT or TRAC RNP (preincubation 15min), 3) 1μL PGA, 4) 15pmol, 30pmol, or 60pmol TRBC RNP (preincubation 15min separately to generate RNP), and 5) 2 million cells per cuvette. After electroporation, 80 μL of plain X-VIVO was added without pipetting, followed by incubation at 37° C. for 20 minutes and transfer to a 48-well plate containing 1 mL of X-VIVO medium with cytokines. Staining conditions 8 days after activation included TCRa / b-BV421 per stain, 5 μL / stain and PI 1:200. Table 9 below lists the donor information.

[0401] [Table 9]

[0402] Example 26. Different homology arm lengths. Plating was performed using X-VIVO without gentamicin, 25 ng / mL IL-7, 50 ng / mL IL-15, b-Me, L-Cys and 1:100 TRANSACT™. Electroporation was performed 43 hours after activation in P3 buffer, code EH115. The template used was the same number of DNA molecules across samples (3.34×10 11) were variable amounts of double-stranded PCR product used to achieve the desired results. The RNP used was 60 pmol with a sgRNA 3:Cas9 ratio of 2.5:1. The order of addition of reagents was as follows: 1) RNP (15 min preincubation), 2) template, 3) 0.5 μL PGA per sample, and 4) 1 million cells per cuvette. After electroporation, 80 μL of plain X-VIVO was added without pipetting, followed by incubation at 37 °C for 20 min and transfer to a 48-well plate containing 1 mL of X-VIVO medium with cytokines. Staining conditions 7 days after activation included TCRa / b-BV421 per stain, 5 μL / stain and PI 1:200. The results are shown in Figure 104A-Figure 105H.

[0403] Example 27. Titration of TRBC-targeting guide RNA and different donor DNA formats. Donor DNA encoding the first 2A peptide, mNeon fluorescent protein, the second 2A peptide, and the TCRa VJ domain was used. T cells were activated in Prime medium containing TRANSACT™ (1:100) + 25 ng / mL IL-7 and 50 ng / mL IL-15. TRAC3 and TRBC22 RNPs were generated by mixing TRAC3 or TRBC22 sgRNA and Cas9 at a 3:1 sgRNA:Cas9 molar ratio and incubating at room temperature for 15 minutes. 2 μL of 100 mg / mL PGA was used per electroporation reaction. A master mix of PGA, TRAC3 RNP, TRBC22 RNP, and donor template was generated for each reaction. Different amounts of TRAC3 RNP (60 pmol or 30 pmol) and / or TRBC22 RNP (60 pmol, 30 pmol, 20 pmol, or 10 pmol) were used to determine the effect of TRBC22 RNP on knock-in efficiency. An appropriate volume of master mix (depending on the pmol of TRAC3 and / or TRBC22 RNP used) was added per well, followed by the addition of 2 x 106 cells per reaction.

[0404] All electroporation conditions were 48 hours after activation in Lonza P3 buffer with code EW113. For sequential electroporations: (1) first electroporation with TRAC3 RNP and donor template (PUC57 vector) using code EW113 (after 48 hours of T cell activation), (2) 24 hours after the first electroporation, TRBC22 RNP was electroporated 24 hours using codes EW113 or EO100. The RNP conditions used were as follows: TRAC3 RNP only 60 pmol or 30 pmol, the following combinations of TRAC3 RNP / TRBC22 RNP: 60 pmol / 30 pmol 60 pmol / 20 pmol, 60 pmol / 10 pmol, 30 pmol / 30 pmol or 30 pmol / 10 pmol. After each electroporation, cells were activated and incubated at 37° C. for 15 minutes (with P3 buffer only).

[0405] The results are shown in Figures 111-123. Figure 111A shows the percentage of Neon positive TRAC+ / -TRBC samples on day 8 for (from left to right): 60 pmol TRAC3 only, 30 pmol TRAC3 + 30 pmol TRBC21, and 30 pmol TRAC3 + 30 pmol TRBC22. Figure 111B shows the percentage of Neon positive samples on day 8 for (from left to right): 60 pmol TRAC3, 60 pmol TRAC3 + 30 pmol TRBC22, 60 pmol TRAC3 + 20 pmol TRBC22, 30 pmol TRAC3, 30 pmol TRAC3 + 30 pmol TRBC22, and 30 pmol TRAC3 + 10 pmol TRBC22. FIG. 112 shows the percentage (FIG. 108A) and number (FIG. 108B) of neon positive samples for (from left to right) 60 pmol TRAC3 RNP only, 30 pmol TRAC3 RNP only, a 60 pmol / 30 pmol mixture of TRAC3 RNP / TRBC22 RNP, a 60 pmol / 20 pmol mixture of TRAC3 RNP / TRBC22 RNP, a 60 pmol / 10 pmol mixture of TRAC3 RNP / TRBC22 RNP, a 30 pmol / 30 pmol mixture of TRAC3 RNP / TRBC22 RNP, and a 30 pmol / 10 pmol mixture of TRAC3 RNP / TRBC22 RNP. Sequential electroporation was tested by first electroporating TRAC3 RNP and donor template (PUC57 vector) using pulse code EW113, followed by TRBC22 RNP 24 hours later using pulse code EW113 or EO100. The data are shown in Figures 109A-C. Figure 109A shows the percentage (or frequency) of Neon positive samples. Figure 109B shows the number of Neon positive samples. Figure 109C shows the frequency of TCRab+Neon negative cells. Following sequential electroporation, cells were cultured in 24-well (24w) or 48-well (48w) plates.

[0406] Example 28. Different donor DNA formats. T cells were activated using Prime medium containing TRANSACT™ (1:100) + 25ng / mL IL-7 and 50ng / mL IL-15. TRAC3 and TRBC22 RNPs were generated by mixing TRAC3 sgRNA (IDT GMP Genes) and Cas9 at a 3:1 sgRNA:Cas9 molar ratio and incubating at room temperature for 15 minutes. 2μL of 100mg / mL PGA was used per electroporation reaction. A master mix of PGA, TRAC3 RNP, and donor template was generated for each reaction. The order of addition was as follows: 1) RNP (pre-incubated at RT for 15 minutes); 2) template; 3) PGA (1μL of 100μg / μL stock); 4) cells (1.5 million per cuvette). Different template formats were tested: PUC57 plasmid, nanoplasmid, and minicircle. Template amounts were normalized to obtain similar numbers of molecules for comparison. All electroporation conditions were 48 hours post activation in Lonza P3 buffer with code EW113. After electroporation, samples were left at 37° C. for 15 minutes (either with P3 buffer alone or with the addition of 75 μL of warmed plain Prime-XV without pipetting) and after this incubation, cells were transferred to 24- or 48-well plates containing Prime-XV medium with cytokines.

[0407] Figure 110C and Figure 11D show the frequency (Figure 110C) and number (Figure 110D) of Neon positive cells. Experiments were performed using only TRAC3 RNP. Templates used were PUC57 vector (GS), nano plasmid (nano) and minicircle (mini).

[0408] Data for pUC57 vs. nano plasmid vs. minicircle DNA on day 6 are shown in Figures 114A-114B. Figure 114A shows the percentage (top graph) and absolute number (bottom graph) of mNeon+TCR+ samples. Figure 114B shows the amount of DNA used in the experiment. Data for pUC57 vs. nano plasmid vs. minicircle DNA on day 8 are shown in Figures 115A-115B. Figure 115A shows the percentage (top graph) and absolute number (bottom graph) of mNeon+TCR+ samples. Figure 115B shows the amount of DNA used in the experiment. Figure 116A shows the percentage of cell viability on day 6 and Figure 116B shows the percentage of cell viability on day 8. The change in knock-in cell number over time from day 6 to day 8 is shown in Figure 117.

[0409] Example 29. pUC57 Plasmid, Minicircle (Mini), Nano Plasmid (Nano) and PCR Templates Donor DNA encoding the first 2A peptide, the mNeon fluorescent protein, the second 2A peptide and the TCRa VJ domain was used.

[0410] Data for pUC57 vs. Nano Plasmid vs. Minicircle DNA on day 6 are shown in Figures 120A-120B. Figure 120A shows the percentage (top graph) and absolute number (bottom graph) of mNeon+TCR+ samples on day 6. Figure 120B shows the amount of DNA used in this experiment. Data for pUC57 vs. Nano Plasmid vs. Minicircle DNA on day 8 are shown in Figures 121A-121B. Figure 125A shows the percentage (top graph) and absolute number (bottom graph) of mNeon+TCR+ samples on day 8. Figure 121B shows the amount of DNA used in this experiment. Sample plots for day 8 of pUC57, Nano Plasmid, Minicircle and PCR samples are shown in Figure 122. Cell viability with equivalent molecules of each template is shown in Figure 123, with data for day 6 shown in the top graph and data for day 8 shown in the bottom graph. Figure 124 shows the change in knock-in cell number over time from day 6 to day 8.

[0411] Example 30. TCR-beta targeting guide RNA and plasmid vs. nanoplasmid templates. Donor DNA encoding the first 2A peptide, the mNeon fluorescent protein, the second 2A peptide and the TCRa VJ domain was used.

[0412] T cell activation conditions: Prime, TRANSACT™ (1:100) + 25 ng / mL IL-7 and 50 ng / mL IL-15. RNPs were generated by mixing sgRNA and Cas9 at a 3:1 sgRNA:Cas9 ratio and incubating at room temperature for 15 minutes. TRAC3, TRBC22 and TRBC19 sgRNAs were resuspended at 200 μM and a master mix for electroporation was generated by mixing: (1) 2 μL of 100 mg / mL PGA per reaction, (2) TRBC22 or TRBC19 RNP, (3) TRAC3 RNP, and (4) donor template. The exact amounts of reagents varied based on pmol of RNP and amount of donor template used per electroporation condition. The following RNP conditions were used: TRAC3 only 60 pmol or 30 pmol, TRAC RNP / TRBC RNP 60 pmol / 30 pmol, 60 pmol / 10 pmol, 30 pmol / 30 pmol (TRBC22 or TRBC19 RNP). For titration of donor templates, the following amounts of PUC57 (GenScript) and nanoplasmid templates were used: PUC57 (GenScript): 8 μg, 6 μg, 4 μg and 2 μg, and nanoplasmid: 10 μg, 8 μg, 6 μg, 4 μg and 2 μg. Using the μg amount of PUC57 (GenScript) as a reference, the same number of nanoplasmid molecules were used for electroporation. Cells were electroporated 48 hours after activation in Lonza P3 buffer containing code EW113. After electroporation, cells were incubated at 37 °C for 15 min (only in P3 buffer) before being transferred to culture plates containing pre-warmed PRIME medium and cytokines.

[0413] Figure 125 shows (from left to right) the percentage of Neon positive samples and (right) the number of Neon positive samples for (from left to right) 60 pmol TRAC3 RNP (GenScript) only with PUC57 template, 60 pmol TRBC22 RNP and 30 pmol TRBC19 RNP (GenScript) with PUC57 template, 60 pmol TRBC22 RNP and 10 pmol TRBC19 RNP (GenScript) with PUC57 template, 30 pmol TRAC3 RNP (GenScript) only with PUC57 template, 30 pmol TRBC22 RNP and 30 pmol TRBC19 RNP (GenScript) with PUC57 template, 30 pmol TRAC3 RNP only with nano plasmid, and 30 pmol TRBC22 RNP and 30 pmol TRBC19 RNP with nano plasmid.

[0414] Figure 126A shows knock-in efficiency at day 5 (left graph) and day 7 (right graph) for GenScript vs. Nanoplasmid template. Samples were electroporated with 30 pmol of TRAC3 RNP and 30 pmol of TRBC22 RNP. Figure 126B shows the number of knock-in cells at day 5 and day 7.

[0415] Day 7 flow cytometry data for TRBC RNP titration (60 pmol) is shown in Figure 127. Day 7 flow cytometry data for TRBC RNP titration (30 pmol) is shown in Figure 128. Day 7 flow cytometry data for PUC57 (GenScript) plasmid titration is shown in Figure 129. Day 7 flow cytometry data for Nanoplasmid titration is shown in Figure 130.

[0416] Example 31. TCR knock-in with pUC57 and nanoplasmid. T cell activation conditions: Prime, TRANSACT™ (1:100) + 25ng / mL IL-7 and 50ng / mL IL-15. RNP+ / -PGA preparations included: TRAC3 RNP and TRBC22 RNP, 200μM and 30pmol of all sgRNAs, respectively; and 15 minutes room temperature incubation for RNP generation (3:1 sgRNA:Cas9) with 2μL of 100mg / mL PGA. Templates used were PUC57 (Neon, WT1 TCR) or Nanoplasmid (Neon). We used an equivalent number of Nanoplasmid molecules with 4μg of PUC57 as a reference. Electroporation was followed by incubation at 37°C for 15 minutes (with P3 buffer only). Electroporation conditions (all 48 hours after activation in Lonza P3 buffer with code EW113) included testing of PGA requirement (PGA: + / - for Neon PUC57, Neon Nano Plasmid and WT1_5213 PUC57 templates); testing of template addition order (RNP / PGA mix added first or last); testing of native (non-codon optimized) vs. codon optimized WT1 TCR (WT1_Ref and WT1_64-9).

[0417] The results of Neon knock-in are shown in Figures 131A-131B. 4 μg of PUC57 plasmid or the equivalent number of molecules of Nano plasmid were used. Figure 131A shows the percentage of Neon positive samples on day 7, and Figure 131B shows the number of Neon positive samples.

[0418] Figure 132A shows the percentage of WT1+ (of total CD8+) samples on day 7. Figure 132B shows the percentage of CD3+ samples among CD8+ cells on day 7. Figure 132C shows the percentage of WT1+ among total CD3+CD8+ cells on day 7. For the "no PGA" condition, the template was added last. 4 μg of PUC57 plasmid was used.

[0419] Figure 133 shows the number of WT1 TCR+ cells on day 7. Conditions graphed, from left to right, are template first, template last, and no PGA. For each condition, data is given for, from left to right, WT1_5213, WT1_Ref_Native, WT1_Ref_CodOpt, WT1_64_9_Native, WT1_64_9_CodOpt.

[0420] Figure 134 shows flow cytometry data for Neon template knock-in at day 7. Figure 135 shows flow cytometry data for WT1_5213 at day 7. Figure 136 shows flow cytometry data for WT1_Ref (all PGAs included) at day 7. Figure 137 shows flow cytometry data for WT1 64_9 (all PGAs included) at day 7.

[0421] [Table 10]

[0422] [Table 11-1] [Table 11-2]

[0423] [Table 12-1] [Table 12-2]

[0424] Example 32. Functional characterization of TCR engineered T cells. This assay determines the potency of TCR T cells by measuring the ability of engineered T cells to induce cytolysis of specific target cells as measured by the specific % loss of target cells using FACS. On the day of the assay, target cells peptide pulsed (3-4 hours) and bystander cells CellTrace staining were prepared in the morning. Killing assays were prepared by incubating T cells with pre-prepared target / bystander cells. Cell killing was measured approximately 18 hours later using FACS. Assay diluent was T cell growth medium (X-VIVO or PRIME-XV + IL-7 and IL-15).

[0425] Preparation of Reagents. Preparation of peptide stock solutions: peptides were diluted to 10 mM in DMSO. CellTrace™ stock solutions were prepared according to the manufacturer's protocol, i.e., the CellTrace™ solution was reconstituted by adding an appropriate volume (e.g., 20 μL) of DMSO (component B) to one vial of CellTrace™ reagent (component A) and mixing. Further dilutions were made in DMSO and stored at ≦−20° C. after reconstitution. LIVE / DEAD™ stock solutions were prepared according to the manufacturer's protocol, i.e., the LIVE / DEAD™ stock solution was reconstituted immediately before use by adding an appropriate volume (e.g., 50 μL) of DMSO (component B) to one vial of LIVE / DEAD™ reagent (component A) and mixing.

[0426] Assay procedure: Peptide dilutions were prepared in target cell growth medium in deep well 96-well plates as shown in Table 13.

[0427] [Table 13]

[0428] Preparation of cells for the assay: All steps were performed in a biosafety cabinet. Preparation of target cells: 0.05M target cells per well; 1) cell viability and cell concentration were determined. Cells were centrifuged in conical tubes at 300 x g for 5 min at ambient temperature and the supernatant was discarded. Cell pellets were resuspended in target cell growth medium in the total volume cell seeding suspension calculated for the assay, and 50 µL of cell suspension per well was seeded into a round-bottom 96-well plate, overlaid with the appropriate peptide dilution, mixed gently by pipetting up and down, and incubated at 37 °C for 3-4 h.

[0429] Bystander cell preparation: This format used 0.05 M bystander cells per well and CellTrace™ Far red was used at a final concentration of 0.1 nM. Cells were centrifuged at 300 x g for 5 min at ambient temperature and diluted to 1 x 10 in 15 or 50 mL conical tubes. 6 The cells were resuspended in PBS at a density of 100 μl / mL. CellTrace (1 μl of a 100 nM working solution in DMSO) was added per mL of cell suspension in PBS. The cells were mixed and incubated for 20 min at room temperature protected from light. Five times the original chromosome volume of culture medium (containing at least 1% protein) was added to the cells and incubated for 5 min. This step removes any free dye remaining in solution. The cells were pelleted by centrifugation (300 × g for 5 min at ambient temperature) and aliquoted at 1 × 10 6 The cells were resuspended in fresh, warmed complete culture medium at a density of 100 / mL. Cells were incubated for at least 10 minutes before analysis to allow for acetic acid hydrolysis of the CellTrace™ reagent.

[0430] Wash target cells: After 3-4 h incubation, cells were washed twice with PBS to remove free peptide. Cells were resuspended in 50 μL of assay diluent ready to be mixed with bystander and effector T cells.

[0431] Transfer bystander cells to plates: Transfer the bystander cell suspension to assay diluent (1 x 10) by centrifugation at 300 x g for 5 min at ambient temperature. 6 / mL density) and 50 μL was added to the plate containing 50 μL of peptide-loaded target cells and mixed by pipetting up and down approximately twice.

[0432] Preparation of effector cells: KI TCR T cells recognizing specific peptides were provided after transfection. KI TCR T cells and non-transfected (or mock) control cells were counted and the volume of cell suspension required was calculated (0.1 x 10 per reaction for a 1:1 T:E ratio). 6 Cells were centrifuged at 300 × g for 5 min at ambient temperature and diluted with assay diluent (1 × 10 6 The cells were resuspended at 100 µg / mL density.

[0433] Transfer effector cells to plates: 100 μL of T cell suspension was added to the plates containing peptide-loaded target cells and bystander cells. Plates were incubated overnight at 37° C. in a humidified incubator.

[0434] Assay readout: The plate was centrifuged at 300 x g for 5 min and 180 μL of the supernatant was transferred to another 96-well plate and stored at -80 °C for later analysis. LIVE / DEAD reconstituted dye was diluted in FACS buffer at a ratio of 1:1000 (e.g., 5 μL of LIVE / DEAD reconstituted dye + 5 mL of FACS), 100 μL of LIVE / DEAD working solution was added to each well and mixed 2-3 times by gently pipetting up and down. The plate was incubated at 4 °C in the dark for 10 min. FcR blocking solution was diluted in FACS buffer at a ratio of 1:20. To wash out the LIVE / DEAD dye, 100 μL of FACS buffer was added to each well and centrifuged at 300 x g for 5 min at room temperature. The supernatant was decanted and 50 μL of FcR blocking solution was added to each well and mixed. The plate was incubated at 4 °C in the dark for 10 min.

[0435] CD8 and CD137 antibody cocktail solutions were prepared by combining CD8, CD137 and FACS buffer to make a 2x staining antibody mix for example for 80 wells: 4000μL FACS buffer + 40μL CD8 + 400μL CD137. At the end of the FcR blocking incubation, 50μL of antibody cocktail was added to each well and mixed by pipetting. The plate was incubated in the dark at 4°C for 30 minutes, then the cells were washed twice to remove free antibody. 100μL of FACS buffer was added to each well, centrifuged at 300×g for 5 minutes at room temperature, and the supernatant was decanted. 200μL of FACS buffer was added to each well, centrifuged at 300×g for 5 minutes at room temperature, then the supernatant was decanted. 100μL of FACS buffer was added to each well and mixed by pipetting up and down approximately 2-3 times.

[0436] Samples were analyzed immediately using BD FACSLyric (gating strategy: lymphocytes>single cells>live cells>non-T (CD8-) and T cells (CD8+):non-T cells: Farred+ / Farred-; T cells: CD137+). FlowJo was used for FACS gating and analysis.

[0437] A flow cytometry-based cell killing assay was used to determine the percent cell killing of T2 target cells when co-incubated with neo-TCR T cells. T2 cells (ATCC, CRL-1992) were preloaded with target peptide WT1 (VLDFAPPGA, SEQ ID NO: 71) (0-100 μM) by incubating the cells in the presence of peptide for 3-4 hours at 37°C, washed twice with PBS, and then exposed to WT1 TCR-T cells in a cell killing assay. No peptide or irrelevant peptide-pulsed target cell populations were used as negative controls. Target cells were then mixed with neo-TCR T cells at a 1:1 ratio for approximately 20 hours to allow T cell-induced cell killing to occur.

[0438] To measure percent cell killing, cell mixtures from each assay well were stained with antibodies to label CD8+ (anti-CD8, Biolegend: 406515) and CD137+ (anti-CD137, Biolegend: 309822) cells, and a viability dye (fixable Aqua dye, Thermofisher: L 34965) to label dead cells. After live cell selection, cells were further divided into CD8+ T cells and CD8- target cells. The % loss of peptide-pulsed target cells per reaction was calculated based on the negative control. Peptide-specific T cell activation during the cell killing assay was also assessed by determining the %CD137+ signal in the CD8+ T cell population.

[0439] FIG. 138 shows a cartoon representation of target cell killing and a graph of percent killing compared to control for neo-TCR and no-TCR.

[0440] Example 33. Electroporation pulse code. Table 14 below shows a summary of the knock-in percentage across the three donors for the various buffers / codes.

[0441] Table 14. Overview of NeoT pulse codes. [Table 14]

[0442] Example 34. Knockout of TCR-alpha or TCR-beta Purified (non-activated) CD8+ T cells were electroporated using Lonza P2 buffer and pulse code EH 100. RNPs were generated by incubating the respective sgRNA and Cas9 (3:1 ratio) for 15 min at room temperature. 60 pmol of RNP was added per electroporation. After electroporation, cells were cultured in X-VIVO medium supplemented with IL-7 and IL-15. Two days after electroporation, cells were harvested and TCR surface expression was analyzed by flow cytometry.

[0443] Example 35. Highly efficient non-viral CRISPR / Cas9-mediated gene editing of human T cells using plasmid donor DNA CRISPR-mediated gene knockout using Cas9-ribonucleoprotein (RNP) delivery into primary human T cells represents a rapid and versatile approach to introduce gene loss-of-function perturbations into this clinically relevant cell type (Schumann et al., 2015; Hendel et al., 2015; Seki and Rutz, 2018; Oh et al., 2019). However, methods for gain-of-function studies and stable expression of therapeutic transgenes in T cells rely mostly on viral delivery techniques that do not allow for precise editing of genes.

[0444] Lentiviruses or retroviruses have been widely used by the research community and have also been applied for the introduction of chimeric antigen receptors (CARs) or T cell receptors (TCRs) in the production of adoptive T cell therapies (Wang and Riviere, 2016; Zhang et al., 2017). Transposon-based gene delivery methods, such as the piggyBac and Sleeping Beauty systems, have been developed as non-viral alternatives (Monjezi et al., 2017; Kebriai et al., 2016; Hudecek and Ivics, 2018). Although these approaches allow highly efficient and stable gene delivery, they insert the transgene into the genome by random integration and are not suitable for precise gene editing. Moreover, the random nature of the integration process poses the risk of insertional mutagenesis (Hacein-Bey-Abina et al., 2008, 2003; Modlich et al., 2009).

[0445] Homologous recombination repair (HDR) of double-stranded breaks introduced by targeted gene editing methods such as transcription activator-like effector nucleases (TALENs), zinc finger nucleases (ZFNs) or CRISPR / Cas9 can be utilized to make deliberate specific changes to genomic sequences, including inserting longer stretches of DNA at defined locations within the T cell genome (Li et al., 2020; Singh et al., 2017). Viral vectors, particularly adeno-associated viruses (AAVs), have been used to deliver donor DNA templates for HDR-mediated targeted gene knock-in in T cells (Sather et al., 2015; Wang et al., 2016; Eyquem et al., 2017; Choi et al., 2019). This approach facilitated the integration of the CAR into the T cell receptor alpha constant (TRAC) region locus (Eyquem et al., 2017), which placed the CAR under the transcriptional control of the endogenous TCR promoter, resulting in improved CAR performance (Eyquem et al., 2017). Several groups subsequently reported high editing efficiency using AAV-based repair templates (Choi et al., 2019; Vakulskas et al., 2018; Dai et al., 2019). However, AAV production and purification not only represent a significant clinical manufacturing challenge (Loo and Wright, 2016; Halbert et al., 2018; Davidsson et al., 2020), but also limit the more widespread use of this approach in the research community.

[0446] Recently, a series of papers demonstrated that linear double-stranded (ds) DNA donor templates can be co-delivered with Cas9-RNP for directional insertion of full-length coding sequences into the T cell genome (Nguyen et al., 2019; Roth et al., 2018; Schober et al., 2019), thus facilitating not only the generation of point mutants but also the targeted integration of one or several expression constructs, including CAR or TCR. Notably, the need to produce and purify linear dsDNA of sufficient quantity and quality, in addition to the modest knock-in efficiency observed with this donor DNA format, constitutes a serious limitation to the utility and scalability of this method. Here, we addressed these challenges by developing an efficient and scalable protocol for CRISPR / Cas9-mediated non-viral gene editing in primary human T cells using readily available plasmid-based donor templates.

[0447] Improved CRISPR-mediated gene knock-in efficiency and cell recovery with plasmid-based homology donors compared to linear DNA templates.

[0448] Based on previous work, including protocols for CRISPR-Cas9-mediated gene perturbation in human and mouse T cells (Seki and Rutz, 2018; Oh et al., 2019), and a report describing the use of linear double-stranded DNA as a repair template (Roth et al., 2018), we set out to develop a robust, efficient, and scalable protocol for non-viral CRISPR / Cas9-mediated gene knock-in in primary human T cells. To circumvent the labor-intensive steps involved in PCR-based generation and purification of linear dsDNA and to facilitate manipulation with sequence-verified templates, we investigated the use of plasmid DNA.

[0449] In addition to traditional plasmid backbones (i.e., pUC57), which are approximately 2.5 kb in size, several smaller circularized DNA backbones have been described for cell engineering applications, including minicircle, midge, and nanoplasmids (Hardee et al., 2017). Commercially available nanoplasmids consist of backbones of less than 0.5 kb (Luke et al., 2009; Williams et al., 2006). Because double-stranded DNA is toxic to T cells, the use of these minimal vectors can maximize the donor element to plasmid backbone sequence ratio and reduce the total amount of DNA required for transfection. We designed a donor template encoding the alpha chain of the NY-ESO1-specific T cell receptor 1G4 (Li et al., 2005), as well as the fluorescent protein mNeonGreen (mNG), which targets the T cell receptor alpha constant region (TRAC).

[0450] Here, the transgene and TRAC homology arms (~500 base pairs, bps) were encoded as linear dsDNA, pUC57 conventional plasmid, or as nanoplasmid (Figure 1A,D,G). Similar to our previous approach to CRISPR / Cas9 knockout in T cells (Seki and Rutz, 2018), we optimized the process separately for CD4 and CD8 T cells, rather than working with a mixed cell population. Here, we transfected human CD8 T cells in PRIME-XV medium supplemented with the cytokines IL-7 and IL-15. + We isolated T cells and activated them with TransAct, a bead-free colloidal polymer nanomatrix conjugated to humanized CD3 and CD28 agonists. +T cells were cultured for 48 hours and then nucleofected with Cas9-RNP containing chemically synthesized single guide (sg) RNA targeting the TRAC locus along with the respective donor DNA template (Lonza Biosciences 4D Nucleofection System). Importantly, all studies were performed using the R691A HiFi-Cas9 variant to minimize CRISPR / Cas9 off-target events (Vakulskas et al., 2018). Furthermore, we titrated the amount of linear DNA and plasmid DNA side-by-side and determined knock-in efficiency, cell viability and cell recovery by flow cytometry 3 days after electroporation.

[0451] Under these conditions, we found that 4 μg of linear DNA resulted in a maximum knock-in rate of 28.8-32.1% across four independent human T cell donors (Figure 140B,C). However, this amount of DNA compromised cell viability and resulted in low T cell recovery (Figure 140B). When using 1 μg of linear DNA instead, the knock-in rate was slightly lower at 13.9-20%, but cell viability and recovery were comparable to transfection with DNA-free Cas 9-RNP (Figure 140B,C). Titration of pUC57 plasmid DNA revealed higher knock-in rates compared to linear dsDNA, ranging from 33.5-44% when as much as 6 μg of plasmid was used (Figure 140E,F). However, this condition resulted in suboptimal viability and cell recovery, although 2 μg of plasmid DNA produced knock-in rates of 24-36.2%, with minimal impairment of cell viability and optimal recovery of edited cells (Figure 140E,F). The nano-plasmid format allowed knock-in rates of 36.2-46.6% with 4 μg of DNA, with minimal impact on cell viability and recovery, when compared to Cas9-RNP only conditions (Figure 140H,I). We also evaluated knock-in efficiency, cell viability and recovery when culturing T cells in RPMI-1640 supplemented with 10% FBS (R10), a more widely used and available culture medium for T cells. Experiments performed in R10 yielded results that were nearly comparable to cultures in PRIME XV medium for linear dsDNA templates, but somewhat lower knock-in rates (29.5-37.4%) for nanoplasmid templates (147A-F), demonstrating that R10 can be used as an alternative to PRIME-XV medium. As previously described (Nguyen et al., 2019), the addition of negatively charged poly-L-glutamic acid (PGA) or one encoding a truncated Cas9 target sequence (tCTS) to the donor template did not affect targeting efficiency (147G,H).

[0452] Our initial studies suggested that the nanoplasmid had favorable qualities for gene targeting in T cells. However, regardless of the format, high amounts of donor DNA compromised T cell viability. Therefore, we sought to better characterize the stress response induced in T cells after exposure to nanoplasmid. We compared cytokine production after overnight culture of CD8 T cells transfected with sgTRAC Cas9-RNP and nanoplasmid or Cas9-RNP alone to unedited T cells. Nanoplasmid transfection (but not Cas9-RNP alone) induced IFN-a, IFN-g, TNF-a, and IL-2 levels (Figure 148A). We also performed RNA sequencing comparing T cells transfected with Cas9-RNP+nanoplasmid or Cas9-RNP alone. Interferon-a and -beta as well as TNF-a and inflammatory response signatures were upregulated in nanoplasmid transfected cells (Figure 148B), consistent with our cytokine data. Importantly, linear DNA and nanoplasmid induced qualitatively and quantitatively similar responses (Figure 148C-H). In conclusion, we demonstrate that plasmid DNA donor templates can upregulate CD8 + We found that this allows for highly efficient CRISPR / Cas9-mediated knock-in in T cells, circumventing the need for production and purification of linear dsDNA. Nanoplasmid vectors generally yielded more consistent results with higher knock-in rates, although traditional plasmid backbones such as pUC57 can be used successfully with careful titration.

[0453] Optimization of CRISPR / Cas9-mediated gene knock-in with plasmid-based donor DNA in CD4 and CD8 T cells

[0454] After confirming the beneficial properties of plasmid-based gene editing, we sought to further optimize this process and extend our protocol to enable modification of CD4+ T cells. Thus, we compared pUC57 and nanoplasmid donor templates with a fixed transgene containing a bicistronic mNG reporter and 1G4 TCR alpha chain, along with TRAC homology arms of different lengths ranging from 0.1 to 2 kb, using CD8+ T cells. Transgene knock-in efficiency increased between 0.1-0.5 kb homology arm lengths, regardless of the backbone used, with a slight improvement in knock-in efficiency observed with nanoplasmids containing homology extended to 2 kb (Figure 141A,B). Importantly, cell viability and cell recovery were comparable for both backbones, except for the 2 kb homology arm pUC57 construct, which significantly impaired cell viability (Figure 141A,B). We conclude that a homology arm length of 0.5 kb was optimal in terms of knock-in efficiency and cell recovery, but longer arms can be used with the nanoplasmid without compromising cell viability (Figure 141A, B).

[0455] The timing of Cas9-RNP / Nanoplasmid delivery after T cell activation is another parameter for optimization. We found that transfection 24 hours after T cell activation dramatically reduced knock-in efficiency compared to later transfection time points (Figure 141C). Cas9-RNP / Nanoplasmid delivery for 48-72 hours resulted in the highest cell recovery (Figure 141C). Finally, we tested several nucleofection pulse codes with Cas9-RNP and Nanoplasmid templates and found that EH115 resulted in the highest targeted gene editing efficiency. Other pulse codes, such as EH111, further increased cell recovery with only a minimal decrease in knock-in efficiency (Figure 141D). The desired application should drive the final selection of nucleofection conditions depending on whether efficiency or cell recovery is a higher priority. For subsequent studies, we used EH115.

[0456] Using the above conditions as a baseline, we cultured human CD4+ cells in PRIME XV medium supplemented with recombinant IL-7 and IL-15. + T cells were isolated and cultured. We also added IL-2 and activated with TransAct for 48 hours. We then nucleofected with TRAC-mNG nanoplasmid donor template and sgTRACCas9-RNP as before and assessed knock-in rates, cell viability and recovery by flow cytometry. We observed knock-in rates of 32.2-41.9 across three independent human T cell donors, with viability and recovery rates consistent with our data from CD8 T cells (Figure 141E, Figure 141F). We concluded that our approach is equally applicable to human CD4 and CD8 T cells.

[0457] Efficient non-viral T cell receptor editing using plasmid DNA donors

[0458] Next, we applied our protocol to TCR editing in T cells. The introduction of a transgenic TCR with the desired antigen specificity also requires knockout of the endogenous TCR to prevent mispairing with its alpha and beta chains. Using our targeting strategy, the insertion of a transgenic TCR containing both alpha and beta chains in the TRAC locus on human chromosome 14 disrupts the endogenous TCR alpha gene. However, the existing TCR beta chain on human chromosome 7 needs to be knocked out separately. Therefore, we designed a single sgTRBC sequence that simultaneously targets the T cell receptor beta chain constant domains 1 and 2 (TRBC1 and TRBC2) (Figure 142A), resulting in a complete loss of TCR expression detected by flow cytometry (Figure 149A). We next designed nanoplasmid donor templates encoding TCRs with known specificities, NY-ESO1-specific 1G4 and CMVA2 / pp65495-503-specific TCR6-2 (Schober et al., 2019) and human CD19CAR (Bloemberg et al., 2020), all targeting the TRAC locus (Figure 142B).

[0459] As previously, we used CD8 + T cells were activated and cultured for 48 h, co-transfected with sgTRAC and sgTRBC-containing Cas9-RNP together with 2 μg of TCR- or CAR-encoding nanoplasmid, and TCR expression was assessed by flow cytometry 5 days later. We detected 1G4 TCR expression on the surface of 44.9–54% of T cells with minimal impact on cell viability (Figure 142C,D). 6 Starting with CD8 T cells, we obtained 0.88–2.88 × 10 61G4 TCR positive cells were recovered (Figure 142D). T cells negative for 1G4 expression did not express endogenous TCR complexes on their surface, demonstrating highly efficient gene knockout (Figure 142C). Transfection with nanoplasmids encoding pp65 TCR and CD20 CAR constructs yielded similar results, with knock-in rates of 44.4-53.3% and 46.3-57.9%, respectively (Figure 142E,G) and 1.7-3.1 x 10 6 The cell recovery of edited cells was (Figure 142F,H). Again, endogenous T cell receptors were knocked out in virtually all T cells (Figure 142E,G).

[0460] When we performed TCR editing in isolated CD4 T cells using the same targeting strategy and TCR or CAR donor template, we observed knock-out and knock-in rates comparable to our results in CD8 T cells (Figure 149B-G). When attempting simultaneous multiplex gene editing such as the TRAC and TRBC knock-in / knock-out approach used here, the occurrence of chromosomal translocations between the cut sites must be considered. We designed a ddPCR assay to quantify all possible translocation events involving the TRAC, TRBC1 and TRBC2 loci (Figure 149H). Translocations between the TRAC and TRBC1 or TRBC2 loci occurred at frequencies between 0.01 and 4.4% depending on the translocation and donor orientation, whereas recombinations of the adjacent TRBC1 and TRBC2 loci (corresponding to a 9.3 kb deletion) occurred at frequencies between 13.1 and 19% (Figure 149I). These figures are consistent with or slightly high...

Claims

1. 1. A method for producing engineered T cells, the method comprising: a) contacting a T cell with a first ribonucleoprotein particle (RNP) and donor DNA under conditions that allow the RNP and donor DNA to enter the T cell, wherein the first RNP comprises a first guide RNA that targets an endogenous TCR-alpha locus, and the donor DNA comprises a nucleic acid sequence that includes genes encoding a polypeptide comprising an exogenous TCR-beta and an exogenous TCR-alpha or a portion thereof; b) incubating the T cells for a period of time; c) culturing the cells in culture for a period of time to allow the donor DNA to be inserted into the endogenous TCR-alpha locus, thereby forming an engineered T cell; Including, the amount of donor DNA is about 0.0004 pmol / μL to about 0.04 pmol / μL, and the donor DNA is on a Nanoplasmid; Methods for generating engineered T cells.

2. The method described in claim 1, wherein the T cell is contacted with a second RNP comprising a second guide RNA that targets an endogenous TCR-beta locus.

3. 2. The method of claim 1, wherein the first RNP comprises a first gene-editing protein and the ratio of the first guide RNA to the first gene-editing protein is between 1:1 and 100:

1.

4. 3. The method of claim 2, wherein the second RNP comprises a second gene-editing protein, and the ratio of the second guide RNA to the second gene-editing protein is between 1:1 and 100:

1.

5. The method of claim 1, wherein the engineered T cells do not express endogenous TCR-beta protein.

6. 2. The method of claim 1, wherein the first guide RNA targets exon 1, 2, or 3 of TRAC: TRAC1 (SEQ ID NO: 7), TRAC2 (SEQ ID NO: 8), TRAC3 (SEQ ID NO: 9), TRAC4 (SEQ ID NO: 10), TRAC5 (SEQ ID NO: 11), TRAC6 (SEQ ID NO: 12), TRAC7 (SEQ ID NO: 13), TRAC8 (SEQ ID NO: 14), TRAC9 (SEQ ID NO: 15), TRAC10 (SEQ ID NO: 16), TRAC11 (SEQ ID NO: 17), TRAC12 (SEQ ID NO: 18), TRAC13 (SEQ ID NO: 19), TRAC14 (SEQ ID NO: 20), TRAC15 (SEQ ID NO: 21), or TRAC16 (SEQ ID NO: 22).

7. 7. The method of claim 6, wherein the first guide RNA comprises a nucleic acid sequence selected from SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, or SEQ ID NO:

22.

8. The second guide RNA is selected from the group consisting of exon 1 of TRBC: TRBC1 (SEQ ID NO: 23), TRBC2 (SEQ ID NO: 24), TRBC3 (SEQ ID NO: 25), TRBC4 (SEQ ID NO: 26), TRBC5 (SEQ ID NO: 27), TRBC6 (SEQ ID NO: 28), TRBC7 (SEQ ID NO: 29), TRBC8 (SEQ ID NO: 30), TRBC9 (SEQ ID NO: 31), TRBC10 (SEQ ID NO: 32), TRBC11 (SEQ ID NO: 33), TRBC12 (SEQ ID NO: 34), TRBC13 (SEQ ID NO: 35), TRBC14 (SEQ ID NO: 36), TRBC15 (SEQ ID NO: 37), TRBC16 (SEQ ID NO: 38), TRBC17 (SEQ ID NO: 39), TRBC18 (SEQ ID NO: 40), TRBC19 (SEQ ID NO: 41), TRBC20 (SEQ ID NO: 42), TRBC21 (SEQ ID NO: 43), TRBC22 (SEQ ID NO: 44), TRBC23 (SEQ ID NO: 45), TRBC24 (SEQ ID NO: 46), TRBC25 (SEQ ID NO: 47), TRBC26 (SEQ ID NO: 48), TRBC27 (SEQ ID NO: 49), TRBC28 (SEQ ID NO: 50), TRBC29 (SEQ ID NO: 51), TRBC30 (SEQ ID NO: 52), TRBC31 (SEQ ID NO: 53), TRBC32 (SEQ ID NO: 54), TRBC33 (SEQ ID NO: 55), TRBC34 (SEQ ID NO: 56), TRBC35 (SEQ ID NO: 57), TRBC36 (SEQ ID NO:

2. The method of claim 1, which targets TRBC14 (SEQ ID NO: 36), TRBC15 (SEQ ID NO: 37), TRBC16 (SEQ ID NO: 38), TRBC17 (SEQ ID NO: 39), TRBC18 (SEQ ID NO: 40), TRBC19 (SEQ ID NO: 41), TRBC20 (SEQ ID NO: 42), TRBC21 (SEQ ID NO: 43), TRBC22 (SEQ ID NO: 44), TRBC23 (SEQ ID NO: 45), TRBC24 (SEQ ID NO: 46), TRBC25 (SEQ ID NO: 47), or TRBC26 (SEQ ID NO: 48).

9. 9. The method of Claim 8, wherein the second guide RNA comprises a nucleic acid sequence selected from SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47, or SEQ ID NO:

48.

10. The method of any one of claims 1 to 9, wherein the conditions that allow the RNPs and the donor DNA to enter the cell comprise electroporation.

11. The method according to any one of claims 1 to 10, wherein the culture medium comprises cytokines, in particular said cytokines comprise IL-2, IL-7 and / or IL-15.

12. The method according to any one of claims 1 to 11, wherein step a) is carried out in the presence of a negatively charged polymer.

13. The method of any one of claims 1 to 12, wherein the amount of RNP is from about 0.2 pmol / μL to about 8 pmol / μL.

14. The method according to any one of claims 1 to 13, wherein the donor DNA comprises a left homology arm and a right homology arm, in particular the left homology arm and the right homology arm are homologous to the endogenous TCR locus.

15. 15. The method of claim 14, wherein the left homology arm is from about 50 bases to about 2000 bases in length, the left homology arm is from about 100 bases to about 1000 bases in length, or the left homology arm is from about 200 bases to about 800 bases in length.

16. 16. The method of claim 14 or 15, wherein the right homology arm is from about 200 bases to about 800 bases in length, from about 250 bases to about 700 bases in length, or from about 250 bases to about 700 bases in length.

17. 17. A method for producing a population of engineered T cells, comprising performing the method of any one of claims 1 to 16 on a population of T cells.

18. 18. The method of claim 17, wherein at least about 50% of said population of T cells is viable after step c).

19. 19. The method of claim 17 or 18, further comprising contacting the cells with a second RNP comprising a guide RNA that targets an endogenous TCR-beta locus, wherein at least about 5% of the population of T cells are recovered as engineered T cells, and wherein the engineered T cells express less than about 20% of the endogenous TCR-beta.

20. 20. An engineered T cell produced by the method of any one of claims 1 to 19.

21. A population of engineered T cells produced by the method of any one of claims 1 to 19.

22. A population of engineered T cells for use in a method of treating cancer in a subject, the method comprising: a) providing a population of T cells generated using the method of any one of claims 17 to 19; b) expanding said population of engineered T cells; and c) administering the expanded population of engineered T cells to the subject; A population of engineered T cells comprising:

23. 22. A pharmaceutical composition comprising the population of engineered T cells of claim 21 and a pharma- ceutically acceptable excipient.

24. A method described in any one of claims 1 to 19, wherein the efficiency of gene insertion is at least 27%.

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